WO2009022104A1

WO2009022104A1 - Fascaplysin derivatives and their use in the treatment of cancer

Info

Publication number: WO2009022104A1
Application number: PCT/GB2008/002678
Authority: WO
Inventors: Bhabatosh Chaudhuri; Sachin Govindrao Mahale
Original assignee: De Montfort University
Current assignee: De Montfort University
Priority date: 2007-08-13
Filing date: 2008-08-07
Publication date: 2009-02-19
Anticipated expiration: 2010-02-13

Abstract

A method of treating cancer comprising administering a compound of Formula (I), (II) or (III) to a patient.

Description

FASCAPLYSIN DERIVATIVES AND THEIR USE IN THE TREATMENT OF CANCER

The present invention relates to chemical compounds, and to their use in the treatment of cancer.

Use of chemotherapeutic anti-cancer agents is one of the standard current treatment protocols for the majority of cancers. However, there is still a need in the art for more effective and less toxic anti-cancer agents.

The phenomenon of progressive and uncontrolled cell growth that eventually leads to tumour development and other proliferative disorders underlines the fundamental role of the cell division cycle in cancer. The normal mitotic cell division cycle consists of four distinct phases: the gap phases G₁ and G₂, the DNA synthesis phase (S) and mitotic (M) phase. A dormant, quiescent cell at G₀ must be re-awakened with the help of growth-promoting factors and ushered into the G₁ phase of the cell cycle. The transition of the cell during the early G₁ to the 171Jd-G₁ phase is driven by growth promoting extra-cellular signals. Towards the end of mid-Gi, the cell crosses the restriction point and enters the late G₁ phase. This is a stage where the cell commits itself to complete a cell division cycle and then prepares itself for the synthesis of genomic DNA that takes place during the S phase of the cell cycle (Planas-Silva & Weinberg 1997; Blagosklonny & Pardee 2002). Once DNA replication is complete, the cell enters the G₂ phase. Misregulation or aberrations at these different stages in the normal cell cycle are responsible for the majority of human malignancies.

All of the major transitions in the cell division cycle, such as commitment to enter the cell cycle, initiation of the S phase and progression from G₂ into M phase, are tightly controlled by the activity of specific cyclin dependent kinases (Cdk-s) along with their regulatory partners, the cyclins (Morgan 1997; Grana and Reddy 1995; Kastan and Bartek 2004; Massague 2004; Murray 2004). There are at least twelve Cdk catalytic subunits (Cdk1-Cdk12) and seventeen cyclins (cyclin A-cyclin L1 ) known, most of which are clearly involved in cell cycle regulation (Pines 1999; Peng et al, 1998; Sherr 1996; Trembley et al, 2003; Guo and Stiller 2004; Sanchez and Dynlacht 2005).

The cyclins, as the name suggests, show a characteristic discontinuous expression over the different phases of the cell cycle. The D-type cyclins (cyclin D1 , D2 and D3) are expressed during early G₁ phase of the cell cycle. Their expression is triggered by the extra-cellular mitogenic signals received from various growth factors. The induction and nuclear localization of D-type cyclins is a consequence of the activation of mitogen-activated protein kinases (MAPK-s) by mitogenic signals. For example, cyclin D1 has been reported to be induced by the oestrogen receptor pathway where the small molecule oestrogen provides the extra-cellular mitogenic signal (Sabbah ef al, 1999; Hong ef al, 1998). D-type cyclins specifically activate cyclin dependent kinases 4 and 6 (Cdk4 and Cdk6). The transition of early Gi cells into late d that allows entry into the S phase is regulated by the activity of Cdk4/Cdk6-cyclin D complexes (Kato 1997). Cdk6 is thought to be a functional homologue of Cdk4, although recent reports indicate that Cdk6 may also actively participate in differentiation (Grossel and Hinds 2006).

Cyclin dependent kinase 4 (Cdk4) is crucially important because a cell's initial commitment to complete a cell division cycle depends on Cdk4 activity. Activation of Cdk4 at Go/d is also essential for the later G^/S transition in the cell cycle, at least in cells that contain a functional copy of pRb. Moreover, many human cancers are characterized by either over-expression of its activating partner cyclin D1 or loss of p16^INK4a which is a natural Cdk4 inhibitory protein (Bartek ef a/, 1996; Weinberg 1995). Although genetic abnormalities in Cdk-s are rare events, the Cdk4 protein is either overexpressed or amplified in a wide range of human tumours (Matsumoto ef al, 1999; Kim ef al, 1998). Even the target of Cdk4, the retinoblastoma protein (pRb), is inactivated in many tumours resulting in excessive cell proliferation.

The natural inhibitors of the cyclin dependent kinases (Cdkl-s) negatively control cell cycle progression. Two distinct families of Cdkls are known. The first family is INK4 (inhibitor of CdM), which includes the proteins, p16^INK4a, p15^INK4b, p18^INK4c and p19^INK4d. They specifically inhibit Cdk4 and Cdk6 but were initially discovered as inhibitors of Cdk4 only. The second family of proteins, which are known to be global inhibitors of Cdk activities, is the CIP/KIP (CDK inhibitory proteins/kinase inhibitory proteins) family and consists of the proteins p21^cιp1, p27^KIP1and p57^KIP2. The p16^INK4 protein, commonly known as p16, is a naturally occurring inhibitor of the cyclin dependent kinases 4 and 6 (Cdk4 and Cdk6), which normally bind to the regulatory D-type cyclins so that they can perform their functional role in the cell cycle. It is estimated that at least 60% of all cancers lack p16 functional activity (Shapiro ef al, 1995; Sakaguchi ef al, 1996). A correlation between pRb function and p16 expression inside the cells is well known and, in most cases, it is inversely proportional to the functional activity of the pRb protein (Tarn et al, 1994; Li et al, 1994). Generally speaking, cells which lack functional p16 contain active pRb. Conversely, when pRb is inactivated by viral oncogenes, p16 is usually over-expressed. In many cancers, either cyclin D1 is overproduced or p16 is inactive (Kataoka et al, 2000; Campbell et al, 2000; Seike et al, 2000).

The most extensively studied substrate for Cdk4/Cdk6 is the product of the tumour suppressor retinoblastoma gene (RB), which acts as a repressor for a number of genes required for GJS transition, and also initiation and completion of DNA synthesis. The phosphorylation of the retinoblastoma protein (pRb) by cyciin D bound Cdk4 (i.e. the Cdk4-cyclin D complex) leads to its functional inactivation and frees the E2F family members to perform their role in transcription. Members of the E2F family initiate a cascade of events that include further phosphorylation of pRb by Cdk2-cyclin E and Cdk2-cyclin A complexes. Cdk4-mediated phosphorylation of pRb compels the cell to commit itself towards completing one full cell division cycle (Bartek ef al, 1996; Lundberg and Weinberg 1998; Figure 1 ).

Many cancers are characterised by abnormalities in the biochemical pathway that pRb controls (Hickman et al, 2002; Chen et al, 2001 ; Bartek et al, 1996; Lundberg and Weinberg 1998). The retinoblastoma protein pRb/p105 is considered to be one of the key regulators of the cell division cycle. When pRb is unphosphorylated, it is active as a growth suppressor protein, whereas one reason for loss of the norma] pRb function in cells is hyper-phosphorylation of the pRb protein by mutated or over-expressed kinases such as cyclin D-Cdk4/6 complexes (Simin ef al, 2004).

Pharmacological inhibition of Cdk-s has been a productive strategy for the design and discovery of novel anti-cancer agents that specifically target the cell cycle, and the number of Cdk inhibitors entering clinical trials is increasing (Senderowicz and Sausville 2000; Knockaert et al, 2002; Sausville 2003; Senderowicz 2003a; Fischer and Gianella-Borradori 2005). Selective Cdk inhibitors are considered to be particularly useful as cancer therapeutics since they may minimise undesirable toxicity or side effects (Garrett and Fattaey 1999; Sausville 2003; Senderowicz 2003; Dai and Grant 2004; Eastman 2004; Liu et al, 2004; Swanton 2004; Hirai et al, 2005; Benson et al, 2005; Senderowicz 2005; Schwartz 2005; Welburn and Endicott 2005).

For example, it has been confirmed that the tumorigenic activity of cyclin D1 during the development or maintenance of breast carcinoma is associated with the Cdk4 kinase activity confirming that Cdk4-cyclin D1 is a crucially important target for cancer therapy (Yu et al, 2006; Landis et al, 2006; Malumbres and Barbacid 2006). The malignant proliferation could be arrested through enzymatic inhibition of Cdk4. This would indicate that a small molecule, which specifically inhibits the Cdk4 enzyme activity in vitro, prevents cell growth and inhibits tumour volume in vivo could be of immense therapeutic value for the treatment of cancer.

Fascaplysin, isolated from a marine sponge (Roll et al, 1988; Soni et al, 2000), is a pentacyclic quaternary salt that inhibits specifically Cdk4 causing G₀ZG₁ arrest of cancer cells. Concomitantly, fascaplysin inhibits the phosphorylation of the cellular pRb protein at Cdk4-specific serine residues confirming specific activity against the Cdk4 enzyme. However, fascaplysin is highly toxic, and the potential for its planar structure to intercalate double-stranded DNA has been suggested as a possible explanation of its toxicity (Hormann et al, 2001 ).

Fascaplysin

Fascaplysin is a selective Cdk4 inhibitor that arrests cycling cells specifically at the G₀ZG₁ boundary which correlates with the accumulation of hypo-phosphorylated pRb. Fascaplysin prevents the initiation of pRb hyper-phosphorylation which is specifically triggered by Cdk4 activity (Paull et al, 1989; Meijer et al, 1997; Alessi et al, 1998; Carlson et al, 1996; Soni et al, 2000; Soni et al, 2001 ).

Fascaplysin was identified in a large screening program as a small molecule that specifically targets enzymatic activity of Cdk4 and inhibits the in vitro phosphorylation of the retinoblastoma protein pRb, the most prominent Cdk4 substrate (Soni et al, 2000). Fascaplysin demonstrates pRb-dependent arrest of U2-OS (osteosarcoma) and HCT-116 (colon carcinoma) cancer cells at the G₀ZGi phase of the cell cycle. It has also shown similar activity against normal lung fibroblast-derived MRC-5 cells in vitro. An analogue of fascaplysin, 1-deoxysecofascaplysin A, has been reported to inhibit cell growth of MCF-7 (human breast cancer) and OVCAR-3 (human ovarian cancer) cell lines (Charan et al, 2004). The isolation of natural fascaplysin analogues from different organisms, the sponge (Fascaplysinopsis reticulate) and other tunicate species, has helped in understanding the structure-activity relationships of this particular class of compounds (Segraves et al, 2004) Recent reports indicate that fascaplysin shows potent activity against angiogenesis of human umbilical vein endothelial cells (HUVEC) in vitro. The anti-angiogenic activity of fascaplysin was also observed in in vivo assays and also could be monitored via the down-regulation of VEGF (Lin et al, 2006).

However, in spite of its potent anti-tumour activity, it is unlikely that fascaplysin will be therapeutically useful as an anticancer agent because it is a highly toxic molecule. The potential for its planar structure to intercalate with double-stranded DNA has been suggested as a possible explanation for its unusual biological activity and toxicity. The DNA binding property of fascaplysin is similar to the structurally related DNA intercalating agents, cryptolepine and ellipticine (Hormann er a/, 2001).

We have explored the possibility of separating the DNA intercalating ability of fascaplysin from its potent Cdk4-specific inhibitory activity (Aubry et al, 2004; Aubry et al, 2005; Aubry et al, 2006; Mahale et al, 2006a; Mahale et al, 2006b; Garcia et al, 2006; Mahale & Chaudhuri 2006) in an attempt to develop non-planar and less toxic compounds based on the structure of fascaplysin. The general chemical strategy that was adopted for removing toxicity (Aubry et al, 2004; Aubry et al, 2005; Aubry et al, 2006) is shown in Figure 2. Specifically, Aubry et al (2004, 2005) describe the synthesis of non-planar analogues of fascaplysin which were shown to be selective inhibitors of CDK4 over CDK2. The most active compound 8f from Aubry et al (2005) was reported to have an IC₅₀ for the inhibition of CDK4 of 50 μM.

Aubry et al (2006) describe the synthesis of non-planar analogues of fascaplysin which were shown to be selective inhibitors of CDK4 over CDK2. The most active compound 9q (referred to herein as CA224) was reported to have an IC₅₀ for the inhibition of CDK4 of 6 μM. Mahale et al (2006a) also describe non-planar analogues of fascaplysin which were predicted to bind to the ATP-binding site of CDK4, and which were shown to be selective inhibitors of CDK4 over CDK2. The most active compound CA224 was shown not to intercalate with double-stranded DNA, and to arrest growth of Calu-1 cancer cells at Go/G1 by inhibiting pRb phosphorylation. Both Aubry et al (2006) and Mahale et al (2006a) concluded that CA224 could be the basis for the development of more potent CDK4 specific inhibitors.

Mahale et al (2006b) describe a number of non-planar analogues of fascaplysin which were shown to be selective inhibitors of CDK4 and not to interact or intercalate with double-stranded DNA. The most active compound CA199 was reported to have an IC₅₀ for the inhibition of CDK4-cyclin D1 of 20 μM, and to inhibit growth of cancer cell lines in a pRb dependent manner at concentrations ranging from 10-40 μM. Mahale et al concluded that CA199 could be the basis for the development of more potent CDK4 specific inhibitors.

Garcia et al (2006) describe the synthesis of non-planar analogues of fascaplysin which were shown to be selective inhibitors of CDK4 over CDK2. The most active compound 3d was reported to have an IC₅₀ for the inhibition of CDK4-cyclin D1 of 11 μM. The relationship between the analogue structure and binding to CDK4 was assessed.

Mahale & Chaudhuri (2006) reviewed the development of CDK inhibitors as anticancer agents and emphasized the importance of inhibition of CDK4-cyclin D1 as a target for cancer therapy. Mahale & Chaudhuri discussed the rational design of CDK-4 specific inhibitors derived from the structure of fascaplysin. The best compounds in three classes of fascaplysin analogues were 7a, 9q (referred to herein as CA224) and 12m (referred to herein as CA199). These compounds were reported to have an IC₅₀ for CDK4 of 50 μM, 6 μM and 20 μM, and a mean IC₅₀ for growth inhibition in a panel of cancer lines of 50 μM, 3.5 μM and 7 μM, respectively.

The listing or discussion of an apparently prior-published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge.

During the in vitro screening of fascaplysin analogues, we identified several compounds with reasonably potent anti-cancer activity, of which the three most potent compounds CA224, AJW089 and DE002 were selected for further evaluation in in vitro and in vivo models.

CA224

Surprisingly, we have found that these analogues manifest not only the expected Cdk4- specific inhibition by blocking at the G₀ZG₁ phase of the cell cycle but also inhibit the G₂/M phase in a Cdk-independent manner. We observed that the compounds were relatively less potent in Calu-1 cells (non-small cell lung carcinoma cells, NSCLC) where the mitotic spindle checkpoint is impaired (Masuda et al, 2003), and we surmised that the relative resistance of Calu-1 cells was probably due to lack of functional tubulin in these cells. Hence we tested the effect of these compounds on tubulin polymerisation in vitro and in vivo. Unexpectedly, we found that the fascaplysin analogues that induce a profound block at G₂/M phase also inhibit tubulin polymerisation both in in vitro cell free assay as well as in a cell-based assay.

Thus these analogues of fascaplysin, a known Cdk4-specifιc inhibitor, do not manifest their cellular activities only via Cdk4. Surprisingly and unexpectedly, we have shown that the anticancer activity of these compounds manifests by targeting multiple sites in the cancer cell division cycle. We have also shown that two of these selected compounds are highly efficacious in inhibiting tumour growth in human xenograft-SCID mice models.

DE002 Series

The biological activity of the DE002 series of compounds is presented in Example 1. DE002 inhibits Cdk4 specifically, which is highly significant due to the crucial role of Cdk4 in the development of human cancers, and we have shown that DE002 does not intercalate with double-stranded DNA thus minimising toxicity. As expected for a Cdk4 inhibitor, DE002 blocks at the G₀ZG₁ phase of the cell division cycle. However, we have also shown that it profoundly blocks cells at the G₂ZM phase at comparatively low concentrations by inhibiting tubulin polymerisation, and induces massive apoptosis in cancer cells as measured by FACS analysis and DAPI staining of the nuclei, which are particularly valuable properties for a potential anticancer compound (Lee et al, 2005;

Komiya et al, 2003; Lopez-Beltran et al, 2007). We have also shown that DE002 selectively induces apoptosis in SV40 large T antigen-transformed cells and not in untransformed normal cells. A colony formation assay is valuable to understand the anti-cancer potential of a test compound (Wu Wei et al, 1983) since the loss of a cancer cell's ability to form a colony could possibly indicate its permanent exit from the cell cycle. The concentration at which DE002 prevents colony formation was found to be noticeably lower than the concentration at which it inhibits cell proliferation in both p53+ and p53-null cancer cells in vitro.

We have also demonstrated that DE002 is a powerful antioxidant, as shown by its ability to prevent H₂O₂-induced cellular reactive oxygen species (ROS) formation in vitro. Antioxidants, such as the flavonoid class of phytochemicals, can be used for cancer therapy, by quenching the oxidant H₂O₂ and preventing ROS formation. Formation of ROS inside the living cells could have detrimental effects on cells, since ROS can induce DNA strand breaks and also can mediate conformational changes in protein molecules. These changes at molecular level could result in many degenerative diseases that include cancer (Izzotti et al, 2006; Butterfield ef al, 1998). Hence the antioxidant property of DE002 is additionally valuable not only for the treatment of cancer but also for its prevention.

Accordingly, we have discovered that not only is DE002 itself a potentially valuable anticancer agent, but analogues based on the DE002 scaffold may be used to improve the therapeutic index of DE002 for the treatment of cancer.

DE002 is a fascaplysin analogue that has a tryptoline (2,3,4,9-tetrahydro-1 H-beta- carboline) structure at the left hand side of the molecule and an unsubstituted phenyl group in the orffto-position at the right hand side of the molecule. Without wishing to be bound by theory, we propose that these features in a fascaplysin analogue are required for the activity of DE002, and define the DE002 series of compounds (Formula I, below). A first aspect of the invention thus provides a compound of Formula I (the "DE002 series") wherein

wherein each of R¹ to R⁴ may independently represent H, aryl, Het¹, halo, CN, NO₂, C_1-12 alkyl, C_1--_I2 alkenyl, C_1-12 alkynyl, C_3-12 cycloalkyl or C_4-12 cycloalkenyl, which latter five groups are optionally substituted by one or more substituents selected from halo, nitro, CN, C_1-6 alkyl, Ci_-6 alkenyl, C_1-6 alkynyl, C_3-8 cycloalkyl (which latter three groups are optionally substituted by one or more substituents selected from OH, =0, halo, C_1-4 alkyl and C_1-4 alkoxy), OR^8a, S(O)_nR^8b, S(O)₂N(R^8c)(R^8d), N(R^8e)S(O)₂R^8f, N(R^δ9)(R^8h) and Het², and which C_3-12 cycloalkyl or C_4--_I2 cycloalkenyl groups may additionally be substituted by =0;

R is either not present, or represents one to four substituents on the fused benzene ring selected from aryl, Het³, halo, CN, NO₂, C_1--_I2 alkyl, C_1--_I2 alkenyl, C_1-12 alkynyl, C_3-12 cycloalkyl or C_4-^ cycloalkenyl, which latter five groups are optionally substituted by one or more substituents selected from halo, nitro, CN, C_1-6 alkyl, C_1-6 alkenyl, C_1-6 alkynyl, C_3-8 cycloalkyl (which latter three groups are optionally substituted by one or more substituents selected from OH, =0, halo, C_1-4 alkyl and C_1-4 alkoxy), 0R^9a, S(0)_pR^9b, S(O)₂N(R^9c)(R^9d), N(R^9e)S(O)₂R^9f, N(R")(R^9h) and Het⁴, and which C_3-12 cycloalkyl or C_4-12 cycloalkenyl groups may additionally be substituted by =O;

R⁶ represents H, C_1--_I2 alkyl, C_3-12 cycloalkyl, C_3-12 cycloalkenyl (which latter three groups are optionally substituted by one or more substituents selected from halo, nitro, CN, C_1-6 alkyl, C_1-6 alkenyl, C_1-6 alkynyl, C_3-8 cycloalkyl (which latter three groups are optionally substituted by one or more substituents selected from OH, =0, halo, Ci_-4 alkyl and C_1-4 alkoxy), OR^lϋa, S(O)_qR^1Ub, S(O)₂N(R^lϋc)(R^iυa), N(R^lue)S(O)₂R >10f N(R¹⁰⁹)(R^10h), aryl and Het⁵, and which C_3-12 cycloalkyl or C_4-12 cycloalkenyl groups may additionally be substituted by =O);

R⁷ is either not present, or represents one to six substituents on the fused tetrahydropyridine ring selected from H, halo, CN, NO₂, C_1-12 alkyl, C_1-12 alkenyl, C_1-12 alkynyl, C_3-12 cycloalkyl or C_4-12 cycloalkenyl, which latter five groups are optionally substituted by one or more substituents selected from halo, nitro, CN, C_1-6 alkyl, C_1-6 alkenyl, C_1-6 alkynyl, C_3-8 cycloalkyl (which latter three groups are optionally substituted by one or more substituents selected from OH, =0, halo, C_1-4 alkyl and C_1-4 alkoxy), OR^1la, S(O)_rR^11b, S(O)₂N(R^11c)(R^11d), N(R^11e)S(O)₂R^11f, N(R^11g)(R^11h), aryl and Het⁶, and which C_3-12 cycloalkyl or C_4--_I2 cycloalkenyl groups may additionally be substituted by

=0;

R^Sa to R^8h, R^9a to R^9h, R^1Oa to R^1Oh and R^11a to R^11h independently represent, at each occurrence,

(a) H,

(b) C_1-I0 alkyl, C_2-10 alkenyl, C_2-10 alkynyl (which latter three groups are optionally substituted by one or more substituents selected from halo, OH, C_1-6 alkoxy, aryl and Het⁷), (c) C_3-10 cycloalkyl, C_4-10 cycloalkenyl (which latter two groups are optionally substituted by one or more substituents selected from halo, OH, =O, C_1-6 alkyl, C_1-6 alkoxy, aryl and Het⁸),

(d) aryl or

(e) Het⁹, provided that R^8b, R^9b, R^1Ob or R^11b does not represent H when n, p, q or r, respectively is 1 or 2;

each aryl independently represents a C_6-10 carbocyclic aromatic group, which group may comprise either one or two rings and may be substituted by one or more substituents selected from

(a) halo,

(b) CN,

(c) C_1-I₂ alkyl, C_1-12 alkenyl, Ci_-12 alkynyl, C_3-12 cycloalkyl or C_4-12 cycloalkenyl, which latter five groups are optionally substituted by one or more substituents selected from halo, nitro, CN, C_1-6 alkyl, C_1-6 alkenyl, C_1-6 alkynyl, C_3-8 cycloalkyl (which latter three groups are optionally substituted by one or more substituents selected from OH,

=O, halo, C_1-4 alkyl and C_1-4 alkoxy), 0R^12a, S(O)_sR^12b, S(O)₂N(R^12c)(R^12d), N(R^12e)S(O)₂R^12f, N(R¹²⁹)(R^12h), phenyl, naphthyl (which latter two groups are optionally susbtituted by one or more substituents selected from OH, halo, Ci_-4 alkyl and C_1-4 alkoxy) and Het¹⁰, and which C_3-12 cycloalkyl or C_4-12 cycloalkenyl groups may additionally be substituted by =0, (d) OR^13a,

(e) S(O)_tR^13b,

(f) S(O)₂N(R^13c)(R^13d),

(g) N(R^13e)S(O)₂R^13f, (h) N(R^13g)(R^13h), (i) phenyl (which latter group is optionally susbtituted by one or more substituents selected from OH, halo, Ci_-4 alkyl and C_1-4 alkoxy) or 0) Het¹¹;

R^12a to R^12h and R^13a to R^13h independently represent, at each occurrence, (a) H,

(b) C_1--_I2 alkyl, C_2--_I2 alkenyl, C_2-12 alkynyl, C_3--_I2 cycloalkyl, C_4-12 cycloalkenyl (which latter five groups are optionally substituted by one or more substituents selected from halo, OH, C_1-6 alkyl, C_3--_I2 cycloalkyl, C_4-12 cycloalkenyl (which latter two groups are optionally substituted by one or more substituents selected from OH, =0, halo, C_1-4 alkyl and C_1-4 alkoxy), C_1-6 alkoxy, NH₂, N(H)-C_1-6 alkyl, N(C_1-6 alkyl)₂, phenyl (which latter group is optionally substituted by one or more substituents selected from OH, halo, C_1-4 alkyl and C_1-4 alkoxy) and Het¹², and which C_3--_I2 cycloalkyl or C_4-12 cycloalkenyl groups may additionally be substituted by =0),

(c) phenyl (which latter group is optionally substituted by one or more substituents selected from OH, CN, halo, C_1-6 alkyl and C_1-6 alkoxy) or

(e) Het¹³, provided that R^13b or R^14b does not represent H when s or t, respectively is 1 or 2;

Het¹ to Het¹³ independently represent 4- to 14-membered heterocyclic or 5- to 10-membered heteroaromatic groups containing one or more heteroatoms selected from oxygen, nitrogen and/or sulfur, which heterocyclic groups may comprise one, two or three rings, and may be substituted by one or more substituents selected from

(a) halo,

(b) CN, (c) C_1-12 alkyl, C_1-12 alkenyl, C_1--_I2 alkynyl, C_3-12 cycloalkyl or C₄.₁₂ cycloalkenyl, which latter five groups are optionally substituted by one or more substituents selected from halo, nitro, CN, C_1-6 alkyl, C_1-6 alkenyl, C_1-6 alkynyl, C_3-8 cycloalkyl (which latter three groups are optionally substituted by one or more substituents selected from OH, =O, halo, C_1-4 alkyl and C_1-4 alkoxy), OR^14a, S(O)_uR^14b, S(O)₂N(R^14c)(R^14d), N(R^14e)S(O)₂R^14f, N(R^14g)(R^14h), phenyl, naphthyl (which latter two groups are optionally susbtituted by one or more substituents selected from OH, halo, C_1-4 alkyl and C_1-4 alkoxy) and Hef, and which C_3--I₂ cycioalkyl or C_4--_I2 cycloalkenyl groups may additionally be substituted by =O,

(d) OR^15a,

(e) =0,

(f) S(O)_vR^15b, (g) S(O)₂N(R^15c)(R^15d),

(h) N(R^15e)S(O)₂R^15f,

(i) N(R^15g)(R^15h),

(j) phenyl (which latter group is optionally susbtituted by one or more substituents selected from OH, halo, C_1-4 alkyl and C_1-4 alkoxy) or (k) Het^b;

R^14a to R^14h and R^14a to R^14h independently represent, at each occurrence,

(a) H,

(b) Ci_-12 a\kyl, C_2-12 a\keny\, C_2-12 aikyny), C_3-12 cycioalky], C_4-I2 cycloalkenyl (which latter five groups are optionally substituted by one or more substituents selected from halo, OH, C_1-6 alkyl, C_3-12 cycioalkyl, C_4-12 cycloalkenyl (which latter two groups are optionally substituted by one or more substituents selected from OH, =0, halo, C_1-4 alkyl and C_1-4 alkoxy), C_1-6 alkoxy, phenyl (which latter group is optionally substituted by one or more substituents selected from OH, halo, C_1-4 alkyl and C_1-4 alkoxy) and Hef, and which C_3-12 cycioalkyl or C_4-12 cycloalkenyl groups may additionally be substituted by =O),

(c) phenyl (which latter group is optionally substituted by one or more substituents selected from OH, halo, C_1-4 alkyl and C_1-4 alkoxy) or

(e) Hef; provided that R^15b or R^16b does not represent H when u or v, respectively is 1 or 2;

n, p, q, r, s, t, u and v independently represent O, 1 or 2;

Hef to Het^d independently represent 5- or 6-membered heterocyclic groups containing one to four heteroatoms selected from oxygen, nitrogen and/or sulfur, which heterocyclic groups may be substituted by one or more substituents selected from halo, =0 and C_1-6 alkyl; and unless otherwise specified

(i) alkyl, alkenyl, alkynyl, cycloalkyl, and cycloalkenyl groups, as well as the alkyl part of alkoxy groups, may be substituted by one or more halo atoms, and (ii) cycloalkyl and cycloalkenyl groups may comprise one or two rings and may additionally be ring-fused to one or two benzene rings;

or a pharmaceutically-acceptable derivative, salt, solvate or prodrug thereof.

Other compounds of Formula I that may be mentioned include compounds of Formula Ib, wherein

wherein, each of R¹ to R⁴ may independently represent H, halo, CN, NO₂, C_1--_I2 alkyl, C_1--_I2 alkenyl, C_1-12 alkynyl, C_3-I2 cycloalkyl or C_4-12 cycloalkenyl, which latter five groups are optionally substituted by one or more substituents selected from halo, nitro, CN, C_1-6 alkyl, Ci_-6 alkenyl, C_1-6 alkynyl, C_3-8 cycloalkyl (which latter three groups are optionally substituted by one or more substituents selected from OH, =0, halo, C_1-4 alkyl and C₁^ alkoxy) or OR^8a;

R⁵ is either not present, or represents one to two substituents on the fused benzene ring selected from halo, CN, NO₂, C_1-I2 alkyl, C_1-12 alkenyl, C_1-12 alkynyl, C_3-12 cycloalkyl or C_4-12 cycloalkenyl, which latter five groups are optionally substituted by one or more substituents selected from halo, nitro, CN, C_1-6 alkyl, C_1-6 alkenyl, C_1-6 alkynyl, C_3-8 cycloalkyl (which latter three groups are optionally substituted by one or more substituents selected from OH, =0, halo, C_1-4 alkyl and C_1-4 alkoxy) or 0R^9a;

R represents H or C_1-3 alkyl;

R is not present; R^8a and R^9a independently represent, at each occurrence,

(a) H,

(b) C_1-6 alkyl, C_2-6 alkenyl, C_2-6 alkynyl (which latter three groups are optionally substituted by one or more substituents selected from halo, OH, C_1-6 alkoxy and aryl), or

(c) C_3-6 cycloalkyl, C₄^ cycloalkenyl (which latter two groups are optionally substituted by one or more substituents selected from halo, OH, =0, C_1-6 alkyl, Ci_-6 alkoxy and aryl);

(a) halo, (b) CN,

(c) C_1-6 alkyl or C_3-6 cycloalkyl which latter two groups are optionally substituted by one or more substituents selected from halo, nitro, CN, C_1-6 alkyl, C_3-8 cycloalkyl (which latter group i.e. optionally substituted by one or more substituents selected from OH,

=0, halo, C_1-4 alkyl and C_1-4 alkoxy) or OR^12a, or (d) 0R^13a;

R^12a and R^13a independently represent, at each occurrence,

(a) H,

(b) C_1-6 alkyl, C_2-6 alkenyl, C_2-6 alkynyl (which latter three groups are optionally substituted by one or more substituents selected from halo, OH, C₁^ alkoxy and aryl), or

(c) C_3-6 cycloalkyl, C_4-6 cycloalkenyl (which latter two groups are optionally substituted by one or more substituents selected from halo, OH, =O, C_1-6 alkyl, C_1-6 alkoxy and aryl);

In an embodiment it may be preferred that R⁵ is either not present, or represents one substituent on the fused benzene ring selected from selected from halo, CN, NO₂, C_1-12 alkyl, C_1--_I2 alkenyl, C_1-12 alkynyl, C_3-12 cycloalkyl or C_4-12 cycloalkenyl, which latter five groups are optionally substituted by one or more substituents selected from halo, nitro, CN, C_1-6 alkyl, C_1-6 alkenyl, C_1-6 alkynyl, C_3-8 cycloalkyl (which latter three groups are optionally substituted by one or more substituents selected from OH, =0, halo, C_1-4 alkyl and C_1-4 alkoxy), 0R^9a. In an embodiment each of R¹ to R⁴ may independently represent H, halo, or C_1-6 alkyl; and

R⁵ is either not present, or represents one substituent on the fused benzene ring selected from selected from halo or C_1-6 alkyl.

As a further embodiment, each of R¹ to R⁴ may independently represent H, halo, or C_1-3 alkyl.

In a more specific embodiment, each of R¹ to R⁴ may independently represent H or halo.

In a further embodiment, wherein R⁵ is not present; and R⁶ represents H.

In a further embodiment, R¹ to R⁴ represent H.

In a still further embodiment, R¹ to R⁴ represent H; R⁵ is not present; R⁶ represents H; and R⁷ is not present.

In a specific preferred embodiment, the compound of Formula I is, or comprises, biphenyl-2-yl-(4,4a,9,9a-tetrahydro-1 H-beta-carbolin-2-yl)-methanone (DE002).

In an embodiment, the compound of Formula I is not (3-Methoxy-phenyl)-(1 , 3,4,9- tetrahydro-beta-carbolin-2-yl)-methanone (CA199).

AJW089

AJW089 is a fascaplysin analogue that is similar to CA224 but has an unsubstituted (unmethylated) central amido group and an unsubstituted phenyl group in the ortho- position. Without wishing to be bound by theory, we propose that these two features in a fascaplysin analogue are required for the activity of AJW089, and define the AJW089 series of compounds (Formula II, below).

A second aspect of the invention thus provides a compound of Formula Il (the "AJW089 series")

wherein each of R¹ to R⁴ may independently represent H, halo, CN, NO₂, CM₂ alkyl, C_1- ₁₂ alkenyl, Ci--_I2 alkynyl, C_3^12 cycloalkyl or C_4-12 cycloalkenyl, which latter five groups are optionally substituted by one or more substituents selected from halo, nitro, CN₁ C_1- ₆ alkyl, C_1-6 alkenyl, C_1-6 alkynyl, C_3-8 cycloalkyl (which latter three groups are optionally substituted by one or more substituents selected from OH, =0, halo, C_1-4 alkyl and C_1-4 alkoxy), OR^7a, S(O)_pR^7b, S(O)₂N(R^7c)(R^7d), N(R^7e)S(O)₂R^7f, N(R^7g)(R^7h), and which C_3-12 cycloalkyl or C_4-12 cycloalkenyl groups may additionally be substituted by =0;

R⁵ is either not present, or represents one to four substituents on the fused benzene ring selected from aryl, Het¹, halo, CN, NO₂, Ci_-12 alkyl, C_1--_I2 alkenyl, C_1-I2 alkynyl, C_3-12 cycloalkyl or C_4-12 cycloalkenyl, which latter five groups are optionally substituted by one or more substituents selected from halo, nitro, CN, C_1-6 alkyl, C_1-6 alkenyl, Ci_-6 alkynyl, C_3-8 cycloalkyl (which latter three groups are optionally substituted by one or more substituents selected from OH, =0, halo, C_1-4 alkyl and C_1-4 alkoxy), 0R^8a, S(O)_qR^8b, S(O)₂N(R^8c)(R^8d), N(R^8e)S(O)₂R^8f, N(R^8g)(R^8h) and Het², and which C_3-12 cycloalkyl or C_4-12 cycloalkenyl groups may additionally be substituted by =0;

R⁶ represents H, C_1-12 alkyl, C_3-12 cycloalkyl, C_3-I2 cycioalkenyl (which latter three groups are optionally substituted by one or more substituents selected from halo, nitro, CN, C_1-6 alkyl, C_1-6 alkenyl, C_1-6 alkynyl, C_3-8 cycloalkyl (which latter three groups are optionally substituted by one or more substituents selected from OH, =O, halo, C_1-4 alkyl and C_1-4 alkoxy), 0R^9a, S(O)_rR^9b, S(O)₂N(R^9c)(R^9d), N(R^)S(O)₂R⁹*, N(R")(R^9h), aryl and Het³, and which C_3-12 cycloalkyl or C_4-12 cycloalkenyl groups may additionally be substituted by =O);

R^7a to R^7h, R^8a to R^8h and R^9a to R^9h, independently represent, at each occurrence, (a) H, (b) C_1-10 alkyl, C_2-10 alkenyl, C_2-i₀ alkynyl (which latter three groups are optionally substituted by one or more substituents selected from halo, OH₁ C^ alkoxy, aryl and Het⁴),

(c) C_3--_I0 cycloalkyl, C₄-₁₀ cycloalkenyl (which latter two groups are optionally substituted by one or more substituents selected from halo, OH, =0,

Ci_-6 alkyl, C_1-6 alkoxy, aryl and Het⁵),

(d) aryl or

(e) Het⁶, provided that R^7b, R^8b or R^9b does not represent H when n, p, q, r or s respectively is 1 or 2;

each aryl independently represents a C_6-I0 carbocyclic aromatic group, which group may comprise either one or two rings and may be substituted by one or more substituents selected from (a) halo,

(b) CN,

(c) C_1-12 alkyl, C_1-12 alkenyl, Ci_-12 alkynyl, C₃.i₂ cycloalkyl or C_4-12 cycloalkenyl, which latter five groups are optionally substituted by one or more substituents selected from halo, nitro, CN, C_1-6 alkyl, Ci_-6 alkenyl, C_1-6 aikyny], C_3-8 cycloalkyl (which latter three groups are optionally substituted by one or more substituents selected from OH, =0, halo, C_1-4 alkyl and C₁^ alkoxy), OR^10a, S(O)_tR^10b, S(O)₂N(R^10c)(R^10d), N(R^10e)S(O)₂R^10f, N(R¹⁰⁹)(R^10h), phenyl, naphthyl (which latter two groups are optionally susbtituted by one or more substituents selected from OH, halo, C_1-4 alkyl and C_1-4 alkoxy) and Het⁷, and which C_3-12 cycloalkyl or C_4-12 cycloalkenyl groups may additionally be substituted by =0,

(d) 0R^11a,

(e) S(O)_uR^11b,

(f) S(O)₂N(R^11c)(R^11d),

(g) N(R^{1 le})S(O)₂R^11f, (h) N(R^11g)(R^{11 h}),

(i) phenyl (which latter group is optionally susbtituted by one or more substituents selected from OH, halo, Ci_-4 alkyl and Ci_-4 alkoxy) or G) Het⁸;

R^1Oa to R^1Oh and R^11a to R^11h independently represent, at each occurrence,

(a) H, (b) Ci_-12 alkyl, C_2--I₂ alkenyl, C_2-I2 alkynyl, C_3-12 cycloalkyl, C_4-12 cycloalkenyl (which latter five groups are optionally substituted by one or more substituents selected from halo, OH, C_1-6 alkyl, C_3--_I2 cycloalkyl, C_4-12 cycloalkenyl (which latter two groups are optionally substituted by one or more substituents selected from OH, =0, halo, Ci_-4 alkyl and C₁^, alkoxy), C_1-6 alkoxy, NH₂, N(H)-C_1-6 alkyl, N(C_1-6 alkyl)₂, phenyl (which latter group is optionally substituted by one or more substituents selected from OH, halo, Ci_₄ alkyl and Ci_-4 alkoxy) and Het⁹, and which C_3-I2 cycloalkyl or C_4-I2 cycloalkenyl groups may additionally be substituted by =0),

(c) phenyl (which latter group is optionally substituted by one or more substituents selected from OH, CN, halo, Ci_-6 alkyl and Ci_-6 alkoxy) or (e) Het¹⁰, provided that R^10b or R^11b does not represent H when t or u, respectively is 1 or 2;

Het¹ to Het¹⁰ independently represent 4- to 14-membered heterocyclic or 5- to 10-membered heteroaromatic groups containing one or more heteroatoms selected from oxygen, nitrogen and/or sulfur, which heterocyclic groups may comprise one, two or three rings, and may be substituted by one or more substituents selected from

(a) halo,

(b) CN, (c) C_M2 alkyl, Ci_-I2 alkenyl, Ci_-12 alkynyl, C_3-12 cycloalkyl or C_4-12 cycloalkenyl, which latter five groups are optionally substituted by one or more substituents selected from halo, nitro, CN, C_1-6 alkyl, C_1-6 alkenyl, C_1-6 alkynyl, C_3-S cycloalkyl (which latter three groups are optionally substituted by one or more substituents selected from OH, =0, halo, C_1-4 alkyl and C_1-4 alkoxy), 0R^12a, S(O)_vR^12b, S(O)₂N(R^12c)(R^12d), N(R^12e)S(O)₂R^12f, N(R^12g)(R^12h), phenyl, naphthyl (which latter two groups are optionally susbtituted by one or more substituents selected from OH, halo, C_1-4 alkyl and C_1-4 alkoxy) and Her³, and which C_3-12 cycloalkyl or C_4-12 cycloalkenyl groups may additionally be substituted by =0, (d) 0R^13a, (e) =0,

(f) S(O)_wR^13b,

(g) S(O)₂N(R^13c)(R^l3d), (h) N(R^13e)S(O)₂R^13f, (i) N(R¹³⁹)(R^13h), (j) phenyl (which latter group is optionally susbtituted by one or more substituents selected from OH, halo, C_1-4 alkyl and C^₄ alkoxy) or (k) Het^b; R^12a to R^12h and R^13a to R^13h independently represent, at each occurrence,

(a) H,

(b) C_1-12 alkyl, C₂.i₂ alkenyl, C_2-I₂ alkynyl, C_3-12 cycloalkyl, C_4-12 cycloalkenyl (which latter five groups are optionally substituted by one or more substituents selected from halo, OH, C_1-6 alkyl, C_3-12 cycloalkyl, C_4-12 cycloalkenyl (which latter two groups are optionally substituted by one or more substituents selected from OH, =0, halo, C_1-4 alkyl and C_1-4 alkoxy), C₁^ alkoxy, phenyl (which latter group is optionally substituted by one or more substituents selected from OH, halo, C_1-4 alkyl and Ci_-4 alkoxy) and Het^c, and which C_3--_I2 cycloalkyl or C_4-12 cycloalkenyl groups may additionally be substituted by =O),

(c) phenyl (which latter group is optionally substituted by one or more substituents selected from OH, halo, C_1-4 alkyl and C_1-4 alkoxy) or (e) Het^d; provided that R^12b or R^13b does not represent H when v or w, respectively is 1 or 2;

n, p, q, r, s, t, u, v and w independently represent O, 1 or 2;

Het^a to Het^d independently represent 5- or 6-merrtbered heterocychc groups containing one to four heteroatoms selected from oxygen, nitrogen and/or sulfur, which heterocyclic groups may be substituted by one or more substituents selected from halo, =O and C_1-6 alkyl; and

unless otherwise specified (i) alkyl, alkenyl, alkynyl, cycloalkyl, and cycloalkenyl groups, as well as the alkyl part of alkoxy groups, may be substituted by one or more halo atoms, and (ii) cycloalkyl and cycloalkenyl groups may comprise one or two rings and may additionally be ring-fused to one or two benzene rings;

or a pharmaceutically-acceptable derivative, salt, solvate or prodrug thereof.

Other compounds of Formula Il that may be mentioned include those in which: each of R¹ to R⁴ may independently represent H, halo, CN, NO₂, Ci_-12 alkyl, C_1-

₁₂ alkenyl, C₁-_I2 alkynyl, C_3-12 cycloalkyl or C_4-12 cycloalkenyl, which latter five groups are optionally substituted by one or more substituents selected from halo, nitro, CN₁ C_1-

₆ alkyl, C_1-6 alkenyl, C_1-6 alkynyl, C_3-8 cycloalkyl (which latter three groups are optionally substituted by one or more substituents selected from OH, =0, halo, C_1-4 alkyl and Ci_-4 alkoxy) or OR^7a;

R⁵ is either not present, or represents one to two substituents on the fused benzene ring selected from halo, CN, NO₂, C₁-₁₂ alkyl, C_1-I2 alkenyl, C_1-12 alkynyl, C₃.-|₂ cycloalkyl or C_4-12 cycloalkenyl, which latter five groups are optionally substituted by one or more substituents selected from halo, nitro, CN, C_1-6 alkyl, C_1-6 alkenyl, C_1-6 alkynyl, C_3-8 cycloalkyl (which latter three groups are optionally substituted by one or more substituents selected from OH, =0, halo, C_1-4 alkyl and Ci_-4 alkoxy) or 0R^8a;

R⁶ represents H;

R^7a and R^8a independently represent, at each occurrence,

(a) H, (b) C_1-6 alkyl, C_2-6 alkenyl, C_2-6 alkynyl (which latter three groups are optionally substituted by one or more substituents selected from halo, OH, C_1-6 alkoxy and aryl), or

(c) C_3-6 cycloalkyl, C_4-6 cycloalkenyl (which latter two groups are optionally substituted by one or more substituents selected from halo, OH, =0, C_1-6 alkyl, C_1-6 alkoxy and aryl);

each aryl independently represents a C_6-10 carbocyclic aromatic group, which group may comprise either one or two rings and may be substituted by one or more substituents selected from (a) halo,

(b) CN,

(c) C_1-6 alkyl or C_3-6 cycloalkyl which latter two groups are optionally substituted by one or more substituents selected from halo, nitro, CN, C_1-6 alkyl, C_3-8 cycloalkyl (which latter group i.e. optionally substituted by one or more substituents selected from OH, =0, halo, C_1-4 alkyl and C_1-4 alkoxy) or 0R^11a, or

(d) 0R^12a;

R^11a and R^12a independently represent, at each occurrence, (a) H, (b) C_1-6 alkyl, C_2-6 alkenyl, C_2-6 alkynyl (which latter three groups are optionally substituted by one or more substituents selected from halo, OH, C_1-6 alkoxy and aryl), or (c) C_3-6 cycloalkyl, C_4-6 cycloalkenyl (which latter two groups are optionally substituted by one or more substituents selected from halo, OH, =0, Ci-e alkyl, C_1-6 alkoxy and aryl).

In an embodiment it may be preferred that R⁵ is either not present, or represents one substituent on the fused benzene ring selected from halo, CN, NO₂, C_1-12 alkyl, C_1-12 alkenyl, Ci_-12 alkynyl, C_3-I2 cycloalkyl or C_4-12 cycloalkenyl, which latter five groups are optionally substituted by one or more substituents selected from halo, nitro, CN, C_1-6 alkyl, C_1-6 alkenyl, C_1-6 alkynyl, C_3-8 cycloalkyl (which latter three groups are optionally substituted by one or more substituents selected from OH, =0, halo, C_1-4 alkyl and C_1-4 alkoxy), 0R^9a.

In an embodiment it may be preferred that R¹ to R⁴ represent H, halo, or C_1-6 alkyl;

R⁵ is either not present, or represents one substituent on the fused benzene ring selected from halo or Ci_-6 alkyl.

In a more specific embodiment each of R¹ to R⁴ may independently represent H, halo, or Ci_-3 alkyl.

In a further specific embodiment each of R¹ to R⁴ may independently represent H or halo.

In a further embodiment R⁵ is not present; and R⁶ represents H.

In a yet more specific embodiment R¹ to R⁴ represent H.

Other compounds of Formula Il that may be mentioned include compounds wherein R¹ to R⁴ represent H;

R⁵ is not present; R⁶ represents H; and

R⁷ is not present.

In a specific preferred embodiment, the compound of Formula Il is, or comprises, biphenyl-2-carboxylic acid [2-(1 H-indol-3-yl)-ethyl]-amide (AJW089).

In an embodiment, the compound of Formula Il is not biphenyI-4-carboxylic acid [2-(1 H- indol-3-yl)-ethyl]-amide. Compounds of Formula I and Formula Il (and Formula III as described below) may contain double bonds and may thus exist as E (entgegen) and Z (zusammen) geometric isomers about each individual double bond. All such isomers and mixtures thereof are included within the scope of the invention.

Compounds of Formula I and Formula Il (and Formula III as described below) may also exhibit tautomerism. All tautomeric forms and mixtures thereof are included within the scope of the invention.

Compounds of Formula I and Formula Il (and Formula III as described below) may also contain one or more asymmetric carbon atoms and may therefore exhibit optical and/or diastereoisomerism. Diastereoisomers may be separated using conventional techniques, e.g. chromatography or fractional crystallisation. The various stereoisomers may be isolated by separation of a racemic or other mixture of the compounds using conventional, e.g. fractional crystallisation or HPLC, techniques. Alternatively the desired optical isomers may be made by reaction of the appropriate optically active starting materials under conditions which will not cause racemisation or epimerisation (i.e. a 'chiral pool' method), by reaction of the appropriate starting material with a 'chiral auxiliary' which can subsequently be removed at a suitable stage, by derivatisation (i.e. a resolution, including a dynamic resolution), for example with a homochiral acid followed by separation of the diastereomeric derivatives by conventional means such as chromatography, or by reaction with an appropriate chiral reagent or chiral catalyst all under conditions known to the skilled person. All stereoisomers and mixtures thereof are included within the scope of the invention.

Unless otherwise specified, Ci_-q alkyl (where q is the upper limit of the range) as defined herein may be straight-chain or, when there is a sufficient number (i.e. a minimum of three) of carbon atoms, be branched-chain, and/or cyclic (so forming, in the case of alkyl, a C_3-q cycloalkyl group). Further, when there is a sufficient number (i.e. a minimum of four) of carbon atoms, such groups may also be part cyclic. Further, unless otherwise specified, such alkyl groups may also be saturated or, when there is a sufficient number (i.e. a minimum of two) of carbon atoms and unless otherwise specified, be unsaturated (forming, for example, a C₂._q alkenyl or a C₂-q alkynyl group).

The term "halo", when used herein, includes fluoro, chloro, bromo and iodo. As used herein, "aryl" groups that may be mentioned include C_6-io aryl groups. Such groups may be monocyclic, bicyclic or tricyclic and have between 6 and 10 ring carbon atoms, in which at least one ring is aromatic. C_6-10 aryl groups include phenyl, naphthyl and the like, such as 1 ,2,3,4-tetrahydronaphthyl, indanyl and indenyl. The point of attachment of aryl groups may be via any atom of the ring system. However, when aryl groups are bicyclic or tricyclic, they are linked to the rest of the molecule via an atom of the aromatic ring.

As used herein, "heteroaryl" groups that may be mentioned include those which have between 6 and 10 members. Such groups may be monocyclic, bicyclic or tricyclic, provided that at least one of the rings is aromatic and wherein at least one (e.g. one to four) of the atoms in the ring system is other than carbon (i.e. a heteroatom). Heteroaryl groups that may be mentioned include benzimidazolyl, benzodioxanyl, benzodioxepinyl, benzodioxolyl (including 1 ,3-benzodioxolyl), benzofuranyl, benzofurazanyl, benzothiazolyl, benzothiadiazolyl (including 2,1 ,3-benzothiadiazolyl), benzoxadiazolyl (including 2,1 ,3-benzoxadiazolyl), benzoxazinyl (including 3,4-dihydro- 2H-1,4-benzoxazinyl), benzoxazolyl, benzimidazolyl, benzomorpholinyl, benzoselena- diazolyl (including 2,1 ,3-benzoselenadiazolyl), benzothienyl, chromanyl, cinnolinyl, furanyl, imidazolyl, imidazo[1,2-a]pyridyl, indazolyl, indoliπyl, indolyi, isobenzofuranyl, isochromanyl, isoindolinyl, isoindolyl, isoquinolinyl, isothiaziolyl, isothiochromanyl, isoxazolyl, naphthyridinyl (including 1 ,5-naphthyridinyl and 1 ,8-naphthyridinyl), oxadiazolyl (including 1 ,2,3-oxadiazolyl, 1 ,2,4-oxadiazolyl and 1 ,3,4-oxadiazolyl), oxazolyl, phenazinyl, phenothiazinyl, phthalazinyl, pteridinyl, purinyl, pyrazinyl, pyrazolyl, pyridazinyl, pyridyl, pyrimidinyl, pyrrolyl, quinazolinyl, quinolinyl, quinolizinyl, quinoxalinyl, tetrahydroisoquinolinyl (including 1 ,2,3,4-tetrahydroisoquinolinyl and 5,6,7,8-tetrahydroisoquinolinyl), tetrahydroquinolinyl (including 1 ,2,3,4- tetrahydroquinolinyl and 5,6,7,8-tetrahydroquinolinyl), thiochromanyl, thienyl, triazolyl (including 1 ,2,3-triazolyl, 1 ,2,4-triazolyl and 1 ,3,4-triazolyl) and the like. Substituents on heteroaryl groups may, where appropriate, be located on any atom in the ring system including a heteroatom. The point of attachment of heteroaryl groups may be via any atom in the ring system including (where appropriate) a heteroatom (such as a nitrogen atom), or an atom on any fused carbocyclic ring that may be present as part of the ring system. However, when heteroaryl groups are bicyclic or tricyclic, they are linked to the rest of the molecule via an atom of the aromatic ring. Heteroaryl groups may also be in the N- or S- oxidised form. Heteroatoms that may be mentioned include include phosphorus, silicon, boron, tellurium, selenium and, preferably, oxygen, nitrogen and sulfur.

Salts which may be conveniently used in therapy include physiologically acceptable base salts, for example, derived from an appropriate base, such as an alkali metal (eg sodium), alkaline earth metal (eg magnesium) salts, ammonium and NX₄ ⁺ (wherein X is C_1-4 alky!) salts. Physiologically acceptable acid salts include hydrochloride, sulphate, mesylate, besylate, phosphate and glutamate.

Such salts may be formed by conventional means, for example by reaction of a free acid or a free base form of a parent compound, e.g. of Formula I or Il (or Formula III as described below) with one or more equivalents of an appropriate acid or base, optionally in a solvent, or in a medium in which the salt is insoluble, followed by removal of said solvent, or said medium, using standard techniques (e.g. in vacuo, by freeze-drying or by filtration). Salts may also be prepared by exchanging a counter-ion of a compound of the invention in the form of a salt with another counter-ion, for example using a suitable ion exchange resin.

Although compounds of Formula I or Formula U (or Formula Ui as described below) and salts thereof may possess pharmacological activity as such, certain pharmaceutically- acceptable (e.g. "protected") derivatives of these compounds may exist or be prepared which may not possess such activity, but may be administered parenterally or orally and thereafter be metabolised in the body to form compounds of Formula I or Il (or

Formula III as described below). Such compounds (which may possess some pharmacological activity, provided that such activity is appreciably lower than that of the "active" compounds to which they are metabolised) may therefore be described as

"prodrugs" of compounds of Formula I or Il (or Formula III as described below).

By a "prodrug", we include compounds that form a compound of Formula I or Il (or Formula III as described below) in an experimentally-detectable amount, within a predetermined time (e.g. about 1 hour), following oral or parenteral administration. All prodrugs of the compounds of Formula I and Formula Il (and Formula III as described below) are included within the scope of the invention.

Furthermore, certain compounds of Formula I or Il (or Formula III as described below) may possess no or minimal pharmacological activity as such, but may be administered parenterally or orally, and thereafter be metabolised in the body to form compounds of formula I that possess pharmacological activity as such. Such compounds (which also includes compounds that may possess some pharmacological activity, but that activity is appreciably lower than that of the "active" compounds of Formula I or Il (or Formula III as described below) to which they are metabolised), may also be described as "prodrugs".

Thus, the compounds of Formula I and Formula Il (and Formula III as described below) and salts thereof may be useful because they possess pharmacological activity, and/or are metabolised in the body following oral or parenteral administration to form compounds which possess pharmacological activity.

The compounds of Formula I and Formula Il (and Formula III as described below) are therapeutic agents which are typically formulated for administration to an individual as a pharmaceutical composition, i.e. together with a pharmaceutically acceptable carrier, diluent or excipient.

Accordingly, a third aspect of the invention provides a pharmaceutical composition comprising a compound of Formula I as defined above in the first aspect of the invention, or a compound of Formula U as defined above in the second aspect of the invention, and a pharmaceutically acceptable carrier, diluent or excipient.

By "pharmaceutically acceptable" is included that the formulation is sterile and pyrogen free. Suitable pharmaceutical carriers are well known in the art of pharmacy.

The carriers) must be "acceptable" in the sense of being compatible with the therapeutic agent and not deleterious to the recipients thereof. Typically, the carriers will be water or saline which will be sterile and pyrogen free; however, other acceptable carriers may be used, including carboxymethyl cellulose, Tween and polyethyleneglycol (PEG). Alternative carriers include nanoparticles and/or lipids/biodegradable polymers (see, Sinha et al (2006) MoI Cancer Ther. 5(8): 1909-17; Yih & Al-fandi (2006) J Cell Biochem. 97(6): 1184-90; Duncan (2006) Nat Rev Cancer. 6(9): 688-701) which may have advantages in overcoming problems of low solubility in the more traditional carriers.

The therapeutic agent will generally be formulated for administration in admixture with a suitable pharmaceutical excipient, diluent or carrier selected with regard to the intended route of administration and standard pharmaceutical practice. In various embodiments, the pharmaceutical compositions or formulations of the invention are for parenteral administration, more particularly for intravenous administration, for example by injection. Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents.

In an alternative embodiment, the pharmaceutical composition is suitable for topical administration to a patient, i.e. for dermal administration of the therapeutic agent.

The therapeutic agent may be formulated for administration orally or by any parenteral route, in the form of a pharmaceutical formulation comprising the active ingredient, optionally in the form of a non-toxic organic, or inorganic, acid, or base, addition salt, in a pharmaceutically acceptable dosage form. For example, the therapeutic agent can be administered orally, buccally or sublingually in the form of tablets, capsules, ovules, elixirs, solutions or suspensions, which may contain flavouring or colouring agents, for immediate-, delayed- or controlled-release applications. The compound of invention may also be administered via intracavernσsal injection.

Tablets may contain excipients such as microcrystalline cellulose, lactose, sodium citrate, calcium carbonate, dibasic calcium phosphate and glycine, disintegrants such as starch (preferably corn, potato or tapioca starch), sodium starch glycollate, croscarmellose sodium and certain complex silicates, and granulation binders such as polyvinylpyrrolidone, hydroxypropylmethylcellulose (HPMC), hydroxy-propylcellulose (HPC), sucrose, gelatin and acacia. Additionally, lubricating agents such as magnesium stearate, stearic acid, glyceryl behenate and talc may be included.

Solid compositions of a similar type may also be employed as fillers in gelatin capsules. Preferred excipients in this regard include lactose, starch, a cellulose, milk sugar or high molecular weight polyethylene glycols. For aqueous suspensions and/or elixirs, the therapeutic agents may be combined with various sweetening or flavouring agents, colouring matter or dyes, with emulsifying and/or suspending agents and with diluents such as water, ethanol, propylene glycol and glycerin, and combinations thereof.

The therapeutic agent can also be administered parenterally, for example, intravenously, intra-artehally, intraperitoneally, intrathecally, intraventricular^, intrastemally, intracranially, intra-musculariy or subcutaneously, or they may be administered by infusion techniques. They are best used in the form of a sterile aqueous solution which may contain other substances, for example, enough salts or glucose to make the solution isotonic with blood. The aqueous solutions should be suitably buffered (preferably to a pH of from 3 to 9), if necessary. The preparation of suitable parenteral formulations under sterile conditions is readily accomplished by standard pharmaceutical techniques weli-known to those skilled in the art.

Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.

In further alternative embodiments, the therapeutic agents may be formulated for administration subcutaneously, rectally, nasally, tracheally, bronchially, by any other parenteral route or via inhalation, in a pharmaceutically acceptable dosage form.

Depending upon the cancer and patient to be treated, as well as the route of administration, the compositions may be administered at varying doses. Preferably, the formulation is a unit dosage containing a daily dose or unit, daily sub-dose or an appropriate fraction thereof, of the active ingredient.

For oral and parenteral administration to human patients, the daily dosage level of the therapeutic agent will usually be from 0.25 to 25 g per adult (i.e. from about 5 to 250 mg/kg), administered in single or divided doses. We have shown that both DE002 and

CA224 are efficacious at 100 mg/kg in SCID mice, and we expect that DE002 will show efficacy at still lower concentrations. It is interesting to note that since the fascaplysin analogues of the invention, such as DE002 and CA224, are relatively non-toxic, they can be used at higher concentrations than are possible for most other anti-cancer compounds. Thus, for example, the tablets or capsules of the therapeutic agent may contain from 0.1, 0.5, 1, 5 or 10 g of therapeutic agent for administration singly or two or more at a time, as appropriate. The physician in any event will determine the actual dosage which will be most suitable for any individual patient and it will vary with the age, weight and response of the particular patient. The above dosages are exemplary of the average case. There can, of course, be individual instances where higher or lower dosage ranges are merited and such are within the scope of this invention.

The amount of a compound in a dose is typically an "effective amount", by which we mean an amount of the compound which confers a therapeutic effect on the treated patient. The effect may be objective (i.e. measurable by some test or marker) or subjective (i.e. the subject gives an indication of or feels an effect).

Preferred pharmaceutical formulations include those in which the active ingredient is present in at least 1% (such as at least 10%, preferably in at least 30% and most preferably in at least 50%) by weight. That is, the ratio of active ingredient to the other components (i.e. the addition of adjuvant, diluent and carrier) of the pharmaceutical composition is at least 1:99 (e.g. at least 10:90, preferably at least 30:70 and most preferably at least 50:50) by weight.

In any event, the physician, veterinarian or skilled person will be able to determine the actual dosage which will be most suitable for an individual patient, which is likely to vary with the route of administration, the type and severity of the condition that is to be treated, as well as the species, age, weight, sex, renal function, hepatic function and response of the particular patient to be treated. The above-mentioned dosages are exemplary of the average case; there can, of course, be individual instances where higher or lower dosage ranges are merited, and such are within the scope of this invention.

The therapeutic agent may be administered intranasally or by inhalation and are conveniently delivered in the form of a dry powder inhaler or an aerosol spray presentation from a pressurised container, pump, spray or nebuliser with the use of a suitable propellant, e.g. dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoro-ethane, a hydrofluoroalkane such as 1 ,1 ,1 ,2-tetrafluoroethane (HFA 134A3 or 1 ,1 ,1 ,2,3,3,3-heptafluoropropane (HFA 227EA3), carbon dioxide or other suitable gas. In the case of a pressurised aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. The pressurised container, pump, spray or nebuliser may contain a solution or suspension of the active compound, e.g. using a mixture of ethanol and the propellant as the solvent, which may additionally contain a lubricant, e.g. sorbitan trioleate. Capsules and cartridges (made, for example, from gelatin) for use in an inhaler or insufflator may be formulated to contain a powder mix of a therapeutic agent and a suitable powder base such as lactose or starch.

Aerosol or dry powder formulations are preferably arranged so that each metered dose or "puff' contains a clinically-useful dose of the therapeutic agent for delivery to the patient. It will be appreciated that the overall daily dose with an aerosol will vary from patient to patient, and may be administered in a single dose or, more usually, in divided doses throughout the day.

Alternatively, the therapeutic agent can be administered in the form of a suppository or pessary, or they may be applied topically in the form of a lotion, solution, cream, ointment or dusting powder. The therapeutic agents may also be transdermal^ administered, for example, by the use of a skin patch. They may also be administered by the ocular route, particularly for treating cancers of the eye such as retinoblastoma. For ophthalmic use, the therapeutic agent can be formulated as mi^'cronised suspensions in isotonic, pH adjusted, sterile saline, or, preferably, as solutions in isotonic, pH adjusted, sterile saline, optionally in combination with a preservative such as a benzylalkonium chloride. Alternatively, they may be formulated in an ointment such as petrolatum.

For application topically to the skin, the therapeutic agent can be formulated as a suitable ointment containing the active compound suspended or dissolved in, for example, a mixture with one or more of the following: mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene polyoxypropylene compound, emulsifying wax and water. Alternatively, they can be formulated as a suitable lotion or cream, suspended or dissolved in, for example, a mixture of one or more of the following: mineral oil, sorbitan monostearate, a polyethylene glycol, liquid paraffin, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.

Formulations suitable for topical administration in the mouth include lozenges comprising the active ingredient in a flavoured basis, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert basis such as gelatin and glycerin, or sucrose and acacia; and mouth-washes comprising the active ingredient in a suitable liquid carrier.

In an embodiment, the therapeutic agent may be delivered using an injectable sustained-release drug delivery system. These are designed specifically to reduce the frequency of injections. An example of such a system is the Nutropin Depot which encapsulates recombinant human growth hormone (rhGH) in biodegradable microspheres that, once injected, release rhGH slowly over a sustained period.

Alternatively, the therapeutic agent may be administered by a surgically implanted device that releases the drug directly to the required site. For example, Vitrasert releases ganciclovir directly into the eye to treat CMV retinitis. The direct application of this toxic agent to the site of disease achieves effective therapy without the drug's significant systemic side-effects.

For veterinary use, a therapeutic agent is administered as a suitably acceptable formulation in accordance with normal veterinary practice and the veterinary surgeon will determine the dosing regimen and route of administration which will be most appropriate for a particular animal.

A fourth aspect of the invention provides a compound of Formula I as defined above in the first aspect of the invention, or a compound of Formula Il as defined above in the second aspect of the invention, for use in medicine.

As described herein, the therapeutic agents of Formula I and Formula Il display a number of properties that make them highly suitable as anticancer agents. Accordingly, a fifth aspect of the invention provides a method of treating cancer in a patient, the method comprising administering to the patient a compound of Formula I as defined above in the first aspect of the invention, or a compound of Formula Il as defined above in the second aspect of the invention.

This aspect of the invention also provides the use of a compound of Formula I as defined above in the first aspect of the invention, or a compound of Formula Il as defined above in the second aspect of the invention, in the preparation of a medicament for treating cancer in a patient. This aspect of the invention also provides a compound of Formula I as defined above in the first aspect of the invention, or a compound of Formula Il as defined above in the second aspect of the invention, for use in treating cancer in a patient.

Further preferences for the treatment of cancer are described below.

CA224

The reason that both Aubry et al (2006) and Mahale et al (2006a) concluded that CA224 could be the basis for the development of more potent CDK4 specific inhibitors, and did not state that CA224 could be used as an anticancer agent, is that its IC₅₀ for Cdk-4 inhibition and for inhibition of cancer cell proliferation were considered to be too high, and thus not clinically optimal for treating cancer (Hannah (2005) Current Molecular Medicine, 5(7): 625-642). Indeed, the Pfizer Cdk4-inhibitor compounds inhibit at nanomolar IC₅₀ concentrations (WO 2005/005426 and WO 2006/024945).

By contrast, we have now identified a number of surprisingly beneficial properties of CA224 that would lead the skilled person to consider CA224 and analogues thereof to be suitable for use in treating cancer.

Specifically, as presented in Example 2, CA224 was identified as the most potent molecule of the CA224 series of compounds (IC₅₀ for Cdk4-cyclin D1 = 5.5 μM). We extensively studied CA224 for its inhibitory action on other Cdks like Cdk2-cyclin A, Cdk2-cyclin E, Cdk1-cyclin B1 and Cdk9-cyclin T1 in vitro, and we found that CA224 inhibits these Cdk-s with an IC₅₀ greater than 500 μM, i.e. CA224 is highly selective for Cdk4-cyclin D1. Interestingly, CA224 inhibits the growth of cancer cells at concentrations (average IC₅₀ in ten cell lines = 3 μM) lower than the concentration at which it inhibits Cdk4-cyclin D1 in vitro. It blocks the cancer cell growth at G0/G1 in a pRb-dependent manner. Surprisingly, we found that CA224 blocks more profoundly at the G₂/M phase of the cell cycle depending on the cell type chosen for the investigation and concentration of the compound used. Further investigations showed that CA224 inhibits tubulin polymerisation in vitro and also acts as an enhancer for tubulin depolymerisation (of tubulin stabilized by paclitaxel) in vivo. The dual mechanism of action of CA224 proved crucial for the block of growth at the G₀ZG₁ phase of the cell cycle of cancer cells (Calu-1) with impaired mitotic spindle checkpoint. The G₀/Gi block seen in Calu-1 cells is imposed as a consequence of Cdk4-cyclin D1 inhibition. It is very likely that the G₀/Gi arrest is a result of the prevention of pRb phosphorylation at Cdk4-specific serine residues, Ser780, Ser795 and Ser807/811. CA224 induces massive apoptosis in cancer cells which are known to be resistant to chemotherapy and significantly reduces the colony formation efficiency (in a dose-dependent manner) of the lung cancer cells, A549 and Calu-1. NSCLC is present in 85% of lung cancer patients and is resistant to chemotherapy (Boyle and J. Ferlay 2005). Thus, induction of apoptosis by CA224 in A549 and Calu-1 cells is highly significant and suggests that CA224 could be used for the treatment of lung cancer. Accordingly, based on these unexpected observations, the inventors now consider that CA224 may be a useful compound for the treatment of cancer, or a lead compound for the development of an improved anticancer agent.

CA224 is a fascaplysin analogue that has an unsubstituted phenyl group in the ortho- position. Without wishing to be bound by theory, we propose that this feature in a fascaplysin analogue is required for the activity of CA224 and, together with a central amido group having a substituent other than H (methyl group preferred), defines the CA224 series of compounds (Formula I, below).

Accordingly, a sixth aspect of the invention provides a method of treating cancer in a patient, the method comprising administering to the patient a compound of Formula III

wherein each of R¹ to R⁴ may independently represent H, halo, CN, NO₂, C_M2 alkyl, C,. 1₂ alkenyl, C_1-I2 alkynyl, C_3-12 cycloalkyl or C_4-12 cycloalkenyl, which latter five groups are optionally substituted by one or more substituents selected from halo, nitro, CN, C_1- ₆ alkyl, Ci_-6 alkenyl, C_1-6 alkynyl, C_3-8 cycloalkyl (which latter three groups are optionally substituted by one or more substituents selected from OH, =0, halo, C_1-4 alkyl and C_1-4 alkoxy), OR^8a, S(O)_pR^8b, S(O)₂N(R^8c)(R^8d), N(R^8e)S(O)₂R^8f, N(R^8g)(R^8h), and which C₃-I₂ cycloalkyl or C_4-I2 cycloalkenyl groups may additionally be substituted by =O;

R⁵ is either not present, or represents one to four substituents on the fused benzene ring selected from aryl, Het¹, halo, CN, NO₂, C_1-12 alkyl, C_1-I₂ alkenyl, C_1-I₂ alkynyl, C_3-12 cycloalkyl or C_4-12 cycloalkenyl, which latter five groups are optionally substituted by one or more substituents selected from halo, nitro, CN, C_1-6 alkyl, C₁^ alkeπyl, Ci_-6 alkynyl, C_3-8 cycioalkyl (which latter three groups are optionally substituted by one or more substituents selected from OH, =O, halo, C_1-4 alkyl and C_1-4 alkoxy), OR^9a, S(O)_qR^9b, S(O)₂N(R^9c)(R^9d), N(R⁹⁶JS(O)₂R⁹*, N(R")(R^9h) and Het², and which C_3-12 cycioalkyl or C_4-I₂ cycloalkenyl groups may additionally be substituted by =0;

R⁶ represents H, C_1--_I2 alkyl, C_3--_I2 cycioalkyl, C_3-12 cycloalkenyl (which latter three groups are optionally substituted by one or more substituents selected from halo, nitro, CN, Ci_-6 alkyl, C_1-6 alkenyl, C_1-6 alkynyl, C_3-s cycioalkyl (which latter three groups are optionally substituted by one or more substituents selected from OH, =0, halo, C_1-4 alkyl and C_1-4 alkoxy), OR^10a, S(O)_rR^10b, S(O)₂N(R^10c)(R^10d), N(R^10e)S(O)₂R^10f, N(R^1θ9)(R^1Oh), aryl and Het³, and which C_3-12 cycioalkyl or C_4-12 cycloalkenyl groups may additionally be substituted by =0);

R⁷ represents C_1-6 alkyl;

R^8a to R^8h, R^9a to R^9h and R^1Oa to R^10h, independently represent, at each occurrence, (a) H, (b) C_1-10 alkyl, C_2-10 alkenyl, C_2-10 alkynyl (which latter three groups are optionally substituted by one or more substituents selected from halo, OH, C_1-6 alkoxy, aryl and Het⁵),

(c) C_3-10 cycioalkyl, C_4-10 cycloalkenyl (which latter two groups are optionally substituted by one or more substituents selected from halo, OH, =O, C_1-6 alkyl, C_1-6 alkoxy, aryl and Het⁶),

(d) aryl or

(e) Het⁷, provided that R^8b, R^9b or R^1Ob does not represent H when n, p, q, r or s respectively is 1 or 2;

each aryl independently represents a C_6-10 carbocyclic aromatic group, which group may comprise either one or two rings and may be substituted by one or more substituents selected from (a) halo, (b) CN,

(c) C_1-12 alkyl, Cv₁₂ alkenyl, C_1-12 alkynyl, C_3-12 cycioalkyl or C_4-12 cycloalkenyl, which latter five groups are optionally substituted by one or more substituents selected from halo, nitro, CN, C₁^ alkyl, C_1-6 alkenyl, C_1-6 alkynyl, C_3-8 cycloalkyl (which latter three groups are optionally substituted by one or more substituents selected from OH, =0, halo, C₁^ alkyl and C_1-4 alkoxy), OR^11a, S(O)_tR^11b, S(O)₂N(R^11c)(R^l1d), N(R^11e)S(O)₂R^11f, N(R^11g)(R^11h), phenyl, naphthyl (which latter two groups are optionally susbtituted by one or more substituents selected from OH, halo, C_1-4 alkyl and C_1-4 alkoxy) and Het⁸, and which C_3-12 cycloalkyl or C_4-12 cycloalkenyl groups may additionally be substituted by =0,

(d) 0R^12a,

(e) S(O)_uR^12b, (f) S(O)₂N(R^12c)(R^12d),

(g) N(R^12e)S(O)₂R^12f,

(h) N(R¹²⁹)(R^12h),

(i) phenyl (which latter group is optionally susbtituted by one or more substituents selected from OH, halo, C_1-4 alkyl and C_1-4 alkoxy) or G) Het⁹;

R^11a to R^11h and R^12a to R^12h independently represent, at each occurrence,

(a) H,

(b) C_1-12 alkyl, C_2-12 alkenyl, C_2-I2 alkynyl, C_3-12 cydoaikyl, C_4-12 cycloalkenyl (which latter five groups are optionally substituted by one or more substituents selected from halo, OH, C_1-6 alkyl, C_3--_I2 cycloalkyl, C_4-12 cycloalkenyl (which latter two groups are optionally substituted by one or more substituents selected from OH, =0, halo, C_1-4 alkyl and Ci_-4 alkoxy), C_1-6 alkoxy, NH₂, N(H)-Ci_-6 alkyl, N(C_1-6 alkyl)₂, phenyl (which latter group is optionally substituted by one or more substituents selected from OH, halo, C_1-4 alkyl and Ci_-4 alkoxy) and Het¹⁰, and which C_3--_I2 cycloalkyl or C_4-I2 cycloalkenyl groups may additionally be substituted by =0),

(c) phenyl (which latter group is optionally substituted by one or more substituents selected from OH, CN, halo, C₁^₃ alkyl and C_1-6 alkoxy) or

(e) Het¹¹, provided that R^12b or R^13b does not represent H when t or u, respectively is 1 or 2;

Het¹ to Het¹¹ independently represent 4- to 14-membered heterocyclic or 5- to 10-membered heteroaromatic groups containing one or more heteroatoms selected from oxygen, nitrogen and/or sulfur, which heterocyclic groups may comprise one, two or three rings, and may be substituted by one or more substituents selected from

(a) halo,

(b) CN, (c) C_1-12 alkyl, C_1-12 alkenyl, C_1-12 alkynyl, C_3-12 cycloalkyl or C_4-12 cycloalkenyl, which latter five groups are optionally substituted by one or more substituents selected from halo, nitro, CN, Ci_-6 alkyl, C_1-6 alkenyl, C_1-6 alkynyl, C_3-8 cycloalkyl (which latter three groups are optionally substituted by one or more substituents selected from OH, =0, halo, C_1-4 alkyl and C_1-4 alkoxy), 0R^13a, S(O)_vR^13b, S(O)₂N(R^13c)(R^13d),

N(R^13e)S(O)₂R^13f, N(R¹³⁹)(R^13h), phenyl, naphthyl (which latter two groups are optionally susbtituted by one or more substituents selected from OH, halo, C_1-4 alkyl and C_1-4 alkoxy) and Hef, and which C_3-12 cycloalkyl or C_4-I2 cycloalkenyl groups may additionally be substituted by =0, (d) 0R^14a,

(e) =0,

(f) S(O)_wR^14b,

(g) S(O)₂N(R^14c)(R^ud), (h) N(R^14e)S(O)₂R^14f, (i) N(R¹⁴⁹)(R^14h),

R^13a to R^13h and R^14a to R^14h independently represent, at each occurrence,

(a) H,

(b) C_1-12 alkyl, C_2-I2 alkenyl, C_2-12 alkynyl, C_3-12 cycloalkyl, C_4-12 cycloalkenyl (which latter five groups are optionally substituted by one or more substituents selected from halo, OH, C_1-6 alkyl, C_3-12 cycloalkyl, C_4-12 cycloalkenyl (which latter two groups are optionally substituted by one or more substituents selected from OH, =0, halo, C_1-4 alkyl and C_1-4 alkoxy), C_1-6 alkoxy, phenyl (which latter group is optionally substituted by one or more substituents selected from OH, halo, C_1-4 alkyl and Ci_-4 alkoxy) and Hef, and which C_3--_I2 cycloalkyl or C_4-12 cycloalkenyl groups may additionally be substituted by =0), (c) phenyl (which latter group is optionally substituted by one or more substituents selected from OH, halo, C_1-4 alkyl and C_1-4 alkoxy) or (e) Het^d; provided that R^13b or R^14b does not represent H when v or w, respectively is 1 or 2;

n, p, q, r, s, t, u, v and w independently represent O, 1 or 2; Het^a to Het^d independently represent 5- or 6-membered heterocyclic groups containing one to four heteroatoms selected from oxygen, nitrogen and/or sulfur, which heterocyclic groups may be substituted by one or more substituents selected from halo, =0 and C_1-6 alkyl; and

unless otherwise specified

or a pharmaceutically-acceptable derivative, salt, solvate or prodrug thereof, wherein the use is treating cancer.

Further compounds of formula III that can be mentioned include ones in which: each of R¹ to R⁴ may independently represent H, halo, CN, NO₂, C_1-12 alkyl, C_1- ₁₂ alkenyl, C_1-12 alkynyl, C_3-I2 cycloalkyl or C_4--_I2 cycloalkenyl, which latter five groups are optionally substituted by one or more substituents selected from halo, nitro, CN, C₁.

₆ alkyl, C_1-6 alkenyl, C_1-6 alkynyl, C_3-8 cycloalkyl (which latter three groups are optionally substituted by one or more substituents selected from OH, =0, halo, C_1-4 alkyl and C_1-4 alkoxy) or OR^8a;

R⁵ is either not present, or represents one to two substituents on the fused benzene ring selected from halo, CN, NO₂, C_1-12 alkyl, C_1-12 alkenyl, C_1--_I2 alkynyl, C_3-12 cycloalkyl or C_4-12 cycloalkenyl, which latter five groups are optionally substituted by one or more substituents selected from halo, nitro, CN, C_1-6 alkyl, C_1-6 alkenyl, C_1-6 alkynyl, C_3-8 cycloalkyl (which latter three groups are optionally substituted by one or more substituents selected from OH, =0, halo, C_1-4 alkyl and C_1-4 alkoxy) or 0R^9a;

R⁶ represents H;

R⁷ represents methyl, ethyl, propyl or isopropyl;

R^8a and R^9a independently represent, at each occurrence, (a) H, (b) C-_I-6 alkyl, C_2-6 alkenyl, C_2-6 alkynyl (which latter three groups are optionally substituted by one or more substituents selected from halo, OH, Ci_-6 alkoxy and aryi), or

(c) C_3-6 cycloalkyl, C_4-6 cycloalkenyl (which latter two groups are optionally substituted by one or more substituents selected from halo, OH, =0,

C_1-6 alkyl, Ci_-6 alkoxy and aryl);

each aryl independently represents a C_6-I0 carbocyclic aromatic group, which group may comprise either one or two rings and may be substituted by one or more substituents selected from

(a) halo,

(b) CN,

(c) C_1-6 alkyl or C_3-6 cycioalkyl which latter two groups are optionally substituted by one or more substituents selected from halo, nitro, CN, C_1-6 alkyl, C_3-8 cycloalkyl (which latter group i.e. optionally substituted by one or more substituents selected from OH, =0, halo, Ci_-4 alkyl and C_1-4 alkoxy) or 0R^11a, or

(d) 0R^12a;

R^11a and R^12a independently represent, at each occurrence, (a) H,

(c) C_3-6 cycloalkyl, C₄^ cycloalkenyl (which latter two groups are optionally substituted by one or more substituents selected from halo, OH, =0,

C_1-6 alkyl, C_1-6 alkoxy and aryl).

In an embodiment it may be preferred that R⁵ is either not present, or represents one substituent on the fused benzene ring selected from halo, CN, NO₂, C_1-12 alkyl, C_1-12 alkenyl, C_1-12 alkynyl, C_3--_I2 cycloalkyl or C_4-12 cycloalkenyl, which latter five groups are optionally substituted by one or more substituents selected from halo, nitro, CN, C_1-6 alkyl, Ci_-6 alkenyl, C_1-6 alkynyl, C_3-8 cycloalkyl (which latter three groups are optionally substituted by one or more substituents selected from OH, =0, halo, C_1-4 alkyl and C_1-4 alkoxy), 0R^9a.

In an embodiment, each of R¹ to R⁴ may independently represent H, halo, or C_1-6 alkyl; and R⁵ is either not present, or represents one substituent on the fused benzene ring selected from selected from halo or Ci_-6 alkyl.

In a more specific embodiment each of R¹ to R⁴ may independently represent H, halo, or Ci-₃ alkyl.

In a yet more specific embodiment each of R¹ to R⁴ may independently represent H or halo.

In a further embodiment, R⁵ is not present; R⁶ represents H; and R⁷ represents methyl or ethyl.

In a still further embodiment R¹ to R⁴ represent H.

In a more specific embodiment R¹ to R⁴ represent H; R⁵ is not present; R⁶ represents H; and R⁷ is methyl.

In a specific preferred embodiment the compound of Formula III is, or comprises, biphenyl-4-carboxylic acid [2-(1 H-indol-3-yl)-ethyl]-methyl-amide (CA224).

This aspect of the invention includes the use of a compound of Formula III as described above in the preparation of a medicament for use in treating cancer in a patient.

The invention also includes a Formula III as described above for use in treating cancer in a patient.

The patient to be treated may be any individual who would benefit from such treatment. Typically and preferably the patient to be treated is a human. However, the methods and compositions of the invention may be used to treat cancer in mammals including agriculturally important animals such as cows, horses, pigs and sheep as well as domestic pets such as cats and dogs. Thus, the methods have uses in both human and veterinary medicine. Due to the multiple sites of action within the cell cycle, a large range of cancers are suitable for treatment by the compounds of Formula I, Il and III, including breast cancer, colorectal cancer, pancreatic cancer, lung cancer, myeloma, glioblastoma or retinoblastoma. Cancer is caused by multi-genetic events (mis-regulation of a single gene rarely, if ever, causes cancer). Therefore, in order for a cancer to be curable, multiple genes/proteins should be targeted. Unlike Cdk4-specific inhibitors or other Cdk-specific inhibitors, the compounds of Formula I₁ Il and III have multiple modes of anticancer activity.

Since we have shown that compounds of Formula I, Il and III are not solely or primarily Cdk4 inhibitors, we now consider the compounds of Formula I₁ Il and III can be used to treat cancers that are not suitable for treatment by Cdk4 inhibitors or that are refractory to Cdk4 inhibition. Cdk4 inhibitor refractory tumours are well known in the art and include drug-resistant tumours. Thus, for example, flavopiridol, a pan-Cdk inhibitor which potently inhbits Cdk4, does not inhibit drug-resistant tumours. Indeed, flavopiridol itself causes resistance in cancer cells - possibly through upregulation of the telomerase catalytic subunit (Incles et al (2003) MoI Pharmacol. 64(5): 1101-8). From our own unpublished observations and from results in ongoing clinical studies, it is clear that Cdk4 inhibitors do not function against cytoxic drug-resistant tumours. This is one of the reasons for the proposal that Cdk-specific inhibitors can only be used effectively in combination with cytotoxic drugs (Grant & Roberts (2003) Drug Resist Updat. 6(1): 15-26). Due to their multi-pronged attack on cancer cells, any resistance to the compounds of Formula I₁ Il and III would only be expected to develop slowly, if at all. Accordingly, we now consider that compounds of Formula I₁ Il and III are therapeutically effective against cytoxic drug-resistant tumours.

As discussed above, Cdk4 inhibitors exert their effect via regulation of pRb, and are not suitable for the treatment of pRb-negative (pRb-mutant or pRb-null) tumours. By contrast, we have shown that compounds of Formula I₁ Il and III are effective against Rb-negative cancer cells in vitro. Accordingly, we now consider that compounds of Formula I, Il and III are therapeutically effective in treating pRb-negative cancers. Rb loss is known to occur in many tumour types. For example, complete Rb-loss is found in retinoblastoma, about 80% of small cell lung cancers, and 2-30% of non-small cell lung cancer (Sherr & McCormick (2002) "The RB and p53 pathways in cancer." Cancer Ce// 2: 103-112). Indeed, most cancers have the potential of becoming pRb-negative. Many malignant Grade III and IV tumours are pRb-negative, independent of the cancer type. As the percentage of cells in a tumour that are pRb-negative increases, the cells would become more and more resistant to Cdk4 inhibition. Even low grade (i.e., Grade I benign tumours) which are pRb-negative will not be treatable by Cdk4 inhibitors.

By a cancer that is pRb-negative we include a cancer that has a high proportion of cells that are pRb-mutant or pRb-null. Typically, a cancer that is pRb-negative has at least 50%, or at least 60%, or at least 70%, preferably at least 80%, more preferably at least 85%, yet more preferably at least 90%, and still more preferably at least 95% of cancer cells that are pRb-mutant or pRb-null. More preferably, a cancer that is pRb-negative has at least 96%, or at least 97%, or at least 98%, or at least 99%, or at least 99.9%, or more of cancer cells that are pRb-mutant or pRb-null. Most preferably, all of the cancer cells in a cancer that is pRb-negative are pRb-mutant or pRb-null.

By a cancer cell that is pRb-mutant we mean that the cancer cell does not produce any functional Rb protein or does so at undetectable levels. In this context, the function of pRb is the ability to act as a repressor of genes required for G-|/S transition, and initiation and completion of DNA synthesis, when unphosphorylated by Cdk4-cyclin D. As is known in the art, this can be measured by ELiSA using antibodies raised against phosphor-specific epitopes phosphorylated only by Cdk4.

By a cancer that is pRb-null we mean that the cancer cell does not produce any Rb protein because it lacks a functional RB gene.

The fact that a cancer cell may be Rb-mutant or Rb-null may be due to either insertional, deletional or point mutational events in the Rb gene. Typically, if both alleles are mutated then the cell is Rb-mutant or Rb-null. It is believed that most mutations found in cancer tissues will be those leading to greatly decreased expression of the Rb gene product. However, mutations leading to non-functional gene products would also lead to a metastatic state. Point mutational events may occur in regulatory regions, such as in the promoter of the gene, leading to loss or diminution of expression of the mRNA. Point mutations may also abolish proper RNA processing, leading to loss of expression of the Rb gene product.

Accordingly, in an embodiment, the method comprises the prior step of determining the Rb status of the cancer before administering the compound of Formula I, II, or III to the patient. In other words, this embodiment of the invention provides a method of treating Rb- negative cancer in a patient, the method comprising: determining that the cancer is Rb-negative; and administering the compound of Formula I, II, or III to the patient.

Methods for determining whether a cancer is Rb-negative are described below.

The invention includes the use of a compound of Formula I, II, or III in the preparation of a medicament for treating cancer in a patient who has previously been determined to have Rb-negative cancer.

We have shown that compounds of Formula I, Il and III up-regulate p53, a known tumour suppressor protein, which exerts its effect by inducing p21. DE002, for example, down-regulates cyclin B1 and Cdk1 in p53+ cancer cells and up-regulates the pan-Cdk inhibitory proteins p21 and p27, providing a mechanistic insight into the block of cell growth in p53+ cells and suggesting that p53+ cells are blocked at G₀IGi, G₂, and M phases of the cell division cycle. Thus, the cancer to be treated may be a p53+ cancer.

All types of cancers may contain both p53-positive (p53+) and p53-negative cells. The p53-negative cells are malignant and are untreatable. As a cancer progresses, all cancers have the potential of becoming p53-negative (p53-null or p53-mutant). Even if a single cell in a cancer was to be p53-negative, and if it was not treatable, then the cancer will not be cured.

We have shown that compounds of Formula I₁ Il and III upregulate cyclin B1 levels in p53-negative (p53-mutant and p53-null) cancer cells indicating that, in these cells, the major block occurs at a post-G₂ phase of the cell cycle. Although we have shown that compounds of Formula I₁ Il and III cause a significant reduction in the colony formation efficiency of both p53+ and p53-negative cancer cells, they are surprisingly and unexpectedly more effective in p53-negative cells. This is a particularly surprising and beneficial property since loss of p53 in tumours is very wide spread and generally causes tumours to be resistant to chemotherapy.

Thus, in a preferred embodiment, the cancer to be treated may be a p53-negative cancer, whether p53-null tumours or containing point mutations in the p53 gene (p53- mutant). As is well known in the art, point mutations in p53 have been found in the majority of cancers, albeit at varying frequencies. Mutations are found in only 10-20% of leukaemias, prostate cancers or hepatocarcinomas, but in as many as 60-70% of ovarian, bladder, head and neck, colon and lung carcinomas. Overall it is estimated that as many as 40-50% of all cancers harbour p53 mutations. In the majority of cases, mutations in one allele are accompanied by loss of the second allele (LOH, loss of heterozygosity). However, in some cancers, p53 inactivation occurs via epigenetic mechanisms including p53 protein degradation in cervical carcinoma mediated by human papilloma virus (HPV) and degradation/inactivation of p53 by mdm-2 in soft tissue sarcomas (Fojo (2002) "p53 as a therapeutic target: unresolved issues on the road to cancer therapy targeting mutant p53" Drug Resistance Updates 5: 209-216).

By a cancer that is p53-negative we include a cancer that has a high proportion of cells that are p53-mutant or p53-null. Typically, a cancer that is p53-negative has at least 50%, or at least 60%, or at least 70%, preferably at least 80%, more preferably at least 85%, yet more preferably at least 90%, and still more preferably at least 95% of cancer cells that are p53-mutant or p53-null. More preferably, a cancer that is p53-negative has at least 96%, or at least 97%, or at least 98%, or at least 99%, or at least 99.9%, or more of cancer cells that are p53-mutant or p53-null. Most preferably, all of the cancer cells in a cancer that is p53-negative are p53-mutant or p53-null.

By a cancer cell that is p53-mutant we mean that the cancer cell does not produce any functional p53 protein or does so at undetectable levels. In this context, the function of p53 is the ability to act as a repressor of p21. As is known the person of skill in the art, p21 function can be measured using the p21 promoter linked to a reporter gene.

By a cancer that is p53-null we mean that the cancer cell does not produce any p53 protein because of the lack of a functional p53 gene.

Accordingly, in an embodiment, the method comprises the prior step of determining the p53 status of the cancer before administering the compound of Formula I₁ II, or III to the patient.

In other words, this embodiment of the invention provides a method of treating p53- negative cancer in a patient, the method comprising: determining that the cancer is p53-negative; and administering a compound of Formula I, II, or III to the patient. The invention thus includes the use of a compound of Formula I₁ II, or III in the preparation of a medicament for treating cancer in a patient who has previously been determined to have p53-negative cancer.

As with Rb₁ the fact that a cancer cell may be p53-mutant or p53-null may be due to either insertional, deletional or point mutational events in the p53 gene. Typically, if both alleles are mutated then the cell is p53-mutant or p53-null. It is believed that most mutations found in cancer tissues will be those leading to greatly decreased expression of the p53 gene product. However, mutations leading to non-functional gene products would also lead to a metastatic state. Point mutational events may occur in regulatory regions, such as in the promoter of the gene, leading to loss or diminution of expression of the mRNA. Point mutations may also abolish proper RNA processing, leading to loss of expression of the p53 gene product.

Typically, determining whether a cancer cell is p53-negative or positive or Rb-negative or positive is carried out by RT-PCR on a sample of the cancer tissue or cancer cells using methods that are now well known in the art. Messenger RNA (mRNA) is isolated from the total RNA of a small section of a tumour tissue flash frozen in liquid nitrogen. RT-PCR using primers specific for the 5' and the 3'-ends of RB or p53 genes is used to isolated cDNA that encodes RB and p53. Tissues which harbour null mutations (i.e. cells which contain homozygous deletions of the genes) will yield no PCR product whereas a product would be obtained from a control tissue that harbours either the wild-type RB or the p53 gene. A tissue harbouring point mutations in the gene (RB or p53) will yield a gene product. DNA sequencing confirms inactivating mutations in the genes. Typically, this is followed by sequencing the p53 or Rb transcript to look for inactivating mutations in the genes. Alternatively, inactivating mutations in the p53 or Rb transcript can be detected using any of the other ways well known in the art. By this route, the person of skill in the art can determine whether a cancer expresses any p53 or Rb, and whether any p53 or Rb thus expressed is functional (i.e. whether the cancer is p53- and/or pRb- negative or positive, as defined).

Alternatively, histochemistry, using commercially available pRb and p53-specific antibodies (from Calbiochem and Santa Cruz Biotechnology, respectively), can be used to confirm null mutations. Tissues, which do not have a functional pRb or p53 protein (because the cells have null mutations of the corresponding genes) will not be stained whereas the proteins will be clearly visualised in tissues that harbour wild-type genes.

It has been observed that Cdk inhibitors, when used in combination with conventional cytotoxic drugs, potentiate cell death in a number of tumour models (Tenzer and

Pruschy 2003; Robson et al, 2005). Cdk inhibitors have been used in combination with several other DNA damaging agents and chemotherapeutic agents like cisplatin, 5- flurouracil, paclitaxel, mitomycin C, doxorubicin and gemcitabine. Highly encouraging outcomes from these different drug combination experiments have been observed in clinical studies. Synergistic effects against tumour growth, sensitization of tumour cells, potentiation of apoptosis and increased cell death are the main effects found after

Cdk inhibitors are used in combination with other anticancer molecules (Lara et al,

2005; Schwartz et al, 1997; Kortmansky et al, 2005).

Most cell cycle inhibitors are being used in the clinic in conjunction with another cytoxic or mechanism-based inhibitor (e.g., the proteasome inhibitor, Velcade). It is appreciated that the compounds of Formula I, Il and III may be effective in the absence of any other anti-cancer compound because they have multiple modes of action. Nevertheless, it may be advantageous to admionister these compounds in conjunction with a further anticancer agent.

Accordingly, a seventh aspect of the invention provides a pharmaceutical composition comprising: (i) a compound of Formula I as defined above in the first aspect of the invention, or a compound of Formula Il as defined above in the second aspect of the invention, or a compound of Formula III as defined above in the sixth aspect of the invention, and (ii) a further anticancer agent, and a pharmaceutically acceptable carrier, diluent or excipient.

The further anticancer agent may be selected from alkylating agents including nitrogen mustards such as mechlorethamine (HN₂), cyclophosphamide, ifosfamide, melphalan (L- sarcolysin) and chlorambucil; ethylenimines and methylmelamines such as hexamethylmelamine, thiotepa; alkyl sulphonates such as busulphan; nitrosoureas such as carmustine (BCNU), lomustine (CCNU), semustine (methyl-CCNU) and streptozocin

(streptozotocin); and triazenes such as decarbazine (DTIC; dimethyltriazenoimidazole- carboxamide); antimetabolites including folic acid analogues such as methotrexate

(amethopterin); pyrimidine analogues such as fluorouracil (5-fluorouracil; 5-FU), floxuridine (fluorodeoxyuridine; FUdR) and cytarabine (cytosine arabinoside); and purine analogues and related inhibitors such as mercaptopurine (6-mercaptopurine; 6-MP), thioguanine (6-thioguanine; TG) and pentostatin (2'-deoxycoformycin); natural products including vinca alkaloids such as vinblastine (VLB) and vincristine; epipodophyllotoxins such as etoposide and teniposide; antibiotics such as dactinomycin (actinomycin D), daunorubicin (daunomycin; rubidomycin), doxorubicin, bleomycin, plicamycin (mithramycin) and mitomycin (mitomycin C); enzymes such as L-asparaginase; and biological response modifiers such as interferon alphenomes; miscellaneous agents including platinum coordination complexes such as cisplatin (c/s-DDP) and carboplatin; anthracenedione such as mitoxantrone and anthracycline; substituted urea such as hydroxyurea; methyl hydrazine derivative such as procarbazine (N-methylhydrazine, MIH); and adrenocortical suppressant such as mitotane (o,p'-DDD) and aminoglutethimide; taxol and analogues/derivatives; cell cycle inhibitors; proteosome inhibitors such as Bortezomib (Velcade^®); signal transductase (e.g. tyrosine kinase) inhibitors such as lmatinib (Glivec^®), COX-2 inhibitors, and hormone agonists/antagonists such as flutamide and tamoxifen.

The clinically used anticancer agents are typically grouped by mechanism of action: Alkylaying agents, Topoisomerase I inhibitors, Topoisomerase Il inhibitors, RNA/DNA antimetabolites, DNA antimetabolites and Antimitotic agents. The US NIH/National Cancer Institute website lists 122 compounds

(http://dtp.nci.nih.gov/docs/cancer/searches/standard_mechanism.html), all of which may be used in conjunction with compounds of Formula I, II, or III. They include Alkylating agents including Asaley, AZQ, BCNU, Busulfan, carboxyphthalatoplatinum, CBDCA, CCNU₁ CHIP, chlorambucil, chlorozotocin, c/s-platinum, clomesone, cyanomorpholinodoxorubicin, cyclodisone, dianhydrogalactitol, fluorodopan, hepsulfam, hycanthone, melphalan, methyl CCNU, mitomycin C, mitozolamide, nitrogen mustard, PCNU, piperazine, piperazinedione, pipobroman, porfiromycin, spirohydantoin mustard, teroxirone, tetraplatin, thio-tepa, triethylenemelamine, uracil nitrogen mustard, Yoshi-864; Anitmitotic agents including allocolchicine, Halichondrin B, colchicine, colchicine derivative, dolastatin 10, maytansine, rhizoxin, taxol, taxol derivative, thiocolchicine, trityl cysteine, vinblastine sulfate, vincristine sulfate; Topoisomerase I Inhibitors including camptothecin, camptotheciπ, Na salt, aminocamptothecin, 20 camptothecin derivatives, morpholinodoxorubicin; Topoisomerase Il Inhibitors including doxorubicin, amonafide, m-AMSA, anthrapyrazole derivative, pyrazoloacridine, bisantrene HCL, daunorubicin, deoxydoxorubicin, mitoxantrone, menogaril, N,N-dibenzyl daunomycin, oxanthrazole, rubidazone, VM-26,

VP-16; RNA/DNA antimetabolites including L-alanosine, 5-azacytidine, 5-fluorouracil, acivicin, 3 aminopterin derivatives, an antifol, Baker's soluble antifol, dichlorallyl lawsone, brequinar, ftorafur (pro-drug), 5,6-dihydro-5-azacytidine, methotrexate, methotrexate derivative, N-(phosphonoacetyl)-L-aspartate (PALA), pyrazofurin, trimetrexate; DNA antimetabolites including, 3-HP, 2'-deoxy-5-fluorouridine, 5-HP, alpha-TGDR, aphidicolin glycinate, ara-C, 5-aza-2'-deoxycytidine, beta-TGDR, cyclocytidine, guanazole, hydroxyurea, inosine glycodialdehyde, macbecin II, pyrazoloimidazole, thioguanine, thiopurine.

It is preferred if the further anticancer agent is selected from cisplatin, carboplatin, 5- flurouracil, paclitaxel, mitomycin C, doxorubicin, gemcitabine, Velcade^®, Glivec^®, COX- 2 inhibitors and mitoxantrone. Indeed, Velcade^®, Glivec^® and COX-2 inhibitors are currently being used as combinations in clinical trials in conjunction with Cdk inhibitors.

An eighth aspect of the invention provides (i) a compound of Formula I as defined above in the first aspect of the invention, or a compound of Formula Il as defined above in the second aspect of the invention, or a compound of Formula III as defined above in the sixth aspect of the invention, and (ii) a further anticancer agent, for use in medicine.

A ninth aspect of the invention provides a method of treating cancer in a patient, the method comprising administering to the patient (i) a compound of Formula I as defined above in the first aspect of the invention, or a compound of Formula Il as defined above in the second aspect of the invention, or a compound of Formula III as defined above in the sixth aspect of the invention, and (ii) a further anticancer agent as defined above in the seventh aspect of the invention.

Typically, the method comprises administering to the patient a pharmaceutical composition as defined above in the seventh aspect of the invention. However, it is appreciated that the compound of Formula I, Il or III and the further anticancer agent may be administered separately. Thus it is appreciated that the compound of Formula I, Il or III and the further anticancer agent can be administered sequentially or (substantially) simultaneously. The may be administered within the same pharmaceutical formulation or medicament or they may be formulated and administered separately.

This aspect of the invention includes the use of (i) a compound of Formula I as defined above in the first aspect of the invention, or a compound of Formula Il as defined above in the second aspect of the invention, or a compound of Formula III as defined above in the sixth aspect of the invention, and (ii) a further anticancer agent as defined above in the seventh aspect of the invention, in the preparation of a medicament for treating cancer in a patient.

The invention also includes the use of a compound of Formula I as defined above in the first aspect of the invention, or a compound of Formula Il as defined above in the second aspect of the invention, or a compound of Formula III as defined above in the sixth aspect of the invention, in the preparation of a medicament for treating cancer in a patient who is administered a further anticancer agent as defined above in the seventh aspect of the invention. Typically the patient is administered the further anticancer agent at the same time as the medicament, although the patient may have been (or will be) administered the further anticancer agent before (or after) receiving the medicament containing the compound of Formula I₁ Il or III.

Then invention further includes the use of a further anticancer agent as defined above in the seventh aspect of the invention in the preparation of a medicament for treating cancer in a patient who is administered a compound of Formula I as defined above in the first aspect of the invention, or a compound of Formula Il as defined above in the second aspect of the invention, or a compound of Formula Ui as defined above in the sixth aspect of the invention. Typically the patient is administered the compound of Formula I, Il or III at the same time as the medicament, although the patient may have been (or will be) administered the compound of Formula I₁ Il or III before (or after) receiving the medicament containing the further anticancer agent.

The invention also includes (i) a compound of Formula I as defined above in the first aspect of the invention, or a compound of Formula Il as defined above in the second aspect of the invention, or a compound of Formula III as defined above in the sixth aspect of the invention, and (ii) a further anticancer agent as defined above in the seventh aspect of the invention, for use in treating cancer in a patient.

General preferences for the cancer to be treated and for the further anticancer agent are as described above. However, when the further anticancer agent has been shown to be particularly effective for a specific cancer type, it is preferred if the compound of Formula I₁ Il or III is used in combination with that further anticancer agent to treat that specific cancer type. It would be desirable to know which of the non-planar fascaplysin analogues have the most optimal properties as an anticancer agent. Accordingly, a tenth aspect of the invention provides a method of identifying an anticancer agent, or a lead compound for the identification of an anticancer agent, the method comprising: providing a candidate compound which is a compound of Formula IV;

wherein each of R¹ to R⁵ may independently represent H, aryl, Het¹, halo, CN, NO₂, C_1-12 alkyl, C_1-12 alkenyl, C_1-12 alkynyl, C_3-12 cycloalkyl or C_4--_I2 cycloalkenyl, which latter five groups are optionally substituted by one or more substituents selected from halo, nitro, CN, C₁-_S alkyl, C_1-6 alkenyl, C_1-6 alkynyl, C_3-8 cycloalkyl (which latter three groups are optionally substituted by one or more substituents selected from OH, =0, halo, Ci_-4 alkyl and C_1-4 alkoxy), OR^9a, S(O)_nR^9b, S(O)₂N(R^9c)(R^9d), N(R^9e)S(O)₂R^9f, N(R")(R^9h) and Het², and which C_3-12 cycloalkyl or C_4-12 cycloalkenyl groups may additionally be substituted by =0;

R⁶ is either not present, or represents one to four substituents on the fused benzene ring selected from aryl, Het³, halo, CN, NO₂, C_1-12 alkyl, C_1-I2 alkenyl, C_1-12 alkynyl, C_3-12 cycloalkyl or C_4-12 cycloalkenyl, which latter five groups are optionally substituted by one or more substituents selected from halo, nitro, CN, C_1-6 alkyl, C_1-6 alkenyl, C_1-6 alkynyl, C_3-8 cycloalkyl (which latter three groups are optionally substituted by one or more substituents selected from OH, =0, halo, C₁^ alkyl and C_1-4 alkoxy), OR^10a, S(O)pR^10fa, S(O)₂N(R^10c)(R^10d), N(R^10e)S(O)₂R^l0f, N(R^1θ9)(R^1Oh) and Het⁴, and which C_3--_I2 cycloalkyl or C_4--_I2 cycloalkenyl groups may additionally be substituted by

=0;

R⁷ represents H, C_1--_I2 alkyl, C_3-12 cycloalkyl, C_3-12 cycloalkenyl (which latter three groups are optionally substituted by one or more substituents selected from halo, nitro, CN, C_1-6 alkyl, C_1-6 alkenyl, C_1-6 alkynyl, C_3-8 cycloalkyl (which latter three groups are optionally substituted by one or more substituents selected from OH, =0, halo, C_1-4 alkyl and C_1-4 alkoxy), OR^11a, S(O)_qR^11b, S(O)₂N(R^11c)(R^11d), N(R^11e)S(O)₂R^11f, N(R^{1 i9})(R^11h), aryl and Het⁵, and which C_3-I2 cycloalkyl or C_4-12 cycloalkenyl groups may additionally be substituted by =0);

R⁸ is either not present, or represents one to six substituents on the fused tetrahydropyridine ring selected from H, halo, CN, NO₂, C_1-I2 alkyl, C_1-12 alkenyl, C_1-I2 alkynyl, C_3-12 cycloalkyl or C_4-12 cycloalkenyl, which latter five groups are optionally substituted by one or more substituents selected from halo, nitro, CN, C_1-6 alkyl, C_1-6 alkenyl, C_1-6 alkynyl, C_3-8 cycloalkyl (which latter three groups are optionally substituted by one or more substituents selected from OH, =0, halo, C_1-4 alkyl and C_1-4 alkoxy), 0R^12a, S(O)_rR^12b, S(O)₂N(R^12c)(R^12d), N(R^12e)S(O)₂R^12f, N(R^12g)(R^12h), aryl and Het⁶, and which C_3-I2 cycloalkyl or C_4-12 cycloalkenyl groups may additionally be substituted by =0;

R^9a to R^9h, R^1Oa to R^1Oh, R^11a to R^11h and R^12a to R^12h independently represent, at each occurrence,

(a) H,

(b) C_1-10 alkyl, C_2-io alkenyl, C_2-10 alkynyl (which latter three groups are optionally substituted by one or more substituents selected from halo, OH, C_1-6 alkoxy, aryl and Het⁷),

(c) C_3--_I0 cycloalkyl, C_4-10 cycloalkenyl (which latter two groups are optionally substituted by one or more substituents selected from halo, OH, =0, C_1-6 alkyl, C_1-6 alkoxy, aryl and Het⁸),

(d) aryl or (e) Het⁹, provided that R^9b, R^1Ob, R^11b or R^12b does not represent H when n, p, q or r, respectively is 1 or 2;

(a) halo,

(b) CN,

(c) C_1-12 alkyl, C_1-12 alkenyl, C_1-12 alkynyl, C_3-12 cycloalkyl or C_4-12 cycloalkenyl, which latter five groups are optionally substituted by one or more substituents selected from halo, nitro, CN, C_1-6 alkyl, C_1-6 alkenyl, C_1-6 alkynyl, C_3-8 cycloalkyl (which latter three groups are optionally substituted by one or more substituents selected from OH, =0, halo, C_1-4 alkyl and C_1-4 alkoxy), OR^13a, S(O)_sR^13b, S(O)₂N(R^13c)(R^13d), N(R^13e)S(O)₂R^13f, N(R¹³⁹)(R^13h), phenyl, naphthyl (which latter two groups are optionally susbtituted by one or more substituents selected from OH₁ halo, C_1-4 alkyl and C₁^ alkoxy) and Het¹⁰, and which C_3-12 cycloalkyl or C_4-I2 cycloalkenyl groups may additionally be substituted by =0,

(d) 0R^14a,

(e) S(O)_tR^14b,

(f) S(O)₂N(R^14c)(R^14d),

(g) N(R^14e)S(O)₂R^14f, (h) N(R^14g)(R^14h),

(i) phenyl (which latter group is optionally susbtituted by one or more substituents selected from OH, halo, C_1-4 alkyl and C_1-4 alkoxy) or G) Het¹¹;

R^13a to R^13h and R^14a to R^14h independently represent, at each occurrence,

(a) H,

(b) C_1-12 alkyl, C_2-12 alkenyl, C_2--_I2 alkynyl, C_3-12 cycloalkyl, C_4-12 cycloalkenyl (which latter five groups are optionally substituted by one or more substituents selected from halo, OH, C_1-6 alkyl, C_3-12 cycloalkyl, C_4-12 cycloalkenyl (which latter two groups are optionally substituted by one or more substituents selected from OH, =O, halo, C_1-4 alkyl and C_1-4 alkoxy), C_1-6 alkoxy, NH₂, N(H)-C_1-6 alkyl, N(C_1-6 alkyl)₂, phenyl (which latter group is optionally substituted by one or more substituents selected from OH, halo, C_1-4 alkyl and C₁^ alkoxy) and Het¹², and which C_3-12 cycloalkyl or C_4-12 cycloalkenyl groups may additionally be substituted by =0), (c) phenyl (which latter group is optionally substituted by one or more substituents selected from OH, CN, halo, C_1-6 alkyl and C_1-6 alkoxy) or (e) Het¹³, provided that R^13b or R^14b does not represent H when s or t, respectively is 1 or 2;

Het¹ to Het¹³ independently represent 4- to 14-membered heterocyclic or 5- to

10-membered heteroaromatic groups containing one or more heteroatoms selected from oxygen, nitrogen and/or sulfur, which heterocyclic groups may comprise one, two or three rings, and may be substituted by one or more substituents selected from (a) halo, (b) CN,

(c) C_1-12 alkyl, C_1-12 alkenyl, Ci_-12 alkynyl, C_3-12 cycloalkyl or C_4-12 cycloalkenyl, which latter five groups are optionally substituted by one or more substituents selected from halo, nitro, CN, C_1-6 alkyl, C_1-6 alkenyl, C_1-6 alkynyl, C_3-8 cycloalkyl (which latter three groups are optionally substituted by one or more substitueπts selected from OH, =0, halo, C_1-4 alkyl and C_1-4 alkoxy), 0R^15a, S(O)_uR^15b, S(O)₂N(R^15c)(R^15d), N(R^15e)S(O)₂R^15f, N(R^15g)(R^15h), phenyl, naphthyl (which latter two groups are optionally susbtituted by one or more substituents selected from OH, halo, C_1-4 alkyl and C_1-4 alkoxy) and Het^a, and which C_3-12 cycloalkyl or C_4-12 cycloalkenyl groups may additionally be substituted by =0,

(d) 0R^16a,

(e) =0, (f) S(O)_vR^16b,

(g) S(O)₂N(R^16c)(R^16d),

(h) N(R^16e)S(O)₂R^16f,

(i) N(R¹⁶⁹)(R^16h),

(j) phenyl (which latter group is optionally susbtituted by one or more substituents selected from OH, halo, C_1-4 alkyl and C_1-4 alkoxy) or

(k) Het^b;

_R ^i5a _{t0 R} ^i5h _{and R} ^i6a _{tø R} ^i6h j_ncj_ep_enc|ently represent, at each occurrence,

(a) H, (b) C_1-12 alkyl, C_2-12 alkenyl, C_2-12 alkynyl, C_3--_I2 cycloalkyl, C_4-I2 cycloalkenyl (which latter five groups are optionally substituted by one or more substituents selected from halo, OH, C_1-6 alkyl, C_3--_I2 cycloalkyl, C_4-12 cycloalkenyl (which latter two groups are optionally substituted by one or more substituents selected from OH, =0, halo, C_1-4 alkyl and C_1-4 alkoxy), C_1-6 alkoxy, phenyl (which latter group is optionally substituted by one or more substituents selected from OH, halo, C_1-4 alkyl and C_1-4 alkoxy) and Hef, and which C_3-12 cycloalkyl or C_4-12 cycloalkenyl groups may additionally be substituted by =O),

(c) phenyl (which latter group is optionally substituted by one or more substituents selected from OH, halo, C_1-4 alkyl and C_1-4 alkoxy) or (e) Het^d; provided that R^15b or R^16b does not represent H when u or v, respectively is 1 or 2;

n, p, q, r, s, t, u and v independently represent O, 1 or 2;

Het^a to Het^d independently represent 5- or 6-membered heterocyclic groups containing one to four heteroatoms selected from oxygen, nitrogen and/or sulfur, which heterocyclic groups may be substituted by one or more substituents selected from halo, =0 and C_1-6 alkyl; and

and determining whether the candidate compound exhibits at least one anticancer activity which is not dependent upon Cdk4 inhibition.

Candidate compounds of Formula IV that could be mentioned include compounds wherein R¹ represents phenyl.

In an embodiment, the candidate compound of Formula IV is a compound of Formula I, as defined in the first aspect of the invention.

In an embodiment, the candidate compound of Formula IV is not (3-Methoxy-phenyl)- (1 ,3,4,9-tetrahydro-D-carbolin-2-yl)-methanone (CA199).

An eleventh aspect of the invention provides a method of identifying an anticancer agent, or a lead compound for the identification of an anticancer agent, the method comprising: providing a candidate compound which is a compound of Formula V

wherein one of R¹ or R² must represent phenyl, and the other substituent may represent H, halo, CN, NO₂, C_1-12 alkyl, C_1-12 alkenyl, C_1-12 alkynyl, C_3-12 cycloalkyl or C_4-12 cycloalkenyl, which latter five groups are optionally substituted by one or more substituents selected from halo, nitro, CN, C₁-_S alkyl, C_1-6 alkenyl, C_1-6 alkynyl, C_3-8 cycloalkyl (which latter three groups are optionally substituted by one or more substituents selected from OH, =0, halo, C₁^ alkyl and C_1-4 alkoxy), OR^9a, S(O)_nR⁹", S(O)₂N(R^9c)(R^9d), N(R^9e)S(O)₂R^9t, N(R")(R^9h), and which C_3-12 cycloalkyl or C_4-12 cycloalkenyl groups may additionally be substituted by =0;

each of R³ to R⁵ may independently represent H, halo, CN, NO₂, C_1-12 alkyl, C_1--_I2 alkenyl, C_1-12 alkynyl, C_3-12 cycloalkyl or C_4-I2 cycloalkenyl, which latter five groups are optionally substituted by one or more substituents selected from halo, nitro, CN, C_1-6 alkyl, C_1-6 alkenyl, C_1-6 alkynyl, C_3-8 cycloalkyl (which latter three groups are optionally substituted by one or more substituents selected from OH, =0, halo, Ci_-4 alkyl and C_1-4 alkoxy), OR^10a, S(O)_pR^10b, S(O)₂N(R^10c)(R^10d), N(R^10e)S(O)₂R^10f, N(R¹°⁹)(R^1Oh), and which C_3-12 cycloalkyl or C_4--_I2 cycloalkenyl groups may additionally be substituted by =0;

R⁶ is either not present, or represents one to four substituents on the fused benzene ring selected from aryl, Het¹, halo, CN, NO₂, C_1--_I2 alkyl, C_1-12 alkenyl, C_1-12 alkynyl, C_3-12 cycloalkyl or C_4-12 cycloalkenyf, which latter five groups are optionally substituted by one or more substituents selected from halo, nitro, CN, Ci_-6 alkyl, Ci_-6 alkenyl, Ci_-6 alkynyl, C_3-8 cycloalkyl (which latter three groups are optionally substituted by one or more substituents selected from OH, =0, halo, Ci_-4 alkyl and Ci_-4 alkoxy), 0R^11a, S(0)_qR^11b, S(O)₂N(R^11c)(R^11d), N(R^11e)S(O)₂R^11f, N(R^{1 i9})(R^11h) and Het², and which C_3-12 cycloalkyl or C_4-12 cycloalkenyl groups may additionally be substituted by =0;

R⁷ represents H, C_1-12 alkyl, C_3--_I2 cycloalkyl, C_3--_I2 cycloalkenyl (which latter three groups are optionally substituted by one or more substituents selected from halo, nitro, CN, Ci_-6 alkyl, Ci_-6 alkenyl, Ci_-6 alkynyl, C_3-8 cycloalkyl (which latter three groups are optionally substituted by one or more substituents selected from OH, =0, halo, Ci_-4 alkyl and Ci_-4 alkoxy), 0R^12a, S(0)_rR^12b, S(O)₂N(R^12c)(R^12d), N(R^12e)S(O)₂R^12f, N(R¹²⁹)(R^12h), aryl and Het³, and which C_3-12 cycloalkyl or C_4--_I2 cycloalkenyl groups may additionally be substituted by =0);

R⁸ represents H, C_1-I2 alkyl, C_3-12 cycloalkyl, C_3-12 cycloalkenyl (which latter three groups are optionally substituted by one or more substituents selected from halo, nitro, CN, C_1-6 alkyl, C_1-6 alkenyl, Ci_-6 alkynyl, C_3-3 cycloalkyl (which latter three groups are optionally substituted by one or more substituents selected from OH, =0, halo, C_1-4 alkyl and C_1-4 alkoxy), OR^13a, S(O)_sR^13b, S(O)₂N(R^13c)(R^13d), N(R^13e)S(O)₂R^13f, N(R^13g)(R^13h), aryl and Het⁴, and which C_3-12 cycloalkyl or C_4-12 cycloalkenyl groups may additionally be substituted by =0);

R^9a to R^9h, R^1Oa to R^10h, R^11a to R^11h, R^12a to R^12h and R^13a to R^13h independently represent, at each occurrence,

(a) H,

(b) C_1-10 alkyl, C_2-I0 alkenyl, C_2-10 alkynyl (which latter three groups are optionally substituted by one or more substituents selected from halo, OH, C_1-6 alkoxy, aryl and

Het⁷),

(c) C_3-10 cycloalkyl, C_4-10 cycloalkenyl (which latter two groups are optionally substituted by one or more substituents selected from halo, OH, =O, C_1-6 alkyl, C_1-6 alkoxy, aryl and Het⁸), (d) aryl or (e) Het⁵, provided that R^9b, R^1Ob, R^11b or R^12b does not represent H when n, p, q, r or s respectively is 1 or 2;

(a) halo,

(b) CN, (c) C_1-12 alkyl, C_1-12 alkenyl, CM₂ alkynyl, C_3-I2 cycloalkyl or C_4-12 cycloalkenyl, which latter five groups are optionally substituted by one or more substituents selected from halo, nitro, CN, Ci_-6 alkyl, C_1-6 alkenyl, C_1-6 alkynyl, C_3-8 cycloalkyl (which latter three groups are optionally substituted by one or more substituents selected from OH, =0, halo, C_1-4 alkyl and C_1-4 alkoxy), 0R^14a, S(O)_tR^14b, S(O)₂N(R^14c)(R^14d), N(R^14e)S(O)₂R^14f, N(R¹⁴⁹)(R^14h), phenyl, naphthyl (which latter two groups are optionally susbtituted by one or more substituents selected from OH, halo, C_1-4 alkyl and C_1-4 alkoxy) and Het⁶, and which C_3--_I2 cycloalkyl or C_4-12 cycloalkenyl groups may additionally be substituted by =0, (d) OR^15a, (e) S(O)_uR^15b,

(f) S(O)₂N(R^15c)(R^15d),

(g) N(R^15e)S(O)₂R^15f, (h) N(R^15g)(R^15h),

(i) phenyl (which latter group is optionally susbtituted by one or more substituents selected from OH, halo, C_1-4 alkyl and C_1-4 alkoxy) or

0) Het⁶;

R^14a to R^14h and R^15a to R^15h independently represent, at each occurrence,

(a) H,

(b) C_1-12 alkyl, C_2-i₂ alkenyl, C_2-12 alkynyl, C_3-12 cycloalkyl, C_4-12 cycloalkenyl (which latter five groups are optionally substituted by one or more substituents selected from halo, OH, C_1-6 alkyl, C_3--_I2 cycloalkyl, C_4-12 cycloalkenyl (which latter two groups are optionally substituted by one or more substituents selected from OH, =0, halo, C₁^ alkyl and C_1-4 alkoxy), C_1-6 alkoxy, NH₂, N(H)-C_1-6 alkyl, N(C_1-6 alkyl)₂, phenyl (which latter group is optionally substituted by one or more substituents selected from OH, halo, C_1-4 alkyl and C_1-4 alkoxy) and Het⁷, and which C_3-12 cycloalkyl or C_4-12 cycloalkenyl groups may additionally be substituted by =0),

(e) Het⁸, provided that R^14b or R^15b does not represent H when t or u, respectively is 1 or 2;

Het¹ to Het⁸ independently represent 4- to 14-membered heterocyclic or 5- to 10-membered heteroaromatic groups containing one or more heteroatoms selected from oxygen, nitrogen and/or sulfur, which heterocyclic groups may comprise one, two or three rings, and may be substituted by one or more substituents selected from (a) halo,

(b) CN,

(c) C_1-12 alkyl, C_1-12 alkenyl, C_1-12 alkynyl, C_3-12 cycloalkyl or C_4-12 cycloalkenyl, which latter five groups are optionally substituted by one or more substituents selected from halo, nitro, CN, C_1-6 alkyl, C_1-6 alkenyl, C_1-6 alkynyl, C_3-8 cycloalkyl (which latter three groups are optionally substituted by one or more substituents selected from OH, =0, halo, C_1-4 alkyl and C_1-4 alkoxy), 0R^16a, S(O)_vR^16b, S(O)₂N(R^16c)(R^16d), N(R^16e)S(O)₂R^16f, N(R^16g)(R^16h), phenyl, naphthyl (which latter two groups are optionally susbtituted by one or more substituents selected from OH, halo, C_1-4 alkyl and C_1-4 alkoxy) and Het^a, and which C_3-12 cycloalkyl or C_4-12 cycloalkenyl groups may additionally be substituted by =0,

(d) 0R^17a,

(e) =0, (f) S(O)_wR^17b,

(g) S(O)₂N(R^17c)(R^17d),

(h) N(R^17e)S(O)₂R^17f,

(i) N(R^17g)(R^17h), G) phenyl (which latter group is optionally susbtituted by one or more substituents selected from OH, halo, C_1-4 alkyl and C_1-4 alkoxy) or (k) Het^b;

_R ^i6a _{t0 R} ^i6h _{and R} ^i7a _{to R} ^i7h j_ncj_ep_encjently represent, at each occurrence, (a) H,

(b) C_1-12 alkyl, C_2-12 alkenyl, C_2-12 alkynyl, C_3-12 cycloalkyl, C_4-12 cycloalkenyl (which latter five groups are optionally substituted by one or more substituents selected from halo, OH, C_1-6 alkyl, C_3-I2 cycloalkyl, C_4-12 cycloalkenyl (which latter two groups are optionally substituted by one or more substituents selected from OH, =0, halo, C_1-4 alkyl and Ci_-4 alkoxy), C_1-6 alkoxy, phenyl (which latter group is optionally substituted by one or more substituents selected from OH, halo, C₁^ alkyl and C₁^ alkoxy) and Hef, and which C_3-12 cycloalkyl or C_4-12 cycloalkenyl groups may additionally be substituted by =O),

(e) Het^d; provided that R^16b or R^17b does not represent H when v or w, respectively is 1 or 2;

n, p, q, r, s, t, u, v and w independently represent O, 1 or 2;

Hef to Het^d independently represent 5- or 6-membered heterocyclic groups containing one to four heteroatoms selected from oxygen, nitrogen and/or sulfur, which heterocyclic groups may be substituted by one or more substituents selected from halo, =0 and C_1-6 alkyl; and

unless otherwise specified

(i) alkyl, alkenyl, alkynyl, cycloalkyl, and cycloalkenyl groups, as well as the alkyl part of alkoxy groups, may be substituted by one or more halo atoms, and (ii) cycloalkyl and cycloalkenyl groups may comprise one or two rings and may additionally be ring-fused to one or two benzene rings; and determining whether the candidate compound exhibits at least one anticancer activity which is not dependent upon Cdk4 inhibition.

Candidate compounds of Formula V that can be mentioned include those compounds wherein R¹ represents phenyl.

In an embodiment, the candidate compound of Formula V is a compound of Formula Il as defined in the second aspect of the invention.

In an embodiment, the compound of Formula V is not biphenyl-4-carboxylic acid [2- ( 1 H-indol-3-yl)-ethyl]-amide.

Other candidate compounds of Formula V that can be mentioned include those compounds wherein R² represents phenyl.

In an embodiment, the candidate compound of Formula V is a compound of Formula III as defined in the sixth aspect of the invention.

In an embodiment, the compound of Formula V is not biphenyl-4-carboxylic acid [2- (1 H-indol-3-yl)-ethyl]-methyl-amide (CA224).

It is appreciated that screening assays which are capable of high throughput operation are particularly preferred.

It is appreciated that in the methods described herein, which may be drug screening methods, a term well known to those skilled in the art, the candidate compound may be a drug-like compound or lead compound for the development of a drug-like compound.

The term "drug-like compound" is well known to those skilled in the art, and may include the meaning of a compound that has characteristics that may make it suitable for use in medicine, for example as the active ingredient in a medicament. Thus, for example, a drug-like compound may be a molecule that may be synthesised by the techniques of organic chemistry, which may be of less than 5000 Daltons, and which may be water-soluble. A drug-like compound may additionally exhibit features of selective interaction with a particular protein or proteins and be bioavailable and/or able to penetrate target cellular membranes or the blood.brain barrier, but it will be appreciated that these features are not essential. The term "lead compound" is similarly well known to those skilled in the art, and may include the meaning that the compound, whilst not itself suitable for use as a drug (for example because it is only weakly potent against its intended target, non-selective in its action, unstable, poorly soluble, difficult to synthesise or has poor bioavailability) may provide a starting-point for the design of other compounds that may have more desirable characteristics.

Accordingly, it is appreciated that the identified compound may be modified, and the modified compound tested for at least one anticancer activity which is not dependent upon Cdk4 inhibition.

Preferably and suitably, the at least one anticancer activity is selected from:

1. Blocking cells at the G₂M phase of the cell division cycle. Preferably greater than 40% of cells should maintain a block at G₂/M (Stark & Taylor (2006) MoI

Biotechnol. 32(3): 227-48).

2. Inducing greater than 20% apoptosis in cancer cells after 72h incubation and/or selectively inducing greater than 30% apoptosis in SV40 large T antigen-transformed (tumorigenic) cells after 48h incubation (Matranga & Shapiro (2002) Cancer Res. 62(6): 1707-17).

3. Inhibition of tubulin polymerisation in cells with an IC₅₀ of less than 2 μM, and more preferably less than 1 μM.

4. Reducing the long-term survival of SV40 large T antigen-transformed cells; cells with an IC₅₀ of less than 1 μM (Matranga & Shapiro (2002) Cancer Res. 62(6): 1707-17).

5. Acting as an anti-oxidant with an in vitro IC₅₀ of less than 50 μM (Watjen et al (2005). J Nutr. 135(3): 525-31 ; Michels et al (2005). Toxicology. 206(3): 337- 48; Bhatia ef a/ (1999) Cancer Lett 147(1-2): 77-84).

6. Downregulating cyclin B1 and Cdk1 and upregulating p21 and p27 in p53+ cancer cells (for cyclin B1 : Yuan et al (2004) Oncogene. 23(34): 5843-52; for

Cdk1 : Wheatley (1997) J Cell Biol. 138(2): 385-93; and for p21 & p27: Don (2001 ) MoI Pharmacol. 59(4): 784-94).

7. Upregulating cyclin B1 levels in p53-mutant and p53-null cancer cells Russo (2006) Cancer Res. 66(14): 7253-60). 8. Inhibiting the growth of Rb-null or Rb-mutant cancer cells in vitro. 9. Reducing the efficiency of colony formation in Rb-null or Rb-mutant cancer cells in vitro.

Methods for testing the candidate compounds for each of these anticancer activities which is not dependent upon Cdk4 inhibition are well known in the art and described below and in the Examples.

Typically, when testing for the ability to block cells at the G₂/M phase of the cell division cycle (#1, above) the cells are synchronised either post G₀/G₁ at d/S (using hydroxyurea, mimosine or thymidine as blocking agents at G₁ZS) or G₂/M (using nocodazole or paclitaxel as blocking agents at G₂/M), and, after release of cells from synchrony, the ability of the candidate compound to effect cell block at G₂/M is assessed. Upon release from synchronisation at G₁ZS, in the presence of a candidate compound having the desired ability, cells proceed forward in the cell cycle and block at G₂ZM. Similarly, upon release from synchronisation at G₂ZM, in the presence of a candidate compound having the desired ability, the cells remain at G₂ZM and do not proceed further through the cell cycle.

A number of methods are known in the art to screen for the ability of a compound to act as an anti-oxidant (#5, above) (e.g., Srinivas et al (2004) MoI Carcinog, 40(4): 201-211 ; Steffan et al (2005) J Pharm Pharmacol 57(2): 233-240). For example, an antioxidative assay may be performed according to the instructions given in the data sheet of H₂DCF diacetate (H₂DCFDA), with some minor modifications (Molecular Probes Cat. No. D399). Cancer cells (i.e. colon cancer LS174T, non-small cell cancer A549 cells) are seeded at a density of 20,000 cellsZ well in a 96-well tissue culture grade black-well plate. Cells are allowed to recover for 24h at 37⁰C in a humidified CO₂ incubator. Cells are incubated with different concentrations of the candidate compound (e.g., 2.5- 80 μM) for 60 min. The compound is removed, cells are briefly washed with PBS, and 10 μM H₂DCFDA in PBS is added. The plate is incubated at 37°C in the CO₂ incubator and after 30 min H₂O₂ is added at a final concentration of 500 μM. The oxidation of intracellular non-fluorescent H₂DCF to highly fluorescent DCF is measured using a fluorimeter (Bio-Tek) using an excitation of 485 nM and emission of 528 nM. Blank values, indicating the fluorescence of the dye in PBS are subtracted from all samples. The % of control is calculated in comparison with fluorescence exhibited by cells in presence of H₂O₂ alone. Alternatively, 50,000 cells/well of H4IIE rat hepatoma cells may be seeded in a 96-well microtitre plate. The cells are allowed to attach and the medium changed. Cells are first incubated with different concentrations of the test compound for 60 min, then the medium containing the compound is removed and the cells washed with PBS twice and fresh medium added. H₂DCFDA is added at a final concentration of 5 μM and the cells are incubated further for 30 min. The oxidation of intracellular non-fluorescent H₂DCF to highly fluorescent DCF can be measured after addition of H₂O₂ (500 μM) at an excitation of 485 nm and an emission of 525 nm to measure the inhibition by the test compound of H₂O₂ mediated ROS formation. Further alternatively, cancer cells may be seeded at a density of 100,000-150,000 cells/well in a 6-well tissue culture grade plate. Cells may be treated in the same way as mentioned above and a qualitative reduction in fluorescence in compound-treated cells may be assessed using fluorescence microscopy (Olympus BH Series).

More soluble analogues of the compounds of Formula IV and IV are likely to result in more efficacious and bio-available compounds. Accordingly, the screening methods may also comprise the additional step of determining the solubility of the identified compound or modified compound in one or more clinically acceptable solvents. Identification of the optimal solvent would be especially useful. Suitable test solvents include phospholipids and nanoparticle technology as discussed above. Many other suitable test solvents are known in the art (see, for example, Remington: The Science and Practice of Pharmacy, 19^th Edition (1995), ISBN: 0-912734-04-3).

In a preferred embodiment, the identified compound or the modified compound is further tested for the ability to inhibit the growth of cancer cells in vitro. Various approaches are known in the art for conducting a cell proliferation assay. For example, trypan blue is a simple way to evaluate cell membrane integrity and thus isolate viable cells which may be counted directly using light microscopy. Alternatively, the uptake and incorporation of radioactive substances, usually tritium-labelled thymidine, or bromodeoxyuridine, could be used as an indicator of the number of viable cells.

Additionally, cell proliferation may be assayed using the colorimetric MTT assay which relies on the reduction of a tetrazolium component MTT by the mitochondria of viable cells.

It is appreciated that it would be especially useful to test the identified compound or the modified compound in vitro for inhibition of growth of drug resistant cancer lines

(paclitaxel, doxorubicin, cisplatin and multi-drug resistant lines) to help in understanding their potential against human cancers for which no treatment is currently available.

In another preferred embodiment, the identified compound or the modified compound is further tested for the ability to reduce the efficiency of colony formation of cancer cells in vitro. This may be used to assess the effect of the candidate compounds on the long term survival of cancer cells. As discussed above, a colony formation assay may be invaluable to understand the anticancer potential of a candidate compound (Wu Wei et al, 1983) since the loss of a cancer cell's ability to form a colony could possibly indicate its permanent exit from the cell cycle. Any colony formation protocol known in the art may be used in the context of the present invention (see, for example, Gohji et al (1987) J Urol. 137(3): 539-43). For example, a typical method of performing a colony formation assay is follows. Cells are plated at a concentration of 500 cells per well in 2 ml of complete medium in 35 mm or 6-well plates. The plates are incubated for a 24 h stabilization period and further incubated with different concentrations of candidate compound for 24 h. Plates are then gently washed with PBS, replaced with fresh complete medium and incubated at 37⁰C. After 10-12 days of incubation (when the colonies are visible), the plates are gently washed with PBS and the cell colonies fixed in methanolracetic acid (2:1) fixative for 20 miπ at room temperature. The plates are washed 2X with 2 ml of distilled water per well and then air dried for 15 min. 1 ml of 1% crystal violet solution is added to each well and the colonies are allowed to stain for 15 min. The staining solution is removed, the plates are washed 3X with distilled water and then air dried for 30 min. The colonies are evaluated by visual counts and the number of colonies in treated cultures expressed as a percentage of the control cultures.

As above, it would be especially useful to test the identified compound or the modified compound in vitro for the ability to reduce the efficiency of colony formation of drug resistant cancer lines. Suitable cancer cell lines include non-small cell lung cancer, pancreatic cancer, colon cancer, prostate cancer, breast cancer and myeloma-derived cell lines.

The screening methods preferably also comprise the further step of assessing the identified compound or modified compound for efficacy in an assay of angiogenesis, tumour invasion and/or cell migration. Additionally, the screening methods may also comprise the further step of assessing the identified compound or modified compound in pharmacokinetic, pharmacodynamic and toxicological studies in animal models. A number of assays for determining the ability of a candidate compound to inhibit angiogenesis are commercially available. Both in vitro and in vivo assays are well known in the art and are described, for example, by Auerbach et a/ (2003). Examples include the in vivo Matrigel plug and corneal neovascularisation assays, in vivo or in vitro chick chorioallantoic membrane (CAM) assays, in vitro cellular (proliferation, migration, tube formation) and organotypic (aortic ring) assays, and the chick aortic arch and Matrigel sponge assays.

It will be understood that it will be desirable to identify compounds that may have anticancer activity in vivo. Thus it will be understood that reagents and conditions used in the screening methods may be chosen to be substantially the same as occur in vivo.

The screening methods preferably also comprise the further step of assessing the identified compound or modified compound for efficacy in an animal model of cancer.

The model may be a model for any of the cancers mentioned above. In a particular embodiment, the animal model of cancer is a mouse model. For example, the animal model of cancer may be a model of an Rb-negative cancer. The model may be a xenograft or allograft model (Voskoglou-Nomikos et a\ (2003) Clinical Cancer Research 9: 4227-4239).

The screening methods of the invention may further comprise the step of synthesising, purifying and/or formulating the identified compound.

Thus the invention also includes a method for preparing an anticancer compound of Formula IV or Formula V, the method comprising identifying a compound using the screening methods described above and synthesising, purifying and/or formulating the identified compound. Moreover, the invention includes a method of making a pharmaceutical composition comprising the step of mixing the compound identified using the screening methods described above with a pharmaceutically acceptable carrier.

The invention is now described in more detail by reference to the following, non- limiting, Figures and Examples.

Figure 1. Systematic representation of the cell division cycle. This figure shows various cyclin dependent kinases and their respective cyclin partners, their positions at different phases of the cell division cycle, where they function and where their activities are required for the controlled progression of the cell cycle.

Figure 2. General strategy used to synthesize non-planar analogues of fascaplysin. Fascaplysin analogues were made by releasing bonds a, b and changing double bond c into a single bond leading to derivatives of the four series of compounds (see, Aubry et al, 2004; Aubry et al, 2005; Aubry et al, 2006).

Figure 3. Representative histogram of a flow cytometric analysis. The histogram obtained after the FACS analysis of NCI-H 1299 asynchronous cells shows the percentage of cells at the different phases of a normal cell cycle.

Figure 4. Representative standard curve of Bradford protein assay showing linear increase in absorbance with increase in BSA concentration.

Figure 5. Coomassie blue staining of Cdk enzymes purified on glutathione- agarose beads. The gel was loaded with lysates from cells co-infected with Cdk4/GST-cyclin D1 (lane 1 ), purified Cdk4/GST-cyclin D1 (lane 2; a distinct band of size 52 kD indicates GST-cycliπ D1), lysates from cells co-infected with Cdk2/GST- cyclin A (lane 3), purified Cdk2/GST-cyclin A (lane 4; a distinct band of size 73 kD indicates GST-cyclin A), lysates from cells co-infected with Cdk2/GST-cyclin E (lane 5), purified Cdk2/GST-cyclin E (lane 6; a distinct band of size 68 kD indicates GST-cyclin E), lysates from cells co-infected with His-Cdk4/GST-cyclin D1 (lane 7), purified His- Cdk4/GST-cyclin D1 (lane 8; a distinct band of size 52 kD indicates GST-cyclin D1) and pre-stained protein molecular weight marker (lane 9). The Cdk component of these complexes was not observed by Coomassie staining because of the relatively low amounts that were expressed. However, we confirmed the presence of the Cdk-s by Western blotting using Cdk-specific antibodies. The presence of the Cdk-s in the holoenzyme complex was later confirmed using Cdk-specific inhibitory compounds.

Figure 6. Western blot analyses of purified Cdk enzymes. Cdk4/GST-cyclin D1, Cdk2/GST-cyclin A, His-Cdk2/GST-cyclin E and His-Cdk4/GST-cyclin D1 were purified on glutathione agarose beads. 30 μg of each sample was loaded on 10% agarose gel in the lanes indicated in the figure above. The resolved proteins were transferred on Immobilon-P membrane and probed with specific antibodies for Cdk4, Cdk2, cyclin D1 and cyclin A. Western analyses confirmed the presence of Cdks and cyclins in the holoenzyme complexes of the active enzymes Figure 7. The effect of Cdk enzyme concentrations on rates of reaction by monitoring ATP depletion. Above graphs show that ATP depletion increases with increasing concentrations of Cdk enzymes, thus increasing the fold difference in relative light units (RLU) between the control and blank reactions.

Figure 8. IC₅₀ determination of flavopiridol, fascapfysin, CINK4 and indirubin 5 suiphonic acid in the in vitro Cdk4-cyclin D1 enzyme assay. The percent enzyme inhibition values represent the mean and standard deviation (from the mean) from three independent experiments.

Figure 9. IC₅₀ determination of flavopiridol, fascaplysin, roscovitine and indirubin 5 suiphonic acid in the in vitro Cdk2-cyclin A enzyme assay. The percent enzyme inhibition values represent the mean and standard deviation (from the mean) from three independent experiments.

Figure 10. IC₅₀ determination of flavopiridol, fascaplysin, roscovitine and indirubin 5 suiphonic acid in the in vitro Cdk2-cyclin E enzyme assay. The percent enzyme inhibition values represent the mean and standard deviation (from the mean) from three independent experiments.

Figure 11. IC₅₀ determination of flavopiridol and fascaplysin in the in vitro Cdk1- cyclin B1 enzyme assay. The percent enzyme inhibition values represent the mean and standard deviation (from the mean) from three independent experiments.

Figure 12. IC₅₀ determination of flavopiridol and fascaplysin in the in vitro Cdk9- cyclin T1 enzyme assay. The percent enzyme inhibition values represent the mean and standard deviation (from the mean) from three independent experiments.

Figure 13. FACS analysis of serum-starved Calu-1 cells released in the presence of CA199 and Western blot analysis of proteins from asynchronous Calu-1 cells treated with DE002. The results support the inhibition of the Cdk4-cyclin D1 enzyme at the cellular level in the mitotic spindle checkpoint-deficient Calu-1 cells after treatment with DE002. Figure 13A shows FACS analyses of untreated or control cells (A), serum-starved cells for 24 h (B), serum-starved ceils released in the presence of DE002 at the IC₅₀ concentration for 24 h (C), and serum-starved cells released in the presence of DE002 at the IC_7O concentration for 24 h (D). Two to three-fold increase in the Gi:S ratio was considered as an indication of the G₀ZG₁ arrest of cells. Figure 13B shows on Western blots the status of pRb phosphorylation in Cdk4-specific serine residues after a 24 h treatment of asynchronously growing Calu-1 cell with DE002. The phospho-specific polyclonal antibodies Ser780, Ser795 and Ser807/811 detect pRb phosphorylated at Serine residues 780, 795 and 807/811 respectively. The monoclonal antibody against pRb (4H) detects the total phosphorylated and unphosphorylated pRb protein. Figure 13B shows proteins from untreated cells (C), from cells treated with IC₅₀ concentration of fascaplysin, 24 h (T1 ), from cells treated with IC₅₀ concentration of DE002, 24 h (T2), and from cells treated with IC₇₀ concentration of DE002, 24 h (T3).

Figure 14. FACS analysis showing that DE002 does not prevent late G1-_b|_Ocked Caiu-1 cells from re-entering into cell cycle, (a) Untreated cells as control, (b) cells treated with 200 μM mimosine for 32 h, (c) cells released in fresh medium for 18 h, (d) cells released in the presence of roscovitine IC₅₀, 18 h, (e) cells released in the presence of [IC₅₀] fascaplysin, 18 h, (f) cells released in the presence of [IC₅₀] DE002, 18 h, (g) cells released in fresh medium, 36 h, (h) cells released in the presence of [IC₅₀] roscovitine, 36 h, (i) cells released in the presence of [IC₅₀] fascaplysin, 36 h, and (j) cells re/eased in the presence of [IC₅₀] DE002, 36 h. Two to three-fold increase in the Gi:S ratio was considered as an indication of the G₀ZG₁ arrest of cells.

Figure 15. Flow cytometric analysis of asynchronous A549 and NCI-H1299 (mitotic spindle checkpoint-proficient, human non-small cell lung carcinoma (NSCLC) cell lines. DE002 treatment resulted in the majority of cells undergoing cell cycle blockage in the G₂ZM phase of the cell cycle (4n DNA content) in both cell lines. The Figure shows untreated A549 cells as control (A); A549 cells after treatment with IC₅₀ concentration of DE002 for 24 h (B); A549 cells after treatment with IC₇₀ concentration of DE002 for 24 h (C); untreated NCI-H 1299 cells as control (D); NCI- HI 299 cells after treatment with IC₅₀ concentration of DE002 for 24 h (E).

Figure 16. FACS analysis of NCI-H358 NSCLC cells, synchronised at G₂/M and G₁ZS and released in the presence of DE002. Nocodazole and hydroxyurea were used to synchronize NCI-H358 cells at the G₂ZM and G₁ZS phases of the cell division cycle. Synchronised cells were released either in fresh medium or in fresh medium containing IC₅₀ concentration of DE002. After release from block in the presence of DE002, cells either maintain the block at G₂/M (after release from the nocodazole block) or move forward in the cell cycle and then get blocked later at the G2ZM phase (after release from the hydroxyurea block). For the nocodazole block experiment, the figure shows untreated cells as control (A); cells treated with 1 μM the nocodazole for 18 h (B); cells treated with 1 μM nocodazole for 18 h and released in fresh medium (C); cells treated with 1 μM nocodazole for 18 h and released in the presence of [IC₅₀] DE002, 12 h (D). For the hydroxyurea synchronization experiment, the figure shows untreated cells as control (E); cells treated with 250 μM hydroxyurea for 18 h (F); cells treated with 250 μM hydroxyurea for 18 h and released in fresh medium (G); cells treated with 250 μM hydroxyurea for 18 h and released in the presence of [IC₅₀] DE002, 18 h (H).

Figure 17. DE002 selectively kills SV40-transformed cells as revealed by FACS analysis. DE002 treatment of BNL CL2 (mouse embryonic normal hepatic) cells for 48 h at IC₅₀ and IC₇₀ concentrations resulted in prominent G2/M arrest with less than 8% cells showing apoptosis. This can be seen through comparison of histograms (A, B and C) in the upper panel of the figure. Untreated cells as control (A); cells treated with IC₅₀ concentration of DE002, 48 h (B); cells treated with IC₇₀ concentration of DE002, 48 h (C). In contrast, BNL SV A.8 (mouse embryonic SV40 transformed hepatic cells; upper panel of the figure) underwent apoptotic cell death upon incubation with DE002. The levels of apoptosis are depicted as the percentage of cells appearing in the SUb-G₁ peak. 43% and 51 % of cells were found in the sub-G1 peak after 48 h exposure to [IC₅₀] DE002 (E) and [IC₇₀] DE002 (F). The untreated control cells (D) do not show any cells in the SUb-G₁ peak.

Figure 18. Selective killing of SV40-transformed mouse embryonic hepatic cells. BNL CL2 (normal) and BNL SV A.8 (SV40-transformed) cells were incubated with different concentrations of DE002 for 48 h. Apoptosis was selectively seen only in SV40-transformed cells but not in the parent line which is the normal counterpart. (A) The left hand graph shows percentage cell death measured by the trypan blue dye exclusion method in the two cell lines (BNL CL2 and BNL SV A.8) after treatment with increasing concentrations of DE002 for 48 h. The right hand graph shows cell growth inhibition of BNL CL2 and BNL SV A.8 cells after treatment with DE002 for 48 h, as measured by the MTT assay. (B) Fluorescence microscopic pictures of DAPI-stained cells captured at 40X magnification. Untreated BNL SV A.8 cells (a); BNL SV A.8 cells treated with [IC₅₀] of DE002 for 48 h (b); Untreated BNL CL2 cells (c); BNL CL2 cells treated with [IC₅₀] of DE002 for 48 h (d). A minimum of 500 nuclei were counted for each sample. The fragmented nuclei and apoptotic cells are indicated with arrows Figure 19. DE002 does not intercalate with pBlueScript plasmid DNA. The ability of DE002 to intercalate DNA was investigated using a topoisomerase I catalysed DNA unwinding assay and compared with the results obtained using fascaplysin and the DNA-intercalating agent, camptothecin. The unwinding/relaxation assays were carried out as described. The final products of topoisomerase I relaxation assay were resolved on a 0.8% agarose gel and were stained with 0.5 mg/ml ethidium bromide in order to visualise with UV illumination. Lane 1 contained the control pBlueScript DNA showing the supercoiled form. Lane 2 contained the control relaxation reaction with topoisomerase I enzyme without any compound. Lanes 3, 4, 5, 6 and 7 contained the topoisomerase I relaxation reaction carried in the presence of camptothecin, fascaplysin and DE002 at the concentrations indicated in the figure.

Figure 20. Topoisomerase I treated plasmid DNA was further subjected to DE002 treatment. The reaction products were resolved on a 0.8% agarose gel and stained with 0.5 mg/ml ethidium bromide in order to visualise DNA with UV illumination. The figure shows relaxed pBlueScript plasmid DNA (lane 1 ), relaxed plasmid DNA treated with 1 μM, 10 μM and 100 μM of DE002 (lanes 2, 3 and 4 respectively) and relaxed plasmid DNA treated with fascaplysin 1 μM (lane 5).

Figure 21. DE002 does not displace ethidium bromide from the minor groove of double-stranded DNA molecules. The ability of fascaplysin, DE002 (a non-planar analogue of fascaplysin) and actinomycin D (a known DNA intercalator) to interact with the minor groove of DNA was determined by the fluorescence-based ethidium bromide displacement assay. The final concentration of ethidium bromide in the assay was 1.3 μM. The Figure shows representative curves with increasing concentrations of fascaplysin (filled squares) actinomycin D (unfilled squares) and DE002 (filled triangles). The results represent means and standard deviations from three independent experiments.

Figure 22. A549 and LS174T cells analysed by Western blotting. The treatment of p53+ cells (A549 and LS174T) for 24 h with DE002, at an IC₅₀ concentration, resulted in the induction of p53, p21CIP1/WAF1 (p21 ) and p27KIP1 (p27) while cyclin B1 and Cdk1 levels were repressed. Equal amounts of total proteins (40 μg as estimated by Bradford protein estimation assays) from cell lysates were resolved on a 10% SDS- polyacrylamide gel and then transferred on to an Immobilon-P Transfer Membrane. The membranes were probed with the following antibodies: mouse monoclonal antibody cyclin B1 (CR-UK, Cat. No. V152) at 1 :1500 dilution to detect cyclin B1 ; rabbit polyclonal antibody Pab 1801 (Santa Cruz Biotechnology, Cat. No. sc-98) at 1 :500 dilution to detect p53; rabbit polyclonal antibody N-20 (Santa Cruz Biotechnology, Cat.

No. sc-469) at 1 :500 dilution to detect p21 ; rabbit polyclonal antibody C-19 (Santa Cruz

Biotechnology, Cat. No. sc-528) at 1:250 dilution to detect p27; mouse monoclonal antibody AC-40 (Sigma-Aldrich, Cat. No. A4700) at 1 :2000 dilution to detect actin. An appropriate HRP conjugated secondary antibody, either anti-mouse (Santa Cruz

Biotechnology Cat. No. sc-2302) at 1: 2500 dilution when a mouse monoclonal was used as the primary antibody, or anti-rabbit (Santa Cruz Biotechnology, Cat. No. sc-

2301) at 1 :2000 dilution when rabbit polyclonals were used as the primary antibody to detect the expression levels of p27, p53, p21 , cyclin B1 and actin. The same levels of actin observed in different lanes confirm that equal amounts of cellular protein were loaded on all gels.

Figure 23. Cell-free tubulin polymerisation assay in vitro. Purified tubulin was used to test the ability of DE002 to inhibit tubulin polymerisation in vitro. The assay measures the increase in optical density as a result of tubulin assembly or polymerisation. Nocodazoie and paclitaxel were used in the assay, as controls, as a known inhibitor and enhancer of tubulin polymerisation, respectively. DE002 was tested at four different concentrations which had already show inhibition of in vitro cell growth. The change in Vmax value was used as an indicator of tubulin — ligand interactions. The polymerisation curves indicate that 2.5 μM, 5 μM, 10 μM and 25 μM concentrations of DE002 reduced the Vmax value from 19 mOD/min (control) to 12.5,

9.2, 3 and 0.5 mOD/min, respectively, in a highly dose dependent manner. The curves shown here represent the average of three independent experiments.

Figure 24. Inhibition of tubulin polymerisation and enhancement of tubulin de- polymerisation in vivo. Tubulin polymerisation assay was performed in A549 (whole cells) after treatment with DE002 for 30 min at the concentrations indicated in the figure. Supernatant and pellet represent unassembled and assembled tubulin, respectively. Tubulin polymerisation is detectable by the increase of tubulin amounts in the pellet and its disappearance from the supernatant. The Western blots show dose- dependent inhibition of tubulin polymerisation after the simultaneous treatment of cells with paclitaxel and DE002. This results in the accumulation of unassembled tubulin in the supernatant. DE002 also acts as an enhancer for tubulin de-polymerisation in a dose-dependent manner when paclitaxel-stabilized tubulin was subjected to DE002 treatment for 30 min. The proteins were resolved on a 10% SDS-polyacrylamide gel and then transferred on to an Immobilon-P Transfer Membrane. The membranes were probed with a specific mouse monoclonal α-tubuiin antibody B-7 (Santa Cruz Biotechnology, Cat. No. sc-5286) at a 1:1000 dilution. HRP conjugated mouse secondary antibody (Santa Cruz Biotechnology, Cat. No. sc-2302) was used at a dilution of 1:2500 to illuminate the protein bands.

Figure 25. Long-term survival of cancer cells after treatment with DE002. A549 and Calu-1 cells were investigated for their long-term survival efficiency after treatment with different concentrations of DE002. The colony formation efficiency is expressed as the percentage of colonies formed in the treated cultures compared with untreated cultures. (A) The representative plates show untreated A549 cells (a); A549 cells treated with 0.25 μM DE002 (b); A549 cells treated with 0.5 μM DE002 (c); A549 cells treated with 1 μM DE002, (d); A549 cells treated with 0.8 μM fascaplysin (IC₅₀) (e); untreated Calu-1 cells (f); Calu-1 cells treated with 1.25 μM DE002 (g); Calu-1 cells treated with 2.5 μM DE002 (h); Calu-1 cells treated with 5 μM DE002 (i); Calu-1 cells treated with 1 μM fascaplysin (IC₅₀) G)-

Figure 26. Long term survival of SV40 transformed mouse embryonic hepatic cells after the treatment with DE002. The cells were treated with different concentrations of DE002 for 24 h and then incubated in drug free medium for 12 days. The colonies were fixed in methanol : acetic acid (2:1 ) and stained with 1% crystal violet. Representative plates were scanned using the gel documentation system. The figure shows BNL CL2 cells (a) untreated cells; (b) treated with DE002, IC₂₀ (0.25 μM); (c) treated with DE002 IC₃₀ (0.4 μM); (d) treated with DE002 IC₅₀ (0.6 μM) and (e) treated with DE002 IC₇₀ (0.9 μM). BNL SV A.8 cells (f) untreated cells; (g) treated with DE002, IC₂₀ (0.25 μM); (h) treated with DE002 IC₃₀ (0.4 μM); (i) treated with DE002 IC₅₀ (0.6 μM) and 0) treated with DE002 IC₇₀ (0.9 μM).

Figure 27. DE002 inhibits H₂O₂-mediated ROS formation in rat hepatoma cells, H4IIE. The oxidation of intracellular non-fluorescent H2DCF to highly fluorescent DCF was measured after addition of H₂O₂ (500 μM) to H4IIE cells. The cells were pre- incubated with different concentrations of DE002 for 60 min in order to assess the antioxidant potential of DE002. IC₅₀ represents the concentration at which 50% of ROS formation was inhibited by DE002 treatment. The results represent means and standard deviations (from the mean value) from three independent experiments.

Figure 28. CA224 does not intercalate DNA. The ability of CA224 to intercalate DNA was investigated using a topoisomerase I catalysed DNA unwinding/relaxation assay and compared with the effects of fascaplysin. DNA relaxation assays were carried out as described. Lane 1 contains the control pBlueScript DNA and shows the supercoiled form as seen as a single band in the figure. Lane 2 contains the control relaxation reaction with topoisomerase I enzyme in the absence of any compound. Lanes 3, 4, 5, 6 and 7 contain the topoisomerase I relaxation reaction carried out in the presence of CA224, camptothecin and fascaplysin at the concentrations indicated in the figure.

Figure 29. CA224 does not displace ethidium bromide from the minor groove of double-stranded DNA. The ability of CA224 to interact with the minor groove of double-stranded DNA was determined by a fluorescence based ethidium bromide displacement assay. The assay was performed as described with increasing concentrations of fascaplysin (filled squares), actinomycin D (unfilled squares) and CA224 (filled triangles). The results represent means and standard deviations from three independent experiments. The IC₅₀ is the concentration of compound at which 50% displacement of bound ethidium bromide is observed.

Figure 3OA and 3OB. FACS analysis and Western blotting in Calu-1 cells (mitotic spindle checkpoint-deficient cells) after treatment with CA224 indicates the inhibition of enzyme Cdk4-cycliπ Df at the cellular level. Figure 3OA shows untreated cells as control (A); cells starved of serum for 24 h (B); serum-starved cells released in the presence of IC₅₀ concentration of CA224 at for 24 h (C); serum-starved cells released in the presence of IC₇₀ concentration of CA224 for 24 h (D). Figure 3OB shows the status of pRb phosphorylation at Cdk4-specific serine residues after cells were treated with CA224 for 24 h. The rabbit polyclonal antibodies Ser780, Ser795 and Ser807/811 detect the phosphorylated pRb at Serine 780, Serine 795 and Serine 807/811 residues while the mouse monoclonal pRb (4H) detects the total phosphorylated and unphosphorylated pRb protein. In Figure 3OB, untreated cells as control (lane C); cells treated with IC₅₀ concentration of fascaplysin, 24 h (lane T1 ); cells treated with IC₅₀ concentration of CA224, 24 h (lane T2) and cells treated with IC₇₀ concentration of CA224, 24 h (lane T3).

Figure 31. Response of the mitotic spindle checkpoint-proficient human lung cancer cell lines A549 and NCI-M 299 to CA224 treatment. Flow cytometric analysis of asynchronous cells show that the majority of cells are arrested in the G₂/M phase of the cell cycle (4n DNA content) in both cell lines. Untreated A549 cells as control (A); cells treated with IC₅₀ concentration of CA224 for 24 h (B); cells treated with IC₇₀ concentration of CA224 for 24 h (C); untreated NCI-H 1299 cells or control (D); cells treated with IC₅₀ concentration of CA224 for 24 h.

Figure 32. The pro-metaphase block induced in NCI-H358 cells with nocodazole and paclitaxel treatment, was maintained by CA224. Cells synchronized by nocodazole and paclitaxel were released either in fresh medium or in fresh medium containing IC₅₀ concentration of CA224 compound exhibit greater tendency of CA224 to block the cell growth at G₂/M. In the figure, (A) control cells; (B) treated with 1 μM nocodazole for 18 h; (C) treated with 1 μM nocodazole for 18 h and released in fresh medium; (D) treated with 1 μM nocodazole for 18 h and released in the presence of CA224, IC₅₀; (E) treated with 50 nM paclitaxel for 18 h; (C) treated with 50 nM paclitaxel for 18 h and released in fresh medium; (D) treated with 50 nM paclitaxel for 18 h and released in the presence of CA224, IC₅₀.

Figure 33. Analysis of NCI-H358 cells using the flow cytometer. Cells synchronized at Gi/S by treatment with hydroxyurea were released either in fresh medium or in fresh medium containing IC₅₀ concentration of CA224. Treated cells exhibit greater tendency towards cell growth block at G₂/M. In the figure, untreated cells act as control (A); cells treated with 250 μM hydroxyurea for 18 h (B); cells treated with 250 μM hydroxyurea for 18 h and released in fresh medium (C); cells treated with 250 μM hydroxyurea for 18 h and released in the presence of IC₅₀ concentration of CA224 (D).

Figure 34. Selective apoptosis in SV-40 transformed cells by CA224, analysed by FACS. BNL CL2 (mouse embryonic normal hepatic cells), when exposed to CA224 for 48 h at IC₅₀ and IC₇₀ concentrations, show prominent G₂/M arrest. As shown, untreated cells (histogram A), cells treated with IC₅₀ concentration of CA224, 48 h (histogram B) and cells treated with IC₇₀ concentration of CA224, 48 h (histogram C). BNL SV A.8 (mouse embryonic SV-40 transformed hepatic cells) underwent apoptotic cell death upon incubation with CA224. The apoptosis was quantified by measuring the percentage of cells that appeared under the sub-G-i peak, 31% and 44% cells were found in the SUb-G₁ peak after 48 h exposure with CA224, at the IC₅₀ (histogram E) and IC₇₀ (histogram F) concentrations, respectively. The untreated cells (D) do not show any apoptosis.

Figure 35. Western blot analysis of p53+ cells, A549 and LS174T. The treatment with CA224 at the IC₅₀ concentration for 24 h resulted in the induction of the tumour suppressor protein, p53. Thereby the Cdk inhibitor, _P21^CIP1/WAF1 (p21 ), was also induced. The levels of p27^KIP1 (p27) were elevated while cyclin B1 and Cdk1 levels were down-regulated.

Figure 36. Western blot analysis of lysates from MiaPaCa-2 cells. CA224 treatment (IC₅₀) of MiaPaCa-2 cells for 24 h did not alter p53, p21 and p27 expression levels. Cdk1 protein levels did not change while cyclin B1 was up-regulated. The phosphorylation status of Cdk1 at Tyr15 remained unaffected after treatment.

Figure 37. In vitro polymerisation assay using purified tubulin. The ability of CA224 to inhibit tubulin polymerisation in vitro was investigated as described. Paclitaxel and nocodazole were used in the assay as a known enhancer and inhibitor of tubulin polymerisation, respectively. CA224 was tested for a range of concentrations at which it shows inhibition of in vitro cell growth. The change in Vmax value was used as an indicator of tubulin — ligand interactions. The polymerisation curves indicate that 2.5 μM, 5 μM, 10 μM and 25 μM concentrations of CA224 reduced the Vmax value from 19 mOD/min (control) to 6.2, 2.1 , 1.1 and 0.4 mOD/min, respectively. The curves shown are average of three independent experiments.

Figure 38. In vivo tubulin polymerisation assay in A549 cells. Western blots show the response of CA224 to tubulin polymerisation in the presence of paclitaxel and the effect of CA224 on paclitaxel-stabilized tubulin. The assay is performed in whole cells (A549) after 30 min compound treatment at the concentrations indicated in Figure. Supernatant and pellet represent unassembled and assembled tubulin respectively. Tubulin polymerisation is detectable by the increase of tubulin in the pellet and its disappearance from the supernatant. Simultaneous treatment of paclitaxel and CA224 show inhibition of tubulin polymerisation by CA224 in a dose-dependent manner and results in accumulation of unassembled tubulin in the supernatants. We observed that CA224 also acts as an enhancer for tubulin de-polymerisation in a dose-dependent manner when paclitaxel-stabilized tubulin was subjected to CA224 treatment.

Figure 39. CA224 activity in colony formation assays. A549 and Calu-1 cells were investigated for their long-term survival efficiency after treatment with different concentrations of CA224. The colony formation efficiency is expressed as the percentage of colonies formed in the treated cultures compared with untreated cultures. (A) The representative plates show untreated A549 cells (a); cells treated with CA224, 3.12 μM (b); cells treated with CA224 6.25 μM (c); cells treated with CA224, 12.5 μM (d); cells treated with fascaplysin 0.8 μM (e); untreated Calu-1 cells (T); cells treated with CA224, 3.12 μM (g); cells treated with CA224 6.25 μM (h); cells treated with CA224, 12.5 μM (i); cells treated with fascaplysin 1 μM G). (B) The curves represent colony formation efficiencies of A549 and Calu-1 cells with increasing concentrations of CA224. All results represent the means and standard deviations of three independent experiments.

Figure 40. Induction of apoptotic cell death analysed by DAPI staining and FACS.

Incubation of A549 cells with increasing concentrations of CA224 for 24 h shows the dose-dependent induction of fragmented nuclei, disrupted cell membrane and apoptotic cell death. (A) The fluorescence microscopic images captured at 40X magnification after staining with DAPI. Untreated A549 cells (a); cells treated with CA224, 1 μM (b); cells treated with CA224, 2.5 μM (c); cells treated with CA224, 5 μM (d); cells treated with CA224, 10 μM (e). The pre-apoptotic and apoptotic cells are indicated with arrows. (B) The percentage apoptosis, induced in three cancer cell lines by treatment with CA224, determined by FACS analysis. The percentage of cells undergoing apoptosis is calculated from the percentage of cells that appear in the SUb-G₁ peak during cell cycle analysis. The number of apoptotic cells increase with the concentration of CA224 used and also the time of incubation.

Figure 41. Tumour growth inhibition curves for CA224, AJW089, DE002 and flavopiridol (used as a positive control) in the in vivo HCT116 xenograft model.

Graphs depict tumour growth inhibition in a group of animals treated with a specific compound which is compared with the untreated (control) group. Tumour sizes were recorded at 2-5 day intervals. Tumour weight (in mg) was estimated according to the formula for a prolate ellipsoid: {Length (mm) x [width (mm)²] x 0.5} assuming specific gravity to be one and π to be three.

Figure 42. Animal weight profile in the HCT116 SCID mice xenograft model. The body weights of animals in different treatment and control groups were monitored by taking measurements daily during the treatment schedule. By considering the body weight at the start of treatment as 100%, the percentage weight loss was calculated on subsequent days of treatment.

Figure 43. Percentage inhibition of tumour growth by CA224 and DE002 at ¹Λ MTD concentrations. SCID mice treated with CA224 and DE002 showed up to 80% tumour growth inhibition in the HCT116 tumour model. Tumour growth in animals treated with compound is calculated as T/C (Treated/Control) x 100% and Growth inhibition Percentage (% Gl) = [100-%T/C]. Both CA224 at 250 mg/kg and DE002 at 250 mg/kg are highly active in this tumour model (see Table 15).

Figure 44. Tumour growth inhibition curves for CA224, AJW089, DE002 and flavopiridol (used as a positive control) in the in vivo NCI-H460 tumour model.

Figure 45. Animal weight profile in the NCI-H460 xenograft model. The body weights of animals in different treatment and control groups were monitored by taking the measurements daily during the treatment schedule. By considering the body weight at the start of the treatment as 100%, the percentage weight loss was calculated on subsequent days of treatments.

Figure 46. Average growth inhibition percentage of CA224, AJW089, DE002 and flavopiridol in NCI-H460 xenograft model. Stars indicate significant tumour growth inhibition. The tumour growth inhibition above 50% was considered as significant activity.

Figure 47. Pictures of NCI-H460 tumour tissues exhibiting the growth inhibition of compounds CA224, DE002, AKW089 and flavopiridol in SCID mice. These pictures of the tumours were obtained after treatment of tumour-bearing animals with

CA224, DE002, AJW089 and flavopiridol for 9 consecutive days. The cultured NCI-

H460 cells were injected subcutaneously into the dorsal side of SCID mice at a tune of

5.3 x 10⁶ cells in a 0.2 ml suspension. When the tumour growth reached about 4-6 mm in diameter (after about 6 days), the animals were randomly divided into eight groups, each containing 6 or 7 mice. The intraperitoneal treatment was continued for 9 consecutive days when animals were sacrificed.

Figure 48. Control and treatment groups of SCID mice showing NCI-H460 tumour growth inhibition following treatment with CA224, DE002, AKW089 and flavopiridol. These pictures were obtained after treatment with CA224, DE002 and flavopiridol. The treatments were continued for 9 consecutive days intraperitoneal^ when tumour growth had reached about 4-6 mm in diameter after about 6 days.

Methods Used in the Examples

Kinase assays

Reagents used for the assays: Purified Cdk enzyme complexes and their appropriate substrates, kinase buffer, DMSO, 5X Pl solution, ATP, Kinase-Glo™ reagent, Cdk inhibitory compounds and distilled water.

Experimental procedure: The extensive screening of synthetic compounds was carried out using different kinase assays: Cdk4/GST cyclin D1 ; Cdk2/cyclin E using GST pRb152 as a substrate, Cdk1/cyclin B1 ; Cdk2/GST-cyclin E using Histone H1 as a substrate and Cdk9/cyclin T1 using GST-CTD or MBP as a substrate. Fascaplysin and its structural analogues, flavopiridol, CINK4, indirubin-5-sulphonic acid-sodium salt and Purvalanol A were dissolved in 100% DMSO as 10 mM stock solutions, aliquoted in micro-centrifuge tubes and stored at -80⁰C. Multiple^" freeze thawing of the 10 mM stocks was avoided for all compounds. Compounds were further diluted in kinase buffer in order to obtain the desired serial dilutions (usually 10-fold) for determination of IC₅₀-S (i.e. the concentrations of compounds at which a kinase activity was inhibited by 50%).

The kinase assays were carried out in a 96-well format in solid white polystyrene plates (Fisher Scientific, Cat. No. DPS-134-050A). The purified enzyme complexes and substrates were diluted in kinase buffer in order to obtain the desired enzyme and substrate concentrations. The kinase assays were performed in a total volume of 50 μl kinase buffer (Table 1 ). Initially 10 μl of kinase buffer containing kinase substrate, i.e. 2 μg of purified GST-pRb152 (for Cdk4-cyclin D1 and Cdk2-cyclin E), 3 μg of Histone H1 (for Cdk2-cyclin A), 10 μg of Histone H1 (for Cdk1-cyclin B1), 2 μg of GST-CTD or MBP (for Cdk9-cyclin T1 ) was added to each well except the wells labelled as substrate blanks. 10 μl of kinase buffer was added to the substrate blank wells. After the addition of a substrate, 10 μl of the diluted compound was added to the respective wells (in order to test a compound at 10 μM concentration, 10 μl of a 50 μM, that is, 5X concentrated stock solution of compound was added). The purified enzymes (in soluble form or on glutathione agarose beads) were diluted in ice cold kinase buffer to obtain 200 ng of enzyme in 20 μl of total volume. ATP solution (10 mM stock solution in sterile distilled water) was diluted to 30 μM in 5X Pl solution The addition of (10 μl) 5X Pl solution containing 30 μM ATP resulted in final concentrations of 10 mM β- glycerophophate, 0.1 mM sodium fluoride, 0.1 mM sodium orthovanadate, 1 mM dithiothreitol and 6 μM ATP. The assays were initiated by adding 200 ng (except 100 ng in case of Cdk1-cyclin B1 and 75 ng in case of Cdk9-cyclin T1 ) of active enzyme complexes per reaction and the plates were incubated for 30 min at 30⁰C in a humidified incubator. All reactions were performed in triplicates. Table 1 shows the combination of substrate, enzyme and test compounds used in the different reactions. After incubation, the reaction was stopped by addition of an equal volume (50 μl) of Kinase-Glo™ reagent. The contents were mixed well and after waiting for 10 min for signal stabilization, the Relative Luminescence Units (RLU) were measured using a Luminometer (either Packard or BMG Labtech) at voltage and gain settings described above.

Table 1. Volumes (in μl) of components used to perform the in vitro kinase assays.

Determination of ICgn concentrations: The measured Relative Luminescence Units (RLU) from the control, blank and test samples were used to determine the ATP depletion in kinase reactions. The RLU from dual blank reactions, where the compound is only added but not the enzyme and substrate, are used to check if the compound has any inhibitory effect on the luciferase catalysed luminescence reaction. None of the compounds affected luciferase activity. We also observed that the ATP depletion in all substrate blanks (reactions containing only GST-pRb152 or Histone H1 or GST-CTD or MBP but no enzyme) was negligible, i.e. less than 1-3%. However, in enzyme blanks (reactions containing enzyme but no substrate) up to 10% ATP depletion was observed. Therefore, the enzyme blanks instead of the substrate blanks were used for calculations and determinations of the IC₅₀-S of the compounds. The mean blank reading was corrected by subtracting the mean control reading and the following formula was used to calculate the % inhibition of enzyme activity.

% inhibition of enzyme activity = (corrected mean blank-test) * 100 corrected mean blank

For each compound, the potency to inhibit enzyme activity was calculated at different concentrations and the IC₅₀ (the concentration at which 50% enzyme activity is inhibited) was calculated by extrapolation. Several Cdk inhibitory compounds reported in the literature, i.e. fascaplysin, flavopiridol, roscovitine CINK4 and indirubin-5- sulphonic acid-sodium salt (Knockaert et al, 2002; Soni et al, 2000; Soni ef a/, 2001; Meijer ef a/, 1997) were used to validate the chemiluminescent assays. The IC₅₀ concentrations of these known compounds determined using this assay compared favourably with the IC₅₀ values published in the literature.

Cell proliferation assay

Maintenance of cell lines

All cell lines were maintained at 37°C in 5% CO₂ in a humidified incubator (Heraeus,

Hera CeW 150). The cancer cell lines were used for screening the fascapiysin analogues in cell proliferation assays, FACS analyses, Western blotting, apoptosis assays, colony formation assays and anti-oxidation assays. Cells with an early passage number were used for all these assays (cells were grown in the culture for a maximum of 10 passages and then fresh vials were revived from liquid nitrogen). For storage in liquid nitrogen, cells were washed and re-suspended in ice-cold cryo- protectant solution (95% fetal bovine serum (FBS) and 5% DMSO). The tubes were transferred to an iso-propanol bath and then placed at -80⁰C in order to achieve controlled freezing of the cells (1°C/min). On the next day the cells were transferred to a liquid nitrogen cryo-can and stored under the liquid phase.

The non-small cell lung carcinoma (NSCLC; a form of cancer which is resistant to chemotherapy) lines NCI-H460 (pRb⁺, p53⁺), A549 (pRb⁺, p53⁺), Calu-1 (pRb⁺, p53- null), NCI-H 1299 (pRb\ p53-null), NCI-H358 (pRb-null, p53-null) were grown in RPMI- 1640 medium, supplemented with 10% FBS and 100 μg/ml Normocin™

The colon carcinoma line LS174T (pRb⁺, p53⁺), the prostate carcinoma line PC3 (pRb⁺, p53-null) and the pancreatic cancer line MiaPaca-2 (pRb⁺, p53-mutant) were cultured in RPMI-1640 medium, supplemented with 10% FBS, 2 mM L Glutamine solution and 100 μg/ml Normocin™.

The BNL CL.2 and BNL SV A.8 (normal mouse embryonic liver cells and its SV40 transformed subline) were grown in RPMI-1640 medium supplemented with 15% FBS, 1 mM sodium pyruvate solution and 1% PSN solution (penicillin, streptomycin and neomycin mixture). The large T antigen of SV40 virus is responsible for functional inactivation of the tumour suppressor proteins pRb and p53.

H4IIE (rat hepatoma cells) were grown in Dulbecco's modified Eagles's medium (DMEM) containing 4.5 g/L glucose and 2 mM L-glutamine, 1% non-essential amino acids, 10% FBS and 100 μg/ml Normocin™.

Cell proliferation assay Reagents used for cell proliferation assays: 2 mg/ml MTT (3-(4,5-Dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide) stock solution (Sigma-Aldrich, Cat. No. M2003), DMSO, trypan blue, trypsin-EDTA solution, sterile PBS, PRMI-1640 or DMEM, FBS, Normocin™ or PSN solution, 100X non essential amino acids solution and 100 mM sodium pyruvate solution.

Experimental procedure: The monolayer of exponentially growing cells representing an asynchronous population was washed with sterile PBS, and dislodged from the culture flasks using trypsinization (Trypsin-EDTA solution treatment at 37°C for few min, incubation time varied with different cell lines). The cells were suspended in medium containing 10% FBS to inhibit the trypsin activity. The viable cell count by the trypan blue dye exclusion method was performed to determine the cell count and viability of cells. 10 μl of cell suspension was mixed with 10 μl of trypan blue solution and the mixture loaded on to a haemocytometer (Neubauer's chamber). The cells from four large squares were counted and an average was used in the following standard formula. The cell count determined using the following formula, was used for further experiments:

Cell number/ml = average from four large squares * dilution factor* 10⁴.

The cells were seeded in 96-well plates (Sarstedt, Cat. No. 83.1835), at densities between 5,000-10,000 cells per well, depending on the doubling time of the individual cell line, in 180 μl of complete growth medium. The wells at the extreme four corners were omitted in order to avoid the edge effect and variations in the assay. The plated cells were allowed to stabilize at 37°C for 24 h in a CO₂ incubator. All the compounds were dissolved in 100% DMSO and 10 rtiM stock solutions were prepared. After 24 h of stabilization, the stock solutions of compounds were diluted in medium without any serum and antibiotics. 20 μl of 1 OX concentrated compounds were added into the wells in triplicates while equivalent amount of DMSO was added to the control (vehicle control) wells. The plates were mixed gently and incubated for a further 48 h. After drug exposure, 50 μl of 2 mg/ml MTT reagent was added and the plates were incubated for 2-4 h at 37°C in the dark. The plates were monitored every 30 min and cells were observed for the formation of blue coloured crystals inside the cells. When more than 95% of cells show blue crystals, the medium containing MTT was removed from the wells. The coloured formazan that formed was dissolved by adding 150 μl of DMSO per well. The plates were mixed gently by placing them on a plate-shaker in the dark. The absorbance was measured at a wavelength of 540 nm using a 96-well plate- reader (Biotek Synergy HT). Each concentration was tested in triplicate and the average of the three readings was calculated. The IC₅₀ concentration of compound was considered as the concentration at which 50% of cell growth was inhibited as compared to the control wells which did not contain any drug. The wells containing medium but no cells were treated as reagent blanks and both the mean control and mean test readings were corrected for the reagent blank before using them to determine the % growth inhibition. The % cell growth inhibition for each concentration of a compound was calculated using the following formula:

% cell growth inhibition = (corrected mean control - corrected mean test) x 100 corrected mean control

The graphs of % inhibition with the increasing concentrations of compounds were plotted using Microsoft Excel and the concentration at which 50% cell growth was inhibited was calculated by extrapolation.

DNA binding studies

The ability of a compound to interact or intercalate with double-stranded DNA molecules was important to distinguish between fascaplysin and its non-planar analogues. The compounds were tested for their affinity for pBlueScript plasmid DNA that was used as a substrate for two independent assays: (a) the ethidium bromide displacement assay and (b) the topoisomerase I catalysed DNA unwinding/relaxation assay. Ethidium bromide displacement assay

Ethidium bromide (EtBr) is a commonly used nucleic acid stain that intercalates with the double-stranded DNA molecules and is also a known chemical mutagen. If excited at 260 nm, ethidium bromide can emit fluorescence at 600 nm. The fluorescence of ethidium bromide increases 25-fold upon binding with double stranded DNA molecules and when the DNA bound ethidium bromide is displaced, quenching of the fluorescence can be observed. The pBlueScript DNA was used for binding studies in this modified assay based on the method previously described by Geall et al, 1999 and Brotz-Oesterhelt et al, 2003. Ethidium bromide was dissolved in sterile distilled water as a 10 mg/ml stock solution and stored at 4°C. The DH5α E. coli cells, transformed with pBlueScript plasmid DNA, were grown in LB medium broth containing 25 μg/ml ampicillin. The plasmid DNA was isolated using the Maxi (QIAGEN-tip 500) preparation protocol following the manufacturer's instructions.

Reagents used for the EtBr displacement assay: 10 mg/ml EtBr solution, purified pBlueScript plasmid DNA, DMSO, EtBr displacement assay buffer, test compounds.

Experimental procedure: The assays were performed in 96-well black plates with c\ear bottom (Costar®). The assay involved addition of 10 μl of 10X concentrated stock solution of compounds (dissolved in DMSO and further diluted in EtBr displacement assay buffer) to 90 μl of reaction mixture containing 6 μg of purified pBlueScript DNA and 1.3 μM ethidium bromide in a EtBr displacement assay buffer with a final pH 7.4. Equivalent amounts of DMSO were added to the vehicle controls. In addition to control samples (DNA + EtBr), test samples (DNA + EtBr + test compound), blank 1 (EtBr only), blank 2 (DNA only), wells containing DNA and compound were also prepared to test any change in the background fluorescence readings. The reduction in relative fluorescence counts was monitored (λexcit = 260 nM, λemiss = 600 nM) and recorded after 1 min equilibration time. Fascaplysin and actinomycin D, which are known to intercalate double stranded DNA molecules, were used as standard compounds in the assay (Hormann et al, 2001 ). The mean control and test readings were corrected by substracting blank readings from them. The % fluorescence in the test samples (in relation with the control samples) was calculated by using following formula:

% fluorescence = corrected mean RFU in control - corrected mean RFU in test * 100 corrected mean RFU in control The graphs of % fluorescence against the concentration were plotted in Excel and the concentration that showed 50% reduction in % fluroscence was considered as the IC₅₀ in the EtBr displacement assay.

Topoisomerase I catalysed DNA relaxation or unwinding assay in vitro

The interconversions of topological isomers of DNA molecules are essential for many vital cellular processes like DNA replication, transcription, chromosomal segregation and recombination. This can be achived by breaking and re-uniting (re-ligating) the DNA strands sequentially. This process is catalysed by a key class of enzymes known as topoisomerases. There are two types of topoisomerases, DNA topoisomerase I (topo I) which acts on only one strand and induces single-strand nicks in the DNA followed by their religation while DNA topoisomerase Il (topo II) acts on both the strands resulting in DNA breaks and sequential reunions (Giles and Sharma 2005; Leppard and Champoux 2005).

Reagents used for DNA unwinding assays: Topoisomerase I enzyme, 10 mg/ml EtBr solution, purified pBlueScript plasmid DNA, agarose, DNA unwinding assay buffer, phenol, chloroform, EDTA, Tris-EDTA buffer, SDS, absolute ethanol, 10X DNA loading buffer and test compounds.

Experimental procedure: A double-stranded plasmid DNA mainly exists in a super- coiled form and a small fraction of it exists in a nicked circular or linear form. The super-coiled DNA when treated with topo I or topo Il enzyme in vitro can become relaxed and be converted into sequential topological isomers which differ in their mobility when separated on an agarose gel. Compounds having affinity for DNA molecules and that can intercalate into DNA can hamper the topoisomerase I catalysed DNA relaxation process, thus allowing the assessment of the ability of compounds to intercalate DNA in an in vitro assay (Antony et al, 2005; Das et al, 2006; Fortune et al, 1998).

The ability of fascaplysin and its structural analogues to intercalate into plasmid DNA was thus determined by a topoisomerase I unwinding/relaxation assay. The pBlueScript plasmid DNA was used for the assay. Each reaction contained 5 nM super-coiled pBlueScript plasmid DNA and 10 units of topoisomerase I active enzyme. To ensure that the assay determines the DNA intercalating property of the compounds and not topoisomerase I inhibition, in a parallel experiment relaxed plasmid DNA was first prepared by treating with topo I enzyme for 30 min and then used as an initial substrate for the assay. DNA relaxation assays were performed in the presence or absence of compounds in 40 μl of DNA unwinding assay buffer. After 30 min incubation at 37°C, reaction mixtures were treated with 3 μl of 250 mM EDTA and extracted with phenol/chloroform. The DNA was dissolved in Tris-EDTA buffer, pH 8. The samples (20 μl) were treated with 2 μl of 2.5% SDS, mixed with 2.5 μl agarose gel- loading buffer (10X) and subjected to electrophoresis on a 0.8% agarose gel without ethidium bromide (separating the DNA in the presence of ethidium bromide would convert the relaxed DNA into the supercoiled form). After the electrophoretic separation, DNA bands were stained with 1 μg/ml ethidium bromide and visualised using a UV illuminator. The compounds were compared with fascaplysin which is a known DNA intercalating molecule. Camptothecin which is a known topoisomerase I inhibitor was used to test the activity and inhibition of the enzyme.

Flow cytometric analyses Information about the normal cell cycle and the effects on the cell cycle due to drug treatment can be assessed by studying the DNA content in drug-treated mammalian cells. Since DNA content of cells at different phases of the cell cycle differ substantially, cells can be differentiated on the basis of the amount of DNA present and hence percentage of cells in different phases of cell cycle can be determined. For example, if cells are stained with nucleic acid specific stains, like propidium iodide, all cells in the d phase should take up the same amount of stain and should fluoresce in a single channel (or single peak). Measurements of DNA content can be performed using a Fluorescence Activated Cell Sorter (FACS). A FACS machine measures the fluorescence from the DNA-binding fluorochrome which infers the total DNA content. The instrument used for these studies was the Beckman-Coulter EPICS® ALTRA™ and the instrument was calibrated before every use with the help of 'fluorescent calibration beads' supplied by Beckman-Coulter. The single peaks for forward scatter, side scatter and the rest of the filters including PMT 4 (excitation 495, emission 637) were calibrated in such a way that the coefficient of variation (CV) falls below 2%. A typical cell cycle picture obtained by FACS analysis is shown in Figure 3.

Reagents used for FACS analyses: Propidium iodide (Sigma-Aldrich, Cat. No. P4170), RNase A (Sigma-Aldrich, Cat. No. R6513), PBS, absolute ethanol, different cell cycle and anticancer compounds and cancer or normal cells cultured in vitro. Experimental procedures: Single cell suspensions for FACS analyses were made after harvesting the cells. Cells were seeded in such a way that no cultures became confluent or super-confluent before cell harvesting.

Serum starvation experiments

If mammalian cells are starved of serum growth factors, their growth tends to be arrested in early G₁ phase of the cell cycle (Soni et al, 2001). In order to block the cells at early Gi phase, cells were serum starved by incubating in medium containing 0.1 % FBS for 24 h. They were then released in complete growth medium supplemented with 10% FBS in the presence or absence of test compound for a further 24 to 48 h. The cells released in the fresh medium were used to check any abnormal effect on cells due to serum starvation.

Mimosine block experiments Mimosine, a non-essential amino acid, inhibits DNA polymerase α in eukyaryotic cells and thereby blocks them at the d/S boundary of the cell cycle (Ji et al, 1997). To block cells at the G₁ZS boundary, cells were seeded in 25 cm² tissue culture flasks. When the cultures became 40-50% confluent, they were treated with mimosine as follows. Mimosine was prepared as a 10 mM stock solution in 100% DMSO, diluted in sterile growth medium without serum and added to cultures at 200 μM as a final concentration for 32 h. Cells were washed twice with fresh medium and then incubated in fresh medium in the absence or presence of test compounds for a further 18 h or 36 h.

Nocodazole block experiments

Nocodazole was dissolved in 100% DMSO as a 10 mM stock solution and then stored at -80⁰C until required. The NCI-H358 (p53-null) cells were seeded in 25 cm² tissue culture flasks. Cells were allowed to grow until 40-50 % confluency had been reached; then nocodazole (diluted in pre-warmed growth medium without FBS) was added to culture flasks at a final concentration 1 μM (a sublethal concentration at which cells can re-enter cell cycle without any damage or apoptosis). The flasks were returned to the incubator and incubated for 18 h. After this, the cells were harvested, fixed and stained with propidium iodide for FACS analysis. The rest of the cells were released in fresh medium in the absence or presence of test compound and incubated for a further 12 h. All samples were harvested, fixed, stained and subjected to FACs analysis.

Paclitaxel block experiments Paclitaxel was dissolved in 100% DMSO as a 10 mM stock solution and further diluted in pre-warmed growth medium without FBS. The paclitaxel block experiments were performed as described above.

Cell harvesting, fixation and staining

Floating cells were collected by centrifugation of the growth medium at 3,000 rpm for 5 min. Attached cells from the flasks were harvested by trypsinization. All the collected cells (floating and attached) were washed once with PBS and counted using a haemocytometer. 1 * 10 ⁶ cells were fixed in 1 ml of 70% chilled (-20⁰C) ethanol for at least 1 h. Cells were stored at -20⁰C until the staining. After the fixation step, cells were centrifuged for 5 min at 3,000 rpm at room temperature and the pellet re- suspended in 1 ml of PBS containing 50 μg/ml propidium iodide and 0.5 mg/ml DNase free Ribonuclease (RNase A Sigma Cat. No. R6513). The cells were stained for 1 h in the dark at 4°C and then analysed by FACS.

Instrument set up and FACS analyses

Cell cycle analyses were performed by FACS. Sterile PBS was used as a sheath fluid during the FACS analyses. After calibration, 1 ml of cells (1 x 10 ⁶CeIIs) were transferred to a round base FACS sample tube. The data rate was kept in the uniform range (15-20) for all samples and was sometimes slightly adjusted in order to achieve around 200/second total number of events. To avoid cell doublets or cell clumps being analysed with the events representing single cells, following analyses were performed on each sample. Cytograms were plotted using ungated data to represent the propidium iodide fluorescence peak signals (PMT 4 peak) on Y axis and the integrated fluorescence signals or the linear fluorescence signals (PMT 4 Lin) on X axis. From this ungated cytogram, all data points representing single cells on the straight line were isolated in a single gate and the gated data further used for plotting a histogram (PMT 4 Lin on X axis) that represents a complete cell cycle. At any point the total number of events was not allowed to exceed 200 events/second. Data acquisition was programmed to collect a minimum of 10,000 events for each sample analysed.

Western blot analyses

Treatment of cells, sample preparation and protein estimation

Reagents used: Cell lysis buffer (Sigma-Aldrich, Cat. No. C-2978), protease inhibitor cocktail (Sigma-Aldrich, Cat. No. P8340), Bradford reagent (BIO-RAD protein assay,

Cat. No. 500-0006), 1 mg/ml BSA solution (Sigma-Aldrich, Cat. No. A9418), PBS, test compounds and sterile distilled water. Experimental procedure: For Western blot analyses, all cells from different cell lines were seeded in 25 cm² tissue culture flasks in complete growth medium. When the culture flasks reached 40-50% confiuency, the cells were treated with the desired concentration of test compound for 24 h. The floating/detached cells were collected by centrifugation and the attached cells were harvested by trypsinization, washed in ice- cold PBS two times and then suspended in the lysis buffer that contained the protease inhibitor cocktail. The cells were incubated at room temperature and allowed to lyse for a period of 2 h. The lysates were centrifuged at 14,000 rpm for 10 min at 4°C and the clear supernatants were assayed for protein content using the Bradford method. For Bradford protein estimation assay, appropriate dilutions of sample and standard (BSA 1 mg/ml solution) were made in sterile distilled water. 10 μl of diluted sample or standard was added to 790 μl of sterile distilled water, then 200 μl of Bradford reagent was added to each tube and mixed well. The tubes were incubated at room temperature for 10 min. 200 μl of mixture from each tube was transferred to a 96-well plate and the absorbance was measured at a wavelength of 595 nm. Each sample and standard was assayed in triplicate. A standard curve of BSA concentration and absorbance was plotted in Excel and the protein concentrations in samples were determined by extrapolation (a representative curve is shown in Figure 4).

SDS-Polvacrylamide Gel Electrophoresis

Reagents used: 6X SDS sample buffer, 30% acrylamide/Bis solution (BIO-RAD, Cat. No. 161-0156), Tris-HCI pH 8 & pH 6.8, SDS, ammonium persulphate (APS), TEMED, pre-stained protein marker, distilled water and butanol.

Experimental protocol: After protein estimation; equal amounts of protein (40 μg for each sample) were mixed with 6X SDS sample buffer and boiled for 7 min. The tubes were centrifuged quickly and the samples were loaded on SDS-polyacrylamide gels (4% stacking gel and 10% running or resolving gel). The 10% running gel was prepared with the following composition of different chemicals. 10% running gel: Distilled water 7.9 ml

30% acrylamide/bis solution (29:1) 6.7 ml

1.5 M Tris-HCI pH 8 5 ml

10 % SDS 200 μl 10 % APS (ammonium persulphate) 200 μl

TEMED 20 μl For each experiment APS solution was made fresh. The components were added in the sequence indicated above and the solution poured between the two glass plates immediately after the addition of APS and TEMED. Dual vertical gel electrophoresis apparatus (BIO-RAD, MINI PROTEIN II™) was used to perform the gel electrophoresis. 1 ml of water saturated butanol was poured on top of the running gel. The gel was allowed to polymerise at room temperature for 30 min. The water- saturated butanol was removed and the upper part of the polymerised gel was washed with distilled water. The 4% stacking gel was prepared using the following composition of chemicals. 4% Stacking gel: Distilled water 2.5 ml

30% acrylamide/bis solution 625 μl

0.5 M Tris-CI pH 6.8 1.05 ml

10% SDS 41.74 μl

10% APS 10 μl TEMED 4.5 μl

An appropriate sized comb was inserted in between the glass plates and the stacking gel was poured immediately after the addition of APS and TEMED. The stacking gel was allowed to polymerise for 30 min at room temperature. 1X SDS page running buffer was prepared and after loading sample on to the gel, the gel was initially run at 150 V for 10 min and then at 110 V for 1 h.

Transfer of proteins on lmmobilon membrane and probing with different antibodies Reagents used for transfer and membrane blocking: Cathode buffer, Anode I buffer, Anode Il buffer, blocking grade non-fat dry milk powder (BIO-RAD, Cat. No. 170 6404), appropriate primary and secondary antibodies, developer solution (Kodak, Cat. No. 190 0943), fixer solution (Kodak, Cat. No. 190 1875) and ECL detection reagent kit (Santa Cruz Biotechnology, Cat. No. sc-2048)

Experimental procedure: After electrophoresis, the gel was soaked in Cathode buffer. Immobilon-P Transfer Membrane (Millipore Cat. No IPVH20200) was cut to the same size as that of the gel and soaked in methanol for 15 s. 3X Whatman^® chromatography papers (Cat. No. 3030335), twice the size of the gel were soaked in Cathode buffer and placed on the blotter (PHASE Blucherst 2-23564 Lubek). The air bubbles were removed by gently rolling a 10 ml pipette over the filter papers. The gel was placed on top of the filter papers. The Immobilon-P membrane was labelled to mark the side on which proteins will be transferred to and then slowly placed on the gel, air bubbles were removed. 3X Whatman^® chromatography papers (the same size as the gel) were soaked in Anode Il buffer and placed on top of the gel. Another a set of 3X Whatman^® chromatography papers were soaked in Anode I buffer and placed on top of the previous Whatman^® papers and air bubbles were removed. The top part of the blotter was carefully placed and the transfer was carried out at a constant current of 0.8 mA/cm² of gel surface (6 mA per gel). BIO-RAD power pack 200 was used to maintain the low voltage required to avoid heating of the gels. The transfer was carried out for 1 h and 30 min at room temperature. After transfer, the membrane was removed and washed in PBS containing 0.1% Tween 20 for 10 min with gentle shaking. The PBS/0.1% Tween 20 was poured off and 10 ml of blocking solution, 5% milk powder in PBS was added to the membrane, and incubated with gentle shaking for 1 h. The membrane was rinsed in PBS/0.2 % Tween 20 solution and washed 1 x 15 min followed by 3 * 5 min in PBS/0.2 % Tween 20 with gentle shaking. The membrane was incubated with primary antibody diluted in 1% milk powder in PBS/0.2% Tween 20 solution.

The membranes were probed with different primary antibodies (at 4°C overnight) as described: C-22 (Santa Cruz Biotechnology, Cat. No. sc-260) at 1 :1000 dilution to detect Cdk4; M-20 (Santa Cruz Biotechnology, Cat. No. sc-718) at 1:1000 dilution to detect cyciin D1 ; Cdk2 human (CR-UK, Cat. No. AN21.2) at 1 :4000 dilution to detect Cdk2; cyciin A (CR-UK, Cat. No.E23.1 ) at 1 :5000 dilution to detect cyciin A; cdc2 (New England Biolabs, Cat. No. 9110) at 1 :1000 to detect Cdk1 ; cyciin B1 (CR-UK, Cat. No. V152) at 1 :1500 dilution to detect cyciin B1 ; Pab 1801 (Santa Cruz Biotechnology, Cat. No. sc-98) at 1 :500 dilution to detect p53; N-20 (Santa Cruz Biotechnology, Cat. No. sc-469) at 1 :500 dilution to detect p21 ; C-19 (Santa Cruz Biotechnology, Cat. No. sc- 528) at 1 :250 dilution to detect p27; Rb (4H1 ) (New England Biolabs, Cat. No. 9309) at 1 :1000 dilution to detect phosphorylated and unphosphorylated full length pRb; Ser780 (New England Biolabs, Cat. No. 9307) at 1 :1000 dilution to detect phosphorylation of pRb at serine residue 780; Ser795 (New England Biolabs, Cat. No. 9301) at 1 :1000 to detect phosphorylation of pRb at serine residue 795 and Ser807/811 (New England Biolabs, Cat. No. 9308) at 1 :1000 dilution to detect phosphorylation of pRb at serine residues 807/81 1; AC-40 (Sigma-AIdrich, Cat. No. A4700) at 1 :2000 dilution to detect actin. After an overnight incubation with primary antibody, the membrane was rinsed briefly with PBS/0.2% Tween 20 and then washed with gentle shaking 3 * 10 min in PBS/0.2% Tween 20. An appropriate secondary antibody is then used. An anti-mouse antibody conjugated to horseradish peroxidase (Santa Cruz Biotechnology Cat. No. sc-

2302) is used at a 1 :2500 dilution in the case of a mouse monoclonal primary antibody or an anti-rabbit antibody conjugated to horseradish peroxidase (Santa Cruz Biotechnology, Cat. No. sc-2301) is used at a 1:2000 dilution in the case of a rabbit polyclonal primary antibody. The secondary antibody was diluted in PBS/0.2% Tween 20 containing 1% milk powder and incubated with the membrane for 1 h 30 min at room temperature with gentle shaking. After probing with secondary antibody, the membrane was washed 2 * 10 min in PBS/0.2% Tween 20 followed by 2 * 10 min in PBS with gentle shaking. Finally the membrane was rinsed with sterile distilled water. Since the secondary antibodies are conjugated to horseradish peroxidase (HRP), the protein bands can be visualised by chemiluminescence using the ECL detection kit (Santa Cruz Biotechnology, Cat. No. sc-2048). The membrane was placed on cling film and 2 ml of ECL detection reagent was added drop-wise onto the membrane. The membrane was incubated with the reagent for 1 min and the excess fluid was removed. The chemiluminescence was detected using either Gel documentation system (BIO- RAD, UNIVERSAL HOOD Il S. N. 76S) or by exposing to Hyperfilm™ ECL™ (Amersham Pharmacia Biotech). Immediately after the treatment with ECL detection reagent, the membrane was exposed to Hyperfilm™ ECL™ for various times (e.g. 30 s to 5 min) and developed.

Western blot analysis to test effect of compounds on tubulin polymerisation and depolymerisation of stabilized tubulin in vivo

The tubulin polymerisation and depolymerisation assays in vivo were performed with some modifications to the basic procedures described previously (Jordan et al, 2002; Giannakakou et. al., 1997; Hua et al, 2001 ). A549 cells were plated at a concentration of 10,000 cells per well in 1 ml complete growth medium in 24-well (15 mm) plates. The plates were incubated for 24 h to allow cell attachment and stabilization. In the first set of experiments, the cells were treated simultaneously with 10 nM paclitaxel and different concentrations of test compound for 30 min. In order to study the effect of compounds on the stabilised form of tubulin, the cells were treated with 10 nM paclitaxel for 30 min. Then the cell monolayer was washed twice with sterile PBS and fresh growth medium containing different concentrations of compound was added. The plates were further incubated for 30 min, the cell monolayer were washed twice with sterile PBS at room temperature and then 100 μl tubulin extraction buffer supplemented with 2 mM phenylmethylsulfonyl fluoride, and a protease inhibitor cocktail (Sigma-Aidrich Cat. No. P8340) was added per well. The plates were incubated for 10 min at room temperature and then the cell suspensions were transferred to microcentrifuge tubes. After a brief and vigorous vortex of the tubes, the cell lysates were incubated at room temperature for 5 min and then centrifuged at 16,000 rpm for 10 min in order to separate the soluble and polymerised tubulin fractions. Each supernatant and pellet fraction was mixed with 6X SDS sample buffer, heated for 7 min at 95°C and resolved on a 10% SDS-polyacrylamide gel. The resolved proteins were Western blotted and tubulin-specific proteins were illuminated with a mouse monoclonal β-tubulin antibody B-7 (Santa Cruz Biotechnology, Cat. No. sc-5286) used at a 1:1000 dilution and HRP-conjugated secondary antibody (Santa Cruz Biotechnology, Cat. No. sc-2302) used at a 1:2500 dilution.

Tubulin polymerisation assay in vitro The purified bovine brain tubulin and the cytoDYNAMIX™ Screen was obtained commercially (Cytoskeleton Inc. Denver USA) and the polymerisation assays were carried out according to the method described previously (Ferlini et al, 2005; Jordan et al, 2002). The tubulin polymerisation assay is based on an adaptation of the original methods of Lee et al, (1997) which demonstrated that light is scattered by microtubules to an extent that is proportional to the concentration of the microtubule polymer in the solution. When the protein tubulin is allowed to polymerise in vitro, the turbidity of the solution increases with time resulting in a typical polymerisation curve representative of three phases of microtubule polymerisation, namely nucleation, growth and steady state equilibrium. Paclitaxel and nocodazole were used in the assay as a known enhancer and inhibitor of tubulin polymerisation, respectively.

Reagents used for tubulin polymerisation: General tubulin buffer, tubulin glycerol buffer, tubulin polymerisation buffer, purified bovine brain tubulin (Cytoskeleton Inc. Denver USA, Cat. No. BK006 / CDS03), 200 mM GTP solution, DMSO and test compounds.

Experimental procedure: The ability of test compounds to inhibit tubulin polymerisation in vitro was determined according to the manufacturer's instructions. The tubulin polymerisation assays were performed in 96-well plates. Prior to starting the assay, the plate was pre-warmed at 37°C for 30 min (warming the plate is essential for higher polymerisation activity as well as reproducible results). Tubulin polymerisation buffer (TP buffer) was prepared by the addition of the following components and then incubation on ice for 10 min: 1.5 ml of general tubulin buffer (80 mM PIPES pH 6.9, 2 mM MgCI₂, 0.5 mM EGTA), 500 μl of tubulin glycerol buffer (15% glycerol in general tubulin buffer) and 20 μl of GTP stock solution (200 mM).

The TP buffer was made fresh each time and used within 4 h of preparation due to unstable nature of GTP. 1 ml of general tubulin buffer was made warm by incubating at 37°C for 10 min and then used for the different dilutions of paclitaxel, nocodazole and test compounds. For control wells (tubulin minus compound), 10 μl of general tubulin buffer was pipetted into two wells. For test reactions, 10 μl of 10 μM paclitaxel,

10 μM nocodazole or 10X concentrations of the test compounds were added to the respective wells. The purified bovine brain tubulin (10 mg/ml) was defrosted by placing it on a room temperature water bath for exactly 1 min or until thawed and then placed on ice (the fast thawing is important because concentrated tubulin may start polymerising). The concentrated tubulin protein was diluted 1 :3.3 in ice cold TP buffer to obtain 3 mg/ml solution. 100 μl of diluted tubulin was added in each well and the plate immediately placed in a temperature regulated Biotech spectrophotometer previously adjusted to 37°C. The absorbance was recorded using the kinetic set-up described below.

Measurement type kinetic, 61 cycles of 1 reading per minute Absorbance wavelength 340 nm (monochrome filter adjusted to 340 nm)

Temperature 37⁰C

Shaking orbital shaking for 5 s (medium)

Designation of blanks is not required because the spectrophotometer was adjusted to read zero at the beginning of the polymerisation reactions. From the polymerisation curves, the change in Vmax of each reaction was calculated and used as an indicator of tubulin/test compound interactions.

Detection of apoptosis using 4'-6-Diamidino-2-phenylindole (DAPO staining The post apoptotic characterstic of cells like degeneration of nuclear membrane and nuclear fragmentation were detected using 4'-6-Diamidino-2-phenylindole (DAPI) staining followed by observations under a fluorescence microscope (Sandra et a/, 2006). DAPI is known to form complexes with natural double-stranded DNA. When DAPI binds to DNA, its fluorescence is strongly enhanced making it possible to observe the nuclear morphology of cells under a fluorescence microscope.

Reagents used for DAPI staining: DAPI (Sigma-Aldrich, Cat. No. D8417), glycerol, acetic acid, ethanol, complete growth medium, PBS, trypsin-EDTA solution.

Experimental procedure: A549 and MiaPaCa-2 cells were seeded at a concentration of 50,000 and 40,000 cells per well respectively, in 6-well (35 mm) plates (Sarstedt, Cat.

No. 83.1839.500) in a total of 2 ml complete growth medium. After a 24 h stabilization period, cells were observed under the microscope for attachment. The plates were further incubated in the presence of different concentrations of test compounds for 24 h. After exposure to drug the cells along with floating cells were collected by trypsinization, washed in sterile PBS and fixed in chilled -20⁰C ethanokacetic acid (3:1 ) fixative for 10 min. The cell suspension was dropped on a clean glass slide in order to break open the cells and then allowed to air dry. The smear formed on the slide was mounted in DAPI staining solution containing 1 μg/ml DAPI dissolved in 50% glycerol and covered with a coverslip. The slides were observed under a fluorescence microscope (Olympus, BX51) and a minimum of 500 nuclei were counted for each sample.

Determination of Mitotic index

The mitotic index (percentage of cells in mitosis) of cultures followed by the treatment with test compound was determined by fluoroscence microscopic observations after staining the cells with DAPI (Augustin, et al, 1997). A549 cells were seeded at a density of 50,000 cells per well in 6 well tissue culture plates. After 24 h, the medium was replaced with fresh medium containing the desired concentration of test compound. 20 h later, attached and floating cells were collected and combined (by trypsinization and centrifugatron) The cells were fixed in chilled (-20⁰C) acetic acid:ethanol (1:3) fixative for 10 min. One drop of cell suspension was dropped on a glass slide in order to disrupt the cell membrane and was allowed to air dry for 10 min. The smear was mounted in a mounting medium containing 1 μg/ml DAPI in 50% glycerol and covered with a coverslip. The slides were observed under a fluorescence microscope and at least 1000 nuclei per slide were observed. The relative percentages of cells in M (mitosis) phase as compared with control cultures were calculated and the mitotic indices were determined.

Colony formation assay

Reagents used for colony formation assays: PBS, complete growth medium, trypsin- EDTA solution, 1% crystal violet solution (Sigma-Aldrich, Cat. No. C3886), methanol, acetic acid, DMSO and distilled water.

Experimental procedure: Long term survival of cancer cells after treatment with the test compound was analysed using a colony formation assay. Cells were plated at a concentration of 500 cells per well in 2 ml of complete medium in 35 mm or 6-well plates. The plates were incubated for a 24 h stabilization period and further incubated with different concentrations of test compound for 24 h. Plates were then gently washed with PBS, replaced with fresh complete medium and incubated at 37°C. After 10-12 days of incubation (when the colonies are visible), the plates were gently washed with PBS and the cell colonies fixed in methanoLacetic acid (2:1 ) fixative for 20 min at room temperature. The plates were washed 2X with 2 ml of distilled water per well and then air dried for 15 min. 1 ml of 1 % crystal violet solution was added to each well and the colonies were allowed to stain for 15 min. The staining solution was removed, the plates were washed 3X with distilled water and then air dried for 30 min. The colonies were evaluated by visual counts and the number of colonies in treated cultures were expressed as a percentage of the control cultures. Representative plates were scanned using the gel documentation system.

Antioxidant assay

Reagents used: H₂DCF diacetate (Molecular probes Cat. No. D399), H₂O₂ DMEM, FBS, L-glutamine, non essential amino acid solution and trypsin-EDTA solution.

Experimental protocol: H4IIE (rat hepatoma cells), 50,000 cells/well were seeded in a 96-well microtitre plate. The cells were allowed to attach and then the medium was changed. Cells were first incubated with different concentrations of test compounds for 60 min, then the medium containing compounds was removed and the cells were washed with PBS twice and fresh medium was then added. H₂DCF diacetate was added at a final concentration of 5 μM and the cells were further incubated for 30 min. The oxidation of intracellular non-fluorescent H₂DCF to highly fluorescent DCF was measured after addition *of H₂O₂ (500 μM) at an excitation of 485 nm and an emission of 525 nm to measure the inhibition by the test compound of H₂O₂ mediated ROS formation (Srinivas et al, 2004; Steffan et al, 2005).

Senescence assay

Senescence is the condition where cells can no longer replicate themselves through processes like entering the normal cell cycle and cellular mitosis. Cells lose the ability to divide during senescence, their physical appearance or morphology also changes during this process and can be observed microscopically. Senescence is also associated with changes in cellular gene expression patterns which results in the detection of β-galactosidase activity by using cellular staining techniques. In these studies β-galactosidase staining of cells was used to detect the senescent cells (Serrano et al, 1997). Reagents used: complete growth medium, trypsin EDTA solution, sterile PBS, DMSO, senescent cell staining kit (Sigma-Aldrich Cat. No. CS0030), glycerol.

Experimental protocol: MiaPaCa-2 (40,000 cells /well) or LS174T (30,000/well) or A549 (25,000/well) cells were seeded in 6-well plates in 2 ml complete growth medium. Cells were incubated for 24 h (to allow for stabilization) followed by 96 h of treatment with test compound (at IC_3O and IC₅₀ concentrations). After compound treatment, the growth medium was removed by aspiration and the cells were washed twice with 1 ml PBS. 1.5 ml of fixation buffer was added to each well and the plates were incubated at room temperature for 10 min. While cells were being fixed, the staining mixture was prepared as per the manufacturer's instructions. The cells were washed 3 times with 1 ml PBS and 1 ml of staining mixture was added to each well. The plates were incubated at 37°C without CO₂ for 48 h. The cells were observed under the microscope and the percentage of cells expressing β-galactosidase was determined. The staining mixture was removed and replaced with 70% glycerol solution and stored at 4°C for long term storage.

Testing of antitumour activity of compounds CA224, AJW089 and DE002 against human xenografts in SCID mice The in vivo efficacy profile of the fascaplysin analogues CA224, AJW089 and DE002 was studied in a human xenograft model. For this purpose, HCT-116 (human colon carcinoma) and NCI-H460 (human non small cell lung carcinoma, NSCLC) cells were xenografted on to SCID mice to create tumours that could be treated with compounds. SCID (severely combined immuno-deficient) mice lacking both T and B immune cells, is an established model to study in vivo efficacy of molecules against human cancers.

Maximum tolerated dose (MTD) finding studies

Swiss albino mice were used to determine the maximum tolerated dose for all three compounds. In this study 6 animals per group were administered with compounds at different doses for five days (Q1 D x 5) via intraperitoneal route. Animals were monitored for weight loss, morbidity symptoms and mortality up to two weeks by the end of treatment. Significant weight loss was considered when mean animal weight dropped by >10% and was considered highly significant when the drop was >20%.

Compounds solubility and dose preparation

Compounds CA224 and DE002 were weighed and mixed with 0.5 % (w/v) carboxymethylcellulose (CMC) and triturated with Tween 20 (secundum artum) with gradual addition of water to make up the final concentration. Care was taken not to exceed > 0.25% of Tween 20 in the final formulation of the compound

The compound AJW089 was partially soluble in 10% polyethyleneglycol 400 (PEG 400). An appropriate quantity of this compound was mixed by gradual addition of 10% PEG solution and was triturated until a homogenous suspension was achieved.

Efficacy study in SCID mice

HCT-116 experiments: A group of 60 SCID (strain-CBySmn.CB17-Pr/ccfc^sc/cy^j, The Jackson Laboratory, Stock # 001803) male mice weighing 18-25 g and 6-8 weeks old were used for the studies. Human colon carcinoma, HCT-116 (ATCC, Cat. No. CCL- 247) cells were grown in McCoy's 5A medium supplemented with 10% FBS (Sigma- Aldrich). 6.6 * 10⁶ of cultured cells in 0.2 ml of suspension were injected subcutaneously into dorsal side of SCID mice. When the tumour growth reached to about 4-6 mm in diameter (about 5 days), the animals were randomly divided into eight groups, each containing 7 mice. The treatments were continued for 9 consecutive days intraperitoneal^.

NCI-H460 experiments: A group of 65 Severely Combined Immune-Deficient (SClD strain-CBySmn.CBI 7-PrZCdC⁵^AJ, The Jackson Laboratory, Stock # 001803) female mice weighing 15-24 g and 6-8 weeks old were used. Human non-small-cell lung carcinoma, NCI-H460 (ATCC, Cat. No. HTB-177) cells grown in RPMI-1640 medium supplemented with 10% FBS (Sigma-Aldrich). The cultured cells were injected subcutaneously into the dorsal side of SCID mice at a tune of 5.3 * 10⁶ cells in 0.2 ml of suspension. When the tumour growth reached about 4-6 mm in diameter (about 6 days), the animals were randomly divided into eight groups, each containing 6 or 7 mice. The treatments were continued for 9 consecutive days intraperitoneally.

Tumour weight measurements: Tumour size was recorded at 2-5 day intervals. Tumour weight (mg) was estimated according to the formula for a prolate ellipsoid: {Length (mm) x [width (mm)²] x 0.5} assuming specific gravity to be one and π to be 3. Tumour growth in compound treated animals is calculated as T/C (Treated/Control) x 100% and Growth inhibition Percent (% Gl) was [100-% T/C] (Tashiro et al 1989; Mohammad et al, 1998; Mohammad et al, 1996).

Body weight measurements: The body weights of animals in different treatment and control groups were monitored by taking the measurements daily during the treatment schedule. By considering the body weight at the start of the treatment as 100%, the percent weight loss was calculated on subsequent days of treatments.

Statistical analysis: Data from each experiment was analysed by Microsoft Excel 2000. Statistically significant differences were identified and analyzed using student t-test for multiple comparisons versus control group (Tashiro et al 1989; Mohammad et al, 1998; Mohammad et al, 1996).

Chemiluminescent-based Cdk assays Sf9 cells, co-infected with baculoviruses carrying the gene of interest, were lysed. Cdk complexes, present in the whole cell lysates, were purified by incubation with glutathione-agarose beads. 10 μl from each sample was resolved on SDS-PAGE and stained with Coomassie blue. Distinct bands representing GST-tagged cyclins and associated Cdks can be seen in Figure 5. The cyclins are tagged with GST (molecular weight 27 kD).

The presence of Cdk and cyclin components in the enzymes Cdk4/GST-cyclin D1 , Cdk2/GST-cyclin A, His-Cdk2/GST-cyclin E and His-Cdk4/GST-cyclin D1 were also confirmed by Western b/otting using Cdk-specifϊc and cycliπ-specific antibodies (Figure 6). As expected; Cdk4, GST-cyclin D1 , Cdk2 and GST-cyclin A proteins were seen as specific bands with molecular sizes 33, 52, 36 and 73 kD respectively. These results confirmed the presence of Cdks and cyclins in the holoenzyme complexes of the active enzymes

Validation of kinase assay using the luminescence method

The ATP concentrations for the standardisation of all kinase assays were kept constant, i.e. the final ATP concentration in the kinase reaction was always 6 μM. The kinase substrates GST-pRb152, Histone H1 , GST-CTD and MBP were used at the concentrations described above. The substrate concentrations were chosen so that less than 5% ATP depletion occurred at a given substrate concentration.

The optimum enzyme concentration was determined by performing titration experiments using a varied set of enzyme concentrations. The optimum concentrations of different enzymes were also determined by testing them for their inhibition with known Cdk inhibitory molecules. In the case of Cdk1-cyclin B1 , the active enzyme complexes were used at 100 ng per reaction according to the manufacturer's instructions. Determination of optimum enzyme concentration

The difference in the numerical fold between the control reactions and substrate blank reactions was calculated in order to determine the enzyme activity (Table 2). Different concentrations of enzymes were used for the standardization assays, which were performed at constant substrate concentration. It was observed that the fold difference between blank and control readings show linear increase with increase in enzyme concentration (Figure 7). A greater than three-fold difference was maintained while assessing the concentration of any enzyme.

Table 2. Representative relative luminescence units (RLU) in kinase reactions with increase of enzyme concentrations. Each kinase reaction was performed in triplicate and the average was used to determine the fold difference.

Inhibition of cyclin dependent kinases (Cdk-s) with known Cdk inhibitors Known cyclin dependent kinase (Cdk) inhibitors (Knockaert et al, 2002; Soni et al, 2000; Soni et al, 2001 ; Meijer et al, 1997) were used as standard controls. Inhibitors were tested at a minimum of 5 different concentrations. The concentration at which 50% enzyme activity was inhibited (IC₅₀) was calculated and compared with the IC₅₀ values published in the literature (Table 3 and Figures 8 to 12). The IC₅₀ concentrations of the known compounds calculated using this assay compare favourably with the IC₅₀ values published in the literature confirming that the assays are accurate.

Table 3. Comparison of experimentally determined IC₅₀ values for Cdk inhibition with published information. The values in bracket have been taken from the published literature on Cdk inhibitors (Knockaert et al, 2002; Soni et al, 2000; Soni et a/, 2001 ; Meijer et al, 1997).

ND = Not Determined NA = Not Available

The results shown in Figures 8 to 12 depict the IC₅₀ curves obtained for different inhibitors in the chemiluminescent-based Cdk assays. The IC₅₀ values which are presented in Table 3 have been extrapolated from these enzyme inhibition graphs (Figures 8 to 12).

Example 1 : Biological activity of the DE002 series of fascaplysin analogues

Chemical structures of compounds of the DE002 series

Molecular modelling and detailed synthetic routes to some compounds of the DE002 series, such as CA199, are described in Aubry et al, (2006). Modifications of this route to synthesize further members of the DE002 series are known to the person of skill in the art.

Chemical synthesis of DE002

Synthesis

a) 0-CICO-C₆H₄-Br, CH₂CI₂, NaOH (4 M, aq, 1eq), 0 ⁰C 15 min then RT. 3 h. b) Pd(PPHs)₄, Toluene, EtOH₁ (HO)₂BC₆H₅, K₂CO₃ (2 M, aq), 90 ⁰C, 24 h.

i) To a suspension of 1,2,3,4-Tetrahydro-β-carboline (1.2mmol) in CH₂CI₂ (3 mL) at 0⁰C was added slowly an aquous solution of sodium hydroxide 4M (1.2 mmol). After 5 min stirring at 0⁰C, 2-benzoyl chloride (1.2 mmol) was added dropwise. The mixture was stirred for 5 min at 0⁰C and then a further 3 h at room temperature. H₂O (20 mL) was added. The two layers were separated and the aqueous phase was extracted with dichloromethane (3 x 20 mL). The organic layer was dried over anhydrous MgSO₄ and evaporated under reduced pressure. The crude product was purified by flash column chromatography on silica. Elution was made successively with ethyl acetate.petroleum ether (40-60°C) = 50:50 and ethyl acetate.

ii) To a stirred solution of B (1 mmol) in toluene (10 mL), under nitrogen, was added K₂CO₃ (1 mmol, 2M aqueous) and Pd(PPh₃)₄ (5 mol %, 0.05 mmol). The solution was stirred for 20 min at room temperature before the addition of a solution of phenyl boronic acid, (1.2 mmol) in EtOH (IO mL). The reaction mixture was heated to 90 ⁰C for 24 h then allowed to cool to room temperature before the addition of H₂O₂ (30%, 1 mL), the reaction mixture was then stirred for a further 1 h. The desired product was extracted into CHCI₃, washed with saturated brine solution (2 x 25 mL) and H₂O (2 x 25 mL), aqueous washings being re-extracted with CH₂CI₂ (3 x 50 mL), the combined organic phases were then dried over anhydrous sodium or magnesium sulphate, filtered and isolated under reduced pressure. The crude product was then purified by flash column chromatography on silica from ethyl acetate - petroleum ether (40-60 ⁰C) to give biphenyl-2-yl(3,4-dihydro-1 H-pyrido[3,4-b]indol-2(9H)-yl)methanone an an off white amorphous solid; 89% yield; δ_H (300 MHz; (CDCI₃) rotomers 1:2.0, (major rotomeή 2.05-2.11 (1H, m), 2.39-2.48 (1H, m), 2.92 (1H, ddd, 12.9, 8.3, 4.6), 3.38 (1H, dt, 13.0, 5.0), 4.31 (1 H, d, 17.0), 5.02 (1 H, d, 17.0), 6.92-7.45 (13H, m), 8.47 (1 H, s); (distinct peaks for minor rotomer) 2.50-2.55 (1 H, m), 2.62-2.69 (1 H, m), 3.67-3.75 (1 H, m), 3.80 (1 H, d, 16.0), 3.84-3.91 (1 H, m), 4.07 (1 H, d, 16.0), 7.72 (1 H, s); δ_c (75 MHz; (CDCI₃) (major rotamer) 21.4 (CH₂), 40.3 (CH₂), 44.9 (CH₂), 107.7, 11 1.1 (CH), 117.8 (CH)₁ 119.3 (CH), 121.6 (CH), 126.6, 127.5 (CH), 127.8 (CH), 128.3 (CH), 128.6 (CH), 129.6, 129.6 (CH), 129.7 (CH), 135.5, 136.3, 138.9, 139.8, 171.2, (minor rotomer) 20.7 (CH₂), 40.3 (CH₂), 44.7 (CH₂), 109.1, 110.8 (CH), 118.0 (CH), 119.5 (CH), 121.7 (CH), 126.9, 127.4 (CH), 127.7 (CH), 127.78 (CH), 128.3 (CH), 128.6 (CH), 129.0, 129.5 (CH), 129.6 (CH), 135.6, 136.1 , 139.0, 139.3, 170.4; m/z (FAB) 352.15760 (M+H⁺ C₂₄H₂₀N₂O requires 352.15756).

The analogues listed in Table 4 were tested in a number of different bio-chemical assays. Their potency to inhibit different Cdks in vitro was evaluated first. This was followed by further analysis on their ability to inhibit cancer cell growth in vitro. The DNA-binding properties of these compounds were also explored.

Table 4 Chemical structures of the tryptoline class of fascaplysin analogues (DE002 series).

CA19

CA161 CA199 8

CA204 CA206 CA205

CA207 CA208 CA209

CA210 CA211 CA212

Inhibition of Cdk4-cyclin D1 enzyme in vitro

All of the compounds in Table 4 were screened in different in vitro assays to study their capacity to inhibit Cdks. All molecules are non-planar and based on the structure of fascaplysin. Selective inhibitors of the Cdk4-cyclin D1 enzyme were identified within this series of analogues. The activity was assayed using the ATP depletion assay as described. In this series of compounds, DE002 was extensively studied since it inhibits Cdk4- cyclin D1 at a relatively low IC₅₀ of approximately -10 μM (Table 5). To ascertain whether the inhibitory activity of these novel compounds was selective for Cdk4-cyclin D1 , we determined the concentrations at which the enzyme Cdk2-cyclin A, Cdk2-cyclin E, Cdk1-cyclin B1 and Cdk9-cycliπ T1 were inhibited by these compounds. As shown in Table 5, the compound DE002 was found to be more than 50-fold more specific towards Cdk4-cyclin D1 than other Cdk-s when IC₅₀ values in all the above Cdk enzyme assays were compared.

Table 5. Activity of the CA199 series of fascapiysin analogues in different in vitro kinase and DNA-binding assays. IC₅₀ values are presented in μM concentration. The IC₅₀ values represent means and standard deviations from three independent experiments.

Cancer cell growth inhibition The results of cell proliferation assays indicated that, from the above series of compounds, DE002 and CA199 were the most potent molecules at the cellular level.

All compounds in this series were tested in four different cancer cell lines (Table 6) for their ability to inhibit cancer cell growth in vitro while the four best compounds were evaluated further in an additional 6 cancer lines (Table 6). The inhibitory effects of compounds were quantified using MTT assay and IC₅₀ values were determined. IC₅₀ is the concentration of a compound at which 50% cell growth was inhibited.

CA199 and DE002 inhibited the growth of cancer cells at low micro-molar concentrations (Table 6) and they were found to be the most active molecules in the series with an average IC₅₀ of 7 μM and 0.75 μM, respectively. The compounds CA198 and CA211 also inhibit the in vitro cell growth at low micro molar concentrations. Interestingly, we observed that the inhibition of cell growth was independent of the presence or absence of the tumour suppressor proteins p53 and pRb (see Table 7 for indication of the p53 and pRb status of the cancer cell lines). High potency of DE002 in a cell line which lacks pRB activity (i.e. NCI-H358 which is pRb-null) would suggest that Cdk4 inhibition may not be the only cellular target for the mechanism of action of these molecules. The presence of ceUular pRb is essential for mediation of the action of Cdk4 inhibitors.

Table 6. Activity of the DE002 series of fascaplysin analogues in cell proliferation assays. IC₅₀ values are in μM. The IC₅₀ values represent means and standard deviations from three independent experiments.

Table 7. The four best compounds in the DE002 series were evaluated in 9 different cancer cell lines and one normal line. IC₅₀ concentrations, expressed in μM, for in vitro cell growth inhibition induced after exposure to CA199, CA211 , CA198 and DE002 for 48 h. All results represent means and standard deviation from three independent experiments. The tissue origin and the p53 or pRb status are indicated in brackets.

Flow cytometric analysis of the effects of compound DE002 on the cell division cycle

Since the in-vitro Cdk4-cyclin D1 and other Cdk enzyme assays had confirmed that DE002 selectively inhibits the holoenzyme Cdk4-cyclinD1 , it should also block the growth of asynchronous cells at the G₀/Gi phase of the cell cycle and maintain the G₀ZG₁ block induced by serum starvation.

Cell lines with varied mitotic spindle checkpoint status (either normal or impaired) and p53 status (either normal or null) were used to investigate the role of DE002 in blocking the cell division cycle. We first examined if DE002 had any role in blocking cells at G(ZG₁ as would be expected if it was a Cdk4 inhibitor. We chose the Calu-1 non-small cell lung carcinoma (NSCLC) cell line for these experiments since we had already obtained preliminary observations that DE002 blocked most, if not all, cancer cells more profoundly at G₂/M rather than at G₀ZG₁ (see below). Since the mitotic spindle checkpoint is partially impaired in Calu-1 cells (Masuda et al, 2003), impairment of the mitotic spindle checkpoint in Calu-1 could have led to the comparatively high IC₅₀ for cell growth inhibition for DE002 (Table 7; Calu-1 IC₅₀ for growth inhibition -2.8 μM compared to an average IC₅₀ of 0.5 μM over nine other cell lines). We therefore considered that if DE002 did possess a Cdk4-inhibitory function it would be better manifested in a cellular environment where DE002's function at G₂/M (i.e. mitosis) was relatively subdued. It was expected that in cells (i.e. Calu-1 ) which lack a fully functional mitotic spindle checkpoint, Cdk4 inhibition would lead to a more measurable block at G₀ZG₁.

DE002 maintains the GnZG₁ block in serum-starved p53-null, mitotic spindle checkpoint impaired Calu-1 cells

Calu-1 cells were treated with IC₅₀ and IC₇₀ concentrations of DE002 after release from cell synchronization. Cells were starved of serum for 24 h using 0.1% FBS. This resulted either in the partial (at IC₅₀) or full maintenance (at IC₇₀) of the G₀ZG₁ block. Since the maintenance of the G₀ZG₁ block after serum starvation requires Cdk4 enzyme to be inactive, these results may indicate that DE002 is likely to inhibit cellular Cdk4 at these concentrations (Figure 13A) thereby maintaining the G₀ZG₁ block. The higher G₁₁-S percentage ratios compared to serum-starved cells, in the cells released from serum starvation in the presence of the IC₇₀ concentration of compound, is because nearly all cells in the S phase present during serum starvation entered the G₂ZM phase after release. However, at the lower concentration (i.e. IC₅₀) we observed a greater tendency towards a G₂ZM block (Figure 13, panel C) as depicted by the increased percentage of cells in G₂ZM. This indicated that DE002, although initially identified as a Cdk4-specific inhibitor in the in vitro enzyme screens, tends to block more profoundly at G₂/M than the G₀ZG₁ phase of the cell cycle.

Effects on the Cdk4-specific pRb phosphorylation status in the mitotic spindle checkpoint impaired Calu-1 cells The relative sensitivity of cell lines to DE002 was probably because of its dual mechanism of action that lies in its simultaneous ability to bind tubulin and inhibit Cdk4- cyclin D1. The tubulin-binding affinity was found to be more pronounced than Cdk4- cyciin D1 inhibition (data shown later in Figure 23).

Since tubulin's function is already impaired in cells which are deficient in the mitotic spindle checkpoint, the Calu-1 cell line was chosen to check Cdk4-specific pRb phosphorylation. The pRb phosphorylation status was tested in DE002-treated Calu-1 cells to seek affirmation of DE002's ability to inhibit cellular Cdk4-cyclin D1 enzyme. The Western blot results obtained after DE002 (IC₅₀) treatment of Calu-1 cells for 24 h show that pRb remains unphosphorylated at serine residues Ser780, Ser795 and Ser807/811 which are specifically phosphorylated by Cdk4 enzyme while pRb levels in DE002 treated cells remain unchanged (Figure 13B). This would indicate that DE002 prevents Cdk4-mediated phosphorylation of pRb. Fascaplysin treatment of Calu-1 cells also result in the inhibition of pRb phosphorylation at Cdk4-specifιc serine residues (Figure 13B) (Soni et al, 2000).

DE002 does not prevent cells blocked at late G₁ phase from re-entering the cell cycle but arrests them at the following GcJG₁ phase

Mimosine, a non-protein amino acid inhibits DNA polymerase α in eukaryotic cells and thereby blocks them at the d/S boundary of the ceil cycle (Ji C et a/, 1997). Since the function of the Cdk4 enzyme is crucial while cells progress through the early Gi phase of the cell cycle, we hypothesised that if DE002 selectively inhibits Cdk4, it would not affect the progression of cells which have already passed early G₁ and are blocked at the G₁ZS boundary. We therefore studied the effect of DE002 on mimosine-treated cells which are blocked at G₁ZS. Calu-1 cells were blocked with mimosine for 32 h and were then released in the presence of (a) fresh medium, (b) DE002, (c) roscovitine (a Cdk2-specific inhibitor) and fascaplysin (a Cdk4-specific inhibitor which also intercalates DNA). We then analysed the cell cycle by FACS after cells were released for 18 h and 36 h from the mimosine block. At the IC₅₀ concentration of DE002, cells re-entered the cell cycle and then underwent arrest at the following G₀ZG₁ phase (Figure 14, compare histograms f and j). In the presence of roscovitine, a Cdk2- specific inhibitor, the cells failed to re-enter the cell cycle and remained blocked at G₁ZS (Figure 14 compare histograms d and h), while the cells released in the presence of fascaplysin progressed from the G₁ZS phase and underwent partial block at the S phase (after 18 h release from the mimosine block) and at the G₂ZM phase (after 36 h release from the mimosine block) of the cell cycle (Figure 14, compare histograms e and i). The fascaplysin induced block at the S and G₂ZM phases, after release of synchronised cells at G₁ZS, is probably because of fascaplysin's innate DNA- intercalating ability and is reminiscent of another known DNA intercalator, cisplatin (Yang and Wang 1999). These results suggest that DE002 is very likely to (a) be a Cdk4-specific inhibitor and (b) have no effect on the Cdk2 enzyme that is active at the late G₁ (i.e. G1/S) phase of the cell cycle, which confirms the results of the earlier in vitro Cdk assays.

ICgn concentration of DE002 induced profound G?ZM block in two asvnchronouslv growing cancer cells, A549 (p53+) and NCI-H 1299 (p53-null)

The two non-small cell lung carcinoma (NSCLC) cell lines, A549 and NCI-H 1299, were used for these studies. In contrast to another NSCLC line, Calu-1 , the mitotic spindle checkpoint is intact in these cells (Masuda et al 2003). Treatment of asynchronously growing A549 (p53+, pRb+) cells with DE002 at an IC₅₀ concentration for 24 h induce a profound block at G₂/M as indicated by the percentage of cells at the G₂/M phase of the cell cycle. As seen in Figure 15, at the IC₅₀ concentration of DE002, 82% of cells appear to be in the G₂/M phase (histogram B) whereas at the IC₇₀ concentration, 59% of cells block at G₂/M (histogram C), 13% cells appear to be apoptotic and 15% cells remain in the G₀IGi phase indicating that, at the higher concentration, DE002 tends to act like a Cdk4 inhibitor partially blocking cells at G₀ZG₁. However, at the lower IC₅₀ concentration, greater tendency towards G₂M block is observed. Incubation of asynchronous NCI-H 1299 (p53-null, pRb+) cells with an IC₅₀ concentration of DE002 also resulted in a large number of cells (50% cells) accumulating at the G₂/M phase of the cell cycle (histogram E).

DE002 maintains nocodazole and paclitaxel induced G₂ZM block in NCI-H358 non- small cell lung carcinoma (NSCLC) cells

In order to obtain a block at G₂ZM under conditions where cells are minimally stressed, a partial G₂ZM block was induced in NCI-H358 (p53-null, pRb-null) cells after treatment with sub-optimal, 1 μm, concentration of nocodazole for only 18 h. When blocked cells were released in fresh medium, they readily re-entered the cell cycle without any apoptosis. However, when released in the presence of DE002 for 12 h, cells not only maintained the G₂ZM block but also more than 50% of G₀ZG₁ and S phase cells moved forward in the cell cycle and entered G₂ZM (Figure 16, compare histograms A, B, C and D). Similar results were obtained when paclitaxel-blocked cells were released in the presence of DE002 for 12 h (results not shown). These observations suggest that, at least in p53-null NCI-H358 cells, DE002 maintains the pro-metaphase block induced by nocadozole or paclitaxel during mitosis. Nocodazole treated cells do enter mitosis but can not form spindles (which are required during the metaphase) because microtubules can not polymerise (TuIu et al, 2006). Paclitaxel arrests the function of microtubule by exerting opposite effect as nocodazole does and forms a complex with microtubules which can not disassemble and perform normal functions (De Brabander et al, 1981).

DE002 blocks NCI-H358 cells at G₂ZM after release from hvdroxyurea-mediated G₁ZS cell synchronization

NCI-H358 cells were treated with 250 μM hydroxyurea for18 h to block cells at the G₁ZS boundary (77% cells observed at G₁ZS; Figure 16, histogram F), at a stage of the cell cycle where Cdk2-specific inhibitors normally act. When released in the presence of DE002, cells proceed from G₁ZS, confirming that DE002 does not inhibit cellular Cdk2 as indicated by the results from the in vitro Cdk2 enzyme assay. Released cells ultimately accumulate at G₂ZM (74%; Figure 16, histogram H). These results again indicate that DE002 has an inherent tendency to induce block at the G₂ZM phase of the cell cycle.

DE002 selectively induces apoptotic cell death in SV40 large T antigen transformed normal mouse embryonic liver cells

SV40 large T antigen inactivates the two tumour suppressor proteins, p53 and pRb, and thereby through inactivation of two major tumour suppressors transforms normal cells into tumorigenic ones (Herzig et al, 1999; Pipas and Levine 2001 ).

We investigated the effect of compound DE002 on normal mouse embryonic hepatic (liver) cells BNL CL2 and its SV40 large T antigen-transformed counterpart BNL SV A.8. The normal cells (BNL CL2), upon 48-h incubations with DE002, exhibited prominent G₂ZM arrest at both IC₅₀ and IC₇₀ concentrations with less than 10% cells detected in the sub-G-, phase (Figure 17, histograms B and C) and more than 50% cells appearing in the G₂ZM phase of the cell cycle. Interestingly, in the SV40- transformed cell line significant apoptotic cell death was observed as determined from the percentage of cells appearing in the sub-G1 phase. After 48 h treatment with an IC₅₀ concentration of DE002, 43% cells were detected in the SUb-G₁ phase (Figure 17 histogram E) indicating a high level of apoptosis. The amount of cells undergoing apoptosis increased further to 51% when the SV40-transformed cells were incubated at the IC₇₀ concentration of DE002 for 48 h (Figure 17, histogram F).

Selective killing of SV40-transformed cells DE002 inhibits the cell growth of BNL CL2 and its SV40 large T antigen-transformed counterpart BNL SV A.8 at similar IC₅₀ and IC₇₀ concentrations (Figure 18, Panel A, right-hand graph). As determined by the MTT assay, IC₅₀ of DE002 in BNL CL2 is -7.0 μM and IC₅₀ of DE002 in BNL SV A.8 is -8.2 μM. IC₇₀ of DE002 in BNL CL2 is 9.8 μM and IC₇₀ of DE002 in BNL SV A.8 is 11 μM (see Table 5 and Figure 18, Panel A, right- hand graph). However, the induction of cell death was remarkably higher in the SV40- transformed line. When analysed by the trypan blue dye exclusion method, approximately 50% cell death was observed in the transformed line BNL SV A.8 at an IC₇₀ concentration of 11 μM (Figure 18A).

The selective killing of transformed cells was again observed when cells were treated with 11 μM DE002 and stained with DAPI. In the transformed line, more than 50% cells showed fragmented nuclei and post-apoptotic morphology while only cell growth was blocked without any cell death in the normal cells (Figure 18B). It is interesting to note that only at high concentrations of DE002 (>80 μM) was apoptosis visible (-20% of cells were apoptotic) in the normal line BNL CL2.

These results indicate that DE002 acts as a strong antiproliferative agent in both normal and SV40-transformed cells. However, DE002 selectively kills SV40- transformed cells at concentrations where normal cells are totally unaffected. This would suggest that cancer cells, which are analogous to SV40-transformed cells, will be much more sensitive to apoptosis than untransformed normal cells when treated with DE002.

Testing DE002 in the Topoisomerase I catalysed DNA unwinding assay Interactions between DE002 and double-stranded DNA molecules were studied using topoisomerase I catalysed DNA unwinding assays (Figure 19). In the first set of experiments, unwinding/relaxation of supercoiled pBlueScript plasmid DNA was carried out in the presence of DE002, fascaplysin and camptothecin. Fascaplysin showed inhibition of DNA unwinding/relaxation catalysed by the enzyme topoisomerase I indicating fascaplysin's DNA-intercalating nature which is very likely due to its planar structure. DE002, a non-planar analogue of the planar molecule fascaplysin, does not show any inhibition of DNA relaxation even at high concentrations as 100 μM (Figure 19, lanes 6-7) when compared with fascaplysin (Figure 19, lanes 4-5). Camptothecin, which is known to inhibit topoisomerase I activity in vitro, was used as a standard to verify the enzyme's activity (Figure 19, lane 3).

To ensure that these results reflected a lack of DNA intercalation rather than an inhibition of topoisomerase I enzyme activity, a second set of experiments was performed using relaxed (i.e. negatively supercoiled) pBlueScript DNA as the initial substrate. For the assay, negatively supercoiled DNA was prepared by relaxing supercoiled plasmid DNA with Topoisomerase I (Figure 20, lane 1). The negatively supercoiled DNA remains relaxed even after treatment with a relatively high 100 μM concentration of DE002 (Figure 20 lanes 2-4) which strongly indicates its non- intercalative nature. Fascaplysin-treated DNA does not remain relaxed clearly indicating that it intercalates DNA (Fig. 5.8, lane 5).

Testing DE002 in the ethidium bromide displacement assay

Results obtained from the ethidium bromide displacement assay indicate that none of the DE002 series of fascaplysin analogues interact with the minor groove of double- stranded DNA because they do not displace the DNA-bound ethidium bromide (Table 5). Representative curves of fascaplysin, DE002 and actinomyciπ D are shown in Figure 21 which exhibits the interactions of these compounds with pBlueScript plasmid DNA. As seen in Figure 21 , 100 μM of DE002 was incapable of displacing 1.3 μM ethidium bromide from pBlueScript DNA. Less than 5% displacement of bound ethidium bromide was observed at 100 μM. In contrast, the DNA-intercaiative drug actinomycin D readily dislodges the bound ethidium bromide (IC₅₀ = 40 μM). Fascaplysin displaces the DNA-bound ethidium bromide at low concentrations (IC₅₀ of ethidium bromide displacement, between 5 and 10 μM). Taken together, these results indicate that if DE002 does in fact bind to DNA, it neither intercalates nor interacts with the minor groove of double-stranded DNA molecules.

Effects of DE002 on the cellular levels of cyclin B1, Cdk1, p53, _P21^CIP1/WAF1 (p21) and p∑^¹ (p27) in two p53+ cell lines

Western blot analysis was performed on A549 (a NSCLC cell line) and LS174T (a colon carcinoma line) cells after treatment for 24 h with an IC₅₀ concentration of DE002. Both cells lines bear functional copies of the tumour suppressor gene p53 and are therefore referred to as p53+ cells. A549 and LS174T contain functional copies of the retinoblastoma protein. Treatment with DE002 results in a 10-fold induction of p53. Concomitantly, the non-specific Cdk inhibitory protein _P21^CIP1/WAF1 (p21 ) is induced since p53 is a transcriptional activator that can activate p21 gene transcription by binding to the p53 protein-specific enhancer sequences in the p21 gene promoter. The levels of another pan-Cdk inhibitor, p27^KIP1 (p27), are also elevated after DE002 treatment (Figure 22).

The proteins Cdk1 and cyclin B1 are down-regulated in treated cells when compared with proteins from untreated control cells. Repression of the expression of the cyclin B1 and Cdk1 proteins (Figure 22) is a possible explanation of the profound G₂/M block observed in A549 cells (portrayed in Figure 15) and LS174T cells (results not shown). Elevated levels of p21 and p27 not only can reinforce the G₂/M block but may also play a major role in the G₀ZG₁ block which is observed in cells treated with higher concentrations (i.e. IC₇₀) of DE002.

Effects of DE002 on the cellular levels of cyclin B1, Cdk1, p53, _P21^CIP1/WAF1 (p21) and p27^KIP1 (p27) in MiaPaCa-2, a p53 mutant pancreatic cell line

DE002 was evaluated for its effect on the cell cycle in MiaPaCa-2 cells. Similar cell cycle results to those obtained from other cancer cell lines (A549, NCI-H1299, NCI- H358, and BNL SV A.8) were obtained. MiaPaCa-2 cells are also arrested at G₂/M phase of the cell cycle followed by the treatment with IC₅₀ and IC₇₀ concentration of DE002 for 24 h. Approximately 52 and 55% cells were found to be blocked at the G₂/M phase using IC₅₀ and IC₇₀ concentrations of DE002, whereas in control cultures only 18% cells appeared in the G₂/M phase (data not shown).

Experiments were performed to check the levels of cell cycle regulatory proteins in MiaPaCa-2 cells (p53 mutated). Western blot results demonstrate that p53, p21 and p27 levels remain unchanged (data not shown) suggesting that the induction of the p21 and p27 proteins seen earlier (Figure 22) in the p53+ cell lines, A549 and LS174T, is likely to be dependent on the presence of a functional p53 protein. Interestingly, the cyclin B1 and cdk1 levels were elevated and phosphorylation of Cdk1 at the residue Tyr15 remains unaffected (data not shown) indicating that Cdk1 -cyclin B1 is still active in the p53 mutated MiaPaCa-2 cells after treatment with DE002. This observation would indicate that, at least in p53-mutant and p53-null cells, the G₂/M block observed after treatment with DE002 must be at a phase of the cell cycle that follows the phase where Cdk1 -cyclin B1 enzyme is usually active. The activity of the Cdk1 -cyclin B1 enzyme is manifested at the G₂-M interface. Therefore, without wishing to be bound by theory, we consider that the profound effects of DE002 occurs post-G₂. This is likely to happen in between the G₂ and the G₀ZG₁ phases of the cell cycle suggesting that DE002 somehow affects the functions of mitosis. Tubulin is known to play a major role in mitosis.

The in vitro inhibition of Tubulin polymerisation by DE002 To distinguish between G₂ versus mitotic arrest, mitotic indices of A549 cells treated with DE002 were calculated after staining of nuclei with DAPI (Shtivelman et al, 2002; Augustin et al, 1997). In A549 cells, DE002 induced profound G₂/M arrest at the IC₅₀ concentration (82% cells in G₂/M, Figure 15). Normally, the mitotic index is >70 for cells at the metaphase. Fluorescence microscopic observations showed less than 20% cells in metaphase (data not shown) suggesting that growth arrest of A549 cells occurs before the metaphase (i.e. at the pro-metaphase stage).

Moreover, FACS analysis of cells, released in the presence of DE002 after cell nocodazole-mediated cell synchronisation, also suggests a pro-metaphase block during the cell cycle (Figure 16 upper panel, histograms A, B, C and D). Similar results were obtained after paclitaxel-mediated cell synchrony. Both nocodazole and paclitaxel are known to block cells at the pro-metaphase (TuIu et al, 2006; De Brabander et al, 1981 ).

The fact that the growth of cancer cells in vitro is inhibited by DE002 at concentrations (IC₅₀ ~7 μM) lower than at which it inhibits the Cdk4-cyclin D1 enzyme (IC₅₀ ~26 μM) suggest that there is another cellular target of DE002 which may be unlinked to its Cdk4-inhibitory activity. The observation that DE002 is ~5-fold less potent in a cell line (i.e. Calu-1) with an impaired mitotic spindle checkpoint than in cell lines (i.e. A549, NCI-H 1299) that have intact mitotic spindle checkpoints suggests a possible role of DE002 as an anti-microtubule agent. We therefore investigated the action of DE002 and other compounds of this series on tubulin polymerisation in vitro. The polymerisation experiments with purified tubulin were performed as described.

The results indicate a strong anti-polymerisation of tubulin activity for DE002 and other fascaplysin analogues while the parent molecule fascaplysin does not show any interaction with tubulin. Representative polymerisation curves of DE002, paclitaxel and nocodazole are shown in Figure 23. That DE002 inhibits the polymerisation of tubulin can be concluded from the dose-dependent decrease in Vmax (mOD/min) and reduction in the final polymer mass (Figure 23). When tested at four different concentrations, DE002 decreased the Vmax from 17 mOD/min to 12.5, 9.2, 3 and 0.5 mOD/min at 2.5, 5, 10 and 25 μM concentrations, respectively. As a consequence of decreased Vmax, up to 80% reduction in final polymer mass has been observed. Nocodazole and paclitaxel were used in the assay as controls (inhibitor and enhancer of tubulin polymerisation, respectively).

Enhancement of tubulin de-polymerisation and inhibition of tubulin polymerisation in the presence ofDE002 in cells

The in vivo cellular experiments to explore the interactions between DE002 and tubulin protein were performed in A549 (lung cancer, NSCLC) cells. The mitotic spindle checkpoint in A549 cells is normal and hence these cells should be sensitive to anti- microtubule agents. The assembled (cytoskeletal) and unassembled (cytosolic) forms of tubulin were determined from their accumulation and disappearance from pellet and supernatant fractions of the cell lysates treated with DE002 and further evaluated with Western blotting. Paclitaxel treatment of A549 cells for 30 min resulted in an accumulation of cytoskeletal tubulin as a consequence of enhanced tubulin polymerisation, while in the presence of DE002 this paclitaxel-mediated polymerisation is inhibited in a dose-dependent manner (Figure 24). More interestingly, when intracellular stabilized tubulin (in paclitaxel treated cells) was subjected to DE002 treatment, DE002 enhanced the tubulin de-polymerisation resulting in disappearance of cytoskeletal tubulin (pellet) form and accumulation of cytosolic tubulin (supernatant) form (Figure 24). Hence, it seems that DE002 not only can inhibit polymerisation of tubulin both in vitro and in living cells but it also enhances the de-polymerisation of tubulin once it is formed.

Clonogenic assays

Effect of DE002 on A549 and Calu-1 cells

We evaluated the effects of DE002 on the colony formation efficiency of A549 and Calu-1 cells. The results from colony formation assays generally give insight into the long-term survival of cancer cells in vitro. A549 cells have the normal mitotic spindle checkpoint and bear functional copies of the pRb and p53 proteins. In contrast, Calu-1 cells have an impaired mitotic spindle checkpoint, contain a functional pRb protein but are p53-null. Since DE002 shows dual mechanism of action at the cellular level (acts as an inhibitor of both Cdk4-cyclin D1 enzyme activity at G₀ZG₁ and tubulin polymerisation at the M phase of the cell division cycle), we explored the possibility that DE002 could perhaps act potently in reducing the long-term survival and colony formation efficiency of both A549 and Calu-1 cells. The results indicate significant reduction in colony formation efficiency in both cell lines (Figure 25). Surprisingly, the colony formation efficiency was inhibited by DE002 at much lower concentrations than at which cell growth was inhibited (average IC₅₀ in colony formation = 3.5 μM while average IC₅₀ in MTT assay was 7 μM; see Figure 16 and Figure 25B). These results indicate that cancer cells suffer a remarkable loss in their ability to survive after treatment with DE002 indicating the potential of this compound in cancer therapy.

DE002 selectively reduces long-term survival in SV40 large T-antigen transformed normal mouse embryonic liver cells BNL-CL2.

The ability of DE002 to reduce colony formation efficiency of mouse embryonic hepatic cells (BNL CL2) and SV40 transformed mouse embryonic hepatic cells (BNL SV A.8) was evaluated in a colony formation assay. DE002 was tested at IC₂O, IC₃₀, IC₅₀ and IC₇₀ concentrations in both the cell lines. These concentrations were determined using MTT cell proliferation assay. The representative plates are shown in Figure 26. The results indicate that even at the low concentrations of DE002 (IC₂o) the efficiency of colony formation is selectively reduced in SV40 transformed cells. Less than 50% of colonies were observed at IC_2O concentrations DE002. At IC₅₀ and IC₇₀ concentrations, more than 90% cells lost their ability to form colonies.

DE002 does not induce senescence in A549 cells

DE002 was tested for its ability to induce senescence in A549 cells. Non-toxic concentrations of DE002 (IC₅₀ and IC₃₀) were used to find out the effect of this compound on senescence. After long-term exposure (96 h) of A459 cells to DE002, the cells were stained for β-galactosidase activity. It was found that β-galactosidase levels in treated A549 cells remained unchanged from untreated cells, indicating that DE002 fails to induce senescence in A549 cells.

DE002 inhibits H₂O₂-induced cellular ROS formation in H4IIE cells The structure of DE002 resembles the structure of an indole derivative. Indoles constitute some of the most powerful antioxidants known in nature (Andreadou et al, 2002; Mor et al, 2003; Olgen et al, 2002)

H4IIE, rat hepatoma cells were used to test the possible antioxidant potential of DE002. It was found that DE002 inhibits 50% of H₂O₂-mediated ROS formation at -20 μM concentration. The results are shown in Figure 27. It was found that the pre- incubation of H4IIE cells with DE002 protects cells from H₂O₂-mediated ROS induction. Formation of reactive oxygen species (ROS) inside the living cells could have detrimental effects on cells, since ROS can induce DNA strand breaks and also can mediate conformational changes in protein molecules. These changes at molecular level could result in many degenerative diseases that include cancer (Izzotti et al, 2006; Butterfield et al, 1998). Hence the antioxidant property of DE002 can have vital importance not only for the treatment of cancer but also for prevention of the disease.

Silibinin, a naturally occurring flavonoid undergoing clinical trials at the National Cancer Institute also acts as an anti-oxidant. DE002 inhibits H₂O₂-mediated ROS formation (IC₅₀ ~20 μM) whereas silibinin does not (IC₅₀ >200 μM) (Dehmlow et al, 1996), H₂O₂ being one of the most powerful pro-oxidants known. It is noteworthy that the well known anti-oxidant, /^-acetylcysteine, has an IC₅₀ >100 μM for inhibition of H₂O₂- mediated ROS formation

Conclusions

DE002, a non-planar analogue of fascaplysin, is a compound that shows novel effects on cancer cells. DE002 was initially identified on the basis of an in vitro screen for the Cdk4 enzyme, and was found to inhibit Cdk4 specifically (it does not inhibit Cdk2, Cdk1 and Cdk9). Besides, Cdk4-specific inhibition, DE002 shows some unique properties.

(1) Unlike fascaplysin, it does not intercalate DNA,

(2) It partially blocks at G₀ZG₁ phase of the cell division cycle as would be expected of a true Cdk4 inhibitor,

(3) It profoundly blocks cells at the G₂/M phase of the cell division cycle at comparatively low concentrations,

(4) It induces massive apoptosis in cancer cells,

(5) It selectively induces apoptosis in SV40 large T antigen-transformed cells and not in untransformed normal cells,

(6) It selectively reduces the long-term survival of SV40 large T antigen-transformed cells with a higher potency than seen in normal cell cultures,

(7) It inhibits tubulin polymerisation in vitro and in vivo which results in the induction of profound G₂/M arrest,

(8) It acts as an anti-oxidant by quenching the biological oxidant H₂O₂ (thus having a similar action to a known flavonoid class of phytochemicals that are being used for cancer therapy). (9) It down-regulates cyclin B1 and Cdk1 in p53+ cancer cells but up-regulates the pan-Cdk inhibitory proteins p21 and p27, providing a mechanistic insight into block of cell growth in p53+ cells, suggesting that p53+ cells block at G0/G1, G2 and M phases of the cell division cycle, (10) It up-regulates cyclin B1 levels in p53-mutant and p53-null cells indicating that in these cancer cells, the major block occurs at a post-G₂ phase,

(11 ) It shows significant reduction in the colony formation efficiency of both p53+ and p53-null cancer cells in vitro.

Hence, DE002 has a profile which is uniquely distinct from any anticancer compound reported in the literature. Thus DE002 and other compounds within the DE002 series having a similar structural scaffold, are clinically useful compounds for the treatment of cancer.

Example 2: Biological activity of the CA224 and AJW089 series of fascaplysin analogues

Introduction

In this Example, we describe the biological activity of further structural analogues of fascaplysin, the CA224 series and the AJW089 series. We again show that fascaplysin's ability to inhibit Cdk4 enzyme specifically can be separated from its deleterious DNA-intercalating characteristic. These analogues manifest the expected Cdk4-specific inhibition by blocking at the G₀ZG₁ phase of the cell cycle but surprisingly also inhibit the G₂/M phase in a Cdk-independent manner. Further investigations showed that these non-planar analogues of fascaplysin inhibit the growth of a panel of 10 cancer cell lines (some of the cell lines are resistant to cancer chemotherapy) at low micromolar concentrations and also exhibit a profound effect on the long-term survival of cancer cells. The Cdk-independent G₂/M block was found to be associated with anti-tubulin activity of these compounds. The most potent analogues of fascaplysin in this series inhibit the polymerisation of tubulin in vitro and enhance tubulin de- polymerisation in vivo.

All of these fascaplysin analogues (Table 8) were tested in a number of different biochemical assays including Cdk enzyme assays and cellular assays (using normal and cancer cell lines). We evaluated their ability to inhibit different Cdks in vitro and to inhibit cancer cell growth in vitro, and we analysed their DNA-binding properties in two independent assays: (a) the ethidium bromide displacement assay and (b) the topoisomerase I catalysed DNA relaxation assay.

Chemical syntheses and molecular modelling of compounds from the CA224 and AJW089 series

Molecular modelling and detailed synthetic routes to some compounds of the CA224 series, including CA224 itself, are described in Aubry et al, (2006). Modifications of this route to synthesize further members of the CA224 series, and the compounds of the AJW089 series are known to the person of skill in the art.

Synthesis of CA224

Synthesis

B

i) a) Ethyl chloroformate, NaOH 4M, CHCI₃, 3h, 95%; b) LiAIH₄, THF, N₂, reflux, 1 h ii) CICOC₆H₄-P-C₆H₅, NaOH_{aq), 4M, CH₂CI₂, O⁰C 15 min, RT-3h, 37-99%;

i) a) To a solution of tryptamine (10.00g, 62.4 mmol) in chloroform (156 ml_) at 0⁰C was added ethylchloroformate (5.97 ml_, 62.4 mmol) and an aqueous solution of NaOH 4M (15.60 ml_, 62.4 mmol). After addition, the mixture was stirred for 3h at room temperature, and then washed with water (150 mL). The two layers were separated and the aqueous phase was extracted with dichloromethane (2 x 150 mL). The chloroform and dichloromethane layers were combined, dried over anhydrous MgSO₄ and evaporated under reduced pressure to give an orange oil. No purification was needed. The oil was dried in vaccuo to give the intermediate compound [i) a)] (13.78g, 95%), as published. i) b) To a solution of [i) a)] (13.78 g, 59.4 mmol) in dry THF (110 mL) under N₂ at 0⁰C was added portionwise LAH (6.76 g, 178 mmol). After the addition was complete the mixture was heated under reflux for 1 h. The reaction was then cooled to O⁰C and the excess of LAH was hydrolysed by adding successively and carefully water (13.25 mL), 15% aqueous solution of NaOH (13.25 mL) and water (3 x 13.25 mL). During these steps it was necessary to add THF (10OmL) to avoid the mixture becoming very thick.

The suspension was filtered and the white solid was washed with THF (30 mL). The organic layer was dried over anhydrous MgSO₄ and evaporated under reduced pressure to give the title compound B (Λ/-ω-methyl tryptamine) (9.24g, 89%) as a beige solid.

ii) To a suspension of Λ/-ω-methyl tryptamine (1.2mmol) in CH₂CI₂ (3 mL) at 0⁰C was added slowly an aquous solution of sodium hydroxide 4M (1.2 mmol). After 5min stirring at 0⁰C a solution of biphenyl-4-carbonyl chloride (1.2 mmol) in CH₂CI₂ (5 mL) was added dropwise. The mixture was stirred for 5 min at O⁰C and for a further 3 h at room temperature. H₂O (20 mL) was added. The two layers were separated and the aqueous phase was extracted with dichloromethane (3 x 20 mL). The organic layers were dried over anhydrous MgSO₄ and evaporated under reduced pressure. The crude product was purified by flash column chromatography on silica. Elution was made successively with ethyl acetate: petroleum ether (40-60 ⁰C) = 50-50 and ethyl acetate to give Biphenyl-4-carboxylic acid [2-(1H-indol-3-yl)-ethyl]-methyl-amide as a beige solid; yield 58%; mp 178 ⁰C. Rotamers 1/1.5 (from the duplicated triplet signal

(¹H) at 3.59 and 3,89 ppm). δ_H (300 MHz; (CDCI₃) δ (major rotamer) 2.95 (2H, distorded t, 6.8), 3.18 (3H, s), 3.59 (2H, t, 6.8), 6.84-7.55 (14H, m), 8.42 (1 H, br s). δ (distinct peaks for minor rotamer) 2.91 (3H, br s), 3.89 (2H, m), 7.72 (1 H, br d, 6.9). ¹³C NMR

(75MHz, CDCI₃) δ (major rotamer) 24.4 (CH₂), 33.1 (CH₃), 51.9 (CH₂), 111.3 (2CH), 111.6, 118.2 (CH), 119.3 (2CH), 122.0 (2CH), 127.0 (2CH), 127.2 (2CH), 127.5, 127.7

(CH), 128.9 (2CH), 135.2, 136.3, 140.5, 142.0, 172.4; δ (distinct peaks for minor rotamer) 23.0 (CH₂), 38.3 (CH₃), 48.6 (CH₂), 112.9, 118.7 (CH), 122.3 (CH), 135.5, 142.3, 171.3m/z (ES⁺) 355 MH⁺ ; (ES^') 353 (M-H)^" ; m/z (FAB⁺) 355 MH⁺ (found : C,

81.34 ; H, 6.07 ; N, 7.84; MH⁺ 355.18103. C₂₄H₂₂N₂O requires C, 81.26 ; H, 6.26 ; N,

7.90; MH 355.18104).

Synthesis of AJW089

Structure

a) 0-CICO-C₆H₄-Br₁ CH₂CI₂, NaOH (4 M, aq, 1eq), 0 ⁰C 15 min then RT. 3 h. b) Pd(PPH₃)₄, Toluene, EtOH, (HO)₂BC₆H₅, K₂CO₃ (2 M, aq), 90 ⁰C, 24 h.

i) To a stirred solution of tryptamine (1.2 mmol) in dichloromethane (10 ml_) at O⁰C was added slowly an aquous solution of sodium hydroxide (4M aqueous, 1.2 mmol). After 5 min stirring at O⁰C was added dropwise 2-benzoyl chloride (1.2 mmol). The mixture was stirred for 5 min at O⁰C and then 3 h at room temperature. H₂O (20 mL) was added and the two layers were separated. The aqueous phase was extracted with dichloromethane (3x20 mL) and the combined organic phases ware dried over anhydrous MgSO₄ and evaporated under reduced pressure. The crude product was purified by flash column chromatography on silica gel. Elution was made successively with ethyl acetate:petroleum ether (50:50) and ethyl acetate, to give B.

ii) To a stirred solution of B (1 mmol) in toluene (10 mL), under nitrogen, was added

K₂CO₃ (1 mmol, 2M aqueous) and Pd(PPh₃)₄ (5 mol %, 0.05 mmol). The solution was stirred for 20 min at room temperature before the addition of a solution of phenyl boronic acid, (1.2 mmol) in EtOH (10 mL). The reaction mixture was heated to 9O⁰C for

24 h, then allowed to cool to room temperature before the addition of H₂O₂ (30%, 1 mL), the reaction mixture was then stirred for a further 1 h. The desired product was extracted into CHCI₃, washed with saturated brine solution (2 x 25 mL) and H₂O (2 x 25 mL), aqueous washings being re-extracted with CH₂CI₂ (3 x 50 mL), the combined organic phases were then dried over anhydrous sodium or magnesium sulphate, filtered and isolated under reduced pressure. The crude product was then purified by flash column chromatography on silica from ethyl acetate - petroleum ether (40-60 ⁰C) to give biphenyl-2-carboxylic acid[2-(1 /-/-indol-3-yl)-ethyl]-amide (0.29 g, 85.5%) as an off-white amorphous solid; u_max(ATR)/cm^"1 3402 (w, indole NH), 3267 and 3056 (amide NH), 1634 and 1520 (amide CO), 739, 699, 661; δ_H (300 MHz; (CD₃)₂SO) 2.75 (2H, dd, 7.6, 7.6, C(8)H), 3.40 (2H, ddd, 7.6, 7.6, 5.7, C(9)H), 7.00 (1 H, ddd, 7.4, 7.4, 1.1), 7.07- 7.12 (2H, m), 7.33-7.54 (11 H₁ m), 8.27 (1H, dd, 5.7, 5.7, N(IO)H), 10.82 (1 H, br-s, N(I)H); δ₁₃ (75 MHz; (CD₃)₂SO) 24.6 (CH₂), 39.7 (CH₂), 111.3 (CH), 111.6, 118.1 (2C, CH), 120.8 (CH), 122.4 (CH), 126.9 (CH), 127.0 (CH), 127.5 (CH), 128.0 (2C, CH), 128.3 (2C, CH), 129.2 (CH), 129.7 (CH), 136.1 , 137.4, 138.9, 140.1 , 169.0; m/z (ES⁺) 341 (MH⁺), (ES^') 339 (M-H^"); m/z (FAB) 341.16529 (M+H⁺ C₂₃H₂₁N₂O requires 341.16539); (Found: C, 81.07; N, 8.12; H, 6.03. C₂₃H₂₀N₂O requires C, 81.15; N, 8.23; H, 5.92.).

Table 8. Chemical structures of the CA224/AJW089 series of compounds. All compounds shown below were soluble in 100% DMSO at a concentration of 10 mM.





CA224 specifically inhibits Cdk4-cyclin D1 enzyme in vitro

All of the compounds shown in Table 8 were screened in different in vitro assays to assess their capacity to inhibit Cdks. The Cdk inhibitory activity was assayed using the ATP depletion assay as described. All of the compounds were shown to inhibit Cdk4- cyclin D1 at relatively high potency when compared to their ability to inhibit other Cdk activities (Table 9). CA224 inhibits Cdk4-cyclin D1 at an IC₅₀ of approximately 6 μM, while compounds AJW089, MS014, AJW099, AJW090 and AJW102 also inhibited Cdk4-cyclin D1 in vitro with relatively high potencies (IC₅₀ of 7 μM, 15 μM, 11 μM, 15 μM and 9 μM, respectively). To ensure that the inhibitory activity of these novel compounds was selective for Cdk4-cyclin D1, we determined the concentrations at which the enzyme Cdk2-cyclin A, Cdk2-cyclin E, Cdk1-cyclin B1 and Cdk9-cyclin T1 were inhibited by these compounds. As shown in Table 9, all of the compounds were selective inhibitors of Cdk4-cyclin D1 , and CA224 displayed more than 10-fold specificity for the inhibition of Cdk4-cyclin D1.

Since the 3D structure of Cdk4 is yet to be determined, the Cdk4 ATP binding site was modelled on the basis of the known crystal structure of the homologous Cdk2 and Cdk6 enzymes. Using this homology model, in silico studies suggest that inhibition of Cdk4 activity by fascaplysin arises from binding to the same amino acid residues to which ATP binds (Aubry et al, 2004; Aubry et al, 2005; Aubry et al, 2006). Thus the non-planar analogues were expected to maintain most of the key interactions thought to occur between fascaplysin and Cdk4. Our biochemical studies using the Cdk4 and Cdk2 in vitro enzyme assays have corroborated that the molecules predicted to be Cdk4-specific inhibitors indeed inhibit Cdk4 but not Cdk2, Cdk1 and Cdk9.

Table 9. Activity of fascaplysin analogues in different in vitro kinase and DNA binding assays. IC₅₀ values are presented in μM concentrations. All the fascaplysin analogues were dissolved in 100% DMSO solution and were further diluted in the kinase assay buffer or the ethidium bromide displacement assay buffer. The IC₅₀ values represent means and standard deviations from three independent experiments.

ND = Not determined.

Cancer cell growth inhibition The results of cell proliferation assays are depicted in Table 10 and indicate that, of the compounds listed in Table 8, CA224 and AJW089 are the most potent in their ability to inhibit the growth of cancer cells. The compounds were initially tested in four different cancer cell lines (LS174T, A549, Calu-1 and PC-3) for their ability to inhibit cancer cell growth in vitro (Table 10). The best four compounds from this series were further evaluated in an additional 6 cancer lines using the MTT assay, MiaPaCa-2, NCI-H460, NCI-H1299, NCI-H358, BNL CL2 and BNL SV A.8, and the results are shown in Table 1 1. CA224 and AJW089 inhibited the growth of cancer cells at low micro-molar concentrations (Table 10 and Table 1 1 ) and were found to be the most active molecules with an average IC₅₀ of 4 μM and 2 μM respectively. CA225 and CA223, which were moderately active in the Cdk4 assay, inhibited cell growth in the range of 10-20 μM and 20-40 μM, respectively.

Interestingly, we observed that the inhibition of cell growth was p53 and pRb independent (see Table 11) implying that the inhibition of cancer cells by these molecules does not depend on the presence or absence of p53 or pRb in the cell line. The pRb-independent action of these compounds would indicate that Cdk4 inhibition may not be the only cellular target for the mechanism of action of these molecules.

Table 10. Activity of the CA224/AJW089 series of fascaplysin analogues in cell proliferation assays. IC₅₀ values are denoted in μM. The in vitro cell growth inhibition was determined by the MTT assay. All compounds including fascaplysin were dissolved in 100% DMSO and the resultant solutions were then further diluted in fresh growth medium just before addition to the cultured cells. The IC₅₀ values represent means and standard deviations from three independent experiments. IC₅₀ is the concentration at which 50% of cell growth was inhibited after treatment with compound.

Cell growth inhibition as determined by MTT assay

Compounds LS174T A549 Calu-1 PC-3

Fascaplysin 0.88 + 0.04 0.69 + 0.03 1.3 + 0.1 0.92 + 0.06

FW2005 88 + 6 71 + 3.5 97 + 5 84.5 + 4

FZW008 58 + 2 51 + 4 68 + 7 49 + 5

MGR010 114 + 6 98 + 5.5 126 + 11 110 + 4

MS008 21 + 3 17 + 2.5 56 + 3.5 24 + 2

MS009 96 + 4.5 86.5 + 5 106 + 7 91 + 3

MS011 60 + 3.5 55 + 2.5 84 + 4 63.5 + 3

AJW136 82.5 + 5 75 + 3 94 + 4.5 74 + 4

AJW137 45 + 2.5 41 + 3 66 + 5 48 + 3.5

AJW129 69 + 3.5 58 + 2 83 + 6 64 + 3

AJW113 50 + 4.5 44 + 3 67 + 5 49 + 3.5

CA225 18 + 1 12 + 1.8 52 + 3.5 15 + 1.5

CA226 106 + 6 103 + 5 146 + 9 92 + 6

CA228 109 + 10 95 + 7.5 139 + 9 85.5 + 5

CA229 112 + 7 110 + 7.5 141 + 11 92.5 + 6

CA230 88 + 6.5 96.2 + 5 136 + 10.5 81.5 + 7

CA233 82 + 2 92 + 4.5 103 + 4 55.5 + 1

CA234 78 + 3.5 95 + 5.5 122 + 8.5 59 + 4

CA237 89 + 6 91 + 6.5 144 + 10 63 + 6

CA238 52 + 2 55 + 3 64 + 3 41 + 1. 5

Table 11. IC₅₀ concentrations, expressed in μM, for in vitro cell growth inhibition of 10 different cell lines exposed to AJW089, CA224, CA225 and CA223 for 48 h.

The cell growth inhibition was determined by the MTT assay. The IC₅₀ values represent means and standard deviations from three independent experiments. IC₅₀ is the concentration at which 50% of cell growth was inhibited after treatment with compound.

Cell lines CA223 CA224 CA225 AJW089

LS174T (colorectal carcinoma; p53+,

42 + 2.5 3.5 + 0.9 18 + 1 3.5 + 0.5 pRb+)

PC-3 (prostate; p53-null, pRb+) 47 + 3 6.2 + 1.1 15 + 1.5 4.5 + 0.7

MiaPaCa-2 (pancreatic; p53His273mut, pRb+) 31 + 2.2 4 + 0.3 10.2 + 0.9 3.7 + 1.1

A549 (NSCLC; p53+, pRb+) 27 + 2.5 3.5 + 0.6 12 + 1.8 2.8 + 0.4

Calu-1 (NSCLC; p53 null, pRb+) 78 + 3 11. 5 + 2.5 52 + 3.5 8.6 + 1

NCI-H460 (NSCLC; p53+, pRb+) 24 + 1.8 2 + 0.3 9.8 + 1 ND

NCI-H 1299 (NSCLC; p53-null, pRb+) 21 + 0.9 2.5 + 0.3 11.5 + 1.6 1.78 H H 0.4

NCI-H358 (NSCLC; p53-null, pRb null) 26 + 2 2.2 + 0.6 14 + 1.4 1 .45 H H 0.2

BNL CL2 (mouse normal hepatic cells) 29 + 2.4 2.6 + 0.4 18 + 2 ND

BNL SV A.8(mouse hepatic; SV-40

32 + 1 mediated transformed cells) 3.8 + 0.35 22 + 1.8 ND ND = Not determined.

DNA binding studies The Topoisomerase I DNA unwinding assay

CA224 was tested for its ability to intercalate DNA molecules using topoisomerase I catalysed unwinding/relaxation assay (Figure 28). Relaxation of supercoiled pBlueScript plasmid DNA was carried out in the presence of fascaplysin, CA224 and camptothecin. Fascaplysin shows inhibition of DNA relaxation catalysed by the enzyme topoisomerase I indicating the intercalating nature of a planar molecule. The non-planar compound, CA224, does not show any inhibition of DNA relaxation even at high concentrations up to 100 μM (Figure 28). To ensure that these results reflect a lack of DNA intercalation rather than an inhibition of topoisomerase I, a second set of experiments was performed using relaxed DNA as the initial substrate (prepared by the treatment of supercoiled pBlueScript plasmid DNA with topoisomerase I) for the assay (data not shown). The relaxed DNA remains as such after treatment with 100 μM concentration of CA224. These results indicate that CA224 does not intercalate with double-stranded DNA and also does not inhibit topoisomerase I activity in vitro up to a concentration of 100 μM, confirming the expected non-intercalative nature of a non- planar compound.

The ethidium bromide displacement assay

All of the compounds listed in Table 8 were tested in the ethidium bromide displacement assay in order to check their ability to interact with the minor groove of double-stranded DNA molecules. The results obtained showed that none of the analogues displace bound ethidium bromide from double-stranded DNA (Table 9). A graph of the representative compound CA224 is shown in Figure 29 along with the results obtained with fascaplysin and actinomycin D, a known DNA intercalator. CA224 (up to 100 μM concentration) was incapable of displacing 1.3 μM ethidium bromide. Less than 5% displacement of bound ethidium bromide was observed at 100 μM. In contrast, the DNA intercalative drug actinomycin D readily dislodged the bound ethidium bromide (IC50 for displacement of ethidium bromide = 3 5 μM). Fascaplysin also dislodged the DNA bound ethidium bromide at low concentrations (IC₅₀ = 5μM). These results indicate that CA224 does not intercalate nor interact with the minor groove of double-stranded DNA molecules (Figures 28 and 29).

Flow cytometric analysis The in vitro enzyme assays confirmed that CA224 selectively inhibits Cdk4-cyclinD1 (Table 9). Thus it should also block the growth of asynchronously growing cells at the G₁ phase of the cell cycle and should maintain the G₀IGi block induced by serum starvation. Cell lines with varied mitotic spindle checkpoint and p53 status were used to investigate the role of CA224 in blocking specific phases of the cell division cycle.

CA224 retains the GnZG₁ block in serum-starved p53-null Calu-1 cells

Treatment of Calu-1 cells, after release from cell synchronization (via serum starvation by growing cells in the presence of 0.1% FBS for 24 h), with IC₅₀ and IC₇₀ concentrations of CA224 resulted in partial and full maintenance of the G0/G1 block, respectively. Since the maintenance of GoZG₁ block after serum starvation requires Cdk4 enzyme to be inactive, these results indicate that CA224 probably inhibits cellular Cdk4 at these concentrations (Figure 30A) and thereby maintain the GoZG₁ block. In Calu-1 cells, the mitotic spindle checkpoint is impaired and since CA224 also inhibits tubulin polymerisation in vitro with higher potency than it inhibits the enzyme Cdk4- cyciin D1 (see below), Calu-1 cells were used to study the Cdk4 inhibitory property of CA224.

The higher G₀ZGi : S ratios in cells released from serum starvation in the presence of compound compared to serum starved cells is because nearly all cells in S phase present during serum starvation enter G₂ZM phase after release (Figure 3OA histogram B and D). However at the lower IC₅₀ concentration, we observed a greater tendency towards a G₂ZM block (Figure 3OA histogram C) depicted by the increased percentage of cells in G₂ZM phase. This would indicate that CA224, although identified as a Cdk4- specific inhibitor in the in vitro enzyme screens, tends to block more profoundly at G₂ZM than at the G₀ZGi phase of the cell cycle.

CA224 at the ICgn concentration induces profound G₂ZM block in two asynchronous cancer cell lines (p53+ A549 and p53-null NCI-H1299 cells) Incubation of A549 cells with CA224 at the IC₅₀ concentration for 24 h induces a profound block at G₂/M as indicated by the percentage of cells at G₂ZM. As seen in Figure 31, at the IC₅₀ concentration of CA224, 89% cells appeared to be arrested in the G₂ZM phase (B) and at the IC₇₀ concentration 91% cells blocked at G₂ZM (C). Upon incubation of NCI-H 1299 (p53-null) with IC₅₀ concentration of CA224 resulted in a large number of cells (71 % cells) accumulating at G₂ZM (E). The profound G₂ZM block is observed both in A549 and NCI-H 1299 cells where the mitotic spindle checkpoint is intact. Nocodazole and paclitaxel induced G?/M block is maintained by CA224 in NCI-H358 lung cancer cells

NCI-H358 (p53-null) cells were treated with nocodazole (1 μM: a sub-optimal concentration) for only 18 h in order to induce a partial block at G₂/M so that treated cells are minimally stressed. The blocked cells were released either in fresh medium for 12 h (they readily re-enter the cell cycle without any apoptosis) or in the presence of CA224 for 12 h (cells not only maintain the G₂/M block but also >50% of G₀ZG₁ and S phase cells enter G₂/M (Figure 32, histograms A,B, C and D).

Similar results were obtained when paclitaxel blocked cells (50 nM paclitaxel for 18 h) were released in the presence of CA224 for 12 h (Figure 32, histograms A, E, F₁ and G). These observations suggest that, at least, in p53-null NCI-H358 cells, CA224 maintains the G₂/M block induced by the anti-mitotic agents, nocodazole and paclitaxel.

CA224 blocks NCI-H358 cells in G?/M after release from hydroxyurea-mediated G1/S cell synchronization

Hydroxyurea (250 μM, 18 h) was used to block cells at the G₁ZS boundary (G₁ZS = 77%; Figure 33, histogram B), at a stage of the cell cycle where Cdk2-specifϊc inhibitors normally act. When released in the presence of CA224, cells proceed from G₁ZS, confirming that CA224 does not inhibit cellular Cdk2. Cells ultimately accumulate at the G₂ZM phase of the cell cycle (G₂ZM = 72%; Figure 33, histogram D). These results again indicate that CA224 has an inherent tendency to induce block at the G₂ZM phase of the cell cycle at least in cells where the mitotic spindle checkpoint is normal. These results also indicate that CA224 does not act at the G 1ZS checkpoint and the G₁ block induced in the mitotic spindle checkpoint-proficient cells is definitely before the G₁ZS boundary (i.e. in the early G₁ phase, which is likely to be at the GOZ G₁ phase where Cdk4 acts).

Selective killing of SV40 large T antigen-transformed normal mouse embryonic liver cells by CA224

SV40 large T antigen inactivates both the tumour suppressor proteins, p53 and pRb, and thereby transforms normal cells into tumorigenic cells.

We used normal mouse embryonic hepatic (liver) cells BNL CL2 and its SV-40 large T antigen-transformed counterpart BNL SV A.8 to study the effects of compound CA224.

The normal cells upon 48 h incubation exhibited prominent G₂ZM arrest at both IC₅₀ and IC_7O concentrations with less than 5% cells detected in the sub-Gi phase (Figure 34, histogram B and C). More than 50% of cells appeared to be in the G2/M phase of the cell cycle. Interestingly, in the SV40-transformed cell line significant apoptotic cell death was observed. After 48 h treatment at IC₅₀ concentration of CA224, 31 % cells were detected in the SUb-G₁ (Figure 34, histogram E) phase indicating 31% of the total cells were apoptotic. The percentage apoptosis increased further from 31 to 44% when cells were incubated at the IC₇₀ concentration for 48 h (Figure 34, histogram F).

The primary characteristic of an ideal anti-cancer compound is to selectively kill rapidly proliferating cancerous cells while leaving the normal cells unaffected. The selective cell death induction in cancer cells by CA224 treatment is very significant. These results suggest that the oncologically transformed cells (BNL SV A.8), which are rapidly proliferating because both the cell cycle checkpoints (G₁ and G₂) are impaired due to pRb and p53 inactivation, undergo massive cell death when the checkpoints are re- installed in the form of G₀ZG₁ and G₂/M blocks induced by CA224 treatment. This could not have been predicted from the published information that CA224 was a Cdk4 inhibitor.

Western blot analysis Effects on the pRb phosphorylation status in Calu-1 cells (mitotic spindle checkpoint impaired)

The sensitivity of cancer cell lines to CA224 was probably because of its dual mechanism of action. Its tubulin binding affinity was found to be more potent than its ability to inhibit Cdk4-cyclin D1. In order to check Cdk4-specific pRb phosphorylation we selected mitotic spindle checkpoint impaired Calu-1 cells. Western blot analysis after CA224 treatment (at IC₅₀ concentration) of Calu-1 cells for 24 h showed that pRb remains unphosphorylated at serine residues Ser780, Ser795 and Ser807/811 which are specifically phosphorylated by the Cdk4 enzyme. The pRb levels in CA224-treated cells remain unchanged (Figure 31 B). We observed that fascaplysin treatment of CaIu- 1 cells also prevented pRb phosphorylation at Cdk4 specific serine residues which has been reported previously (Soni et al 2000). The results indicate that CA224 treatment of Calu-1 cells does not alter the pRb expression levels but actively prevents the phosphorylation of pRb protein at the serine residues, which are known to be phosphorylated by the enzyme Cdk4. Since pRb is a tumour suppressor protein, its inactivating phosphorylations by Cdk4 enzyme could lead to excessive cell proliferation and prevention of these phosphorylations is the mechanism by which Cdk4 inhibitors exert their anti-cancer effects. Effects on the cellular level of cvclin B1. Cdk1. p53, p21CIP1/WAF1 (p21) and p27KIP1 (p27); analyses in p53+ cells

Western analyses of p53+ cells, A549 and LS174T, after treatment with CA224 (at the IC₅₀ concentration) for 24 h demonstrated more than 10-fold induction of p53 protein and thereby the Cdk inhibitor p21CIP1/WAF1 (p21) was also probably induced. The p27KIP1 (p27) levels were also elevated due to CA224 treatment (Figure 35). The proteins Cdk1 and cyclin B1 were downregulated in the treated cells compared with untreated control cells. Repression of cyclin B1 and Cdk1 and elevated levels of p21 and p27 is a possible explanation for the G₂/M block in A549 and LS174T cells that bear functional copies of the tumour suppressor gene p53 (Figure 35). The tumour suppressor protein, p53, plays a crucial role in controlling cell proliferation. p53 can induce p21 which is a cell cycle inhibitory protein, and thereby blocking cell growth. Intracellular accumulation of p53 can also result in cyclin B1 depletion which could result in the G₂/M arrest of cells.

Effects on the cellular level of cvclin B1. Cdk1 , p53, p21 CIP1/WAF1 (p21 ) and P27KIP1 (p27); Western blot analyses of p53-mutant cells

CA224 was analysed for its effects on the cell cycle of MiaPaCa-2 cells which are p53 mutated. FACS analysis shows that CA224 also blocks MiaPaCa-2 cells at the G₂/M phase of the cell cycle. At the IC₅₀ concentration of CA224, approximately 48% cells were found in G₂/M while in control cultures (i.e. cells that were untreated) only 16% cells appeared in G₂/M (data not shown).

When a Western blot analysis was performed to check the levels of specific proteins in MiaPaCa-2 cells (Figure 36), we found that p53, p21 and p27 levels remain unchanged, indicating that p21 and p27 induction is probably p53-dependent. Interestingly, the cyclin B1 and cdk1 levels were elevated and phosphorylation of Cdk1 at the residue Tyr15 remain unaffected indicating that Cdk1 -cyclin B1 is still active in p53-mutated MiaPaCa-2 cells after CA224 treatment.

Tubulin polymerisation assays

Cell free tubulin polymerisation assays in vitro indicate inhibition of tubulin polymerisation by novel fascaplysin analogues including CA224 CA224 inhibits growth of cancer cells in vitro at concentrations that are lower than the concentration for inhibition of the enzyme Cdk4-cyclin D1. FACS analyses (shown above) and mitotic index experiments (results not shown) have already confirmed that CA224 blocks cell growth at the pro-metaphase of the cell cycle. In addition, the resistance of cells containing an impaired mitotic spindle checkpoint to CA224- mediated growth inhibition suggests that CA224 has another cellular target other than Cdk4. It is possible that it has a role as an anti-microtubule agent. We investigated the action of CA224 and other analogues on tubulin polymerisation in vitro. The experiments with purified tubulin were performed as described. The results indicate strong anti-tubulin activity of CA224 and other fascaplysin analogues, whereas fascaplysin itself does not show any interaction with the tubulin protein (Figure 37). Representative polymerisation curves for CA224, paclitaxel and nocodazole are shown in the figure. CA224 inhibits the polymerisation of tubulin which can be concluded from the dose-dependent decrease in Vmax (mOD/min) and reduction in the final polymer mass (Figure 37). When tested at four different concentrations, CA224 decreased the Vmax from 17 mOD/min to 6.2, 2.1 , 1.1 and 0.4 mOD/min at 2.5, 5, 10 and 25 μM, respectively. As a consequence of decreased Vmax, up to 80% reduction in final polymer mass is observed.

CA224 inhibits paclitaxel-mediated tubulin polymerisation and enhances tubulin de- polymerisation in vivo The tubulin polymerisation experiments performed in A549 cells (NSCLC cells with an intact mitotic spindle checkpoint) confirm the observation that CA224 inhibits polymerisation of tubulin. Moreover, CA224 also enhances the depolymerisation of the stabilized tubulin protein. The polymerised and depolymerised (soluble) forms of tubulin can be perceived from the accumulation or disappearance (observed by Western blotting) of tubulin protein from the pellets and vice versa from the supernatant fractions of lysed cells treated with CA224 (at IC₅₀ concentrations). In the first set of experiments where cells were treated with CA224 in the presence of the microtubule- stabilizing agent paclitaxel, CA224 shows prevention of tubulin polymerisation (mediated by paclitaxel) in a dose dependent manner (Figure 38). In a second set of experiments where intracellular tubulin was first stabilised with paclitaxel and then subjected to CA224 treatment, results show enhancement of tubulin de-polymerisation with increasing concentrations of CA224 (Figure 38).

Colony formation assays

The possible advantages of CA224's dual mechanism of action in killing cancer cells were explored in the context of long-term survival of cells. The assay monitors the ability of cells to form colonies in vitro in the presence of the fascaplysin derivative compounds. A549 (mitotic spindle checkpoint normal, pRb+) and Calu-1 (mitotic spindle checkpoint impaired, pRb+) cells were used in the colony formation assays. Surprisingly and unexpectedly, we found that the colony formation efficiency of both A549 and Calu-1 cells is significantly reduced on CA224 treatment (Figure 39). The concentration at which 50% colony formation efficiency is observed is comparatively lower than the IC₅₀ concentration for cell growth inhibition in the MTT assay, indicating that large number of cells lose the ability to form colonies or do not survive for a long time after CA224 treatment. For the A549 cell line, 50% colony formation efficiency was observed at an average of 1.4 μM (cf. cell growth inhibition IC₅₀ = 3.5 μM) and for Calu-1 cells 50% colony formation efficiency was observed at an average of 3 μM (cf. cell growth inhibition IC₅₀ = 11.5 μM). These values are a measure of the colony formation efficiency of A540 and Calu-1 cells in the presence of CA224 (Table 11 ; Figure 39)

Induction of cell death

Cancer cell lines NCI-H460, NCI-H 1299 and NCI-H358, when treated with CA224 for 24, 48 and 72 h, showed dose-dependent as well as time-dependent increase in percentage of apoptotic cells. In FACS analysis, the percentage of apoptotic cells is determined by the number of ce/is appearing in the sub-Gi peak. In order to confirm that treated cells are indeed undergoing apoptosis, the SUb-G₁ peak in these cells was compared to that in control cultures. The percentage apoptosis in three different cell lines is presented in Figure 4OB. The results indicate that long-term exposure (72 h) of cells to CA224 can induce massive cell death both in p53+ and p53-null cancer cells.

A549 cells treated with different concentrations of CA224 for 24 h were analysed by nuclear staining (i.e. DAPI staining). We found that with increasing concentrations of CA224, the number of cells undergoing apoptosis gradually increase. Apoptotic morphology of treated cells is shown in Figure 40A. The fragmented nucleus, disrupted cell and nuclear membranes, formation of apoptotic bodies which indicate apoptotic cell death were observed in treated cells. Less than 5% apoptosis was detected in control cultures.

Conclusions

We report the inhibition of cancer cell growth by a series of compounds (shown in Table 8) based on the structure of fascaplysin. They specifically target Cdk4-cyclin D1 enzyme inhibition and also prevent tubulin polymerisation. Unlike fascaplysin, these compounds are non-planar and do not interact or intercalate with double-stranded DNA. Hence, they are less toxic at the cellular level. CA224 was identified as the most potent molecule (IC₅₀ for Cdk4-cyclin D1=5.5 μM) in the CA224 series of compounds and was extensively studied for its inhibitory action on other Cdks like Cdk2-cyclin A, Cdk2-cyclin E₁ Cdk1-cyclin B1 and Cdk9-cyclin T1 in vitro. It was found that CA224 inhibits Cdk4-cyclin D1 specifically at low micro-molar concentrations. The IC₅₀-S in other Cdk assays are greater than 500 μM.

However, we also identified a number of additional properties of CA224 that could not have been predicted from its known activity as a Cdk4 inhibitor. CA224 • inhibits the growth of cancer cells at concentrations (average IC₅₀ in ten cell lines = 3 μM) lower than the concentration at which it inhibits Cdk4-cyclin D1 in vitro;

• blocks cancer cell growth at G₀ZG₁ (in a pRb-dependent manner) but more profoundly at the G₂/M phase of the cell cycle; • inhibits tubulin polymerisation in vitro and also acts as an enhancer for tubulin de-polymerisation (of paclitaxel stabilized tubulin) in vivo;

• induces massive apoptosis in cancer cells which are resistant to chemotherapy; and

• significantly reduces the colony formation efficiency (in a dose-dependent manner) of the lung cancer cells, A549 and Calu-1.

Taken together, these findings transform CA224 from a compound that could be the basis for the development of more potent CDK4 specific inhibitors (Aubry et al 2006; Mahale et al 2006a) into a compound that is suitable itself for use as an anticancer agent. Moreover, AJW089 and other analogues of the CA224/ AJW089 series are also expected to have these novel and unexpectedly beneficial properties and hence be of use as anticancer agents.

Example 3: Anti-tumour activity of CA224, AJW089 and DE002 against xenografts human cancer cells in SCID mice

Testing in animal models

The results shown in Examples 1 and 2 indicate that CA224, AJW089 and DE002 are the three most potent molecules which inhibit cancer cell growth (Table 12). These three compounds were therefore chosen to test their activity against models of human cancers in vivo. Table 12. IC₅₀-S of CA224, AJW089 and DE002 in nine different cancer cell lines and a normal cell line.

ND = Not determined.

SCID (severely combined jmmuno-deficient) mice lacking both T and B immune cells are an established modef system to study the in vivo efficacy of molecules against human cancers (Bankert et at, 2002; Kelland 2004). The human tumour cells can grow in SCID mice without any graft rejection because the mice are immuno-deficient. Human tumour cells grown in cell culture or a small piece of tumour of human origin can be transplanted subcutaneously and grown for a few days to form a small tumour. The small tumours can then be treated with potential anti-cancer compounds for evaluation of their in vivo efficacy.

The in vivo activity of CA224, DE002, AJW089 and flavopiridol was tested against xenografts derived from two human cancer cell lines: (a) the colon cancer line HCT116 cells and (b) the non-small cell lung carcinoma NCI-H460) cells.

Maximum tolerated dose (MTD)

In toxicology, MTD is the highest daily dose of a compound that does not cause over- toxicity (which sometimes can translate to death) in laboratory mice or rats. The studies which allowed determination of MTD were performed in Swiss albino mice for two weeks. The concentration at which the three compounds would be tested in vivo was thus ascertained. Loss in animal body weight was considered as a measure of over-toxicity for the test compound. The concentration of the compound at which >10% weight loss was observed was determined and designated as MTD. However, since a weight loss of up to 20% of the initial weight is usually harmless and animals usually recover once the treatment is stopped, the MTD figures used were an underestimation of the true value.

As shown in Table 13, the toxicity results indicated that the MTD for CA224 and DE002 in mice was -1000 mg/kg. For AJW089 the MTD was found to be -500mg/kg. CA224 and DE002 were tested at two different concentrations, VA of MTD and Vi of MTD. AJW089 was tested at lower than VA of MTD concentrations due to its poor solubility.

Table 13. Toxicity and MTD findings study in Swiss albino mice. Each group contained 6 mice and were injected with one dose intra-peritoneally (i.p.) on 5 consecutive days, using different concentrations of CA224, AJW089 and DE002. The body weight of the animals was then monitored for two weeks and the drop in relative mean animal weight was calculated.

^* Indicates a drop of >10% in mean body weight of the animals at the end of the two- week period after dosing on five consecutive days. ND = Not determined

CA224 and DE002 significantly inhibit the growth ofHCT116 xenografts

Effects on HCT116 tumour growth inhibition

Flavopiridol is a known Cdk inhibitor and its ability to inhibit tumour growth in vivo has been widely reported (Zhai et a/, 2002; Drees et al, 1997; Senderowicz and Sausville 2000). Hence flavopiridol was used as a standard compound in the in vivo experiments. The compounds CA224 and DE002 showed statistically significant (p<0.05) tumour growth inhibition (Table 14; Figure 41 and Figure 43) at Vz MTD as well as V* MTD concentrations. CA224 and DE002 exhibited approximately 80% tumour growth inhibition as compared to the untreated group. These results indicate their strong anti-tumour properties in vivo. Although AJW089 treated animals had on average noticeably smaller tumours than the controls, this reduction of tumour growth was not significant. A lower concentration of AJW089 was used due to its poor solubility in the solvent, which led to decreased bioavailability for AJW089, thus contributing to its apparent in vivo inactivity.

Table 14. Tumour weights (in mg) from SCID mice, control and experimental groups that bear the HCT116 human colon tumour cell xenografts. Tumour size was recorded at 2-5 day intervals. Tumour weight (mg) was estimated according to the formula for a prolate ellipsoid: {Length (mm) x [width (mm)²] x 0.5} assuming specific gravity to be one and π to be three. AVG = Average; STDEV = standard deviation; SEM = standard error of mean.

AD = Animal died

Effect on animal body weight due to compound treatment. The weight loss observed in treated animals was found to be <10% of the starting weights of the animals (Figure 42). This loss of weight can be considered to be statistically insignificant indicating that compound treatment cause no major toxicity or harm to the animals. The body weight of the animals at the beginning of treatment was measured and this was considered to be 100%. The percentage weight loss or gain was calculated using the initial weight as reference.

Determination of in vivo efficacy

The percentage tumour growth inhibition (throughout the treatment) using V« MTD concentrations of CA224 and DE002 was calculated and presented in Figure 43. From the results, we concluded that CA224 and DE002 are highly efficacious against tumour growth in vivo at a YA MTD concentration. The different degrees of in vivo efficacy of the compounds were assigned using the parameters described in Table 15.

Table 15. Correlation of percentage tumour growth and the activity of a potential anti-cancer compound. Based on the values of percentage tumour growth inhibition, the in vivo efficacies of the different anti-cancer compounds are usually affixed.

In vivo experiments in the SCID mice-NCI-H460 tumour model

Effects of compounds CA224, DE002 and AJW089 on NCI-H460 tumour growth NCI-H460 is a drug resistant lung cancer cell line derived from NSCLC, a form of cancer which is mostly untreatable using the drugs currently available in the clinic. NCI-H460 cells demonstrate aggressive growth and spread of cancer cells in vivo in SCID mice. Most anti-cancer compounds which inhibit cancer cell growth in vitro fail to inhibit NCI-H460 tumours in vivo due to the aggressive nature of these tumours.

We therefore tested the three best compounds, CA224, DE002 and AJW089, for their in vivo efficacy in the SCID mice-NCI-H460 model. Flavopiridol, the pan-Cdk inhibitor which is effective in a variety of mouse tumour models, was used as the standard compound in these in vivo experiments. CA224 and DE002 both proved to be highly efficacious against NCI-H460 at Vz MTD and ^ΛA MTD concentrations. Again, at the concentrations used, AJW089 failed to exhibit significant activity in this particular in vivo model (Table 16; Figure 44) due to its low solubility in the solvent and hence low bioavailability.

Table 16. Tumour weights (in mg) from SCID mice, control and experimental groups that bear the NCI-H460 tumour transplants. Tumour size was recorded at 2-5 day intervals. Tumour weight (mg) was estimated according to the formula for a prolate ellipsoid: {Length (mm) x [width (mm)²] x 0.5} assuming specific gravity to be one and π to be three. STDEV = standard deviation; SEM = standard error of mean.

AD = Animal died Effect on animal body weight

CA224 and AJW089 treatment did not show any significant weight loss in the animals, but DE002 treatment resulted in weight loss slightly above 10%. A weight loss which is below 20% of the initial weight is relatively harmless and animals can recover once the treatment is stopped. Figure 45 shows percentage mean animal weights during the period when animals were treated with compounds.

In vivo efficacy determination in the SCID mice-NCI~H460 tumour model

The percentage tumour growth inhibition after compound treatment was calculated. The tumour growth inhibition values were derived from the data presented above. The results indicate more that 75% tumour growth inhibition following CA224 and DE002 treatment (Figure 46). After the experimental schedule, the animals were sacrificed and photographs were taken to demonstrate in situ tumour growth inhibition (Figure 48). Representative animals from each group were dissected and tumour tissues (Figure 47) were photographed and preserved for further experiments.

Conclusions

CA224 and DE002 are highly efficacious against human tumours derived from HCT116 and NCI-H460 cell in in vivo. The two compounds show minimal toxicity in animal models and can be tolerated up to 1000 mg/kg concentrations. CA224 and DE002 have proven their efficacy as a new class of anticancer compounds via these studies. The use of more soluble salts of AJW089, or the use of different solvents, is expected to show that this compound is also a highly effective anticancer agent in vivo.

Additional References

Alessi, F., Quarta, S., Savio, M., Riva, F., Rossi, L., Stivala, L.A., Scovassi, A.I., Meijer, L., and Prosperi, E. 1998. The cyclin-dependent kinase inhibitors olomoucine and roscovitine arrest human fibroblasts in G1 phase by specific inhibition of CDK2 kinase activity. Exp Cell Res, 245, 8-18. Andreadou, I., Tasouli, A., Bofilis, E., Chrysselis, M., Rekka, E., Tsantili-Kakoulidou, A., lliodromitis, E., Siatra T., Kremastino, D., T. 2002. Antioxidant Activity of Novel Indole Derivatives and Protection of the Myocardial Damage in Rabbits. Chem Pharm Bull, 50: 165-168.

Antony, S., Kohlhagen, G., Agama, K., Jayaraman, M., Cao, S., Durrani, F.A., Rustum, Y.M., Cushman, M., and Pommier, Y. 2005. Cellular topoisomerase I inhibition and antiproliferative activity by MJ-lll-65 (NSC 706744), an indenoisoquinoline topoisomerase I poison. MoI Pharmacol, 67: 523-530.

Aubry, C, Jenkins, P. R., Mahale, S., Chaudhuri, B., Marechal, J-D., and Sutcliffe, M.J. 2004. New fascaplysin-based CDK4-specific inhibitors: design synthesis and biological activity. Chem Commun, 15: 1696-1697. Aubry, C, Patel, A., Mahale, S., Chaudhuri, B., Marechal, J.-D., Sutcliffe, M.J., and Jenkins, P. R. 2005. The design and synthesis of novel 3-[2-indol-1-yl-ethyl]-1 H-indole derivatives as selective inhibitors of CDK4. Tetrahedron Letters, 46: 1423-1425.

Aubry, C, Wilson.A.J., Jenkins, P.R., Mahale, S., Chaudhuri, B., Marechal, J.-D., and Sutcliffe, M.J. 2006. Design, synthesis and biological activity of new CDK4-specific inhibitors, based on fascaplysin. Org Biomol Chem, 4(5): 787-801.

Augustin, E., Wheatley, D. N., Lamb, J., Konopa, J.1996. Imidazoacridinones arrest cell-cycle progression in the G2 phase of L1210 cells. Cancer Chemother Pharmacol, 38: 39-44. Bartek, J., Bartkova, J., and Lukas, J. 1996. The retinoblastoma protein pathway and the restriction point. Curr Opin Cell Biol, 8: 805-814.

Bankert, R.B., Hess, S. D., Egilmez, N. K 2002. SCID mouse models to study human cancer pathogenesis and approaches to therapy: potential, limitations, and future directions. Front Biosci, 7: c44-62. Benson, C₁ Kaye, S., Workman, P., Garrett, M., Walton, M., and de Bono J. 2005. Clinical anticancer drug development: targeting the cyclin-dependent kinases. Br J Cancer 92: 7-12.

Blagosklonny, M. V., and Pardee, A. B. 2002. The restriction point of the cell cycle. Cell Cycle, 1(2): 103-110. Brotz-Oesterhelt, H., Knezevic, I., Bartel, S., Lampe, T., Warnecke-Eberz, U., Ziegelbauer, K., Habich, D., Labischinski, H. 2003. Specific and potent inhibition of NAD+ dependent DNA ligase by pyridochromanones. J Biol Chem, 278: 39435-39442.

Butterfield, D.A., Koppal, T., Howard, B., Subramaniam, R., Hall, N., Hensley, K., Yatin, S., Allen, K., Aksenov, M., Aksenova, M., Carney, J. 1998. Structural and functional changes in proteins induced by free radical-mediated oxidative stress and protective action of the antioxidants N-tert-butyl-alpha-phenylnitrone and vitamin E. Ann N Y Acad Sci, 854: 448-62.

Campbell, I., Magliocco, A., Moyana, T., Zheng, C₁ Xiang, J. 2000. Adenovirus- mediated p16INK4 gene transfer significantly suppresses human breast cancer growth. Cancer Gene Ther, 7(9): 1270-1278.

Carlson, B.A., Dubay, M. M., Sausville, E.A., Brizuela, L., and Worland, PJ. 1996. Flavopiridol induces G1 arrest with inhibition of cyclin dependent kinase (CDK) 2 and Cdk4 in human breast carcinoma cells. Cancer Res, 56: 2973-2978.

Charan, R.D., McKee, T.C., and Boyd, M. R. 2004. Cytotoxic alkaloid from the marine sponge thorectandra. Nat Prod Res, 18: 225-229.

Chen, J.T., Chen, Y.C, Chen, CY. and Wang, Y.C. 2001. Loss of p16 and/or pRb protein expression in NSCLC. An immunohistochemical and prognostic study. Lung Cancer, 31(2-3): 163-170.

Dai, Y., and Grant, S. 2004. Small molecule inhibitors targeting cyclin dependent kinases as anticancer agents. Curr Oncol Rep, 6: 123-130.

Das, B. B., Sen, N., Roy, A., Dasgupta, S.B., Ganguly, A., Chandra, B., Dinda, M. B., and Majumder H. K. 2006. Differential induction of Leishmania donovani bi-subunit topoisomerase I-DNA cleavage complex by selected flavones and camptothecin: activity of flavones against camptothecin-resistant topoisomerase I. Nucleic Acids Research, 34(4): 1121-1132.

De Brabander M., Geuens, G., Nuydens, R., Willebrords, R., J. De Mey, J. 1981. Taxol Induces the Assembly of Free Microtubules in Living Cells and Blocks the Organizing Capacity of the Centrosomes and Kinetochores. PNAS, 78 (9 ): 5608-5612. Dehmlow, C, Murawski, N., de Groot, H. 1996. Scavenging of reactive oxygen species and inhibition of arachidonic acid metabolism by silibinin in human cells. Life Sci, 58(18): 1591-600.

Drees M, Dengler WA₁ Roth T, Labonte H, Mayo J, Malspeis L₁ Grever M₁ Sausville EA₁ Fiebig HH. Flavopiridol (L86-8275): selective antitumor activity in vitro and activity in vivo for prostate carcinoma cells. 1997. Clin Cancer Res. 3(2):273-9.

Eastman, A. 2004. Cell cycle checkpoints and their impact on anticancer therapeutic strategies. J Cell Biochem, 91 : 223-231.

Ferlini, C₁ Raspaglio, G., Mozzetti, S., Cicchillitti, L., Filippetti, F., GaIIo, D., Fattorusso, C.Campiani, G. and Scambia, G. 2005. The seco-taxane IDN5390 is able to target class III beta-tubulin and to overcome paclitaxel resistance. Cancer Res, 65(6): 2397- 2405.

Fischer, P.M., and Gianella-Borradori, A. 2005. Recent progress in the discovery and development of cyclin-dependent kinase inhibitors. Expert Opin Investig Drugs, 14: 457-477.

Garrett, M. D., and Fattaey, A. 1999. CDK inhibition and cancer therapy. Curr Opin Genet Dev, 9: 104-111.

Geall, A.J., Eaton, M.A., Baker, T., Catterall, C, and Blagbrough, I.S. 1999. The regiochemical distribution of positive charges along cholesterol polyamine carbamates plays significant roles in modulating DNA binding affinity and lipofection. FEBS Letters, 459: 337-342.

Giannakakou, P., Sackett, D.L., Kang, Y.K., Zhan, Z., Buters, J.T., Fojo, T., Poruchynsky, M.S. 1997. Paclitaxel-resistant human ovarian cancer cells have mutant beta-tubulins that exhibit impaired paclitaxel-driven polymerization. J Biol Chem,. 272(27): 17118-17125.

Giles, G. I., and Sharma, P. R. 2005. Topoisomerase enzymes as the therapeutic targets for cancer chemotherapy. Med Chem, 1(4): 383-394.

Grana, X., and Reddy, E.P. 1995. Cell cycle control in mammalian cells: role of cyclins, cyclin dependent kinases (Cdks), growth suppressor genes and cyclin dependent kinase inhibitors (CKIs). Oncogene, 11 : 211-219.

Green, D.R., Evan, G.I. 2002. A matter of life and death. Cancer Cell, 1(1 ):19-30.

Grossel, M. J., and Hinds, P.W. 2006. Beyond the cell cycle: A new role for Cdk6 in differentiation. J Cell Biochem, 97: 485-493.

Guo, Z., and Stiller, J. W. 2004. Comparative genomics of cyclin-dependent kinases suggest co-evolution of the RNAP Il C-terminal domain and CTD-directed CDKs. BMC Genomics, 5: 69-81.

Herzig, M., Novatchkova, M., Christofori, G. 1999. An unexpected role for p53 in augmenting SV40 large T antigen-mediated tumorigenesis. Biol Chem, 380(2): 203-11.

Hickman, E. S., Moroni, M. C, Helin, K. 2002. The role of p53 and pRB in apoptosis and cancer. Curr Opin Genet Dev, 12(1 ): 60-76.

Hirai, H., Kawanishi, N., and Iwasawa, Y. 2005. Recent advances in the development of selective small molecule inhibitors for cyclin dependent kinases. Curr. Top. Med. Chem, 5: 167-179.

Hong, J., Shah, N.N., Thomas, T.J., GaIIo, M.A., Yurkow, EJ. , Thomas, T. 1998. Differential effects of estradiol and its analogs on cyclin D1 and CDK4 expression in estrogen receptor positive MCF-7 and estrogen receptor-transfected MCF-10AEwt5 cells. Oncol Rep, 5(5): 1025-33. Hormann, A., Chaudhuri, B., and Fretz, H. 2001. DNA binding properties of the marine sponge pigment Fascaplysin. Bioorg. Med. Chem, 9: 917-921.

Hua, X.H., Genini, D., Gussio, R., Tawatao, R., Shih, H., Kipps, TJ. , Carson, D.A. and Leoni, LM. 2001. Biochemical genetic analysis of lndanocine resistannce in human leukemia. Cancer Res, 61(19): 7248-7254.

Izzotti, A., Bagnis, A., Sacca, S. C. 2006. The role of oxidative stress in glaucoma. Mutat Res, 12(2): 105-14.

Ji, C, Marnett, L. J., and Pietenpol, J.A. 1997. Cell cycle re-entry following chemically induced cell cycle synchronization leads to elevated p3 and p21 protein levels. Oncogene, 15(22): 2749-2753.

Jordan, M.A, Ojima, I₁ Rosas, F., Distefano, M., Wilson, L. and Scambia, G. 2002. Effects of novel taxanes SB-T-1213 and IDN5109 on tubulin polymerization and mitosis. Chem Biol, 9: 93-101.

Kastan, M. B., and Bartek J. 2004. Cell-cycle checkpoints and cancer. Nature, 432: 316-323.

Kataoka, M., Wiehle, S., Spitz, F., Schumacher, G., Roth, J.A., Cristiano, RJ. 2000. Down-regulation of bcl-2 is associated with p16lNK4-mediated apoptosis in non-small cell lung cancer cells. Oncogene, 19(12): 1589-1595.

Kato, J. Y. 1997. Control of G1 progression by D-type cyclins: key event for cell proliferation. Leukemia, 11 Suppl 3: 347-351.

Kelland, L.R. 2004. Of mice and men: values and liabilities of the athymic nude mouse model in anticancer drug development. Eur J Cancer, 40 (6): 827-36.

Kim, H., Ham, E.K., Kim, Y.I., Chi, J.G., Lee, H.S., Park, S.H., Jung, Y.M., Myung, N. K., Lee, MJ., Jang, JJ. 1998. Overexpression of cyclin D1 and cdk4 in tumorigenesis of sporadic hepatoblastomas. Cancer Lett, 131(2): 177-83.

Knockaert, M., Grrengard, P., and Meijer, L. 2002. Pharmacological inhibitors of cyclin dependent kinases. Trends Pharmacol Sci, 23: 417-425.

Kortmansky, J., Shah, M.A., Kaubisch, A., Weyerbacher, A., Yi, S., Tong, W., Sowers, R., Gonen, M., O'reilly, E., Kemeny, N., llson, D.I., Saltz, L.B., Maki, R.G., Kelsen, D. P., Schwartz, G. K. 2005. Phase I trial of the cyclin-dependent kinase inhibitor and protein kinase C inhibitor 7-hydroxystaurosporine in combination with Fluorouracil in patients with advanced solid tumors. J Clin Oncol, 23(9): 1875-1884.

Landis, M.W., Pawlyk, B.S., Li, T., Sicinski, P., and Hinds P.W. 2006. Cyclin D1- dependent kinase activity in murine development and mammary tumorigenesis. Cancer Cell, 9: 13-22.

Lara, P.N. Jr, Mack, P.C., Synold, T., Frankel, P., Longmate, J., Gumerlock, P.H., Doroshow, J. H., Gandara, D. R. 2005. The cyclin-dependent kinase inhibitor UCN-01 plus cisplatin in advanced solid tumors: a California cancer consortium phase I pharmacokinetic and molecular correlative trial. Clin Cancer Res, 11(12): 4444-4450. Lawrie, A.M., Noble, M. E., Tunnah, P., Brown, N. R., Johnson, L.N., and Endicott, J.A. 1997. Protein kinase inhibition by staurosporine revealed in details of the molecular interactions. Nat Struct Biol, 4: 796-801.

Lee, J. C. and Timasheff, S.N. 1997. In vitro reconstitution of calf brain microtubules: effects of solution variables. Biochemistry, 16: 1754-1764. Lee, J.W., Moon, MJ., Min, H.Y., Chung, HJ., Park, EJ., Park, HJ., Hong, J.Y., Kim, Y.C., Lee, S. K. 2005. Induction of apoptosis by a novel indirubin-5-nitro-3'-monoxime, a CDK inhibitor, in human lung cancer cells. Bioorg Med Chem Lett, 15(17):3948-52. Leppard, J. B., and Champoux, J.J. 2005. Human DNA topoisomerase I: relaxation, roles and damage control. Chromosoma, 114(2) 75-85.

Li Y, Nichols MA, Shay JW, Xiong Y. 1994. Transcriptional repression of the D-type cyclin-dependent kinase inhibitor p16 by the retinoblastoma susceptibility gene product pRb. Cancer Res, 54(23): 6078-6082.

Lin, J., Yan, XJ. , Chen, H. M. 2006. Fascaplysin, a selective CDK4 inhibitor, exhibit anti-angiogenic activity in vitro and in vivo. Cancer Chemother Pharmacol, [Epub ahead of print].

Liu, M. C₁ Marshall, J. L., and Pestell, R.G. 2004. Novel strategies in cancer therapeutics: targeting enzymes involved in cell cycle regulation and cellular proliferation. Curr Cancer Drug Targets, 4: 403-424.

Lundberg, A.S., and Weinberg, R.A. 1998. Functional inactivation of retinoblastoma protein requires sequential modification by at least two distinct cyclin-cdk complexes. MoI Cell Biol, 18: 753-761. Mahale S. and Chaudhuri B. Development of CDK Inhibitors as Anticancer Agents: From Rational Design to CDK4-Specific Inhibition. 2006. In CRC press 'Enzyme Inhibitors Series' volume 3, Inhibitors of Cyclin-dependent Kinases as Anti-tumor Agents (editors Paul J. Smith and Eddy W. Yue), Chapter 6.

Mahale, S., Aubry, C, James-Wilson, A., Jenkins, P. R., Marechal, J. D., Sutcliffe, M. J., and Chaudhuri B. 2006a. CA224, a non-planar analogue of fascaplysin, inhibits Cdk4 but not Cdk2 and arrests cells at G0/G1 inhibiting pRB phosphorylation. Bioorg Med Chem Lett, 16(16): 4272-4278.

Mahale, S., Aubry, C, Jenkins, P.R, Marechal, J. D., Sutcliffe, M. J., Chaudhuri B. 2006b. Inhibition of cancer cell growth by cyclin dependent kinase 4 inhibitors synthesized based on the structure of fascaplysin. Bioorg Chem, 34: 287-297.

Malumbres, M., and Barbacid M. 2006. Is Cyclin D1-CDK4 kinase a bona fide cancer target? Cancer Cell, 9: 2-4.

Massague J. 2004. G1 cell-cycle control and cancer. Nature, 432: 298-306.

Masuda, A., Maeno, K., Nakagawa, T., Saito, H., Takahashi, T. 2003. Association between mitotic spindle checkpoint impairment and susceptibility to the induction of apoptosis by anti-microtubule agents in human lung cancers. Am J Pathol, 163(3): 1109-1116.

Matsumoto, M., Furihata, M., Ishikawa, T., Ohtsuki, Y., Ogoshi, S. 1999. Comparison of deregulated expression of cyclin D1 and cyclin E with that of cyclin-dependent kinase 4 (CDK4) and CDK2 in human oesophageal squamous cell carcinoma. Br J Cancer, 80(1-2): 256-61.

Meijer, L., Borgne, A., Mulner, O., Chong, J. P., Blow, JJ. , Inagaki, N., Inagaki, M., Delcros, J. G., and Moulinoux, J. P. 1997. Biochemical and cellular effects of roscovitine, a potent and selective inhibitor of the cyclin-dependent kinases cdc2, cdk2 and cdk5. Eur J Biochem, 243: 527-536.

Mohammad, R.M., Dugan, M. C₁ Mohamed, A.N., Almatchy, V.P., Flake, T.M., Dergham, ST., Shields, A.F., Al-Katib, A.A., Vaitkevicius, V.K., Sarkar, F.H. 1998. Establishment of a human pancreatic tumor xenograft model: potential application for preclinical evaluation of novel therapeutic agents. Pancreas, 16(1 ): 19-25. Mohammad, R.M., Mohamed, A.N., Hamdan, M. Y., Vo, T., Chen, B., Katato, K., Abubakr, Y.A., Dugan, M. C, al-Katib, A. 1996. Establishment of a human B-CLL xenograft model: utility as a preclinical therapeutic model. Leukemia, 10(1): 130-7. Mor, M., Spadoni, G., Diamantini, G., Bedini, A., Tarzia, G., Silva, C₁ Vacondio, F., Rivara, M., Plazzi, P.V., Franceschini, D., Zusso, M., Giusti, P. 2003. Antioxidant and cryoprotective activity of indole derivatives related to melatonin. Adv Exp Med Biol, 527: 567-575. Morgan, D. O. 1997. Cyclin-dependent kinases: engines, clocks and microprocessors. Annu Rev Cell Dev Biol, 13: 261-291.

Murray A.W. 2004. Recycling the cell cycle: cyclins revisited. Cell, 116: 221-234.

Olgen, S., Coban, T. 2002. Synthesis and antioxidant properties of novel N-substituted indole-2-carboxamide and indole-3-acetamide derivatives. Arch Pharm (Weinheim), 335(7): 331-8.

Paull, K.D., Shoemaker, R.H., Hodes, L., Monks, A., Scudiero, D.A., Rubinstein, L., Plowman, J., and Boyd, M. R. 1989) Display and analysis of patterns of differential activity of drugs against human tumor cell lines: development of mean graph and COMPARE algorithm. J Natl Cancer Inst, 81 : 1088-1092. Peng, J., Zhu, Y., Milton, J. T., and Price, D. H. 1998. Identification of multiple cyclin subunits of human P-TEFB. Genes Dev, 12: 755-762.

Pipas, J. M., Levine, A.J. 2001. Role of T antigen interactions with p53 in tumorigenesis. Semin Cancer Biol, 11 (1): 23-30

Pines, J. 1999. Four-dimensional control of the cell cycle. Nat. Cell. Biol, 1 : E73-79. Planas-Silva, M. D., and Weinberg, R. A. 1997. The restriction point and control of cell proliferation. Curr Opin Cell Biol, 9(6): 768-772.

Robson, T., Worthington, J., McKeown, S. R., and Hirst, D. G. 2005. Radiogenic therapy: novel approaches for enhancing tumor radiosensitivity. Technol Cancer Res Treat, 4: 343-361. Roll, D.M., Ireland, CM., Lu H.S.M., Clardy, J. 1988. Fascaplysin, an unusual antimicrobial pigment from the maring sponge Fascaplysinopsis sp. J Org Chem, 53 (14): 3276-3278.

Sabbah, M., Courilleau, D., Mester, J., Redeuilh, G. 1999. Estrogen induction of the cyciin D1 promoter: involvement of a cAMP response-like element. Proc Natl Acad Sci U S A, 96(20): 11217-22.

Sakaguchi, M., Fujii, Y., Hirabayashi, H., Yoon, H. E., Komoto, Y., Oue, T., Kusafuka, T., Okada, A., Matsuda, H. 1996. Inversely correlated expression of p16 and Rb protein in non-small cell lung cancers: an immunohistochemical study, lnt J Cancer, 65(4): 442-5. Sanchez, I., and Dynlacht, B. D. 2005. New insights into cyclins, CDKs, and cell cycle control. Semin. Cell Dev. Biol, 16: 311-321.

Sandra, F., Hendarmin, L., Nakao, Y., Nakamura, N. and Nakamura, S. 2006. Inhibition of Akt and MAPK pathways elevated potential of TNFalpha in inducing apoptosis in ameloblastoma. Oral Oncol, 42: 39-45. Sangfelt, O., Erickson, S., Castro, J., Heiden, T., Gustafsson, A., Einhorn, S., Grander, D. 1999. Molecular mechanisms underlying interferon -alpha-induced G0/G1 arrest: CKI mediated regulation of G1 Cdk complexes and activation of pocket proteins. Oncogene, 18(18): 2798-2810.

Sausville E. A. 2003. Cyclin-dependent kinase modulators studied at the NCI: pre- clinical and clinical studies. Curr Med Chem Anticancer Agents, 3: 47-56. Schwartz, G. K., Farsi, K., Maslak, P., Kelsen, D. P., Spriggs, D. 1997. Potentiation of apoptosis by flavopiridol in mitomycin-C-treated gastric and breast cancer cells. Clin Cancer Res, 3(9): 1467-1472.

Schwartz, G. K. 2005. Development of cell cycle active drugs for the treatment of gastrointestinal cancers: a new approach to cancer therapy. J Clin Oncol 23: 4499- 4508.

Sherr, C. J. 1996. Cancer cell cycles. Science, 274 (5293): 1672-1677.

Segraves, N.L., Robinson, S.J., Garcia, D., Said, S.A., Fu, X., Schmitz, F.J., Pietraszkiewicz, H., Valeriote, F.A., and Crews, P. 2004. Comparison of fascaplysin and related alkaloids: a study of structures, cytotoxicities, and sources. J Nat Prod, 67: 783-792.

Seike, M., Gemma, A., Hosoya, Y., Hemmi, S., Taniguchi, Y., Fukuda, Y., Yamanaka, N., Kudoh, S. 2000. Increase in the frequency of p16INK4 gene inactivation by hypermethylation in lung cancer during the process of metastasis and its relation to the status of p53. Clin Cancer Res, 6(11 ): 4307-4313.

Senderowicz, A. M., and Sausville, E.A. 2000. Preclinical and clinical development of cyclin dependent kinase modulators. J Natl Cancer Inst, 92: 376-387.

Senderowicz A.M. 2003. Small-molecule cyclin-dependent kinase modulators. Oncogene, 22: 6609-6620. Senderowicz A. M. 2003a. Novel direct and indirect cyclin-dependent kinase modulators for the prevention and treatment of human neoplasms. Cancer Chemother. Pharmacol, 52 Suppl. 1 : S61-73.

Senderowicz A.M. 2005. Inhibitors of cyclin-dependent kinase modulators for cancer therapy. Prog. Drug Res, 63: 183-206. Serrano, M., Lin, A. W., McCurrach, M. E., Beach, D., Lowe, S.W. 1997. Induction of the p16INK4a senescence gene as a new therapeutic strategy for the treatment of rheumatoid arthritis. Cell, 1997 88(5): 593-602.

Shapiro, G.I., Edwards, CD., Kobzik, L., Godleski, J., Richards, W., Sugarbaker, D. J., Rollins, B.J. 1995. Cancer Res, 55(3): 505-9. Shtivelman, E., Sussman, J., Stokoe, D. 2002. A Role for Pl 3-Kinase and PKB Activity in the G2/M Phase of the Cell Cycle. Current Biology, 12: 919-924.

Simin, K., Wu, H., Lu, L₁ Pinkel, D., Albertson, D., Cardiff, R.D., Van Dyke, T. 2004. pRb inactivation in mammary cells reveals common mechanisms for tumor initiation and progression in divergent epithelia. PLoS Biol, 2(2): E22. Soni, R., Muller, L., Furet, P., Schoepfer, J., Stephan, C₁ Zumstein-Mecker, S., Fretz, H. and Chaudhuri, B. 2000. Inhibition of cyclin dependent kinase 4 (Cdk4) by fascaplysin, a marine natural product. Biochem Biophys Res Commun, 275: 877-884.

Soni, R., O'Reilly, T., Furet, P., Muller, L., Stephan, C, Zumstein-Mecker, S., Fretz, H., Fabbro, D., and Chaudhuri, B. 2001. Selective in vivo and in vitro effects of a small molecule inhibitor of cyclin dependent kinase 4. J Nat Cancer Inst, 93: 436-446.

Srinivas, P., Gopinath, G., Banerji, A., Dinakar, A., Srinivas, G. 2004. Plumbagin induces reactive oxygen species, which mediate apoptosis in human cervical cancer cells. MoI Carcinog, 40(4): 201-211.

Stark, G. R., and Taylor, W.R. 2006. Control of the G2/M transition. MoI Biotechnol, 32(3): 227-248.

Steffan, B., Watjen, W., Michels, G., Niering, P., Wray, V., Ebel, R., Edrada, R., Kahl, R., Proksch, P. 2005. Polyphenols from plants used in traditional Indonesian medicine (Jamu): uptake and antioxidative effects in rat H4IIE hepatoma cells. J Pharm Pharmacol, 57(2): 233-240.

Swanton, C. 2004. Cell-cycle targeted therapies. Lancet. Oncol. 5: 27-36.

Tarn, S. W., Shay, J.W., Pagano, M. 1994. Differential expression and cell cycle regulation of the cyclin-dependent kinase 4 inhibitor p16lnk4. Cancer Res, 54(22): 5816-5820.

Tashiro, T., Inaba, M., Kobayashi, T., Sakurai, Y., Maruo, K., Ohnishi, Y., Ueyama, Y, Nomura, T. 1989. Responsiveness of human lung cancer/nude mouse to antitumor agents in a model using clinically equivalent doses. Cancer Chemother Pharmacol, 24(3): 187-92.

Tenzer, A., and Pruschy, M. 2003. Potentiation of DNA-damage-induced cytotoxicity by G2 checkpoint abrogators. Curr Med Chem Anticancer Agents, 3: 35-46.

Trembley, J. H., Hu, D., Slaughter, C.A., Lahti, J. M., and Kidd, VJ. 2003. Casein kinase 2 interacts with cyclin-dependent kinase 11 (CDK11 ) in vivo and phosphorylates both the RNA polymerase Il carboxyl-terminal domain and CDK11 in vitro. J Biol Chem, 278: 2265-2270.

TuIu, U., Fagerstrom, C, Ferenz, N., Wadsworth, P. 2006. Molecular requirements for kinetochore-associated microtubule formation in mammalian cells. Curr Biol., 16(5): 536-41 Vermeulen, K., Van Bockstaele, D. R., Berneman, Z.N. 2003. The cell cycle: a review of regulation, deregulation and therapeutic targets in cancer. Cell Prolif, 36(3): 131-49.

Weinberg R.A. 1995. The retinoblastoma protein and cell cycle control. Cell, 81 : 323- 330.

Welburn, J. P., and Endicott, J.A. 2005. Inhibition of the cell cycle with chemical inhibitors: a targeted approach. Semin. Cell Dev Biol 16: 369-381.

Yang, X.L., Wang, A.H. 1999. Structural studies of atom-specific anticancer drugs acting on DNA. Pharmacol Ther, 83(3): 81-215.

Yu, Q., Sicinska, E., Geng, Y., Ahnstrom, M., Zagozdzon, A., Kong, Y., Gardner, h., Kiyokawa, H., Harris, L.N., Stal, O., and Sicinski., P. 2006. Requirment of CDK4 kinase function in breast cancer. Cancer Cell, 9: 23-32.

Zhai, S., Senderowicz, A.M., Sausville, E.A., and Figg, W.D. 2002. Flavopiridol, a novel cyclin-dependent kinase inhibitor, in clinical development. Ann Pharmacother, 36: 905- 911.

Claims

1. A compound of Formula I wherein

wherein each of R¹ to R⁴ may independently represent H, aryl, Het\ halo, CN₁ NO₂, C-_M2 alkyl, C_1-12 alkenyl, C_1-12 alkynyl, C_3-12 cycloalkyl or C_4-12 cycloalkenyl, which latter five groups are optionally substituted by one or more substituents selected from halo, nitro, CN, C_1-6 alkyl, C_1-6 alkenyl, C₁^ alkynyl, C_3-8 cycloalkyl (which latter three groups are optionally substituted by one or more substituents selected from OH, =0, halo, C_1-4 alkyl and C_1-4 alkoxy), OR⁸³, S(O)_nR⁸⁵, S(O)₂N(R^8c)(R^8d), N(R⁸⁶JS(O)₂R⁸', N(R^8g)(R^8h) and Het², and which C_3-12 cycloalkyl or C_4-12 cycloalkenyl groups may additionally be substituted by =0;

R⁵ is either not present, or represents one to four substituents on the fused benzene ring selected from aryl, Het³, halo, CN, NO₂, C_1-I2 alkyl, C_1-12 alkenyl, C_1-12 alkynyl, C_3--_I2 cycloalkyl or C_4-12 cycloalkenyl, which latter five groups are optionally substituted by one or more substituents selected from halo, nitro, CN₁ C_1-6 alkyl, C_1-6 alkenyl, C_1-6 alkynyl, C_3-8 cycloalkyl (which latter three groups are optionally substituted by one or more substituents selected from OH, =0, halo, C_1-4 alkyl and C_1-4 alkoxy), 0R^9a, S(O)_pR^9b, S(O)₂N(R^9c)(R^9d), N(R^9β)S(O)₂R^9f, N(R")(R^9h) and Het⁴, and which C_3-12 cycloalkyl or C_4-I2 cycloalkenyl groups may additionally be substituted by =0;

R⁶ represents H, Ci_-12 alkyl, C_3-12 cycloalkyl, C_3-12 cycloalkenyl (which latter three groups are optionally substituted by one or more substituents selected from halo, nitro, CN, C_1-6 alkyl, C_1-6 alkenyl, C_1-6 alkynyl, C_3-8 cycloalkyl (which latter three groups are optionally substituted by one or more substituents selected from OH₁ =0, halo, C_1-4 alkyl and C_1-4 alkoxy), OR^10a, S(O)_qR^10b, S(O)₂N(R^10cχR^10d), N(R^10β)S(O)₂R^10f, N(R¹⁰⁹)(R^10h), aryl and Het⁵, and which C_3-I2 cycloalkyl or C_4-I2 cycloalkenyl groups may additionally be substituted by =0);

R⁷ is either not present, or represents one to six substituents on the fused tetrahydropyridine ring selected from H₁ halo, CN, NO₂, C_1--_I2 alkyl, Ci_-12 alkenyl, C_1-12 alkynyl, C_3-I₂ cycloalkyl or C_4--_I2 cycloalkenyl, which latter five groups are optionally substituted by one or more substituents selected from halo, nitro, CN₁ Ci_-6 alkyl, Ci_-6 alkenyl, C_1-6 alkynyl, C_3-8 cycloalkyl (which latter three groups are optionally substituted by one or more substituents selected from OH, =O, halo, C_1-4 alkyl and C₁^ alkoxy), OR^11a, S(O)_rR^11b, S(O)₂N(R^11c)(R^11d), N(R^11e)S(O)₂R^11f, N(R¹ⁱ⁹)(R^11h), aryl and Het⁶, and which C_3-12 cycloalkyl or C_4-12 cycloalkenyl groups may additionally be substituted by

=0;

R^8a to R^8h, R^9a to R^9h, R^1Oa to R^1Oh and R^11a to R^11h independently represent, at each occurrence,

(a) H,

(b) C_1-I0 alkyl, C_2-io alkenyl, C_2-1O alkynyl (which latter three groups are optionally substituted by one or more substituents selected from halo, OH, C_1-6 alkoxy, aryl and Het⁷), (c) C_3--_I0 cycloalkyl, C_4-10 cycloalkenyl (which latter two groups are optionally substituted by one or more substituents selected from halo, OH, =0, C_1-6 alkyl, C_1-6 alkoxy, aryl and Het⁸),

(d) aryl or

(a) halo,

(b) CN,

(c) C_1-12 alkyl, C_1-I2 alkenyl, C_1-12 alkynyl, C_3-12 cycloalkyl or C_4--_I2 cycloalkenyl, which latter five groups are optionally substituted by one or more substituents selected from halo, nitro, CN, C_1-6 alkyl, C_1-6 alkenyl, C_1-6 alkynyl, C_3-8 cycloalkyl (which latter three groups are optionally substituted by one or more substituents selected from OH,

=0, halo, C_1-4 alkyl and CM alkoxy), OR^12a, S(O)_sR^12b, S(O)₂N(R^12c)(R^12d), N(R^12β)S(O)₂R^12f, N(R^12g)(R^12h), phenyl, naphthyl (which latter two groups are optionally susbtituted by one or more substituents selected from OH₁ halo, C_1-4 alkyl and Ci_-4 alkoxy) and Het¹⁰, and which C3-₁₂ cycloalkyl or C_4-12 cycloalkenyl groups may additionally be substituted by =0, (d) OR^13a,

(e) S(O)₁R¹³",

(f) S(O)₂N(R^13c)(R^13d),

(g) N(R^13e)S(O)₂R^13f, (h) N(R^13g)(R^13h), (i) phenyl (which latter group is optionally susbtituted by one or more substituents selected from OH, halo, Ci_-4 alkyl and C_1-4 alkoxy) or (j) Het¹¹;

_R ^i2a _{t0 R} ^i2h _{and R} ^i3a _{t0 R} ^i3h independently represent, at each occurrence, (a) H,

(b) C_1-12 alkyl, C_2-I2 alkenyl, C_2-I2 alkynyl, C₃-i₂ cycloalkyl, C_4-12 cycloalkenyl (which latter five groups are optionally substituted by one or more substituents selected from halo, OH, C_1-S alkyl, C_3-12 cycloalkyl, C_4-12 cycloalkenyl (which latter two groups are optionally substituted by one or more substituents selected from OH, =O, ha/o, C_1-4 alkyl and C_1-4 alkoxy), C_1-6 alkoxy, NH₂, N(H)-C_1-6 alkyl, N(C_1-6 alkyl)₂, phenyl (which latter group is optionally substituted by one or more substituents selected from OH, halo, C_1-4 alkyl and Ci_-4 alkoxy) and Het¹², and which C_3-I2 cycloalkyl or C_4-I2 cycloalkenyl groups may additionally be substituted by =0),

(c) phenyl (which latter group is optionally substituted by one or more substituents selected from OH, CN, halo, Ci_-6 alkyl and C_1-6 alkoxy) or

(a) halo,

(b) CN, (c) C₁.₁₂ alkyl, C_M2 alkenyl, C_1-I2 alkynyl, C_3-12 cycloalkyl or C_4-12 cycloalkenyl, which latter five groups are optionally substituted by one or more substituents selected from halo, nitro, CN, Ci_-6 alkyl, Ci_-6 alkenyl, Ci_-6 alkynyl, C_3-8 cycloalkyl (which latter three groups are optionally substituted by one or more substituents selected from OH, =0, halo, C₁-, alkyl and C_1-4 alkoxy), OR^14a, S(O)_uR^14b, S(O)₂N(R^14c)(R^14d), N(R^14e)S(O)₂R^14f, N(R¹⁴⁹)(R^14h), phenyl, naphthyl (which latter two groups are optionally susbtituted by one or more substituents selected from OH, halo, Ci_-4 alkyl and C_1-4 alkoxy) and Het^a, and which C3-12 cycloalkyl or C_4-i₂ cycloalkenyl groups may additionally be substituted by =0,

(d) 0R^15a,

(e) =0,

(f) S(O)_vR^15b, (g) S(O)₂N(R^15c)(R^15d),

(h) N(R^15e)S(O)₂R^15f,

(i) N(R¹⁵⁹)(R^15h),

O) phenyl (which latter group is optionally susbtituted by one or more substituents selected from OH, halo, Ci_-4 alkyl and C_M alkoxy) or (k) Het^b;

R^14a to R^14h and R^14a to R^14h independently represent, at each occurrence,

(a) H,

(b) C_1-12 alkyl, C_2-12 alkenyl, C_2-12 alkynyl, C_3-12 cycloalkyl, C_4-12 cycloalkenyl (which latter five groups are optionally substituted by one or more substituents selected from halo, OH, C_1-6 alkyl, C_3-12 cycloalkyl, C_4-12 cycloalkenyl (which latter two groups are optionally substituted by one or more substituents selected from OH, =0, halo, C_1-4 alkyl and C_1-4 alkoxy), C_1-6 alkoxy, phenyl (which latter group is optionally substituted by one or more substituents selected from OH, halo, C_1-4 alkyl and C_1-4 alkoxy) and Het^c, and which C_3-I2 cycloalkyl or C_4-I2 cycloalkenyl groups may additionally be substituted by =O),

(c) phenyl (which latter group is optionally substituted by one or more substituents selected from OH, halo, Ci_-4 alkyl and C_1-4 alkoxy) or

(e) Het^d; provided that R^1Sb or R^16b does not represent H when u or v, respectively is 1 or 2;

n, p, q, r, s, t, u and v independently represent O₁ 1 or 2;

or a pharmaceutically-acceptable derivative, salt, solvate or prodrug thereof.

2. A compound according to Claim 1 , having Formula Ib

wherein each of R¹ to R⁴ may independently represent H, halo, CN, NO₂, Ci-₁₂ alkyl, d.

₁₂ alkenyl, C_1-I2 alkynyl, C_3-12 cycloalkyl or C_4-12 cycloalkenyl, which latter five groups are optionally substituted by one or more substituents selected from halo, nitro, CN, C_1- ₆ alkyl, C_1-6 alkenyl, C_1-6 alkynyl, C_3-8 cycloalkyl (which latter three groups are optionally substituted by one or more substituents selected from OH, =0, halo, C_1-4 alkyl and C_1-4 alkoxy) or OR^8a;

R⁵ is either not present, or represents one to two substituents on the fused benzene ring selected from halo, CN, NO₂, C_1-12 alkyl, C_1-I2 alkenyl, C_1--_I2 alkynyl, C_3-I2 cycloalkyl or C_4-I2 cycloalkenyl, which latter five groups are optionally substituted by one or more substituents selected from halo, nitro, CN, C_1-6 alkyl, C_1-6 alkenyl, Ci_-6 alkynyl, C_3-8 cycloalkyl (which latter three groups are optionally substituted by one or more substituents selected from OH, =0, halo, C_1-4 alkyl and C_1-4 alkoxy) or OR^9a;

R⁶ represents H or C_1-3 alkyl;

R⁷ is not present; R^8a and R^9a independently represent, at each occurrence,

(a) H,

(b) C1-6 alky!, C_2-6 alkenyl, C_2-6 alkynyl (which latter three groups are optionally substituted by one or more substituents selected from halo, OH, C_1-6 alkoxy and aryl), or

(c) C_3-6 cycloalkyl, C_4-6 cycloalkenyl (which latter two groups are optionally substituted by one or more substituents selected from halo, OH, =0, C_1-6 alkyl, C₁-6 alkoxy and aryl);

(a) halo,

(b) CN, (c) Ci_-6 alkyl or C_3-6 cycloalkyl which latter two groups are optionally substituted by one or more substituents selected from halo, nitro, CN, Ct_-6 alkyl, C_3-B cycloalkyl (which latter group i.e. optionally substituted by one or more substituents selected from OH, =0, halo, Ci_-4 alkyl and C_1-4 alkoxy) or 0R^12a, or

(d) 0R^13a;

R^12a and R^13a independently represent, at each occurrence,

(a) H,

3. A compound according to Claim 1 or Claim 2, wherein R⁵ is either not present, or represents one substituent on the fused benzene ring selected from selected from halo, CN, NO₂, C_1-12 alkyl, C_1-12 alkenyl, C_1-12 alkynyl, C_3-12 cycloalkyl or C_4-12 cycloalkenyl, which latter five groups are optionally substituted by one or more substituents selected from halo, nitro, CN, C_1-6 alkyl, C_1-6 alkenyl, C_1-6 alkynyl, C_3-β cycloalkyl (which latter three groups are optionally substituted by one or more substituents selected from OH, =0, halo, Ci_-4 alkyl and C_1-4 alkoxy), 0R^9a.

4. A compound according to any of the preceding claims, wherein each of R¹ to R⁴ may independently represent H, halo, or C_1-6 alkyl; and

R⁵ is either not present, or represents one substituent on the fused benzene ring selected from selected from halo or Ci_-6 alkyl.

5. A compound according to any of the preceding claims, wherein each of R¹ to R⁴ may independently represent H, halo, or C_1-3 alkyl.

6. A compound according to any of the preceding claims, wherein each of R¹ to R⁴ may independently represent H or halo.

7. A compound according to any of the preceding claims, wherein R⁵ is not present, and R⁶ represents H.

8. A compound according to any of the preceding claims, wherein R¹ to R⁴ represent H.

9. A compound according to any of the preceding claims, wherein R¹ to R⁴ represent H; R⁵ is not present;

R⁶ represents H; and R⁷ is not present.

10. The compound biphenyl-2-yl-(4,4a,9,9a-tetrahydro-1H-beta-carbolin-2-yl)- methanone.

11. A compound of Formula II,

wherein each of R¹ to R⁴ may independently represent H, halo, CN, NO₂, C_t-I2 alkyl, d. ₁₂ alkenyl, C_1-12 alkynyl, C_3-12 cycloalkyl or C_4-12 cycloalkenyl, which latter five groups are optionally substituted by one or more substituents selected from halo, nitro, CN, C₁. ₆ alkyl, C_1-6 alkenyl, C^ alkynyl, C_3-8 cycloalkyl (which latter three groups are optionally substituted by one or more substituents selected from OH₁ =0, halo, C_1-4 alkyl and C_1-4 alkoxy), OR^7a, S(O)_pR^7b, S(O)₂N(R^7c)(R^7d), N(R^7e)S(O)₂R^7f, N(R⁷⁹)(R^7h), and which C_3-12 cycloalkyl or C_4-12 cycloalkenyl groups may additionally be substituted by =0;

R⁵ is either not present, or represents one to four substituents on the fused benzene ring selected from aryl, Het¹, halo, CN, NO₂, C_1-12 alkyl, C_1-12 alkenyl, C_1-12 alkynyl, C₃-_I2 cycloalkyl or C_4-12 cycloalkenyl, which latter five groups are optionally substituted by one or more substituents selected from halo, nitro, CN, C_1-6 alkyl, C_1-6 alkenyl, C_1-6 alkynyl, C_3-S cycloalkyl (which latter three groups are optionally substituted by one or more substituents selected from OH, =0, halo, C_1-4 alkyl and C_1-4 alkoxy), 0R^8a, S(O)_qR^8b, S(O)₂N(R^8c)(R^8d), N(R^8β)S(O)₂R^8f, N(R^8g)(R^8h) and Het², and which C_3-12 cycloalkyl or C_4-12 cycloalkenyl groups may additionally be substituted by =0;

R⁶ represents H, Ci_-I2 alkyl, C_3-12 cycloalkyl, C_3-12 cycloalkenyl (which latter three groups are optionally substituted by one or more substituents selected from halo, nitro, CN, C_1-6 alkyl, C_1-6 alkenyl, C_1-6 alkynyl, C_3-8 cycloalkyl (which latter three groups are optionally substituted by one or more substituents selected from OH, =O, halo, C_1-4 alkyl and C_1-4 alkoxy), 0R^9a, S(O)_rR^9b, S(O)₂N(R^9c)(R^9d), N(R⁹⁶JS(O)₂R⁹¹, N(R")(R^9h), aryl and Het³, and which C_3-12 cycloalkyl or C_4-12 cycloalkenyl groups may additionally be substituted by =0);

R^7a to R^7h, R^8a to R⁸" and R^9a to R^9h, independently represent, at each occurrence, (a) H, (b) C_1-10 alkyl, C_2-10 alkenyl, C_2-1O alkynyl (which latter three groups are optionally substituted by one or more substituents selected from halo, OH, C_1-6 alkoxy, aryl and Het⁴),

(c) C_3-10 cycloalkyl, C_4-io cycloalkenyl (which latter two groups are optionally substituted by one or more substituents selected from halo, OH₁ =O, C_1-6 alkyl, C-_I-6 alkoxy, aryl and Het⁵),

(d) aryl or

each aryl independently represents a C_6-1O carbocyclic aromatic group, which group may comprise either one or two rings and may be substituted by one or more substituents selected from

(a) halo,

(b) CN₁

(c) C_1-12 alkyl, C_1-12 alkenyl, C₁.1₂ alkynyl, C_3-I2 cycloalkyl or C_4-12 cycloalkenyl, which latter five groups are optionally substituted by one or more substituents selected from halo, nitro, CN, Ci_-6 alkyl, C_1-6 alkenyl, C_1-6 alkynyl, C_3-β cycloalkyl (which latter three groups are optionally substituted by one or more substituents selected from OH₁ =0, halo, C_1-4 alkyl and C_1-4 alkoxy), OR^10a, S(O),R^10b, S(O)₂N(R^10c)(R^10d), N(R^10e)S(O)₂R^10f, N(R^1Og)(R^1Oh), phenyl, naphthyl (which latter two groups are optionally susbtituted by one or more substituents selected from OH, halo, C₁^ alkyl and C_1-4 alkoxy) and Het⁷, and which C_3-12 cycloalkyl or C_4-12 cycloalkenyl groups may additionally be substituted by =0,

(d) 0R^11a,

(e) S(O)_uR^11b, (f) S(O)₂N(R^11o)(R^11d),

(g) N(R^11e)S(O)₂R^11f,

(h) N(R^11g)(R^11h),

(i) phenyl (which latter group is optionally susbtituted by one or more substituents selected from OH, halo, C_1-4 alkyl and C₁^ alkoxy) or G) Het⁸;

_R ⁱo^a _{t0 R} ⁱo^h _{and R} ^iia _{tø R}n^h j_nc|_ep_encj_entiy represent, at each occurrence,

(a) H,

(b) Ci_-12 alkyl, C_2-12 alkenyl, C₂.₁₂ alkynyl, C_3--_I2 cycloalkyl, C_4-I2 cycloalkenyl (which latter five groups are optionally substituted by one or more substituents selected from halo, OH, Ci_-6 alkyl, C_3-I2 cycloalkyl, C_4-12 cycloalkenyl (which latter two groups are optionally substituted by one or more substituents selected from OH, =O, halo, C_1-4 alkyl and C_1-4 alkoxy), C_1-6 alkoxy, NH₂, N(H)-C_1-6 alkyl, N(Ci_-6 alkyl)₂, phenyl (which latter group is optionally substituted by one or more substituents selected from OH, halo, C_1-4 alkyl and C_1-4 alkoxy) and Het⁹, and which C_3--_I2 cycloalkyl or C_4-12 cycloalkenyl groups may additionally be substituted by =0), (c) phenyl (which latter group is optionally substituted by one or more substituents selected from OH, CN, halo, Ci_-6 alkyl and C_1-6 alkoxy) or

(e) Het¹⁰, provided that R^10b or R^11b does not represent H when t or u, respectively is 1 or 2;

Het¹ to Het¹⁰ independently represent 4- to 14-membered heterocyclic or 5- to 10-membered heteroaromatic groups containing one or more heteroatoms selected from oxygen, nitrogen and/or sulfur, which heterocyclic groups may comprise one, two or three rings, and may be substituted by one or more substituents selected from (a) halo,

(b) CN₁

(c) C_1-12 alkyl, C1.12 alkenyl, C_1-I2 alkynyl, C3-12 cycloalkyl or C_4-I2 cycloalkenyl, which latter five groups are optionally substituted by one or more substituents selected from halo, nitro, CN, C_1-6 alkyl, C_1-6 alkenyl, C_1-6 alkynyl, C_3-8 cycloalkyl (which latter three groups are optionally substituted by one or more substituents selected from OH, =0, halo, C_1-4 alkyl and C_1-4 alkoxy), OR^12a, S(O)_vR^12b, S(O)₂N(R^12c)(R^12d), N(R^12e)S(O)₂R^12f, N(R^12g)(R^12h), phenyl, naphthyl (which latter two groups are optionally susbtituted by one or more substituents selected from OH, halo, C_1-4 alkyl and C_1-4 alkoxy) and Het^a, and which C_3-12 cycloalkyl or C_4-I2 cycloalkenyl groups may additionally be substituted by =0,

(d) OR¹³³,

(e) =0,

(f) S(O)_wR^13b,

(g) S(O)₂N(R^13c)(R^13d), (h) N(R^13e)S(O)₂R^13f,

(i) N(R^13g)(R^13h),

(j) phenyl (which latter group is optionally susbtituted by one or more substituents selected from OH₁ halo, C_1-4 alkyl and C_1-4 alkoxy) or (k) Het^b;

R^12a to R^12h and R^13a to R^13h independently represent, at each occurrence,

(a) H,

(b) C_1-12 alkyl, C_2-12 alkenyl, C_2-12 alkynyl, C_3--_I2 cycloalkyl, C_4--_I2 cycloalkenyl (which latter five groups are optionally substituted by one or more substituents selected from halo, OH, C_1-6 alkyl, C_3-12 cycloalkyl, C_4-12 cycloalkenyl (which latter two groups are optionally substituted by one or more substituents selected from OH, =0, halo, C_1-4 alkyl and C_1-4 alkoxy), C_1-6 alkoxy, phenyl (which latter group is optionally substituted by one or more substituents selected from OH, halo, C_1-4 alkyl and C_1-4 alkoxy) and Het^c, and which C_3-12 cycloalkyl or C_4-I? cycloalkenyl groups may additionally be substituted by O)₁

(c) phenyl (which latter group is optionally substituted by one or more substituents selected from OH, halo, C_1-4 alkyl and Ci_-4 alkoxy) or (e) Het^d; provided that R^12b or R^13b does not represent H when v or w, respectively is 1 or 2;

n, p, q, r, s, t, u, v and w independently represent O, 1 or 2;

Het^a to Het^d independently represent 5- or 6-membered heterocyclic groups containing one to four heteroatoms selected from oxygen, nitrogen and/or sulfur, which heterocyclic groups may be substituted by one or more substituents selected from halo, =0 and C₁-S alkyl; and

unless otherwise specified

or a pharmaceutically-acceptable derivative, salt, solvate or prodrug thereof.

12. A compound according to Claim 11 , wherein

each of R¹ to R⁴ may independently represent H, halo, CN, NO₂, C_1-12 alkyl, C_1- ₁₂ alkenyl, CM₂ alkynyl, C_3-I2 cycloalkyl or C_4-12 cycloalkenyl, which latter five groups are optionally substituted by one or more substituents selected from halo, nitro, CN, C_1- ₆ alkyl, C_1-6 alkenyl, C_1-6 alkynyl, C₃^ cycloalkyl (which latter three groups are optionally substituted by one or more substituents selected from OH, =0, halo, C_1-4 alkyl and Ci_-4 alkoxy) or 0R^7a;

R⁵ is either not present, or represents one to two substituents on the fused benzene ring selected from halo, CN, NO₂, C_1-12 alkyl, C_1-I2 alkenyl, C₁.^ alkynyl, C_3-I₂ cycloalkyl or C₄.i₂ cycloalkenyl, which latter five groups are optionally substituted by one or more substituents selected from halo, nitro, CN, C_1-6 alkyl, C_1-6 alkenyl, C_1-6 alkynyl, C_3-8 cycloalkyl (which latter three groups are optionally substituted by one or more substituents selected from OH₁ =0, halo, C_1-4 alkyl and C_1-4 alkoxy) or OR^8a;

R⁶ represents H;

R^7a and R⁸⁸ independently represent, at each occurrence,

(a) H,

(b) C₁^ alkyl, C_2-6 alkenyl, C_2-6 alkynyl (which latter three groups are optionally substituted by one or more substituents selected from halo, OH, C_1-6 alkoxy and aryl), or

(a) halo,

(b) CN, (c) Ci_-6 alkyl or C_3-6 cycloalkyl which latter two groups are optionally substituted by one or more substituents selected from halo, nitro, CN, C_1-6 alkyl, C_3-8 cycloalkyl (which latter group i.e. optionally substituted by one or more substituents selected from OH, =0, halo, C_1-4 alkyl and C_1-4 alkoxy) or 0R^11a, or

(d) 0R^12a;

R^11a and R^12a independently represent, at each occurrence,

(a) H,

(b) Ci_-6 alkyl, C_2-6 alkenyl, C_2-6 alkynyl (which latter three groups are optionally substituted by one or more substituents selected from halo, OH, C_1-6 alkoxy and aryl), or

(c) C_3-6 cycloalkyl, C_4-6 cycloalkenyl (which latter two groups are optionally substituted by one or more substituents selected from halo, OH, =0, C_1-6 alkyl, Ci_-6 alkoxy and aryl).

13. A compound according to Claim 11 or Claim 12, wherein R⁵ is either not present, or represents one substituent on the fused benzene ring selected from halo,

CN, NO₂, C_1-12 alkyl, C_1-12 alkenyl, C_1--_I2 alkynyl, C_3-12 cycloalkyl or C_4-12 cycloalkenyl, which latter five groups are optionally substituted by one or more substituents selected from halo, nitro, CN₁ Ci_-6 alkyl, C_1-6 alkenyl, C₁^ alkynyl, C_3-8 cycloalkyl (which latter three groups are optionally substituted by one or more substituents selected from OH, =0, halo, Ci_-4 alkyl and C_1-4 alkoxy), OR^9a.

14. A compound according to any of Claims 11-13, wherein R¹ to R⁴ represent H, halo, or C_1-6 alkyl;

R⁵ is either not present, or represents one substituent on the fused benzene ring selected from halo or C_1-6 alkyl.

15. A compound according to any of Claims 11-14, wherein each of R¹ to R⁴ may independently represent H, halo, or C₁^ alkyl.

16. A compound according to any of Claims 11-15, wherein each of R¹ to R⁴ may independently represent H or halo.

17. A compound according to any of Claims 11-16, wherein R⁵ is not present; and R⁶ represents H.

18. A compound according to any of Claims 11-17, wherein R¹ to R⁴ represent H.

19. A compound according to any of Claims 11-18, wherein R¹ to R⁴ represent H; R⁵ is not present;

R⁶ represents H; and R⁷ is not present.

20. The compound biphenyl-2-carboxylic acid [2-(1 H-indol-3-yl)-ethyl]-amide.

21. A pharmaceutical composition comprising a compound of Formula I as defined in any of Claims 1-10, or a compound of Formula Il as defined in any of Claims 11-20, and a pharmaceutically acceptable carrier, diluent or excipient.

22. A compound of Formula I as defined in any of Claims 1-10, or a compound of Formula Il as defined in any of Claims 11-20, for use in medicine.

23. A method of treating cancer in a patient, the method comprising administering a compound of Formula I as defined in any of Claims 1-10, or a compound of Formula Il as defined in any of Claims 11-20, to the patient.

24. A method of treating cancer in a patient, the method comprising administering to the patient a compound of Formula III,

wherein

each of R¹ to R⁴ may independently represent H, halo, CN, NO₂, CM₂ aikyl, CM₂ alkenyl, CM₂ alkynyl, C_3--_I2 cycloalkyl or C_4-I₂ cycloalkenyl, which latter five groups are optionally substituted by one or more substituents selected from halo, nitro, CN₁ Ci_-6 alkyl, C^₆ alkenyl, Ci_-6 alkynyl, Cs_-8 cycloalkyl (which latter three groups are optionally substituted by one or more substituents selected from OH, =0, halo, Ci_-4 alkyl and Ci_-4 alkoxy), OR^8a, S(O)_pR^8b, S(O)₂N(R^8c)(R^8d), N(R⁸⁶JS(O)₂R*. N(R^δ9)(R^8h), and which C_3-12 cycloalkyl or C_4-I2 cycloalkenyl groups may additionally be substituted by =0;

R^s is either not present, or represents one to four substituents on the fused benzene ring selected from aryl, Het\ halo, CN, NO₂, Ci_-12 alkyl, Ci_-12 alkenyl, CM₂ alkynyl, C_3-I2 cycloalkyl or C_4-I2 cycloalkenyl, which latter five groups are optionally substituted by one or more substituents selected from halo, nitro, CN, Ci_-6 alkyl, Ci_-6 alkenyl, C_1-6 alkynyl, C_3-S cycloalkyl (which latter three groups are optionally substituted by one or more substituents selected from OH, =0, halo, Ci_-4 alkyl and C_1-4 alkoxy), OR^9a, S(O)_qR^9b, S(O)₂N(R^9c)(R^9d), N(R⁹⁶JS(O)₂R* N(R")(R^9h) and Het², and which C_3-12 cycloalkyl or C_4-12 cycloalkenyl groups may additionally be substituted by =0;

R⁶ represents H, CM₂ alkyl, C_3-I2 cycloalkyl, C_3-I2 cycloalkenyl (which latter three groups are optionally substituted by one or more substituents selected from halo, nitro, CN, C_1-6 alkyl, C_1-6 alkenyl, C_1-6 alkynyl, C_3-β cycloalkyl (which latter three groups are optionally substituted by one or more substituents selected from OH, =0, halo, Ci_-4 alkyl and C_1-4 alkoxy), OR^10a, S(O)_rR^10b, S(O)₂N(R^10c)(R^10d), N(R^10e)S(O)₂R^10f, N(R¹⁰⁹)(R^10h), aryl and Het³, and which C₃.₁₂ cycloalkyl or C₄.₁₂ cycloalkenyl groups may additionally be substituted by =0);

R⁷ represents C_1-6 alkyl;

R^8a to R^8h, R^9a to R^9h and R^1Oa to R^1Oh, independently represent, at each occurrence, (a) H, (b) C_1-10 alkyl, C_2-1O alkenyl, C_2-i₀ alkynyl (which latter three groups are optionally substituted by one or more substituents selected from halo, OH, Ci_-6 alkoxy, aryl and Het⁵),

(c) C_3-10 cycloalkyl, C_4-10 cycloalkenyl (which latter two groups are optionally substituted by one or more substituents selected from halo, OH, =0, C_1-6 alkyl, Ci_-6 alkoxy, aryl and Het⁶),

(d) aryl or

each aryl independently represents a Ce_-10 carbocyclic aromatic group, which group may comprise either one or two rings and may be substituted by one or more substituents selected from (a) halo, (b) CN,

(c) C_1-12 alkyl, Ci_-12 alkenyl, C_1--_I2 alkynyl, C_3-i₂ cycloalkyl or C_4-I2 cycloalkenyl, which latter five groups are optionally substituted by one or more substituents selected from halo, nitro, CN, C_1-6 alkyl, Ci_-6 alkenyl, C_1-6 alkynyl, C_3-8 cycloalkyl (which latter three groups are optionally substituted by one or more substituents selected from OH, =0, halo, C_1-4 alkyl and Ci_-4 alkoxy), OR^11a, S(O),R^11b, S(O)₂N(R^11c)(R^11d), N(R^11e)S(O)₂R^11f, N(R^11g)(R^11h), phenyl, naphthyl (which latter two groups are optionally susbtituted by one or more substituents selected from OH, halo, Ci_-4 alkyl and Ci_-4 alkoxy) and Het⁸, and which C_3--_I2 cycloalkyl or C_4-12 cycloalkenyl groups may additionally be substituted by =0, (d) 0R^12a,

(e) S(O)_uR^12b,

(f) S(O)₂N(R^12c)(R^12d), (g) N(R^12e)S(O)₂R^12f, (h) N(R^12g)(R^12h),

(i) phenyl (which latter group is optionally susbtituted by one or more substituents selected from OH, halo, Ci_-4 alkyl and Ci_-4 alkoxy) or G) Het⁹;

_R ⁱⁱa _{t0 R} ^iih _{and R} ^i2a _{t0 R} ^i2h j_nc|_ep_encj_entiy represent, at each occurrence,

(a) H,

(b) C_1-12 alkyl, C_2-i2 alkenyl, C_2-12 alkynyl, C_3-I2 cycloalkyl, C_4-12 cycloalkenyl (which latter five groups are optionally substituted by one or more substituents selected from halo, OH, Ci_-6 alkyl, C_3-12 cycloalkyl, C_4-I₂ cycloalkenyl (which latter two groups are optionally substituted by one or more substituents selected from OH, =0, halo, C_1-4 alkyl and C_1-4 alkoxy), C_1-6 alkoxy, NH₂, N(H)-C_1-6 alkyl, N(C_1-6 alkyl)₂₎ phenyl (which latter group is optionally substituted by one or more substituents selected from OH, halo, Ci_-4 alkyl and C_1-4 alkoxy) and Het¹⁰, and which C_3-12 cycloalkyl or C_4-I2 cycloalkenyl groups may additionally be substituted by =0),

(c) phenyl (which latter group is optionally substituted by one or more substituents selected from OH, CN, halo, Ci_-6 alkyl and Ci_-6 alkoxy) or

(a) halo,

(b) CN,

(c) Ci_-12 alkyl, C_1-I2 alkenyl, C_1-I2 alkynyl, C_3-12 cycloalkyl or C_4-12 cycloalkenyl, which latter five groups are optionally substituted by one or more substituents selected from halo, nitro, CN, Ci_-6 alkyl, C_1-6 alkenyl, Ci_-6 alkynyl, C_3-8 cycloalkyl (which latter three groups are optionally substituted by one or more substituents selected from OH, =0, halo, Ci_-4 alkyl and C_1-4 alkoxy), 0R^13a, S(O)_vR^13b, S(O)₂N(R^13c)(R^13d), N(R^13e)S(O)₂R^13f, N(R^13g)(R^13h), phenyl, naphthyl (which latter two groups are optionally susbtituted by one or more substituents selected from OH, halo, C_1-4 alkyl and C_1-4 alkoxy) and Het^a, and which C_3-I2 cycloalkyl or C_4-I2 cycloalkenyl groups may additionally be substituted by =0,

(d) 0R^14a, (e) =0,

(f) S(O)_wR^14b,

(g) S(O)₂N(R^14c)(R^14d), (h) N(R^14β)S(O)₂R^14f, (i) N(R¹⁴⁹)(R^14h),

0) phenyl (which latter group is optionally susbtituted by one or more substituents selected from OH, halo, C_1-4 alkyl and C_1-4 alkoxy) or (k) Het^b;

R^13a to R^13h and R^14a to R^14h independently represent, at each occurrence,

(a) H,

(b) C_1-12 alkyl, C_2-I2 alkenyl, C₂.i₂ alkynyl, C_3-12 cycloalkyl, C_4-12 cycloalkenyl (which latter five groups are optionally substituted by one or more substituents selected from halo, OH, C_1-6 alkyl, C_3-12 cycloalkyl, C_4-12 cycloalkenyl (which latter two groups are optionally substituted by one or more substituents selected from OH, =0, halo, C_1-4 alkyl and Ci_-4 alkoxy), C_1-6 alkoxy, phenyl (which latter group is optionally substituted by one or more substituents selected from OH, halo, C_1-4 alkyl and C_1-4 alkoxy) and Hef, and which C_3-I2 cycloalkyl or C_4-I2 cycloalkenyl groups may additionally be substituted by =O), (c) phenyl (which latter group is optionally substituted by one or more substituents selected from OH, halo, C_1-4 alkyl and C_1-4 alkoxy) or (e) Het^d; provided that R^13b or R^14b does not represent H when v or w, respectively is 1 or 2;

n, p, q, r, s, t, u, v and w independently represent 0, 1 or 2;

unless otherwise specified

(i) alkyl, alkenyl, alkynyl, cycloalkyl, and cycloalkenyl groups, as well as the alkyl part of alkoxy groups, may be substituted by one or more halo atoms, and (ii) cycloalkyl and cycloalkenyl groups may comprise one or two rings and may additionally be ring-fused to one or two benzene rings; or a pharmaceutically-acceptable derivative, salt, solvate or prodrug thereof.

25. A methods according to Claim 24, wherein

each of R¹ to R⁴ may independently represent H, halo, CN, NO₂, C_1-I2 alkyl, C_1- i₂ alkenyl, C_1-12 alkynyl, C_3-12 cycloalkyl or C_-M2 cycloalkenyl, which latter five groups are optionally substituted by one or more substituents selected from halo, nitro, CN, C_1- ₆ alkyl, C_1-6 alkenyl, C_1-6 alkynyl, C_3-8 cycloalkyl (which latter three groups are optionally substituted by one or more substituents selected from OH, =0, halo, C_1-4 alkyl and C_1-4 alkoxy) or OR^8a;

R⁵ is either not present, or represents one to two substituents on the fused benzene ring selected from halo, CN, NO₂, C_1-12 alkyl, Ci_-12 alkenyl, C_1-12 alkynyl, C_3--_I2 cycloalkyl or C_4-12 cycloalkenyl, which latter five groups are optionally substituted by one or more substituents selected from halo, nitro, CN, C_1-6 alkyl, C_1-6 alkenyl, C_1-6 alkynyl, C_3-6 cycloalkyl (which latter three groups are optionally substituted by one or more substituents selected from OH, =0, halo, C_1-4 alkyl and C_1-4 alkoxy) or 0R^9a;

R⁶ represents H;

R⁷ represents methyl, ethyl, propyl or isopropyl;

R^8a and R^9a independently represent, at each occurrence,

each aryl independently represents a C_6-Io carbocyclic aromatic group, which group may comprise either one or two rings and may be substituted by one or more substituents selected from (a) halo,

(b) CN, (c) C_1-6 alkyl or C_3-6 cycloalkyl which latter two groups are optionally substituted by one or more substituents selected from halo, nitro, CN, Ci_-6 alkyl, C_3-8 cycloalkyl (which latter group i.e. optionally substituted by one or more substituents selected from OH, =0, halo, Ci_-4 alkyl and C_1-4 alkoxy) or OR^11a, or (d) OR^12a;

R^11a and R^12a independently represent, at each occurrence,

(a) H,

(b) Ci-₆ alkyl, C₂-e alkenyl, C_2-6 alkynyl (which latter three groups are optionally substituted by one or more substituents selected from halo, OH, C_1-6 alkoxy and aryl), or

(c) C_3-6 cycloalkyl, C_4-6 cycloalkenyl (which latter two groups are optionally substituted by one or more substituents selected from halo, OH, =0, C_1-6 alkyl, C_1-6 alkoxy and aryl).

26. A method according to Claim 24 or Claim 25, wherein R⁵ is either not present, or represents one substituent on the fused benzene ring selected from halo, CN, NO₂, C_1-I2 alkyl, C_1-I2 alkenyl, C_1--_I2 alkynyl, C_3-12 cycloalkyl or C_4-12 cycloalkenyl, which latter five groups are optionally substituted by one or more substituents selected from halo, nitro, CN, C_1-6 alkyl, C_1-6 alkenyl, C_1-6 alkynyl, C_3-8 cycloalkyl (which latter three groups are optionally substituted by one or more substituents selected from OH, =0, halo, C_1-4 alkyl and C_1-4 alkoxy), 0R^9a.

27. A method according to any of Claims 24-26, wherein each of R¹ to R⁴ may independently represent H, halo, or C_1-6 alkyl; and

28. A method according to any of Claims 24-27, wherein each of R¹ to R⁴ may independently represent H₁ halo, or C_1-3 alkyl.

29. A method according to any of Claims 24-28, wherein each of R¹ to R⁴ may independently represent H or halo.

30. A method according to any of Claims 24-29, wherein R⁵ is not present; R⁶ represents H; and

R⁷ represents methyl or ethyl.

31. A method according to any of Claims 24-30, wherein R¹ to R⁴ represent H.

32. A method according to any of Claims 24-31 , wherein R¹ to R⁴ represent H;

R⁵ is not present; R⁶ represents H; and R⁷ is methyl.

33. A method according to Claim 24 wherein the compound is biphenyl-4-carboxylic acid [2-(1 H-indol-3-yl)-ethyl]-methyl-amide.

34. A method of treating cancer according to any of Claims 23-33 wherein the patient is a human patient.

35. A method of treating cancer according to any of Claims 23-34 wherein the cancer to be treated is refractory to Cdk4 inhibition.

36. A method of treating cancer according to any of Claims 23-35 wherein the cancer is a cytoxic drug-resistant cancer.

37. A method according to Claim 36 wherein the cytoxic drug-resistant cancer is selected from breast cancer, colorectal cancer, pancreatic cancer, lung cancer, myeloma, glioblastoma and retinoblastoma.

38. A method according to Claim 36 or 37 further comprising the prior step of determining whether the cancer is cytoxic drug-resistant.

39. A method of treating cancer according to any of Claims 23-35 wherein the cancer is p53-negative.

40. A method according to Claim 39 wherein the p53-negative cancer is selected from leukaemia, prostate cancer, soft tissue sarcoma, hepatocarcinoma, or ovarian, bladder, head and neck, colon, cervical and lung carcinoma .

41. A method according to Claim 39 or 40 further comprising the prior step of determining whether the cancer is p53-negative.

42. A method of treating cancer according to any of Claims 23-35 wherein the cancer is retinoblastoma (Rb) negative.

43. A method according to Claim 42 wherein the Rb-negative cancer is selected from retinoblastoma, small cell lung cancer and non-small cell lung cancer.

44. A method according to Claim 42 or 43 further comprising the prior step of determining whether the cancer is Rb-negative.

45. Use of a compound of Formula I as defined in any of Claims 1-10, or a compound of Formula Il as defined in any of Claims 11-20, or a compound of Formula III as defined in any of Claims 24-33, in the preparation of a medicament for treating cancer in a patient.

46. Use according to Claim 44 wherein the cancer is as defined in any of Claims 35-37, 39-40 and 42-43.

47. Use according to Claim 45 or 46 wherein the patient to be treated is one who has been determined to have a cytotoxic drug resistant cancer.

48. Use according to Claim 45 or 46 wherein the patient to be treated is one who has been determined to have a p53-negative cancer.

49. Use according to any of Claims 45 or 46 wherein the patient to be treated is one who has been determined to have an Rb-negative cancer.

50. A compound of Formula I as defined in any of Claims 1-10, or a compound of Formula Il as defined in any of Claims 11-20, or a compound of Formula III as defined in any of Claims 24-33, for use in treating cancer in a patient.

51. A compound according to Claim 50 wherein the cancer is as defined in any of Claims 35-37, 39-40 and 42-43.

52. A pharmaceutical composition comprising (i) a compound of Formula I as defined in any of Claims 1-10, or a compound of Formula Il as defined in any of Claims 11-20, or a compound of Formula III as defined in any of Claims 24-33; and (ii) a further anticancer agent, and a pharmaceutically acceptable carrier, diluent or excipient, wherein the further anticancer agent is selected from cisplatin, carboplatin, 5- flurouracil, paclitaxel, mitomycin C₁ doxorubicin and gemcitabine, Velcade^®, Glivec^®, a COX-2 inhibitor and mitoxantrone.

53. A method of treating cancer in a patient, the method comprising administering (i) a compound of Formula I as defined in any of Claims 1-10, or a compound of Formula Il as defined in any of Claims 11-20, or a compound of Formula III as defined in any of Claims 24-33, and (ii) a further anticancer agent as defined in Claim 52, to the patient.

54. Use of (i) a compound of Formula I as defined in any of Claims 1-10, or a compound of Formula Il as defined in any of Claims 11-20, or a compound of Formula III as defined in any of Claims 24-33, and (ii) a further anticancer agent as defined in Claim 52, in the preparation of a medicament for treating cancer in a patient.

55. Use of a compound of Formula I as defined in any of Claims 1-10, or a compound of Formula Il as defined in any of Claims 11-20, or a compound of Formula III as defined in any of Claims 24-33, in the preparation of a medicament for treating cancer in a patient who is administered a further anticancer agent as defined in Claim 52.

56. Use of a further anticancer agent as defined in Claim 52 in the preparation of a medicament for treating cancer in a patient who is administered a compound of Formula I as defined in any of Claims 1-10, or a compound of Formula Il as defined in any of Claims 11-20, or a compound of Formula 111 as defined in any of Claims 24-33.

57. A method or a use according to any of Claims 53-56 wherein the cancer is as defined in any of Claims 35-37, 39-40 and 42-43.

58. A method of identifying an anticancer agent, or a lead compound for the identification of an anticancer agent, the method comprising: providing a candidate compound which is a compound of Formula IV;

wherein

each of R¹ to R⁵ may independently represent H, aryl, Het\ halo, CN, NO₂, C_1-12 alkyl, C_1-12 alkenyl, C_1-12 alkynyl, C_3-I2 cycloaikyl or C_4-12 cycloalkenyl, which latter five groups are optionally substituted by one or more substituents selected from halo, nitro, CN, C_1-6 alkyl, C_1-6 alkenyl, C_1-6 alkynyl, C_3-8 cycloaikyl (which latter three groups are optionally substituted by one or more substituents selected from OH, =O, halo, C_1-4 alkyl and C_1-4 alkoxy), OR^9a, S(O)_nR⁹", S(O)₂N(R^9c)(R^9d), N(R^9e)S(O)₂R^9f, N(R^9g)(R^9h) and Het², and which C_3-12 cycloaikyl or C_4-12 cycloalkenyl groups may additionally be substituted by =0;

R⁶ is either not present, or represents one to four substituents on the fused benzene ring selected from aryl, Het³, halo, CN, NO₂, C_1-I2 alkyl, C_1-12 alkenyl, Ci-i₂ alkynyl, C_3-I2 cycloaikyl or C_4--_I2 cycloalkenyl, which latter five groups are optionally substituted by one or more substituents selected from halo, nitro, CN, C_1-6 alky/, C_1-6 alkenyl, C_1-6 alkynyl, C_3-8 cycloaikyl (which latter three groups are optionally substituted by one or more substituents selected from OH, =0, halo, C_1-4 alkyl and C_1-4 alkoxy), OR^10a, S(O)_pR^10b, S(O)₂N(R^10o)(R^10d), N(R^10e)S(O)₂R^10f, N(R^10g)(R^10h) and Het⁴, and which C₃-_I2 cycloaikyl or C_4-12 cycloalkenyl groups may additionally be substituted by

=0;

R⁷ represents H, C_1-I2 alkyl, C_3-12 cycloaikyl, C_3-I2 cycloalkenyl (which latter three groups are optionally substituted by one or more substituents selected from halo, nitro, CN, C_1-6 alkyl, C_1-6 alkenyl, C_1-6 alkynyl, C_3-8 cycloaikyl (which latter three groups are optionally substituted by one or more substituents selected from OH, =0, halo, C_1-4 alkyl and C_1-4 alkoxy), 0R^11a, S(O)_qR^11b, S(O)₂N(R^11c)(R^11d), N(R^11θ)S(O)₂R^11f,

N(R¹ⁱ⁹)(R^11h), aryl and Het⁵, and which C_3-12 cycloaikyl or C_4-12 cycloalkenyl groups may additionally be substituted by =0); R⁸ is either not present, or represents one to six substituents on the fused tetrahydropyridine ring selected from H, halo, CN, NO₂, C_1-12 alkyl, C_1-12 alkenyl,

C_1-12 alkynyl, C3-₁₂ cycloalkyl or C_4-12 cycloalkenyl, which latter five groups are optionally substituted by one or more substituents selected from halo, nitro, CN, C_1-6 alkyl, C₁^ alkenyl, Ci_-6 alkynyl, C_3-B cycloalkyl (which latter three groups are optionally substituted by one or more substituents selected from OH, =0, halo, C_1-4 alkyl and C_1-4 alkoxy), OR^12a, S(O)_rR^12b, S(O)₂N(R^12c)(R^12d), N(R^12θ)S(O)₂R^12f, N(R^12g)(R^12h), aryl and Het⁶, and which C₃-₁₂ cycloalkyl or C_4-I₂ cycloalkenyl groups may additionally be substituted by =0;

R^9a to R^9h, R^1Oa to R^10h, R^11a to R^11h and R^12a to R^12h independently represent, at each occurrence,

(a) H,

(b) C-_I-10 alkyl, C_2-io alkenyl, C_2-10 alkynyl (which latter three groups are optionally substituted by one or more substituents selected from halo, OH, Ci_-6 alkoxy, aryl and

Het⁷),

(c) C₃.₁t) cycloalkyl, C_4-10 cycloalkenyl (which latter two groups are optionally substituted by one or more substituents selected from halo, OH, =0, C₁-₆ alkyl, C₁-₆ alkoxy, aryl and Het⁸), (d) aryl or (e) Het⁹, provided that R^9b, R^10b, R^11b or R^12b does not represent H when n, p, q or r, respectively is 1 or 2;

each aryl independently represents a C_6-Io carbocyclic aromatic group, which group may comprise either one or two rings and may be substituted by one or more substituents selected from

(a) halo,

(b) CN, (c) CM₂ alkyl, C_1-12 alkenyl, C_1-12 alkynyl, C_3-12 cycloalkyl or C_4-I2 cycloalkenyl, which latter five groups are optionally substituted by one or more substituents selected from halo, nitro, CN, Ci_-6 alkyl, C_1-6 alkenyl, Ci_-6 alkynyl, C_3-8 cycloalkyl (which latter three groups are optionally substituted by one or more substituents selected from OH, =0, halo, C1.4 alkyl and C_1-4 alkoxy), 0R^13a, S(O)_sR^13b, S(O)₂N(R^13c)(R^13d), N(R^13e)S(O)₂R^13f, N(R¹³⁹)(R^13h), phenyl, naphthyl (which latter two groups are optionally susbtituted by one or more substituents selected from OH, halo, Ci_-4 alkyl and C₁-₄ alkoxy) and Het¹⁰, and which C_3-12 cycloalkyl or C_4-I2 cycloalkenyl groups may additionally be substituted by =0,

(d) OR^14a,

(e) S(O),R^14b, (f) S(O)₂N(R^14c)(R^14d), (g) N(R^14β)S(O)₂R^14f, (h) N(R¹⁴⁹)(R^14h),

(i) phenyl (which latter group is optionally susbtituted by one or more substituents selected from OH, halo, Ci_-4 alkyl and C_1-4 alkoxy) or G) Het¹¹;

R^13a to R^13h and R^14a to R^14h independently represent, at each occurrence,

(a) H,

(b) C_1-12 alkyl, C₂-_I2 alkenyl, C_2-I2 alkynyl, C_3-12 cycloalkyl, C_4-12 cycloalkenyl (which latter five groups are optionally substituted by one or more substituents selected from halo, OH, C_1-6 alkyl, C_3-I2 cycloalkyl, C_4-I2 cycloalkenyl (which latter two groups are optionally substituted by one or more substituents selected from OH, =0, halo, Ci_-4 alkyl and C₁^ alkoxy), C_1-6 alkoxy, NH₂, N(H)-C_1-6 alkyl, N(C_1-6 alkyl)₂, phenyl (which latter group is optionally substituted by one or more substituents selected from OH, halo, Ci_-4 alkyl and C_1-4 alkoxy) and Het¹², and which C_3-12 cycloalkyl or C_4-12 cycloalkenyl groups may additionally be substituted by =0),

(a) halo,

(b) CN,

(c) C_1-12 alkyl, C_1-I2 alkenyl, C_1-12 alkynyl, C_3-12 cycloalkyl or C_4-12 cycloalkenyl, which latter five groups are optionally substituted by one or more substituents selected from halo, nitro, CN, C_1-6 alkyl, C_1-6 alkenyl, C_1-6 alkynyl, C_3-8 cycloalkyl (which latter three groups are optionally substituted by one or more substituents selected from OH,

=0, halo, C_1-4 alkyl and C_1-4 alkoxy), 0R^15a, S(O)_uR^15b, S(O)₂N(R^15c)(R^15d), N(R^15e)S(O)₂R^15f, N(R^15g)(R^15h), phenyl, naphthyl (which latter two groups are optionally susbtituted by one or more substituents selected from OH, halo, Ci_-4 alkyl and Ci_-4 alkoxy) and Het^a, and which C_3-12 cycloalkyl or C_4-12 cycloalkenyl groups may additionally be substituted by =0, (d) OR^16a,

(e) =0,

(f) S(O)_vR^16b,

(g) S(O)₂N(R^16c)(R^16d), (h) N(R^16e)S(O)₂R^16f, (i) N(R¹⁶⁹)(R^16h),

(j) phenyl (which latter group is optionally susbtituted by one or more substituents selected from OH, halo, Ci_-4 alkyl and Ci_-4 alkoxy) or (k) Het^b;

R^15a to R^15h and R^16a to R^16h independently represent, at each occurrence,

(a) H,

(b) Ci_-I2 alkyl, C_2-I2 alkenyl, CM₂ alkynyl, C_3-I2 cycloalkyl, C_4-I2 cycloalkenyl (which latter five groups are optionally substituted by one or more substituents selected from halo, OH, C_1-6 alkyl, C_3-12 cycloalkyl, C_4-I2 cycloalkenyl (which latter two groups are optionally substituted by one or more substituents selected from OH₁ =O, halo, Ci_-4 alkyl and Ci_-4 alkoxy), C_1-6 alkoxy, phenyl (which latter group is optionally substituted by one or more substituents selected from OH, halo, C^ alkyl and Ci_-4 alkoxy) and Het^c, and which C_3-I2 cycloalkyl or C_4-I2 cycloalkenyl groups may additionally be substituted by =O), (c) phenyl (which latter group is optionally substituted by one or more substituents selected from OH, halo, Ci_-4 alkyl and Ci_-4 alkoxy) or (e) Het^d; provided that R^15b or R^16b does not represent H when u or v, respectively is 1 or 2;

n, p, q, r, s, t, u and v independently represent 0, 1 or 2;

Het^a to Het^d independently represent 5- or 6-membered heterocyclic groups containing one to four heteroatoms selected from oxygen, nitrogen and/or sulfur, which heterocyclic groups may be substituted by one or more substituents selected from halo, =O and Ci_-6 alkyl; and

59. A method according to Claim 57 wherein, in the compound of Formula IV, R¹ represents phenyl.

60. A method according to Claim 57 or 58, wherein the compound of Formula IV is a compound of Formula I, as defined in Claims 1-10.

61. A method according to any of Claims 57-59, wherein the compound of Formula IV is not (3-Methoxy-phenyl)-(1 ,3,4,9-tetrahydro-D-carbolin-2-yl)-methanone.

62. A method of identifying an anticancer agent, or a lead compound for the identification of an anticancer agent, the method comprising: providing a candidate compound which is a compound of Formula V

wherein

one of R¹ or R² must represent phenyl, and the other substituent may represent H, halo, CN, NO₂, C_M2 alkyl, C_1-I2 alkenyl, C_1-12 alkynyl, C_3-12 cycloalkyl or C_4-I₂ cycloalkenyl, which latter five groups are optionally substituted by one or more substituents selected from halo, nitro, CN, C₁^ alkyl, C_1-6 alkenyl, C_1-6 alkynyl, C_3-8 cycloalkyl (which latter three groups are optionally substituted by one or more substituents selected from OH, =0, halo, C_1-4 alkyl and C_1-4 alkoxy), OR^9a, S(O)_nR^9b, S(O)₂N(R^9c)(R^9d), N(R^9β)S(O)₂R^9f, N(R")(R^9h), and which C₃.₁₂ cycloalkyl or C_4-I2 cycloalkenyl groups may additionally be substituted by =0;

each of R³ to R⁵ may independently represent H₁ halo, CN, NO₂, C_1-I2 alkyl, C_1- ₁₂ alkenyl, C_1-12 alkynyl, C_3-I₂ cycloalkyl or C₄-₁₂ cycloalkenyl, which latter five groups are optionally substituted by one or more substituents selected from halo, nitro, CN, C₁.

₆ alkyl, Ci_-6 alkenyl, C_1-6 alkynyl, C_3-8 cycloalkyl (which latter three groups are optionally substituted by one or more substituents selected from OH, =O, halo, C_1-4 alkyl and C_1-4 alkoxy), OR^10a, S(O)_pR^10b, S(O)₂N(R^10c)(R^10d), N(R^10e)S(O)₂R^10f, N(R¹°⁹)(R^10h), and which C_3--_I2 cycloalkyl or C_4--_I2 cycloalkenyl groups may additionally be substituted by

=0;

R⁶ is either not present, or represents one to four substituents on the fused benzene ring selected from aryl, Het¹, halo, CN, NO₂, CM₂ alkyl, C_1-12 alkenyl, CM₂ alkynyl, C_3-12 cycloalkyl or C_4-12 cycloalkenyl, which latter five groups are optionally substituted by one or more substituents selected from halo, nitro, CN, Ci_-6 alkyl, Ci_-6 alkenyl, Ci_-6 alkynyl, C_3-B cycloalkyl (which latter three groups are optionally substituted by one or more substituents selected from OH, =0, halo, Ci_-4 alkyl and Ci_-4 alkoxy), 0R^11a, S(0)_qR^m, S(0)₂N(R^11c)(R^11d), N(R^11e)S(O)₂R^11f, N(R^11g)(R^11h) and Het², and which C_3-I2 cycloalkyl or C_4-I2 cycloalkenyl groups may additionally be substituted by =0;

R⁷ represents H, C_M2 alkyl, C_3-I2 cycloalkyl, C_3-I2 cycloalkenyl (which latter three groups are optionally substituted by one or more substituents selected from halo, nitro, CN,

alkenyl, Ci_-6 alkynyl, C_3-8 cycloalkyl (which latter three groups are optionally substituted by one or more substituents selected from OH, =0, halo, Ci_-4 alkyl and C_1-4 alkoxy), 0R^12a, S(0)_rR^12b, S(O)₂N(R^12c)(R^12d), N(R^12e)S(O)₂R^12f, N(R^12g)(R^12h), aryl and Het³, and which C_3-12 cycloalkyl or C_4-12 cycloalkenyl groups may additionally be substituted by =0);

R⁸ represents H, C_M2 alkyl, C_3-I2 cycloalkyl, C_3-I2 cycloalkenyl (which latter three groups are optionally substituted by one or more substituents selected from halo, nitro, CN, C_1-6 alkyl, C_1-6 alkenyl, C_1-6 alkynyl, C_3-8 cycloalkyl (which latter three groups are optionally substituted by one or more substituents selected from OH, =0, halo, C_1-4 alkyl and C_1-4 alkoxy), 0R^13a, S(O)_sR^13b, S(O)₂N(R^13c)(R^13d), N(R^13e)S(O)₂R^13f, N(R^13g)(R^13h), aryl and Het⁴, and which C_3-12 cycloalkyl or C_4-12 cycloalkenyl groups may additionally be substituted by =0); R^9a to R^9h, R^1Oa to R^1Oh, R^11a to R^11h, R^12a to R^12h and R^13a to R^13h independently represent, at each occurrence, (a) H, (b) Ci_-10 alkyl, C_2-i₀ alkenyl, C_2-10 alkynyl (which latter three groups are optionally substituted by one or more substituents selected from halo, OH, Ci_-6 alkoxy, aryl and Het⁷),

(c) C_3-10 cycloalkyl, C_4--_I0 cycloalkenyl (which latter two groups are optionally substituted by one or more substituents selected from halo, OH, =0, Ci_-6 alkyl, C_1-6 alkoxy, aryl and Het⁸),

(d) aryl or

(e) Het⁵, provided that R^9b, R^1Ob, R^11b or R^12b does not represent H when n, p, q, r or s respectively is 1 or 2;

each aryl independently represents a Ce-₁₀ carbocyclic aromatic group, which group may comprise either one or two rings and may be substituted by one or more substituents selected from (a) halo, (b) CN,

(c) C_1-12 alkyl, C_1^12 alkenyl, C_1-I2 alkynyl, C_3-12 cycloalkyl or C_4-12 cycloalkenyl, which latter five groups are optionally substituted by one or more substituents selected from halo, nitro, CN, C_1-6 alkyl, C_1-6 alkenyl, C_1-6 alkynyl, C_3-8 cycloalkyl (which latter three groups are optionally substituted by one or more substituents selected from OH, =0, halo, C_1-4 alkyl and C_M alkoxy), OR^14a, S(O),R^14b, S(O)₂N(R^14c)(R^14d), N(R^14e)S(O)₂R^14f, N(R¹⁴⁹)(R^14h), phenyl, naphthyl (which latter two groups are optionally susbtituted by one or more substituents selected from OH, halo, C₁^ alkyl and Ci_-4 alkoxy) and Het⁶, and which C_3-12 cycloalkyl or C_4--_I2 cycloalkenyl groups may additionally be substituted by =0, (d) OR^15a,

(e) S(O)_uR^15b,

(f) S(O)₂N(R^15c)(R^15d),

(g) N(R^15β)S(O)₂R^15f, (h) N(R^15g)(R^15h), (i) phenyl (which latter group is optionally susbtituted by one or more substituents selected from OH, halo, C_1-4 alkyl and C_1-4 alkoxy) or

(j) Het⁶; R^14a to R^14h and R^15a to R^15h independently represent, at each occurrence,

(a) H,

(b) C1-12 alkyl, C_2-I₂ alkenyl, C_2-I2 alkynyl, C_3-12 cycloalkyl, C_4-i₂ cycloalkenyl (which latter five groups are optionally substituted by one or more substituents selected from halo, OH, C_1-6 alkyl, C_3-12 cycloalkyl, C_4-12 cycloalkenyl (which latter two groups are optionally substituted by one or more substituents selected from OH, =0, halo, C_1-4 alkyl and C_1-4 alkoxy), C_1-6 alkoxy, NH₂, N(H)-Ci_-6 alkyl, N(C_1-6 alkyl)₂, phenyl (which latter group is optionally substituted by one or more substituents selected from OH, halo, C_1-4 alkyl and C_1-4 alkoxy) and Het⁷, and which C_3-12 cycloalkyl or C_4-12 cycloalkenyl groups may additionally be substituted by =O),

Het¹ to Het⁸ independently represent 4- to 14-membered heterocyclic or 5- to 10-membered heteroaromatic groups containing one or more heteroatoms selected from oxygen, nitrogen and/or sulfur, which heterocyclic groups may comprise one, two or three rings, and may be substituted by one or more substituents selected from

(a) halo,

(b) CN,

(c) C_1-I2 alkyl, Ci_-12 alkenyl, C_1-12 alkynyl, C_3-12 cycloalkyl or C_4--I₂ cycloalkenyl, which latter five groups are optionally substituted by one or more substituents selected from halo, nitro, CN₁ C_1-6 alkyl, Ci_-6 alkenyl, Ci_-6 alkynyl, C_3-8 cycloalkyl (which latter three groups are optionally substituted by one or more substituents selected from OH, =0, halo, Ci_-4 alkyl and Ci_-4 alkoxy), 0R^16a, S(O)_vR^16b, S(O)₂N(R^16c)(R^16d), N(R^16e)S(O)₂R^16f, N(R^16g)(R^16h), phenyl, naphthyl (which latter two groups are optionally susbtituted by one or more substituents selected from OH, halo,

alkyl and Ci_-4 alkoxy) and Het^a, and which C_3-12 cycloalkyl or C_4-12 cycloalkenyl groups may additionally be substituted by =0,

(d) 0R^17a,

(e) =0,

(f) S(O)_wR^17b, (g) S(O)₂N(R^17c)(R^17d), (h) N(R^17e)S(O)₂R^17f, (i) N(R^17g)(R^17h), (j) phenyl (which latter group is optionally susbtituted by one or more substituents selected from OH, halo, Ci_-4 alkyl and Ci_-4 alkoxy) or (k) Het^b;

R^16a to R^16h and R^17a to R^17h independently represent, at each occurrence,

(a) H,

(b) Ci_-12 alkyl, C_2-i2 alkenyl, C_2-I2 alkynyl, C_3-I2 cycloalkyl, C_4-I2 cycloalkenyl (which latter five groups are optionally substituted by one or more substituents selected from halo, OH, Ci-6 alkyl, C_3-I₂ cycloalkyl, C_4-I2 cycloalkenyl (which latter two groups are optionally substituted by one or more substituents selected from OH, =0, halo, Ci_-4 alkyl and Ci_-4 alkoxy), C_1-6 alkoxy, phenyl (which latter group is optionally substituted by one or more substituents selected from OH, halo, Ci_-4 alkyl and C₁-₄ alkoxy) and Het^c, and which C_3-I₂ cycloalkyl or C_4-I2 cycloalkenyl groups may additionally be substituted by =0), (c) phenyl (which latter group is optionally substituted by one or more substituents selected from OH, halo, Ci_-4 alkyl and Ci_-4 alkoxy) or (e) Het^d; provided that R^16b or R^17b does not represent H when v or w, respectively is 1 or 2;

n, p, q, r, s, t, u, v and w independently represent 0, 1 or 2;

Het^a to Het^d independently represent 5- or 6-membered heterocyclic groups containing one to four heteroatoms selected from oxygen, nitrogen and/or sulfur, which heterocyclic groups may be substituted by one or more substituents selected from halo, =0 and Ci_-6 alkyl; and

unless otherwise specified

63. A method according to according to Claim 61 , wherein, in the compound of

Formula V, R¹ represents phenyl.

64. A method according to Claim 61 or 62, wherein the compound of Formula V is a compound of Formula Il as defined in Claims 1-20.

65. A method according to Claim 61 , wherein, in the compound of Formula V, R² represents phenyl.

66. A method according to Claim 61 or Claim 64, wherein the compound of Formula V is a compound of Formula III as defined in Claims 23-33.

67. A method according to any of Claims 60, 64 and 65 wherein the compound of Formula V is not biphenyl-4-carboxylic acid [2-(1 H-indol-3-yl)-ethyl]-methyl-amide.

68. A method according to any of Claims 57-66 wherein the identified compound is modified, and the modified compound is tested for at least one anticancer activity which is not dependent upon Cdk4 inhibition.

69. A method according to any of Claims 57-67 wherein the at least one anticancer activity is selected from blocking cells at the G₂/M phase of the cell division cycle; inducing apoptosis in cancer cells; inducing apoptosis in SV40 large T antigen- transformed cells; inhibition of tubulin polymerisation; reducing the long-term survival of SV40 large T antigen-transformed cells; acting as an anti-oxidant; downregulating cyclin B1 and Cdk1 and upregulating p21 and p27 in p53+ cancer cells; upregulating cyclin B1 levels in p53-mutant and p53-null cancer cells; inhibiting the growth of Rb-null or Rb-mutant cancer cells in vitro; and reducing the efficiency of colony formation in Rb-null or Rb-mutant cancer cells in vitro.

70. A method according to any of Claims 57-68 further comprising the step of determining an optimal solvent for the identified compound or the modified compound.

71. A method according to any of Claims 57-69 wherein the identified compound or the modified compound is tested for the ability to inhibit the growth of cancer cells in vitro.

72. A method according to any of Claims 57-70 wherein the identified compound or the modified compound is tested for the ability to reduce the reducing the efficiency of colony formation of cancer cells in vitro.

73. A method according to any of Claims 57-71 wherein the identified compound or the modified compound is tested for efficacy in an animal model of cancer.

74. A method according to Claim 72 wherein the animal model of cancer is a model of an Rb-negative cancer.

75. A method according to any of Claims 57-73 further comprising the step of synthesising, purifying and/or formulating the identified compound or the modified compound.

76. A method for preparing an anticancer compound of Formula IV or Formula V, the method comprising identifying a compound using the method according to any of Claims 57-73 and synthesising, purifying and/or formulating the identified compound.

77. A method of making a pharmaceutical composition comprising mixing the compound identified using a method according to any of Claims 57-73 with a pharmaceutically acceptable carrier, excipient or diluent.