HK1101399B

HK1101399B - Inhibitors of cyclin-dependent kinases, compositions and uses related thereto

Info

Publication number: HK1101399B
Application number: HK07109307.2A
Authority: HK
Inventors: Nicholas Bockovich; Arthur F. Kluge; Chris Oalmann; Krishna K. Murthi; Siya Ram; Zhongguo Wang; Jianxing Huang
Original assignee: Agennix Usa Inc.
Priority date: 2003-12-23
Filing date: 2004-12-21
Publication date: 2009-12-04

Description

Cyclin dependent kinase inhibitors and compositions and related uses thereof

I. Field of the invention

The present invention relates generally to compounds useful as inhibitors of cyclin dependent kinases (cdks), pharmaceutical compositions containing such compounds, methods of formulating pharmaceutical compositions, or methods of treating cancer, or proliferative diseases or other diseases with such compounds, as well as intermediates and processes for preparing such compounds.

Background of the invention

One of the most important and fundamental processes in biology is cell division mediated by the cell cycle. This process ensures the controlled production of progeny cells with specific biological functions. It is a highly regulated phenomenon, responding to cellular signals of various intracellular and external sources. The complex network of tumor promoting and suppressor gene products is a key component of this cell signaling process. Overexpression of tumor promoting components or subsequent loss of tumor suppressor products leads to uncontrolled cell proliferation and tumor production (Pardee, Science 246: 603-608, 1989.) cyclin-dependent kinases play a key role in the regulation of the cell cycle machinery. These complexes consist of two components: catalytic subunits (kinases) and regulatory subunits (cyclins). To date, nine kinase subunits (cyclin-dependent kinases 1-9) and several regulatory subunits (cyclins A-H, K, N and T) have been identified. Each kinase is associated with a specific regulatory ligand, which constitutes the active catalytic moiety. Each transition of the cell cycle is regulated by a specific cyclin-dependent kinase complex: (ii) modulation of G1/S by cyclin dependent kinase 2/cyclin E, cyclin dependent kinase 4/cyclin D1 and cyclin dependent kinase 6/cyclin D2; (ii) modulates S/G2 by cyclin dependent kinase 2/cyclin a and cyclin dependent kinase/cyclin a; G2/M is regulated by cyclin-dependent kinase/cyclin D. The synergistic activity of these kinases directs individual cells through the process of replication, ensuring the viability of each progeny batch (Sherr, Cell 73: 1059-.

There is increasing evidence that tumor development is associated with cyclin-dependent kinase-related dysfunction. Over-expression of cyclin-regulated proteins and subsequent kinase hyperactivity has been associated with several cancers (Jiang, Proc. Natl. Acad. Sci. USA 90: 9026-. Endogenous, highly specific protein inhibitors of cyclin-dependent kinases have recently been found to have a significant effect on cell proliferation (Kamb et al, Science 264; 436. 440, 1994; Beach, Nature 336: 701-704, 1993). These include p 16. sup. INK4 (cyclin dependent kinase 4/D1 inhibitor), p21CIP1 (non-specific cyclin dependent kinase inhibitor) and p27KIP1 (cyclin dependent kinase 2/E inhibitor). The recent crystal structure of p27, which binds to cyclin-dependent kinase 2/A, shows how these proteins effectively inhibit kinase activity by multiple interactions with cyclin-dependent kinase complexes (Pavletich, Nature 382: 325-. These proteins help regulate the cell cycle by specifically interacting with their corresponding cyclin-dependent kinase complexes. Cells lacking these inhibitors are prone to uncontrolled growth and tumor formation. These evidences have led to a positive search for small molecule inhibitor therapeutics of the cdk family.

Summary of the invention

The present invention describes compounds which are potent inhibitors of kinases enzymes known as cyclin dependent kinases. The present invention provides methods of treating cancer or other proliferative diseases or other diseases by administering a therapeutically effective amount of at least one compound of the present invention, or its isomer, prodrug, tautomer, pharmaceutically acceptable salt, N-oxide or stereoisomer thereof. The invention also provides methods of treating cancer or other proliferative diseases or other disorders by administering a therapeutically effective combination of at least one compound of the invention and another anti-cancer or anti-proliferative drug.

In certain embodiments, the present invention provides compounds having the structure of formula I or an isomer, prodrug, tautomer, pharmaceutically acceptable salt, N-oxide, or stereoisomer thereof:

wherein

R₈Represents a substituted or unsubstituted heterocycle or a substituted or unsubstituted morpholino, substituted or unsubstituted piperazinyl or substituted or unsubstituted cyclohexyl group;

f represents (CH)₂)_nWherein n is an integer from 1 to 6, in some embodiments n is 1;

q represents a substituted or unsubstituted secondary amino substituent, a substituted or unsubstituted tertiary amino substituent, or a substituted or unsubstituted nitrogen-containing heterocycle.

In some embodiments, n is 1.

In some embodiments, Q in formula I represents a tertiary amino substituent, such as a dialkylamine. In some embodiments, Q in formula I represents a substituted or unsubstituted nitrogen-containing heterocycle such as morpholine, piperidine, piperazine, or pyrrolidine. In some embodiments, Q represents a nitrogen-containing heteroaryl ring, a tertiary amino substituent, or a substituted or unsubstituted nitrogen-containing heterocycle.

In some embodiments, R₈Represents:

wherein Z is O or NR "; and is

R' represents H or lower alkyl.

In some embodiments, the compounds having the structure of formula I do not include one or more of the following compounds:

in some embodiments, the compounds of formula I include one or more of the compounds listed in the tables. For example, the compounds of formula I may include one or more of compounds B1-B20 and C2.

As noted above, in some embodiments, suitable substituents, at each occurrence, can independently include alkyl, oxo, acylamino, hydroxy, carbonyl, sulfonyl, ester, amide, NR ", hydroxyalkyl, alkoxyalkyl, aryl, heterocyclyl, cycloalkyl, or oligo (ethylene glycol). In some embodiments, when Q represents a secondary amino substituent, suitable substituents include alkyl, alkoxyalkyl, hydroxyalkyl and hydroxyalkoxyalkyl. One skilled in the art will readily recognize that the listed substituents are not exhaustive and that many other suitable substituents may be used.

In certain embodiments, the present invention provides a compound having the structure of formula II or an isomer, prodrug, tautomer, pharmaceutically acceptable salt, N-oxide, or stereoisomer thereof:

wherein

B represents M_nR₈；

Ar represents an aryl or heteroaryl ring;

v represents O, S or N-CN;

w represents O, S or NR ";

r' independently at each occurrence represents H, lower alkyl or a metal counterion;

r "independently at each occurrence represents H or lower alkyl;

R₅representative H, P (═ O) (OR')₂Or M_nQ；

R₆Represents H, OH or M_nQ, provided that R is₅And R₆Only one of them represents H;

R₇represents H, halogen, hydroxy, lower alkyl or lower alkoxy;

R₈represents a substituted or unsubstituted alkyl, alkenyl, alkynyl, alkoxy, aryl, heteroaryl, cycloalkyl, heterocyclyl or amine;

m independently at each occurrence represents a substituted or unsubstituted methylene group (including C (═ O) and C (═ S)), NR ", O, S, S (O) or S (O)₂)；

When present in B, n represents an integer from 1 to 4, when present in R₅Wherein n represents an integer of 0 to 6 when present in R₆Wherein n represents an integer of 1 to 3; and is

Q represents substituted or unsubstituted: tertiary amino substituents or nitrogen-containing heterocycles.

In some embodiments, R₈Represents a substituted or unsubstituted morpholino, piperazinyl or cyclohexyl group.

In some embodiments, R "represents H.

In some embodiments, R₅Represents M_nQ。

In some embodiments, M attached to Q represents CH₂、S(O₂) C (═ S), or C (═ O).

In some embodiments, the linkage is to QM represents CH₂。

In some embodiments, M attached to Q represents C (═ O).

In some embodiments, M attached to Q represents a substituted NR ".

In some embodiments, Q represents a substituted or unsubstituted nitrogen-containing heterocycle.

In some embodiments, Q represents a substituted or unsubstituted tertiary amino group.

In some embodiments, R₈Represents a substituted or unsubstituted morpholino, piperazinyl or cyclohexyl group. In some embodiments, R "represents H. And in some embodiments, at least one occurrence of M represents CH₂Or substituted NR ", or when attached to Q, represents CH₂、S(O₂) C (═ S), or C (═ O).

In some embodiments, Q represents a substituted or unsubstituted nitrogen-containing heteroaryl ring. In some embodiments, Q represents a substituted or unsubstituted nitrogen-containing heterocycle. In some embodiments, Q represents a substituted or unsubstituted tertiary amino group. In some embodiments, Q represents a substituted or unsubstituted secondary amino group.

In certain embodiments, the present invention relates to compounds having the following structure or an isomer, prodrug, tautomer, pharmaceutically acceptable salt, N-oxide, or stereoisomer thereof:

C1。

C5。

in some embodiments, the invention provides one or more compounds described herein in purified or synthetic form.

wherein

B represents M_nR₈；

Ar represents an aryl or heteroaryl ring;

v represents O, S or N-CN;

w represents O, S, S (O)₂)、C(＝O)、C(＝S)、CH₂Or NR ";

r "independently at each occurrence represents H or lower alkyl;

r' "represents H or optionally substituted lower alkyl;

R₅represents M_nJK；

R₆Represents H, OH or M_nQ；

R₇Represents H, halogen, hydroxy, lower alkyl or lower alkoxy;

R₈represents a substituted or unsubstituted alkyl, alkenyl, or alkynyl group,Alkynyl, alkoxy, aryl, heteroaryl, cycloalkyl, heterocyclyl or amine;

j represents C (═ O), C (═ S) or SO₂；

K represents OR ', N (R')₂Or N (R') SO₂R”；

When present in B, n represents an integer from 1 to 7, when present in R₅Wherein n represents an integer of 0 to 6 when present in R₆Wherein n represents an integer of 1 to 3; and is

Q represents substituted or unsubstituted: a nitrogen-containing heteroaryl ring, a secondary amino substituent, a tertiary amino substituent, or a nitrogen-containing heterocycle.

In some embodiments, R₈Represents a substituted or unsubstituted morpholino, piperazinyl or cyclohexyl group. In some embodiments, R "represents H.

In some embodiments, M attached to Q represents CH₂Substituted NR', S (O)₂) C (═ S), or C (═ O).

In some embodiments, R "represents H.

In some embodiments, R₅Represents M_nQ。

In some embodiments, M attached to Q is C (═ O).

In some embodiments, M attached to Q represents CH₂。

In some embodiments, M attached to Q represents a substituted NR ".

In some embodiments, Q represents a substituted or unsubstituted tertiary amino substituent.

In some embodiments, the substituents at each occurrence independently comprise alkyl, oxo, acylamino, hydroxy, carbonyl, sulfonyl, ester, amide, NR ", hydroxyalkyl, alkoxyalkyl, aryl, heterocyclyl, cycloalkyl, or oligo (ethylene glycol).

wherein

B represents M_nR₈；

Ar represents an aryl or heteroaryl ring;

v represents O, S or N-CN;

w represents O, S, S (O)₂)、C(＝O)、C(＝S)、CH₂Or NR ";

r "independently at each occurrence represents H or lower alkyl;

r' "represents H or optionally substituted lower alkyl;

R₅representative H, P (═ O) (OR')₂、M_nJK or M_nQ；

R₇independently at each occurrence, represents H, halogen, hydroxy, lower alkyl or lower alkoxy;

j represents C (═ O), C (═ S) or SO₂；

K represents OR ', N (R')₂Or N (R') SO₂R”；

M independently at each occurrence represents a substituted or unsubstituted methylene group (including C (═ O) and C (═ S)), NR ", O, S, S (O), S (O)₂) Or CH₂；

Q represents substituted or unsubstituted: a nitrogen-containing heteroaryl ring, a secondary amino substituent, a tertiary amino substituent, or a nitrogen-containing heterocycle;

with the following conditions: excluding compounds having the structure of formula IIa:

wherein

W and Z independently represent O or NR ";

r "independently at each occurrence represents H or lower alkyl;

R₅representative H, P (═ O) (OR')₂Or M_nQ；

R₇independently at each occurrence, represents H, halogen, lower alkyl or lower alkoxy; m independently at each occurrence represents a substituted or unsubstituted methylene group (including C (═ S) and C (═ O)), NR ", O, S, S (O), S (O)₂)；

n represents an integer of 1 to 5; and is

Q represents a nitrogen-containing heteroaryl ring, a tertiary amino substituent, or a substituted or unsubstituted nitrogen-containing heterocycle.

In some embodiments, Q in formula IIa represents a tertiary amino substituent, such as a dialkylamine. In some embodiments, Q in formula IIa represents a substituted or unsubstituted nitrogen-containing heterocycle, such as morpholine, piperidine, piperazine, or pyrrolidine. In some embodiments, Q represents a nitrogen-containing heteroaryl ring, a tertiary amino substituent, or a substituted or unsubstituted nitrogen-containing heterocycle.

In some embodiments, in formula II,

b represents M_nR₈；

Ar represents an aryl or heteroaryl ring;

v represents O, S or N-CN;

w represents C (═ O), C (═ S), S (O)₂) Or CH₂；

R' independently at each occurrence represents H, lower alkyl, a metal counterion or an alkaline earth metal counterion;

r "independently at each occurrence represents H or lower alkyl;

r' "represents H or optionally substituted lower alkyl;

R₅representative H, P (═ O) (OR')₂、M_nJK or M_nQ；

R₇represents H, halogen, hydroxy, lower alkyl or lower alkoxy;

j represents C (═ O), C (═ S) or SO₂；

K represents OR ', N (R')₂Or N (R') SO₂R”；

M independently at each occurrence represents a substituted or unsubstituted methylene group (including C (═ S) and C (═ O)), NR ", O, S, S (O) or S (O)₂)；

In some embodiments, Q represents a tertiary amino substituent, such as a dialkylamine, or a substituted or unsubstituted nitrogen-containing heterocycle, such as morpholine, piperidine, piperazine, or pyrrolidine.

In some embodiments, in formula II,

b represents M_nR₈；

Ar represents an aryl or heteroaryl ring;

v represents O, S or N-CN;

w represents O, S, S (O)₂)、C(＝O)、C(＝S)、CH₂Or NR ";

r "independently at each occurrence represents H or lower alkyl;

r' "represents H or optionally substituted lower alkyl;

R₅representative H, P (═ O) (OR')₂、M_nJK or M_nQ；

R₇represents H, halogen, hydroxy, lower alkyl or lower alkoxy;

j represents C (═ O), C (═ S) or SO₂；

K represents OR ', N (R')₂Or N (R') SO₂R”；

Q represents a substituted or unsubstituted secondary amino substituent.

In some embodiments, in formula II,

b represents M_nR₈；

Ar represents an aryl or heteroaryl ring;

v represents O, S or N-CN;

w represents O, S, S (O)₂)、C(＝O)、C(＝S)、CH₂Or NR ";

r "independently at each occurrence represents H or lower alkyl;

r' "represents H or optionally substituted lower alkyl;

R₅represents M_nJK, provided that R₅Is not CH₂COOH；

R₆Represents H, OH or M_nQ；

R₇Represents H, halogen, hydroxy, lower alkyl or lower alkoxy;

j represents C (═ O), C (═ S) or SO₂；

K represents OR ', N (R')₂Or N (R') SO₂R”；

In some embodiments, in formula II, Q is a substituted or unsubstituted nitrogen-containing heteroaryl ring and R is₈May represent a substituted or unsubstituted morpholino, piperazinyl or cyclohexyl group. In formula II, R "may represent H.

M may also represent CH₂. In some embodiments, in formula II, W represents CH₂And at least one M represents a substituted NR'.

In some embodiments, in formula II, Q represents a substituted or unsubstituted secondary amino group. In some embodiments, in formula II, Q represents a substituted or unsubstituted tertiary amino group. In some embodiments, in formula II, Q represents a substituted or unsubstituted nitrogen-containing heterocycle.

In some embodiments, in formula II, Q represents substituted or unsubstituted: a nitrogen-containing heteroaryl ring, a tertiary amino substituent, or a nitrogen-containing heterocycle.

In some embodiments, in formula II, R₅Represents M_nQ, and Q represents substituted or unsubstituted: a nitrogen-containing heteroaryl ring, a tertiary amino substituent, or a nitrogen-containing heterocycle.

In some embodiments, in formula II, Q represents a substituted or unsubstituted tertiary amino group.

In some embodiments, in formula II, Q represents a substituted or unsubstituted nitrogen-containing heterocycle.

In some embodiments, in formula II, R₅Represents M_nQ, and Q represents a substituted or unsubstituted secondary amino group.

In some embodiments, in formula II, R₅Represents M_nQ, and Q represents a substituted or unsubstituted nitrogen-containing heteroaryl ring.

In some embodiments, in formula II, R₈Represents a substituted or unsubstituted morpholino, piperazinyl or cyclohexyl group.

In some embodiments, in formula II, R "represents H.

In some embodiments, in formula II, W represents CH₂。

In some embodiments, in formula II, when attached to Q, M is CH₂、S(O₂) C (═ S), or C (═ O).

In some embodiments, in formula II, when attached to Q, M is CH₂。

In some embodiments, in formula II, M attached to Q is CH₂、S(O₂) C (═ S), or C (═ O).

In some embodiments, in formula II, V is O, M represents NH, and R₈Has the following structure:

wherein Z represents O or NR'.

In some embodiments, AR represents a phenyl ring, and all occurrences of R are₆And R₇Represents H.

In certain embodiments, the present invention provides a compound having the structure of formula V or a prodrug, isomer, tautomer, pharmaceutically acceptable salt, N-oxide, or stereoisomer thereof:

wherein

R₈Represents a substituted or unsubstituted heterocyclic ring;

q represents substituted or unsubstituted: a secondary amino substituent, a tertiary amino substituent, or a substituted or unsubstituted nitrogen-containing heterocycle.

As noted above, in some embodiments, R₈May represent a morpholino or piperazinyl ring.

In some embodiments, Q may represent piperazine, morpholine, piperidine, pyridine, pyrrole, oxazole, isoxazole, imidazole or pyrazole, as described above.

Some embodiments include a compound selected from a34, a36, a37, a44, a46, and a76-a82, or a prodrug, isomer, tautomer, pharmaceutically acceptable salt, N-oxide, or stereoisomer thereof.

Some embodiments include a compound selected from a47, a49, a51, and a82, or a prodrug, isomer, tautomer, pharmaceutically acceptable salt, N-oxide, or stereoisomer thereof.

In some embodiments, W represents O, S (O)₂)、C(＝O)、C(＝S)、S、CH₂Or NR'.

As noted above, in some embodiments, W represents substituted or unsubstituted: a nitrogen-containing heteroaryl ring, a tertiary amino substituent, or a nitrogen-containing heterocycle.

In certain embodiments, suitable substituents, at each occurrence, can independently include alkyl, oxo, acylamino, hydroxy, carbonyl, sulfonyl, ester, amide, NR ", hydroxyalkyl, alkoxyalkyl, aryl, heterocyclyl, cycloalkyl, or oligo (ethylene glycol). In certain embodiments, wherein Q represents a secondary amino substituent, suitable substituents include alkyl, alkoxyalkyl, hydroxyalkyl, and hydroxyalkoxyalkyl. Those skilled in the art will readily recognize that the list of substituents is not exhaustive and that many other suitable substituents may be used.

Certain embodiments may include pharmaceutical compositions comprising a pharmaceutically acceptable excipient and any type of compound disclosed herein, while certain embodiments include methods of treating a hyperproliferative disorder comprising administering to an animal any type of compound disclosed herein.

In certain embodiments, the compounds disclosed herein can be used in methods of inhibiting cell proliferation, such methods comprising contacting a cell with a compound of the type disclosed herein; or to a method of treating a viral infection, such as an infection caused by Human Immunodeficiency Virus (HIV), such method comprising administering to a mammal a compound of the type disclosed herein. Certain embodiments include methods of treating or preventing chemotherapy or radiation therapy-induced alopecia, such methods comprising administering to a mammal a compound of the type disclosed herein in combination with one or more chemotherapy or radiation therapy. The compounds disclosed herein may also be used for the preparation of a medicament.

The compounds of the invention are also useful in the treatment of disorders such as hyperproliferative disorders. The compounds may be administered to a human or animal. The compounds of the invention are useful for inhibiting cell proliferation, for example by contacting a proliferating cell with the compound. The compounds of the present invention are also useful for treating viral infections by administering the compounds to a mammal.

The drugs formed using the compounds of the present invention are useful as drugs for treating or preventing disorders (e.g., hyperproliferative disorders), viral infections, chemotherapy-induced alopecia, diseases associated with cyclin-dependent kinase activity, and the like. The medicament may be for treating any of the conditions described herein.

Various methods of using the compounds of the present invention are available. For example, methods may be employed to inhibit cyclin dependent kinases, comprising administering to a host in need of such treatment a therapeutically effective amount of any of the compounds. Methods may also be employed for treating a condition associated with a cyclin dependent kinase, comprising administering to a host in need of such treatment a therapeutically effective amount of the compound. The invention also relates to methods of treating humans or other animals. In some embodiments, a composition comprising a therapeutically effective amount of one or more compounds of the present invention can be used to treat a human or other animal.

In certain embodiments, the present invention provides a novel pharmaceutical composition comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of a compound of formula (I) or (II) or any other compound disclosed herein, or an isomer, prodrug, tautomer, pharmaceutically acceptable salt, N-oxide, or stereoisomer thereof.

In another embodiment, the present invention provides a novel method of treating cancer or other proliferative diseases or other diseases comprising administering to a host in need of such treatment a therapeutically effective amount of a compound of formula (I) or (II) or any other compound disclosed herein, or an isomer, prodrug, tautomer, pharmaceutically acceptable salt, N-oxide or stereoisomer thereof.

In another embodiment, the present invention provides a novel method of treating cancer or other proliferative diseases or other diseases comprising administering to a host in need of such treatment a therapeutically effective amount of: (a) a compound of formula (I) or (II) or any other compound disclosed herein, or an isomer, prodrug, tautomer, pharmaceutically acceptable salt, N-oxide, or stereoisomer thereof; and (b) at least one compound selected from the group consisting of an anticancer agent and an antiproliferative agent.

As described herein, the inhibitors of the invention inhibit the cell cycle machinery and are therefore useful in regulating cell cycle progression and ultimately controlling cell growth and differentiation. Such compounds are useful for treating a disease associated with excessive cell proliferation in a patient, such as cancer, psoriasis, immune diseases involving unwanted leukocyte proliferation; can be used for treating restenosis and other smooth muscle cell diseases. Such compounds are also useful for inhibiting human immunodeficiency virus type I (HIV-I) transcription (Wang et al, J.virology 75: 7266-.

Also described herein are compounds of the invention useful for the preparation of medicaments useful for the treatment of diseases such as those discussed herein.

Brief description of the drawings

Figure 1 shows the effect of exposure to compound a37 in the following cases: (a) cell cycle analysis of HCT-116 cells by PIFACS analysis; (b) induction of PARP cleavage.

FIG. 2 responds with (a) dose; and (b) time course illustrates the irreversible effect of compound a37 on HCT-116 tumor cell clonogenic (clongeneic) survival.

FIG. 3 is a graph showing the irreversible effect of Compound B16 on clonogenic survival of HCT-116 tumor cells, expressed as a time course.

FIG. 4 shows the reduced viability of arrested tumor (HCT-116) cells exposed to Compound A37 compared to arrested normal (IMR90) cells exposed to the same compound.

FIG. 5 represents the results obtained from a HCT-116 xenograft tumor assay using various compounds of the invention.

FIG. 6 shows the results of an A2780 xenograft tumor assay using Compound A37, from (a) the time course of tumor size at various doses; and (b) table representation of significant metrics in the experiment.

FIG. 7 shows the results of a PC3 xenograft tumor assay with Compound A37, from (a) the time course of tumor size at various doses; and (b) significance scale in the test.

FIG. 8 shows the results of an A2780 xenograft tumor assay using Compound B16, from (a) the time course of tumor size at various doses; and (b) significance scale in the test.

FIG. 9 shows CDK₂Example of results obtained with cyclin E bound to a CM 5-inhibitor loaded chip. K_DCalculated from these data, equal to 8, 0+/-2, 8 nM.

Detailed description of exemplary embodiments

The present invention relates to novel cyclin dependent kinase inhibitors (cdks), particularly but not exclusively inhibitors of cdk/cyclin complexes. As described herein, the inhibitors of the invention inhibit the cell cycle machinery and are therefore useful in regulating cell cycle progression and ultimately controlling cell growth and differentiation. Such compounds are useful for treating diseases associated with excessive cell proliferation in a patient, for example, treating cancer, psoriasis, immune diseases involving unwanted leukocyte proliferation; can be used to treat restenosis and other smooth muscle cell disorders, etc., as discussed in more detail below.

In one embodiment, the present invention provides compounds having the structure of formula I, including isomers, prodrugs, tautomers, pharmaceutically acceptable salts, N-oxides, or stereoisomers thereof:

wherein

In some embodiments, R₈Represents:

wherein Z is O or NR "; and is

R' represents H or lower alkyl.

In another embodiment, the present invention also provides compounds having the structure of formula II, including isomers, prodrugs, tautomers, pharmaceutically acceptable salts, N-oxides, or stereoisomers thereof:

wherein

B represents M_nR₈；

Ar represents an aryl or heteroaryl ring such as a phenyl ring;

v represents O, S or N-CN, preferably O or S;

w represents O, S, S (O)₂)、C(＝O)、C(＝S)、CH₂Or NR ";

r' independently for each occurrence represents H, lower alkyl or a metal counterion such as an alkali or alkaline earth metal counterion;

r "independently for each occurrence represents H or lower alkyl, preferably H;

r' "represents H or optionally substituted lower alkyl, preferably with a substituent selected from ester, amide, acylamino or acyloxy;

R₅representative H, P (═ O) (OR')₂、M_nJK or M_nQ；

R₆Represents H, OH or M_nQ, with the preferred proviso that R₅And R₆One and only one represents H;

at each occurrence, R₇Independently represents H, halogen, hydroxy, lower alkyl, such as methyl, or lower alkoxy, such as methoxy;

j represents C (═ O), C (═ S) or SO₂；

K represents OR ', NR ' OR N (R ') SO₂R”；

At each occurrence, M independently represents a substituted or unsubstituted methylene group (e.g., substituted with lower alkyl, oxo, hydroxy, etc.),NR', O, S, S (O) or S (O)₂) Preferably NR' or CH₂Or when bound to W or Q, represents CH₂、S(O₂) C (═ S) or C (═ O);

when present in B, n represents an integer from 0 to 10, preferably from 1 to 7 or even from 1 to 4, in R₅When present, represents an integer of 0 to 6, in R₆Represents an integer of 1 to 3; and

q represents substituted or unsubstituted: a nitrogen-containing heteroaryl ring such as pyrrole, tetrazole, oxazole, oxadiazole, isoxazole, imidazole, or pyrazole; secondary amino substituents such as monoalkylamines, arylalkylamines, heteroarylalkylamines; tertiary amino substituents such as dialkylamines; or a nitrogen-containing heterocycle such as morpholine, piperidine, piperazine, pyridine or pyrrolidine.

In certain embodiments, when K represents N (R') SO₂R "represents a lower alkyl group.

In certain embodiments, when R₅Is M_nAt JK, R₅Is not CH₂COOH。

In certain embodiments, suitable substituents, at each occurrence, independently include alkyl, oxo, hydroxy, alkoxy, hydroxy-alkoxy, carbonyl, sulfonyl, ester, amide, NR ", alkyl halide, acylamino, or substituted or unsubstituted aryl, heteroaryl, heterocyclyl, cycloalkyl, oligo (ethylene glycol), and the like. It will be apparent to those skilled in the art that aryl and heteroaryl groups may be substituted with any suitable substituent including any of those listed above.

In certain embodiments, R₈Represents any of the following substituents: alkyl, alkenyl, alkynyl, alkoxy, hydroxy-alkoxy, aryl, amine or heteroaryl. In certain embodiments, any of the foregoing substituents may be optionally substituted with any of the substituents described, or even with halogen, -CN, N₃、NO₂Or haloalkyl substitution. Other suitable substituents may also include, for example, cyclohexyl, ═ O, carbonyl, sulfonyl, carboxyl, and the like,Sulfonic acid groups (sulfoxyl), amides, heterocycles, esters or ethers.

In certain embodiments, when in R₅Is present and is bonded to R₈When attached, M is at least once substituted NR'.

In certain embodiments, including any of the above embodiments, R₈Having the form:

wherein Z represents O or NR'. In certain embodiments, R₈Represents morpholino or cyclohexyl. In certain such embodiments, M_nIs NR', NH being preferred. In certain embodiments, V is O.

In certain embodiments, W represents CH₂. In certain such embodiments, M is substituted NR "at least once.

In certain embodiments, wherein R' "is present and is substituted lower alkyl, the lower alkyl is substituted with 1-3 (preferably 1) substituents selected from: lower alkyl, lower haloalkyl, NR₈R_8a、NR”C(O)R₈、＝O、COR₈、CO₂R₈、NR”CO₂R₈、C(O)NR₈R_8a、NR”C(O)NR₈R_8a、NR”C(S)NR₈R_8a、C(S)NR₈R_8a、NR”SO₂NR₈R_8a、SO₂NR₈R_8a、NR’′SO₂R_8a、SO₂R_8a、NR’′SO₂R_8aC substituted by 0-5R', C_3-10Carbocyclic ring, and containing 1-4 heteroatoms selected from O, N and S and 0-3R₈Substituted 5-to 10-membered heterocyclic ring, wherein R₈Representative H, C_1-4Haloalkyl, NR_8aR_8a、NR”C(O)OR_8a、NR”C(O)R_8a、COR_8a、CO₂R_8a、CONR_8aR_8a、NHC(O)NR_8aR_8a、NHC(S)NR_8aR_8a、SO₂NR_8aR_8a、SO₂R_8a、C_1-4Alkyl, phenyl, benzyl, C optionally substituted with 0-3R'_2-10Alkenyl-substituted C_5-10Alkyl, C substituted by 0-3R')_2-10Alkynyl, - (CF)₂)_mCF₃C substituted by 0-5R', C_3-10Carbocycle, and a 5-to 10-membered heterocycle containing 1-4 heteroatoms selected from O, N and S substituted with 0-3R' "; at each occurrence, R_8aIndependently represent a group selected from H, lower alkyl, phenyl and benzyl.

In certain embodiments, R '"comprises an amino acid residue such as a valine or glycine residue, e.g., R'" is a lower alkyl residue substituted with an amino acid residue via an amide or ester bond.

In a preferred embodiment, R₅W and R₆Adjacent to each other (ortho position) on Ar, preferably not adjacent to the bond to the tricyclic mother nucleus (ortho position).

In certain embodiments, V represents S or N-CN. In certain embodiments, Ar represents a heteroaryl ring.

In certain embodiments of formula II, W represents O, S or NR ". In certain embodiments, R₅Representative H, P (═ O) (OR')₂Or M_nAnd Q. In certain embodiments, each occurrence of R₇Independently represents halogen, hydroxy, lower alkyl such as methyl or lower alkoxy such as methoxy. In certain embodiments, when present in R₅When n represents an integer of 0 to 5, preferably 1 to 5, more preferably 2 to 4.

In certain embodiments of formula II, W represents O, CH₂C (═ O), C (═ S), or SO₂. In certain embodiments, R₅Represents M_nJK or M_nAnd Q. In certain embodiments, R₆And R₇Represents H. In some instancesIn one embodiment, M represents C (═ O) or CH₂. In certain embodiments, it is preferred that n be 1, while in other embodiments, n may be 0. In certain embodiments, it is preferred that J is C (═ O), K is OR 'OR N (R') SO₂And R' is adopted. In certain embodiments, N (R') SO₂R' is NHSO₂R”。

In certain embodiments, Q represents a substituted or unsubstituted nitrogen-containing heteroaryl ring. In certain embodiments, Q represents a substituted or unsubstituted heteroaromatic ring, such as a5 or 6 membered ring containing at least two nitrogen atoms. In certain embodiments, Q may be a substituted or unsubstituted tetrazole or oxadiazole. In certain embodiments, Q may be a substituted or unsubstituted pyridine, piperidine, or piperazine.

In certain embodiments, Q represents a secondary amino substituent. In certain such embodiments, the substituents on the secondary amino substituents are selected from the group consisting of alkyl, alkoxyalkyl, hydroxyalkyl, and hydroxyalkoxyalkyl.

In certain embodiments of formula II, W represents C (═ O), SO₂Or C (═ S), R₆And R₇Represents H, R₅Represents M_nQ, wherein n represents 0 and Q represents a substituted or unsubstituted nitrogen-containing heteroaromatic ring. In certain embodiments, W represents CH₂，R₆And R₇Represents H, R₅Represents M_nQ, wherein n represents 0 and Q represents a substituted or unsubstituted nitrogen-containing heteroaromatic ring.

In certain embodiments, W represents S, O or NR', R₆And R₇Represents H, R₅Represents M_nJK, wherein N is an integer from 1 to 3, J is C (═ O), K is OR 'OR N (R') SO₂R”。

In certain embodiments, W represents S, O or NR', R₆And R₇Represents H, R₅Represents M_nQ, wherein n is an integer of 1 to 3, and Q is a substituted or unsubstituted 5-membered nitrogen-containing heterocyclic ring. In such embodiments, preferably n is 1. In certain embodiments, Q contains at least two nitrogen atoms.

In certain embodiments, W represents S, O or NR', R₆And R₇Represents H, R₅Represents M_nQ, wherein n represents an integer of 1 to 3, and Q is a substituted or unsubstituted 6-membered nitrogen-containing heterocyclic ring. In certain such embodiments, n is 2, M_nRepresents CH₂C(＝O)。

In certain embodiments, W represents O, S or NR', R₆And R₇Represents H, R₅Represents M_nQ, wherein M is CH₂N is an integer of 1 to 3, and Q is a substituted or unsubstituted nitrogen-containing heterocycle.

In certain embodiments, wherein Q represents a substituted nitrogen-containing heterocycle, e.g., piperazine, morpholine, piperidine, pyridine, thiazole, oxadiazole, tetrazole, pyrrole, or the like, suitable substituents include substituted or unsubstituted alkyl, amino-alkyl, alkoxy, aralkyl (e.g., benzyl), aryl (e.g., phenyl), and heteroaryl such as oxazolyl, piperazinyl, pyridyl, pyrrolyl. In certain such embodiments, wherein Q contains a nitrogen not attached to M, the nitrogen is substituted, for example, with such a substituent.

C1。

C5。

in certain embodiments of formula II, the present invention provides compounds having the structure of formula IIa, including isomers, prodrugs, tautomers, pharmaceutically acceptable salts, N-oxides, or stereoisomers thereof:

wherein

W and Z independently represent O or NR ";

r' independently at each occurrence represents H, a lower alkyl group or a metal counterion, for example an alkali or alkaline earth metal counterion;

r "independently for each occurrence represents H or lower alkyl, preferably H;

R₅representative H, P (═ O) (OR')₂Or M_nQ；

at each occurrence, R₇Independently represent hydrogen, halogen, lower alkyl such as methyl, or lower alkoxy such as methoxy;

at each occurrence, M independently represents a substituted or unsubstituted methylene group (e.g., substituted with lower alkyl, oxo, hydroxy, etc.), NR', O, S, S (O), or S (O)₂) Preferably CH₂Or when bound to W or Q, represents CH₂、S(O₂) C (═ S) or C (═ O);

when present in R₅When n represents an integer from 1 to 5, preferably from 2 to 4, when present in R₆When n represents an integer of 1 to 3; and

q represents a nitrogen-containing heteroaryl ring such as pyrrole, oxazole, isoxazole, imidazole or pyrazole, a tertiary amino substituent such as dialkylamine, or a substituted or unsubstituted nitrogen-containing heterocycle such as morpholine, piperidine, piperazine or pyrrolidine.

In certain embodiments, Q represents a tertiary amino substituent such as a dialkylamine. In certain embodiments, Q represents a substituted or unsubstituted nitrogen-containing heterocycle such as morpholine, piperidine, piperazine, or pyrrolidine. In certain embodiments, Q represents a nitrogen-containing heteroaromatic ring, a tertiary amino substituent, or a substituted or unsubstituted nitrogen-containing heterocycle.

In certain embodiments, the compound having the structure of formula II excludes compounds having the structure of formula IIa.

Exemplary compounds of formula II and IIa include those shown in table B.

The present invention also provides compounds having a structure selected from the group consisting of: a3, a7 to a29, a31, a33 to a37, a40, a41, a44 to a47, a49, a51, a56, a57, a65, a69 to a82, C1, C2 and C5, including isomers, prodrugs, tautomers, pharmaceutically acceptable salts, N-oxides or stereoisomers thereof. In certain embodiments, the present invention provides compounds having structure a37, including isomers, prodrugs, tautomers, pharmaceutically acceptable salts, N-oxides, or stereoisomers thereof.

In another embodiment, the present invention provides an isolated prodrug or pharmaceutically acceptable salt of the metabolite of compound a 37. One preferred such embodiment is a prodrug or a pharmaceutically acceptable salt of compound a68 or C5.

In another embodiment, the present invention provides a compound having a structure selected from the group consisting of: b1 to B20, C1, C2 and C5, including isomers, prodrugs, tautomers, pharmaceutically acceptable salts, N-oxides or stereoisomers thereof. In a preferred embodiment, the present invention provides compounds having the structure B16 or C5, including isomers, prodrugs, tautomers, pharmaceutically acceptable salts, N-oxides, or stereoisomers thereof. In another embodiment, the present invention provides compounds having the structure B3, including isomers, prodrugs, tautomers, pharmaceutically acceptable salts, N-oxides, or stereoisomers thereof.

In another embodiment, the present invention provides an isolated prodrug or pharmaceutically acceptable salt of the metabolite of compound B16. One preferred such embodiment is a prodrug or a pharmaceutically acceptable salt of compound B3.

wherein

R₈Represents a substituted or unsubstituted heterocyclic ring; and

In other embodiments of the invention, the compounds shown in tables C, D and E are exemplary compounds and the invention includes isomers, prodrugs, tautomers, pharmaceutically acceptable salts, N-oxides, or stereoisomers of the compounds described herein.

In another embodiment, the present invention provides a novel pharmaceutical composition comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of a compound of formula I, II or IIa or any of the compounds disclosed herein, e.g., C5, or an isomer, prodrug, tautomer, pharmaceutically acceptable salt, N-oxide or stereoisomer thereof. In a preferred embodiment, such pharmaceutical compositions comprise a therapeutically effective amount of a compound selected from the group consisting of: a1 to a82, B1 to B20, and C1 to C5, or an isomer, prodrug, tautomer, pharmaceutically acceptable salt, N-oxide, or stereoisomer thereof. In another embodiment, such pharmaceutical compositions comprise a therapeutically effective amount of a prodrug or pharmaceutically acceptable salt of a metabolite of compound a37 or B16, preferably a metabolite having the structure a68 or C5.

In another embodiment, the present invention provides a novel method of treating cancer, or other proliferative diseases or other diseases, including any of the diseases or conditions described below, comprising administering to a host in need of such treatment a therapeutically effective amount of a compound of formula I, II or IIa or any of the compounds disclosed herein, or an isomer, prodrug, tautomer, pharmaceutically acceptable salt, N-oxide or stereoisomer thereof. In certain embodiments, at least one compound selected from an anti-cancer drug and an antiproliferative drug may be administered in combination with a compound of formula I, II or IIa or any of the compounds disclosed herein, or an isomer, prodrug, tautomer, pharmaceutically acceptable salt, N-oxide, or stereoisomer thereof. In a preferred embodiment, such a method of treatment comprises the appropriate administration of a therapeutically effective amount of a compound selected from the group consisting of: a1 to a82, B1 to B20, and C1 to C5, or an isomer, prodrug, tautomer, pharmaceutically acceptable salt, N-oxide, or stereoisomer thereof. The term co-administration as used herein includes such therapies: wherein the two therapeutic agents are administered in combination in a single formulation, for example in separate formulations, simultaneously or at different times, or additionally to the patient as part of a treatment regimen.

In another embodiment, the present invention provides a method of formulating a pharmaceutical composition comprising a therapeutically effective amount of a compound of formula I, II or IIa or any of the compounds disclosed herein, or an isomer, prodrug, tautomer, pharmaceutically acceptable salt, N-oxide, or stereoisomer thereof, and optionally a pharmaceutically acceptable carrier. A1 to a82, B1 to B20, and C1 to C5, or an isomer, prodrug, tautomer, pharmaceutically acceptable salt, N-oxide, or stereoisomer thereof. In another embodiment, such pharmaceutical compositions comprise a therapeutically effective amount of a prodrug or pharmaceutically acceptable salt of a metabolite of compound a37 or B16, preferably a metabolite having the structure a68 or C5.

In still other embodiments, the pharmaceutical compositions of the invention are used to treat diseases, such as cancer and other proliferative diseases or other diseases including any of the diseases or conditions described below.

In certain embodiments of the invention in which substituted groups are used, suitable substituents may include, for example, halogen, hydroxy, carbonyl (e.g., ketone, aldehyde, carboxy, ester, acyl), thiocarbonyl (e.g., thioester, thioacetate, thiocarbamate), alkoxy, phosphoryl (e.g., phosphonate, phosphinate), phosphate, phosphonate, phosphinate, amino-alkyl, amido, amidine, imine, cyano, nitro, azido, mercapto, alkylthio, ether, -CF₃Alkyl, alkenyl, alkynyl, cycloalkyl, alkoxy, silyl, sulfonyl (e.g., sulfate, sulfonamido, sulfamoyl, sulfonate), heterocyclyl, aralkyl (e.g., benzyl), or an aromatic or heteroaromatic moiety (e.g., phenyl, oxazolyl, piperazinyl, pyridyl, pyrrolyl). Such substituents may themselves be substituted or unsubstituted.

ii.definition

The following terms and expressions are used herein with the indicated meanings. The compounds of the invention may contain asymmetrically substituted carbon atoms and may thus be isolated in optically active form or as racemates. It is well known in the art how to prepare optically active forms, for example by resolution of the racemic form or by synthesis from optically active starting materials. Except where specific stereochemistry or isomeric forms are specifically indicated, all chirality, diastereomers, racemates and all geometric isomers of the structure are intended to be included. All processes for the preparation of the compounds of the invention and intermediates prepared therein are considered to be part of the invention.

Bag of the inventionIncluding all isotopes of atoms occurring in compounds of the invention. Isotopes include those atoms having the same atomic number but different mass numbers. In general non-limiting examples, isotopes of hydrogen include tritium and deuterium. Isotopes of carbon include¹²C and¹⁴C。

the term "alkyl" is intended to include both branched and straight chain saturated aliphatic hydrocarbon groups having the stated number of carbon atoms. Examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, n-pentyl, and sec-pentyl. Further, the term includes unsubstituted and substituted alkyl groups, the latter meaning that the alkyl moiety has one or more hydrogen substituents replaced by, but not limited to: halogen, hydroxy, carbonyl, alkoxy, ester, ether, cyano, phosphoryl, amino, imino, amido, mercapto, alkylthio, thioester, sulfonyl, nitro, heterocyclyl (heterocyclic), aryl or heteroaryl. Those skilled in the art will also appreciate that the substituted moiety itself may also be substituted as appropriate. The term "lower alkyl" refers to those alkyl groups having 1 to 6 carbon atoms, preferably 1 to 4 carbon atoms, and the term "lower alkoxy" refers to such lower alkyl groups attached to an oxygen atom. In certain embodiments, it is preferred that the alkyl substituent be a lower alkyl substituent.

The term "halo" or "halogen" as used herein refers to fluoro, chloro, bromo and iodo.

The term "aryl" shall denote an aromatic moiety such as, but not limited to, phenyl, indanyl or naphthyl.

The terms "cycloalkyl" and "bicycloalkyl" shall denote any stable ring system, which may be saturated or partially unsaturated. Examples of such groups include, but are not limited to, cyclopropyl, cyclopentyl, cyclohexyl, norbornyl, bicyclo [22] nonane, adamantyl, or tetrahydronaphthyl (1, 2, 3, 4-tetrahydronaphthalene).

As used herein, "carbocycle" or "carbocyclyl" shall mean any stable 3-to 7-membered monocyclic or bicyclic, or 7-to 13-membered bicyclic or tricyclic ring, wherein any ring may be saturated, partially unsaturated, or aromatic. Examples of such carbocycles include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, adamantyl, cyclooctyl, [3.0] bicyclooctane, [4.0] bicyclononane, [4.0] bicyclodecane (decalin), [2.2] bicyclooctane, fluorenyl, phenyl, naphthyl, indanyl, adamantyl, or tetrahydronaphthyl (1, 2, 3, 4-tetrahydronaphthalene).

The term "heterocycle" or "heterocyclic ring system" as used herein shall mean a stable 5-to 7-membered monocyclic or bicyclic, or 7-to 10-membered bicyclic heterocyclic ring (aromatic/heteroaryl) which is saturated, partially unsaturated or unsaturated, consisting of carbon atoms and 1 to 4 heteroatoms independently selected from N, O and S and includes any bicyclic group in which any heterocyclic ring defined above is fused to a benzene ring. The nitrogen and sulfur heteroatoms may optionally be oxidized. The heterocyclic ring may be attached to its pendant group at any heteroatom or carbon atom that results in a stable structure. The heterocyclic ring described herein may be substituted on a carbon or nitrogen atom if the resulting compound is stable. If specified, the nitrogen of the heterocyclic ring may optionally be quaternized. In certain embodiments, when the total number of S and O atoms of the heterocycle exceeds 1, then these heteroatoms need not be adjacent to each other. Preferably, the total number of S atoms in the heterocyclic ring is not more than 1. The term "heteroaromatic ring system" as used herein shall mean a stable 5-to 7-membered monocyclic or bicyclic, or 7-to 10-membered bicyclic, heterocyclic aromatic ring consisting of carbon atoms and 1 to 4 heteroatoms independently selected from N, O and S. Preferably, the total number of S and O atoms on the aromatic heterocycle is not more than 1. Examples of heterocycles include, but are not limited to, 1H-indazole, 2-pyrrolidinone, 2H16H dithiazinyl, 2H-pyrrolyl, 3H-indolyl, 4-piperidinonyl, 4 aH-carbazole, 4H-quinolizinyl, 6H-1, 2, 5-thiadiazinyl, acridinyl, azocinyl, benzimidazolyl, benzofuranyl, benzothiophenyl, benzoxazolyl, benzothiazolyl, benzotriazolyl, benzotetrazolyl, benzisoxazolyl, benzisothiazolyl, benzimidazolonyl, carbazolonyl, carbazolyl, 4 aH-carbazolyl, P-carbolinyl, chromanyl, benzopyranyl, cinnolinyl, decahydroquinolinyl, 2H, 6H dithiazinyl, dihydrofuro [2, 3-b ] tetrahydrofuran, Furyl, furazanyl, imidazolidinyl, imidazolinyl, imidazolyl, 1H-indazolyl, indolinyl (indolynyl), indolinyl, indolizinyl, indolyl, isobenzofuranyl (isobenzofuranyl), isobenzodihydropyranyl, isoindolyl, isoquinolyl, isothiazolyl, isoxazolyl, morpholinyl, 1, 5-diazananyl, octahydroisoquinolyl, oxadiazolyl, 1, 2, 3-oxadiazolyl, 1, 2, 4-oxadiazolyl, 1, 2, 5-oxadiazolyl, 1, 3, 4-oxadiazolyl, oxazolidinyl, oxazolyl, oxazolidinyl, pyrimidyl (perimidyl), phenanthridinyl, phenanthrolinyl, phenazinyl, phenothiazinyl, thiophenechol-yl (phenoxathiinyl), phenoxazinyl, 2, 3-diazananyl, naphtyl, indolinyl, indolizinyl, isothiazolinyl, morpholinyl, 1, 5-oxadiazolyl, 1, 3, 4-oxadiazolyl, oxazolyl, oxazolidinyl, peri, Piperazinyl, piperidinyl, pteridinyl, piperidinonyl, 4-piperidinonyl, pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl, pyridooxazole, pyridoimidazole, pyridothiazole, pyridyl (pyridinyl), pyridyl, pyrimidinyl, pyrrolidinyl, pyrrolinyl, pyrrolyl, quinazolinyl, quinolyl, 4H-quinolizinyl, quinoxalinyl, quinuclidinyl, carbolinyl, tetrahydrofuranyl, tetrahydroisoquinolinyl, tetrahydroquinolinyl, 6H-1, 2, 5-thiadiazinyl, 1, 2, 3-thiadiazolyl, 1, 2, 4-thiadiazolyl, 1, 2, 5-thiadiazolyl, 1, 3, 4-thiadiazolyl, thianthrenyl, thiazolyl, thienyl, thienothiazolyl, thienooxazolyl, thienoimidazolyl, pyrazoyl, pyridazinyl, quinolizyl, pyridoxazolyl, pyrazolinyl, pyridazolyl, Thienyl (thiophenyl), triazinyl, 1, 2, 3-triazolyl, 1, 2, 4-triazolyl, 1, 2, 5-triazolyl, 1, 3, 4-triazolyl, xanthenyl. Preferred heterocycles include, but are not limited to, pyridyl, furyl, thienyl, pyrrolyl, pyrazolyl, imidazolyl, indolyl, benzimidazolyl, 1H-indazolyl, oxazolidinyl, benzotriazolyl, benzisoxazolyl, oxindolyl, benzoxazolinyl or isatinoyl. Also included are fused ring and spiro compounds containing, for example, the above heterocycles.

"pharmaceutically acceptable salts" as used herein refers to derivatives of the compounds disclosed herein wherein the parent compound is modified by making acid or base salts thereof. Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; basic or organic salts of acidic residues such as carboxylic acids; and the like. Pharmaceutically acceptable salts include the conventional non-toxic salts or the quaternary ammonium salts of the parent compound formed, for example, with non-toxic inorganic or organic acids.

For example, such conventional non-toxic salts include those derived from inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, sulfamic acid, phosphoric acid, nitric acid, and the like; and salts prepared with, for example, the following organic acids: acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethanedisulfonic, oxalic, isethionic and the like.

The pharmaceutically acceptable salts of the present invention can be synthesized by conventional chemical methods using the parent compound, which contains a basic or acidic moiety. In general, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or an organic solvent, or a mixture of the two; generally, nonaqueous media such as ether, EtOAc, ethanol, isopropanol, or acetonitrile are preferred. Suitable salts are listed in Remington's Pharmaceutical Sciences, 18 th edition, Mack Publishing Company, Easton, PA, 1990, page 1445, the contents of which are incorporated herein by reference.

The phrase "pharmaceutically acceptable" is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

The term "prodrug" as used herein is intended to include any covalently bonded carriers that release the active parent drug of the present invention in vivo when such prodrug is administered to a mammalian patient. Because prodrugs are known to increase many desirable properties of drugs (i.e., solubility, bioavailability, manufacturability, etc.), the compounds of the invention may be delivered as prodrugs. Accordingly, the present invention is intended to include prodrugs of the claimed compounds, methods of releasing such prodrugs and compositions containing such prodrugs. Prodrugs of the invention are prepared by modifying functional groups in the compound, which modifications are cleaved, either by routine manipulation or in vivo, to the parent compound. Prodrugs include compounds of the present invention wherein a hydroxy, amino, or mercapto group, respectively, is bonded to any group that, when the prodrug of the present invention is administered to a mammalian subject, dissociates to form a free hydroxy, free amino, or free mercapto group. Examples of prodrugs include, but are not limited to, acetate, formate, and benzoate derivatives of alcohol and amine functional groups in the compounds of the present invention.

The term "pure" or "purified" means that other organic molecules, especially impurities such as by-products or degradation products, are substantially absent from the molecule. In some embodiments, a "pure" or "purified" compound is an organic compound having a composition of at least 80% by weight, more preferably 95-99% by weight, and most preferably at least 99.8% by weight (e.g., excluding molecules such as water, buffers, excipients, etc., that may be present in a pharmaceutical formulation of the compound).

"substituted" shall mean that one or more hydrogens on the atom being replaced with the indicated group, with the proviso that the indicated atom's normal valency is not exceeded, and that the substitution results in a stable compound. When the substituent is a ketone or oxo (i.e., ═ O) group, then 2 hydrogens on the atom are replaced. No ketone/oxo substituents are present on the aromatic moiety. Exemplary substituents include, for example, alkyl, perfluoroalkyl (e.g., trifluoromethyl), halogen, hydroxy, carbonyl (e.g., carboxy, alkoxycarbonyl, formyl, or acyl), thiocarbonyl (e.g., thioester, thioacetate, or thioformate), alkoxy, phosphoryl, phosphate, phosphonate, phosphinate, amino, amido, amidine, imine, cyano, nitro, azido, mercapto, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, carbocyclyl, heterocyclyl, aralkyl, heteroaralkyl, or an aromatic or heteroaromatic moiety. It will be appreciated by those skilled in the art that substituents such as heterocyclyl, aryl, alkyl and the like may themselves be substituted if appropriate.

The term "therapeutically effective amount" of a compound of the invention means an amount effective to inhibit a class of enzymes known as cyclin dependent kinases, or to treat cancer or other proliferative diseases or other disease symptoms in a host.

The term "anti-cancer" or "anti-proliferative" agent as used herein includes, but is not limited to, altretamine, busulfan, chlorambucil, cyclophosphamide, ifosfamide, mechlorethamine, melphalan, thiotepa, cladribine, fluorouracil, floxuridine, gemcitabine, thioguanine, pentostatin, methotrexate, 6-mercaptopurine, cytarabine, carmustine, lomustine, streptozocin, carboplatin, cisplatin, oxaliplatin, iproplatin, tetraplatin, lobaplatin, JM216, JM335, fludarabine, aminoglutethimide, flutamide, goserelin, leuprolide, megestrol acetate, cyproterone acetate, tamoxifen, anastrozole, bicalutamide, dexamethasone, diethylstilbestrol, prednisone, bleomycin, dactinomycin, doxorubicin, idarubicin, mitoxantrone, losoxanthrone, mitomycin-c, Plicamycin, paclitaxel, docetaxel, topotecan, irinotecan, 9-aminocamptothecin (camptothecan), 9-nitrocamptothecin, GS-211, JM 118, etoposide, teniposide, vinblastine, vincristine, vinorelbine, procarbazine, asparaginase, pemetrexed, octreotide, estramustine, and hydroxyurea.

Dosage and formulation iii

Cancer or proliferative diseases or other diseases can be treated by any method that results in the active agent being in contact with the site of action of the agent in the mammal by administering a cyclin dependent kinase inhibitor of the invention. They may be administered as individual therapeutic agents or in combination with each other by any conventional method known for use in combination with drugs. The chemical properties of the inhibitors described herein impart good solubility characteristics to the compounds, making them suitable for administration in intravenous, topical, oral formulations, and other formulations discussed in more detail below. They may be administered alone, but are preferably administered with a selected pharmaceutical carrier based on the chosen route of administration and standard pharmaceutical practice. Suitable carriers (vehicles) and their formulations are discussed, for example, in the book Remington's Pharmaceutical Sciences (Remington's Pharmaceutical Sciences. Mack Publishing Company, Easton, Pa., USA 1985).

In another aspect, the invention provides pharmaceutically acceptable compositions comprising a therapeutically effective amount of one or more compounds of the invention, e.g., as described above, together with one or more pharmaceutically acceptable carriers (additives) and/or diluents. As described in detail below, the pharmaceutical compositions of the present invention may be formulated, inter alia, into solid or liquid administration forms, including those suitable for the following routes of administration: (1) oral administration, e.g. drenches (aqueous or non-aqueous solutions or suspensions), tablets, single-use bolus injections (boluses), powders, granules, tongue pastes; (2) parenteral administration, e.g., by subcutaneous, intramuscular, or intravenous injection, e.g., sterile solution or suspension; (3) topical application, such as creams, ointments or sprays for the skin; or (4) intravaginally or intrarectally, such as pessaries, creams, or foaming agents. In certain embodiments, the pharmaceutical formulation may be non-pyrogenic, i.e., does not raise the body temperature of the patient.

Wetting agents, emulsifiers and lubricants, for example, sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating materials, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be added to the composition.

Examples of pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, and the like; (2) oil-soluble antioxidants such as ascorbyl palmitate, Butylated Hydroxyanisole (BHA), Butylated Hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

The dose administered naturally varies with the following known factors: such as the pharmacodynamic properties of the particular drug, its mode and route of administration; age, health and weight of the subject; the nature and extent of the symptoms; the kind of co-therapy; the frequency of treatment; and the desired effect. It is contemplated that the daily dosage of the active ingredient may be from about 0.001 to about 1000 mg/kg body weight, with a preferred dosage being from about 0.1 to about 30 mg/kg.

Compositions suitable for administration are in the form of a unit containing from about 1mg to about 100mg of active ingredient. In these pharmaceutical compositions, the active ingredient will generally comprise about 0.95% by weight based on the total weight of the composition. The active ingredient may be administered orally in solid dosage forms such as capsules, tablets and powders, or in liquid dosage forms such as elixirs, syrups and suspensions. It may also be administered parenterally in sterile liquid dosage forms.

The formulations of the present invention include those suitable for oral, nasal, topical (including buccal and sublingual), rectal, vaginal and/or parenteral administration. The amount of active ingredient that may be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated, the particular mode of administration. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form is generally that amount of inhibitor which achieves a therapeutic effect. Typically, the amount of active ingredient is from about 1% to about 99%, preferably from about 5% to about 70%, most preferably from about 10% to about 30%, by 100%.

The process for preparing these formulations or compositions comprises the step of admixing a compound of the present invention with a carrier and optionally one or more accessory ingredients. In general, such formulations are prepared by uniformly and intimately admixing the inhibitors of the invention with liquid carriers or finely divided solid carriers or both, and then shaping the product as necessary.

Formulations of the invention suitable for oral administration may be in the form of: capsules, cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), powders, granules, or aqueous or non-aqueous liquid solutions or suspensions, or oil-in-water or water-in-oil liquid emulsions, or elixirs or syrups, or pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia), and/or mouthwashes and the like, each containing a predetermined amount of a compound of the present invention as an active ingredient. The inhibitors of the invention may also be administered in a single bolus injection (bolus), dry electuary or patch.

In solid dosage forms of the invention for oral administration (capsules, tablets, pills, dragees, powders, granules and the like), the active ingredient is mixed with one or more pharmaceutically acceptable carriers such as sodium citrate or dicalcium phosphate and/or any of the following: (1) fillers or supplements such as starch, lactose, sucrose, glucose, mannitol, and/or silicic acid; (2) binding agents such as carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and/or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarders such as paraffin; (6) absorption promoters such as quaternary ammonium compounds; (7) wetting agents such as cetyl alcohol and glycerol monostearate; (8) absorbents such as kaolin and bentonite clays; (9) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate and mixtures thereof; and (10) a colorant. In the case of capsules, tablets and pills, such pharmaceutical compositions may also comprise buffering agents. Solid compositions of the same type using such excipients as lactose and high molecular weight polyethylene glycols and the like may also be used as fillers for filling soft and hard gelatin capsules.

Tablets may be compressed or molded, optionally with one or more accessory ingredients. Compressed tablets may be prepared with binders (for example, gelatin or hydroxypropylmethyl cellulose), lubricants, inert diluents, preservatives, disintegrating agents (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agents. Molded tablets may be made by molding in a suitable machine the inhibitor powder mixture moistened with an inert liquid diluent.

Solid dosage forms of the tablets and other pharmaceutical compositions of the present invention, such as dragees, capsules, pills and granules, can optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They may be dosage forms providing slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose, other polymeric matrix materials, liposomes and/or microspheres in various proportions to provide the desired release profile. They may be sterilized, for example, by filtration through a bacteria-retaining filter, or by incorporating sterilizing agents into sterile solid compositions which may be dissolved in sterile water or some other sterile injectable medium immediately prior to use. These compositions may also optionally contain opacifying agents and may be of a type that they release the active ingredient(s) only, or preferentially, in certain parts of the gastrointestinal tract, optionally in a protracted manner. Examples of implant compositions that can be used include polymeric substances and waxes. The active ingredient may also be in microencapsulated form, if appropriate together with one or more of the above-mentioned excipients.

Liquid dosage forms for oral administration of the compounds of the invention include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredient, the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1, 3-butylene glycol, oils (especially cottonseed, groundnut, corn, germ (germ), olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols, fatty acid esters of sorbitan, and mixtures thereof.

In addition to inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.

Suspensions, in addition to the active inhibitors of the invention, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar, tragacanth, and mixtures thereof.

Suppository formulations of the pharmaceutical compositions of the present invention for rectal or vaginal administration may be provided which may be prepared by mixing one or more compounds of the present invention with one or more suitable non-irritating excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax or a salicylate, and which are solid at room temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity to release the active inhibitor.

Formulations of the invention suitable for vaginal administration include pessaries, tampons, creams, gels, pastes, foams or spray formulations containing such carriers as are known in the art to be appropriate.

Dosage forms for topical or transdermal administration of the compounds of the present invention include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. The active compound may be mixed under sterile conditions with a pharmaceutically acceptable carrier and any preservatives, buffers, or propellants which may be required.

In addition to the active prenyltransferase inhibitors, ointments, pastes, creams and gels may contain excipients, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide or mixtures thereof.

Powders and sprays can contain, in addition to a compound of the invention, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicate and polyamide powder, or mixtures of these substances. Sprays can also contain conventional propellants, such as chlorofluorocarbons and volatile unsubstituted hydrocarbons, such as butane and propane.

Transdermal patches also have the advantage of providing controlled release of the compounds of the invention into the body. Such dosage forms may be prepared by dissolving or dispersing the inhibitor of the present invention in a suitable medium. Absorption enhancers may also be used to increase the flux of the drug across the skin. The rate of such flux can be controlled by providing a rate controlling membrane, or dispersing the compound of the present invention in a polymer matrix or gel.

Ophthalmic formulations, ocular ointments, powders, solutions, and the like are also included within the scope of the present invention.

Pharmaceutical compositions of the invention suitable for parenteral administration comprising one or more inhibitors of the invention in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, may contain antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.

Examples of suitable aqueous and nonaqueous carriers which may be employed in the pharmaceutical compositions of the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the dispersion and by the use of surfactants,

these compositions may also contain adjuvants such as preserving, wetting, emulsifying, and dispersing agents. Various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol sorbic acid, and the like can be added to ensure prevention of the action of microorganisms. It may also be desirable to include isotonic agents, for example, sugars, sodium chloride and the like in the compositions. In addition, prolonged absorption of the injectable pharmaceutical form can be brought about by the addition of agents delaying absorption, for example, aluminum monostearate and gelatin.

In some cases, to prolong the efficacy of the inhibitor, it is desirable to slow the absorption of the inhibitor by subcutaneous or intramuscular injection. This can be achieved by using liquid suspensions of crystalline or amorphous materials which have poor water solubility. Thus, the rate of absorption of the inhibitor depends on its rate of dissolution, which in turn depends on crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered inhibitor form is achieved by dissolving or suspending the inhibitor in an oil carrier.

Injectable depot forms are prepared by forming a microencapsulated backbone of the subject inhibitors in a biodegradable polymer, such as polylactide-polyglycolide. Depending on the ratio of drug to polymer and the nature of the particular polymer used, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly (orthoesters) and poly (anhydrides). Long acting injectable formulations can also be prepared by entrapping the drug in liposomes or microemulsions compatible with body tissues.

When the compounds of the present invention are administered as drugs to humans and animals, they may be administered alone or as a pharmaceutical composition containing, for example, 0.1 to 99.5% (more preferably 0.5 to 90%) of the active ingredient in combination with a pharmaceutically acceptable carrier.

The formulations of the present invention may be administered orally, parenterally, topically or rectally. They are naturally administered in a form suitable for each route of administration. For example, they are administered in the form of tablets or capsules; administration by injection, inhalation, eye lotion, ointment, suppository, etc.; administration by injection, infusion or inhalation; topically administered by lotion or ointment; and rectally by suppository. Oral administration is preferred.

The phrases "parenteral administration" and "parenterally administered" as used herein refer to modes of parenteral and topical administration, typically by injection, including, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion.

The phrases "systemic administration," "administered systemically," "administered peripherally," and "administered peripherally" as used herein refer to the administration of a compound, drug, or other substance that does not directly enter the central nervous system, e.g., subcutaneous administration, such that it enters the patient's body and thus undergoes metabolism and other similar processes.

Regardless of the route of administration chosen, the CDK inhibitors used in the subject methods may be used in a suitable hydrated form, and/or the pharmaceutical compositions of the present invention may be formulated into pharmaceutically acceptable dosage forms by conventional methods known to those skilled in the art.

Gelatin capsules contain the active ingredient and carrier powders such as lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, and the like. Compressed tablets may be prepared with similar diluents. Tablets and capsules can be made into sustained release products that provide continuous release of the drug over several hours. The compressed tablets may be sugar-coated or film-coated to mask any unpleasant taste and to insulate the tablet from air, or enteric-coated for selective disintegration of the tablet in the gastrointestinal tract. Similar solid compositions may also be used as fillers for filling soft and hard gelatin capsules; preferred materials in this connection also include lactose and high molecular weight polyethylene glycols. Preferred formulations are soft gelatin capsules of oil, e.g. olive oil, Miglyol or Capmul solutions or suspensions. Antioxidants may be added as appropriate to prevent long-term degradation.

Liquid dosage forms for oral administration may contain coloring and flavoring agents to increase patient compliance. In general, water, suitable oils, saline, ethanol, aqueous dextrose and related sugar solutions, glycols such as propylene glycol or polyethylene glycol, or mixtures of these materials are suitable carriers for parenteral solutions.

The above disclosed compounds may be formulated with physiological buffers or sterile water into sterile solutions of the active ingredient or its salt in free form for intravenous administration. Sugar-containing carrier liquids (e.g., ringer's lactate or other glucose solutions) may be used if desired, provided that the total sugar content does not cause undesirable levels of lactic acidosis. Intravenous administration may be by bolus injection of a single bolus (preferably several times daily) or by continuous infusion over a sustained period of time. The total dose of the preferred single bolus or infusion varies substantially according to the physical condition of the patient; generally, they are generally from about 25mg/kg to about 250 mg/kg.

Preferably, the solutions for parenteral administration contain a water-soluble salt of the active ingredient, a suitable stabilizer and, where appropriate, a buffer substance. Antioxidants such as sodium bisulfite, sodium sulfite or ascorbic acid, alone or in combination, are suitable stabilizers. Citric acid and its salts and sodium EDTA are also used. In addition, parenteral solutions may contain preservatives, such as benzalkonium chloride, methyl or propyl paraben, chlorobutanol. Suitable pharmaceutical carriers are described in the standard reference texts in the art, Remington's pharmaceutical Sciences, 18 th edition, Mack Publishing Company, Easton, PA, 1990, the contents of which are incorporated herein by reference.

Formulations, solutions and other formulations using the compounds of tables A, B and/or C can be prepared according to the methods described in PCT application WO 03/033499 and/or WO04/092139, the teachings of which are incorporated herein by reference.

Therapeutic applications

Since cdks generally play a key role in regulating cell proliferation, the compounds disclosed herein can be used as reversible cytostatics and can be used to treat any disease process characterized by abnormal cell proliferation such as hyperproliferative diseases, including cancer, benign prostate hyperplasia, familial adenomatous polyposis, neurofibromatosis, psoriasis, fungal infections, endotoxic repair, hypertrophic scar formation, inflammatory bowel disease, transplant rejection, vascular smooth muscle cell proliferation associated with atherosclerosis, psoriasis, pulmonary fibrosis, arthritis, glomerulonephritis, restenosis following angioplasty or vascular surgery, and other post-operative stenosis and restenosis. See, for example, U.S. patent nos. 6,114,365 and 6,107,305.

It is contemplated that the compounds disclosed herein may be effective in the treatment of proliferative or hyperproliferative diseases such as cancer, autoimmune diseases, viral diseases, fungal diseases, neurodegenerative diseases, and cardiovascular diseases.

More specifically, the compounds disclosed herein are effective in treating a variety of cancers including (but not limited to) the following: cancers including bladder cancer, breast cancer, colon cancer, kidney cancer, liver cancer, lung cancer including small cell lung cancer, esophageal cancer, gallbladder cancer, ovarian cancer, pancreatic cancer, stomach cancer, cervical cancer, thyroid cancer, prostate cancer, and skin cancer including squamous cell carcinoma; hematopoietic lymphoid tumors including leukemia, acute lymphocytic leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, hairy cell lymphoma and Burkett's lymphoma; hematopoietic myeloid tumors including acute and chronic myelogenous leukemia, myelodysplastic syndrome, and promyelocytic leukemia; tumors of mesenchymal origin, including fibrosarcoma and rhabdomyosarcoma; tumors of the central and peripheral nervous system, including astrocytomas, neuroblastomas, gliomas, and schwannomas; and other tumors including melanoma, seminoma, teratocarcinoma, osteosarcoma, xenoderoma pimentosum, keratocotanthoma, thyroid follicular cancer and kaposi's sarcoma.

Since cdk5 was newly found to be involved in tau phosphorylation (J. biochem, 117, 741-749(1995)), it was suggested that the compounds disclosed herein could also be effective in the treatment of Alzheimer's disease.

The compounds disclosed herein can induce or inhibit apoptosis. In a variety of human diseases, the apoptotic response is abnormal. The compounds described herein as modulators of apoptosis are effective in treating cancer (including but not limited to those species described above), viral infections (including but not limited to herpes virus, poxviruses, Epstein-Barr virus, Sindbis virus and adenovirus), preventing the development of AIDS in individuals infected with HIV, autoimmune diseases (including but not limited to systemic lupus erythematosus, autoimmune-mediated glomerulonephritis, rheumatoid arthritis, psoriasis, inflammatory bowel disease and autoimmune diabetes), neurodegenerative diseases (including but not limited to alzheimer's disease, dementia associated with AIDS, parkinson's disease, amyotrophic lateral sclerosis, retinitis pigmentosa, spinal muscular atrophy and cerebellar degeneration), myelodysplastic syndrome, aplastic anemia, ischemic injury associated with myocardial infarction, ischemic injury, inflammatory bowel disease, and autoimmune diabetes), neurodegenerative diseases (including but not limited to alzheimer's disease, parkinson's disease, amyotrophic lateral sclerosis, retinitis, myelogenous atrophy and cerebellar degeneration), Stroke and reperfusion injury, cardiac arrhythmia, atherosclerosis, toxin-induced or alcohol-related liver disease, hematologic disorders (including but not limited to chronic anemia and aplastic anemia), degenerative diseases of the skeletal system (including but not limited to osteoporosis and arthritis) aspirin allergic rhinosinusitis, cystic fibrosis, multiple sclerosis, kidney disease, and cancer pain.

The compounds disclosed herein as cdks inhibitors may modulate the levels of cellular RNA and DNA synthesis. Thus, these agents are effective in treating viral infections (including but not limited to HIV, human papilloma virus, herpes virus, poxviruses, Epstein-Barr virus, Sindbis virus and adenovirus).

The compounds disclosed herein are also effective in the chemical prevention of cancer. Chemoprevention is defined as inhibiting the development of aggressive cancers by blocking the initiation of mutagenic events, or by blocking the development of pre-malignant cells that have been damaged or inhibiting tumor recurrence.

The compounds disclosed herein are also effective in inhibiting tumor angiogenesis and metastasis.

The compounds disclosed herein are also useful in preventing hair loss that commonly occurs with many traditional chemotherapy regimens. For example, CDK inhibitors of the present invention may be used to inhibit cell proliferation in hair follicles, thus protecting them from the attack of cytotoxic drugs targeting proliferating cells.

The compounds disclosed herein may also be useful as inhibitors of other protein kinases: such as protein kinase C, her2, raf 1, MEK1, MAP kinase, EGF receptor, PDGF receptor, IGF receptor, PI3 kinase, wee kinase, Src, Abl, and thus are effective in treating diseases associated with other protein kinases.

The compounds of the present invention may also be used in combination (simultaneous or sequential administration) with known anti-cancer treatments such as radiotherapy or with the following cytostatic or cytotoxic agents: such as but not limited to DNA acting drugs such as cisplatin or doxorubicin; topoisomerase II inhibitors such as etoposide; topoisomerase I inhibitors such as CPT-11 or topotecan; tubulin-acting drugs such as paclitaxel, docetaxel or epothilones; hormonal agents such as tamoxifen; thymidylate synthase inhibitors such as 5-fluorouracil; and antimetabolites such as methotrexate. In such combinations, the compounds and formulations of the present invention are effective in preventing or reducing the incidence of hair loss, which is typically caused by radiation therapy or chemotherapy.

If formulated in fixed doses, such combination products are treated with the compounds of the present invention and other active agents within the dosage ranges described below or within their approved dosage ranges. For example, the cdc2 inhibitor olomoucine was found to induce apoptosis in conjunction with known cytotoxic drugs (j. cell sci., 108, 2897 (1995)). Where a combined preparation is not appropriate, the compounds described herein may also be administered sequentially with known anti-cancer or cytotoxic drugs. The invention is not limited by the order of administration; the compounds described herein may be administered before or after administration of known anti-cancer or cytotoxic drugs. For example, the cytotoxic activity of the cyclin-dependent kinase inhibitor flavopiridol is affected by the order of administration with the anti-cancer drug. Cancer Research, 57, 3375 (1997).

All of the embodiments described herein apply to all of the different aspects of the present invention. Any described embodiment may be freely combined with one or more other such embodiments, where appropriate. Various embodiments of the compounds of the present invention, of conditions amenable to treatment with such compounds, and of methods of treatment using the compounds of the present invention may be freely combined with each other.

Specific embodiments of the present invention are described in more detail herein. However, these are illustrative embodiments and should not be construed as limiting in any way.

Equivalent scheme

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the scope of the claims. Those skilled in the art will also recognize that all combinations of the embodiments or features of the claims described herein are within the scope of the invention.

v. Synthesis

The compounds of the invention can be synthesized using the methods described below and synthetic methods known in the art of synthetic organic chemistry, or derivatives thereof as recognized by those skilled in the art. Preferred methods include, but are not limited to, those described below. Each reference cited below is incorporated herein by reference.

Key intermediates for the preparation of some of the compounds disclosed herein are pyrazole aminonitriles, aminocarboxamides and aminoesters. Methods for the preparation of these intermediates have been published in the chemical literature and several methods are outlined in scheme a (a.o. abdelhamid et al, j.heterocyclic. chem.1984, 21, 1049), B (c.c. cheng and r.k.robins, j.org.chem.1956, 21, 1240), C (p.schmidt and j.draey, helv.chem.acta 1956, 39, 986). See also Tominaga et al, j.heterocyclic. chem.1990, 27, 775 and PCT application numbers WO 00/21926 and WO 99/54308. A variety of hydrazine and aldehyde starting materials are commercially available or can be prepared by standard organic conversion methods. The substituent Ar used below represents an aromatic ring, substituted to form (conform) or converted to the corresponding aryl substituent of some of the compounds disclosed herein. It is also possible to use CH in the presence of a base₂(CN)₂Treating PrCOCl with PCl₅Treating the resulting compound and then reacting the product with ArNHNH₂Reacted to prepare some compounds.

Procedure A

Procedure B

Procedure C

As shown in scheme D, the aminonitrile II can be converted to the pyrazolo [3, 4-D ] of the invention]A pyrimidine. In general, aminocarboxamides are acylated by treatment with a suitable base such as triethylamine, optionally in the presence of a suitable solvent such as dichloromethane, followed by acylation with a compound of the formula ArCH₂The COX acid halide is preferably acid chloride treated to provide the carboxamido nitrile V. Alternatively, the aminonitrile II can be reacted with the general formula ArCH in the presence of a suitable base and a coupling reagent in a suitable solvent₂CO₂The carboxylic acid coupling of H can produce the carboxamido nitrile V. A review of the coupling of amines and carboxylic acids has been made (Klaussnew and Bodansky, Synthesis1972, 453-463) and one skilled in the art will recognize that there are a variety of reagents that can be used to effect this coupling.

Procedure D

The conversion of the carboxamido nitrile V to the compounds of the invention can be accomplished by treatment with excess hydrogen peroxide in a solvent, preferably water, an alcohol or a water-alcohol mixture, in the presence of a suitable base, preferably a metal hydroxide or alkoxide base, at a temperature of from about 0 ℃ up to 100 ℃.

Alternatively, the catalyst may be prepared by reaction in a concentrated, strong acid, preferably 85% H₃PO₄Preferably, the carboxyamidonitrile V is converted to the compound of the invention by heating for about 1 hour. Scheme E shows another method for preparing compounds of the present invention. In a suitable solventThe agent is preferably a lower alkanol, preferably at the boiling point of the solvent, with an excess of an ArCH of the formula in which R is, for example, lower alkyl₂CO₂The ester of R and an excess of base, preferably a metal lower alcoholate, treat the aminoimide III to give the compounds of the invention. Many aryl acetates are commercially available or can be prepared by reaction in the presence of an acid catalyst such as H₂SO₄Or by esterifying a commercially available arylacetic acid in the presence of p-TsOH in one step with an excess of the alcohol ROH, preferably ethanol or methanol, as solvent at reflux. Alternatively, preferably in a solvent such as CH₂Cl₂With a catalyst such as DMAP, a coupling reagent such as DCC can be used.

Procedure E

Phenylacetic acids can be prepared by acid or base hydrolysis of aryl acetonitrile, which can be prepared by treatment of aryl halides with CN-, preferably in a solvent such as DMF, MeOH, EtOH, water, DMSO, or mixtures thereof. Further examples of aryl acetates can be prepared with aryl carboxylic acids under conditions of Arndt-Eistert (Meier and Zeller, Angew. chem. int. Ed. Engl.1975, 14, 32) or related homologation reactions.

Can be prepared by reaction with an excess of ArCH₂CN nitrile and sodium are reacted to convert the amino ester of formula IV to the compound of the invention.

Procedure F

Preferably, the reaction is carried out without heating of the solvent (neat).

Pyrazolo [3, 4-d]Pyrimidinones can be further prepared as described below to provide other compounds of the present invention. Electrophilic aromatic substitution reactions can be carried out on the Ar group to introduce substituents. Such reactions include, but are not limited to, nitration, acylation (F)riedel-Crafts), halo, alkylation (Friedel-Crafts), chloromethylation, sulfonation and aminomethylation (Mannich reaction). Those skilled in the art of organic synthesis are familiar with the conditions under which these reactions are carried out, and typically involve electrophilic reactions with aryl or heteroaryl substrates in the presence of a catalyst. In the case of nitration or Mannich reactions, the preferred catalyst is a protic acid which acts as a solvent, in which the electrophile is generated in situ from the niter or amine and the carbonyl component, respectively. For other electrophilic aromatic substitution reactions, preferred catalysts are Lewis acids, including but not limited to FeX₃、AlX₃And ZnX₂Wherein X is halogen.

The compound having an amino group prepared above may be derivatized by reaction with an electrophile including, but not limited to, an acid halide, an acid anhydride, an isocyanate, a chloroformate, a sulfonyl halide, an alkyl halide, a lactone, or an ester. Those skilled in the art of organic synthesis are familiar with the operating conditions of these addition reactions, which generally involve the addition of electrophiles to nucleophiles, preferably in solution, at temperatures from 0 ℃ to room temperature. It may be necessary to add a base. It should be noted that the products of these reactions may also react with certain electrophiles at the nitrogen of the pyrimidinone (N5). The resulting functional groups (amide, carbamate, etc.) are less stable to basic hydrolysis than the desired anilino or aliphatic groups and can be cleaved back to pyrimidinones with H on N5.

Reacting a compound having an amine group with a reagent such as a haloacyl halide, an α, β -unsaturated acyl halide, or a halosulfonyl halide provides an intermediate that can be reacted with a nucleophile such as a primary or secondary amine, a diamine, an alkoxide, an aminoalcohol, or a thiol.

The compounds having carboxyl groups prepared above can be derivatized by activation and reaction with nucleophiles including, but not limited to, amines and alcohols to give amides and esters, respectively. A review of the coupling reactions of amines and carboxylic acids with carbodiimides has been made (Klaussnew and Bodansky, Synthesis1972, 453) -463) and those skilled in the art will recognize that a variety of other reagents and protecting Groups may be required to effect this reaction to mask the reactive functional Groups (Greenand Wuts, "Protective Groups in Organic Synthesis", second edition, John Wiley & Sons, 1991). The above is a description of the preparation of esters with acids. These amides and esters can be reduced to amines and alcohols with suitable hydride reducing agents.

Compounds having amino groups prepared above can be derivatized, preferably in the presence of a base, by conversion to electrophiles by activation with phosgene or phosgene equivalents (Tetrahedron: Asymmetry 1995, 61, 745; J.org.chem.1994, 59, 1937) and then reaction with nucleophiles including, but not limited to, amines, alcohols and sulfonamides to give urea, carbamates and sulfonylureas, respectively. Those skilled in the art of organic synthesis are familiar with the operating conditions of these reactions and the hazards associated with handling phosgene and phosgene equivalents, and therefore all appropriate precautions should be taken.

Other transformations that may be required to prepare the compounds of the invention include reducing ketones, aldehydes, esters, acids, amides or reductive amination using aluminum and borohydride reagents (J.Seyden-Penne, "reduction by the aluminum and Borohydrides in Organic Synthesis" VCH Publishers, Inc., 1991) and oxidizing groups including, but not limited to, alcohols, aldehydes, alkenes, thioethers, sulfoxides and heteroaryl groups (Milos Hudlick, "oxidation in Organic Chemistry" American Chemical Society, 1990).

Reducing functional groups such as alkene, alkyne, nitrogen, nitro or cyano groups can be accomplished by catalytic hydrogenation or by reduction by dissolving metals. Further preparation of intermediates containing electrophilic linking sites to the compounds of the present invention can be accomplished by displacement with nucleophiles including, but not limited to, CN-, amines, alkoxides, thiols, or carbanions. Other compounds of the invention may also be prepared by coupling aryl halides or triflates with the appropriate boronic acids or stannanes (Stille, J.K., Angew. chem. int. Ed. Engl.1986, 25, 508; Suzulki, A. pure appl. chem.1985, 57, 1749). The compounds having carbonyl groups prepared above can be further derivatized by reaction with nucleophiles to give secondary alcohols. Such nucleophiles include, but are not limited to, grignard reagents, alkyl lithium, alkenyl lithium, and alkynyl lithium reagents, as well as allyl stannanes, silanes, and the like. The compounds prepared as described above can also be further prepared by rearrangement, for example, by Beckmann (Gawley, org. react.1988, 35, 1) or other rearrangements.

The compounds prepared above can also be prepared by direct metallation to prepare organomagnesium or organolithium species (Beak and Meyers, Acc. chem. Res.1986, 19, 356-363; Beak and Snieckus, Acc. chem. Res.1982, 15, 306-312; Katritzky, Lam and Sentutta, Prog Heterococcus. chem.1989, 11, 1-29) or by aryl halides by lithium-halogen exchange (Parham and Bradsher, Acc. chem. Res.1982, 15, 300-305).

Methods of preparing the compounds of formula II, IIa, and certain other compounds disclosed herein are provided in scheme 1 and can be used to prepare the compounds of the invention. Substituent Z, R₅、R₆And R₇Represent substituents described in formula II, or represent substituents that can be converted to those substituents using standard organic conversion methods. P represents a suitable protecting group. Examples of the protective group include carboxylic acid esters, silyl ethers of alcohols, acetals of aldehydes, and ketals of ketones. There have been reviews of the field of protecting group chemistry (Greeue, T.W.; Wuts, P.G.M.protective Groups in organic Synthesis, 2 nd edition; Wiley: New York, 1991). Catalytic hydrogenation is used to reduce the nitro group of the dimethyl nitrophthalate to an amine. The aniline is acylated with acetic anhydride and pyridine base. The resulting mixture of acetamide 2 and acetophenone is treated with a strong base at elevated temperature in a suitable solvent to afford the desired triketone 3. Known to those skilled in the art are, for example, Kilgore et al, Industrial and Engineering Chemistry 34: 494 497, 1946. Treating the triketone with hydrazine at elevated temperature in a suitable solvent to provide indeno [1, 2-c ]]A pyrazolone ring system.

Known to the person skilled in the art are, for example, Lemke et al, J.Heterocyclic chem.19: 1335 1340, 1982; mosher and Soeder, j.heterocyclic chem.8: 855-59, 1971; hrnciar and Svanygova, Collection, Czech, chem, Commun.59: 2734-40, 1994. The amide is deacylated by heating with a strong acid in a suitable solvent to give aniline 4. The aniline is acylated with an acid chloride under standard conditions in a suitable solvent to give the desired product 5.

Scheme 1

An alternative method for preparing the compounds of the present invention is shown in scheme 2. The triketone 3 intermediate can be deacylated with a strong acid and acylated with the appropriate acid chloride using methods known to those skilled in the art. The trione 6 can then be converted to the indeno [1, 2-c ] pyrazolone ring system using the same conditions described in scheme 1 above.

Scheme 2

An alternative method for preparing triketone 6 of scheme 2 is as follows Rotberg and Oshkaya, zh. 84-87, 1972; zh, organ, khim.9: 25482550, 1973, condensation with 1, 3-dione 6a and 3-nitrophthalic anhydride. When not commercially available, the skilled person can readily prepare the 1, 3-diketones by acetylating the necessary methyl ketone or trifluoroacetylating it. Reduction of the resulting nitro derivative to the aniline 6b can be accomplished by a variety of methods including catalytic hydrogenation, treatment with zinc or iron under acidic conditions, or treatment with other reducing agents such as sodium dithionite or stannous chloride. Aniline 6c can then be converted to the indeno [1, 2-c ] pyrazolone of the present invention by acylation followed by treatment with hydrazine as in scheme 2 above.

Another method for preparing an indeno [1, 2-c ] pyrazolone ring system is shown in scheme 3. Dimethyl hydrazine is reacted with 3-acetylpyridine without the addition of a solvent to give the hydrazone 7. This product was worked up in a similar manner as described in scheme 1 to give the desired intermediate 8.

Scheme 3

Alternatively, 6b may be treated with an activated acylated N-aminomorpholine or piperazine ring, such as nitrophenylcarbamate. Those skilled in the art know the following information according to rappport, j.org.chem.49: 2948 other methods for the preparation of similar intermediates are described in 2953, 1984. This intermediate is processed through a sequence similar to that described in scheme 1.

While the foregoing schemes describe general synthetic routes wherein W is oxygen, those skilled in the art can envision and implement the synthesis of other compounds of the invention wherein W is not oxygen, according to the general route contents. For example, where W is selected from S, S (O)₂)、C(＝O)、C(＝S)、CH₂And NR'.

Other features of the present invention will become apparent in the course of the following description of exemplary embodiments, which are provided to illustrate the present invention and by no means limit it.

v. examples

The compounds of tables A, B and C can be synthesized as shown in PCT applications WO 03/033499 and/or WO04/092139, the teachings of which are incorporated herein by reference.

Assay protocol and results

The biological activity and utility of the compounds of the invention are demonstrated by one or more assays, including those described in more detail below:

assay 1. inhibition of cell cycle progression by compounds of the invention was determined using propidium iodide and BrdU (see figure 1 and table 2 for results).

Assay 2. A wide range of 60 cell lines derived from various human tumors, represented by the NCI series (panel) exposed to the compounds of the invention, were reduced in viability (results are shown in Table 3).

Assay 3. irreversible effect of the compounds of the invention on cells in the clonogenic cell survival assay (results are shown in table 3, figure 2 and figure 3).

Assay 4. evaluation of the reduction in viability of HCT-116 and IMR90 cells exposed to the compounds of the invention was performed using the Calcein AM assay (results are shown in tables 3 and 6).

Assay 5 viability of arrested tumor cells exposed to the compounds of the invention was inhibited, whereas arrested normal cells were not (results are shown in table 4 and figure 4).

Assay 6. inhibitory activity of compounds of the invention in certain kinase biochemical assays (results see tables 5 and 6).

The activity of compounds in xenograft tumor models was determined 7 (results see figures 5, 6, 7 and 8).

The affinity of the compounds for certain target proteins was determined 8 (see figure 9 for results).

The antiviral activity of the compounds of the invention was determined 9 (results are shown in table 7).

Measurement 1: cell cycle analysis with propidium iodide and BrdU

The percentage of cells in the G1, S and G2/M phases of the cell cycle was determined by staining the DNA with propidium iodide and the number of cells was quantified by flow cytometry using 2N or 4N DNA complement. Changes in cell distribution corresponding to the cell cycle exposed to Cdk inhibitors were assessed using this method.

Cell staining method for propidium iodide

3 batches of HCT-116 cells (100,000 cells/batch) were cultured in the presence of test compounds in T-25 flasks as per Table 1 below. The analysis was performed at 24, 48 and 72 hours. The adherent cells were collected by trypsinization, combined with floating cells in Falcon 12 × 75 flow cell tubes and harvested by centrifugation. The cell pellet was decanted from the medium, 100. mu.l of PI stain was added, and the cells were incubated at 37 ℃ for 20-25 minA clock. Preferably the cell count does not exceed 2X 10⁶-4×10⁶And/ml. An equal volume (100. mu.l) of PI salt was then added to the cells, which were then incubated at 4 ℃ for 1-2 hours. Stained cells were analyzed on a Becton Dickinson FACScan flow cytometer. The samples were protected from light. Figure 1 shows that apoptosis and nuclear re-replication demonstrated end-stage arrest of cells at G1 and G2 when exposed to compound a 37. Similar results were found for certain other compounds of the invention, including compound B16.

Determination of DNA binding BrdU

This method measures the percentage of cells that bind the nucleotide analog BrdU of newly synthesized DNA as the cells progress through the S phase of the cell cycle. Inhibition of BrdU binding was used to measure the effect of Cdk inhibitors on S-phase progression and DNA replication.

Method for BrdU labeling

3 batches of HCT-116 cells (100,000 cells/batch) were seeded in a T25 flask and incubated with the test compounds above. The analysis was performed at 24, 48 and 72 hours. BrdU was added to each T-25 flask, the stock from 10mg/ml to 10 u M final concentration, at 37 degrees C, will be cells were incubated for 16-18 hours. Cells were then prepared for Flow cytometry analysis as follows according to the manufacturer's protocol (BrdU Flow kit, BD-Pharmingen catalog # 2354 KK):

cells were harvested (adherent and floating) from T25 flasks, added directly to a Falcon 12X 75 flow cell tube as above, and subsequently fixed and permeabilized (30 min, room temperature) with 100. mu.l of Cytofix/Cytoperm buffer. The cells were then washed with 1ml of Perm wash buffer and the cell pellet was resuspended in 100. mu.l of Cytoperm Plus buffer and incubated on ice for 10 minutes. The cells were then washed with 1ml of Perm wash buffer and the immobilisation repeated in 100. mu.l Cytofix/Cyto Perm buffer for 10 minutes at room temperature. The cells were then washed with 1ml of Perm wash buffer. Next, the cells were treated with 100. mu.l DNase at 37 ℃ for 1 hour to expose bound BrdU, followed by another wash step with 1ml Perm wash buffer. The presence of bound BrdU was demonstrated with alpha-BrdU-FITC antibody (50. mu.l of a 1:50 dilution of antibody Perm wash buffer). The cells were protected from light and incubated at room temperature for 20-30 minutes. After incubation, cells were washed with 1ml of Perm wash buffer, resuspended in 300. mu.l of 2% FBS in PBS and analyzed by flow cytometry. The results are listed in table 2 as the concentration of compound that inhibits 50% BrdU binding (μ M).

And (3) determination 2: evaluation of Cdk inhibitors for inhibition of NCI series of human tumor cell lines

The evaluation of compounds with 60 cell line lines at the national cancer institute provided a wealth of information on the efficacy in relation to a variety of tumor types and genetic backgrounds. Included in this series are cell lines derived from leukemia, melanoma, lung, colon, brain, ovarian, breast, prostate and renal cancers. The use of this series provides a measure of the potency of a compound in cells in which there are alterations in many of the genes involved in tumour transformation, including p53, Her2/Neu as well as those involved in metabolism and those causing multi-drug resistance. The activity of the compounds can be assessed using data from these cell lines obtained using the following protocol.

The results of the NCI series of assays are listed in table 3 (NCI series), represented by two metrics that provide data: (a) the median value of the whole-cell series Mean-curve (Mean-Graph Mid-point) -Mean IC50 was calculated, except that IC50(μ M) was less than 10nM, which in this evaluation equaled 10 nM; (b) the compounds inhibited the activity of the adriamycin-resistant cell line (ADR-res), IC50(μ M).

Other compounds of the invention exhibit the following activity in NCI assays: (i) compound a 37: mean-curve median value <50nM and IC50 for inhibition of ADR-res cell growth <100 μ M; (ii) compound B16: mean-curve median value <50nM and IC50 for inhibition of ADR-res cell growth <10 μ M.

In vitro cancer screening method

Cells were grown in RPMI-164010% FCS and plated in 96-well microtiter plates at densities ranging from 5,000 to 40,000 cells/well. At 37 deg.C, 5% CO₂Next, the plate was incubated for 24 hours. Media containing twice the desired final concentration of compound (5 doses spanning 4 orders of magnitude (log)) was prepared and 100 μ l was added to each well containing 100 μ l of media and cells to give the desired final concentration. Plates were then incubated for an additional 48 hours.

The effect of compounds on cell viability was determined using a Sulforhodamine (Sulforhodamine) b (srb) assay that measures total protein. Cells were fixed with cold TCA to a final concentration of 10% and incubated for 60min at 4 ℃. The supernatant was discarded, and each plate was washed 5 times with water and air-dried. To each well was added a 4% (w/v) SRB/1% acetic acid solution, and each plate was incubated at room temperature for 10 minutes. Each plate was washed 5 times with 1% acetic acid and air dried. Bound dye was dissolved with 10mM trizma base and absorbance read at 515nM on a plate reader.

Measurement 3: protocol for clonogenic cell survival assay using HCT-116 cells

The assay was used to determine the concentration of compounds that resulted in irreversible loss of viability after exposure to a period of time. Typically, cells are exposed to compound for 1, 2, or 5 days and then transferred to compound-free growth medium. After a number of consecutive incubations in compound-free medium, the number of colonies recovered was counted and the number of viable cells was estimated.

The results of this survival assay for various compounds of the invention are listed in table 3 (clones) as the concentration of compound that inhibits 50% colony recovery (IC50) (μ M). FIG. 2 shows irreversible inhibition of cellular activity of HCT-116 cells by Compound A37 and the time course of such inhibition, with an IC50<50nM when exposed to Compound A for 24 hours. In the same assay, compound B16 showed an IC50<100nM, whereas within 30 to 60min, IC50 reached 100nM (fig. 3).

Method of measuring cell survival following exposure to a compound

The medium (RPMI-1640, 10% FCS, penicillin/streptomycin) was pre-warmed to 37 ℃ in a water bath. At 37 deg.C, 5% CO₂Next, the cells were incubated and allowed to grow. By digestion with pancreatinThe cells in the plates were recovered and counted using a hemocytometer. In a 15cm tissue culture dish, 25ml of medium, 1X 10 cells were inoculated⁴And (4) cells. 14 plates were set up for each test compound and incubated overnight at 37 ℃. The compounds were diluted to 7 concentrations with culture medium and the medium in the cells was replaced with the medium containing the test compound. Two plates and two control plates without compound were set for each concentration of test compound. Plates were incubated for 24, 48 or 74 hours as above, medium removed, replaced with fresh medium, and plates were incubated for an additional 7 days and washed with PBS. Colonies were stained with crystal violet solution (0.4% crystal violet, 20% ethanol) for 5 minutes, washed twice with distilled water and counted.

Measurement 4: production of calcein AM in the absence of serum proteins and serum proteins Evaluation of Cdk inhibitors by Retention assay

The potency of Cdk inhibitors as measured by loss of cell viability was determined using the calcein AM assay (molecular probe). Calcein AM is a substrate for intracellular esterases that divide only in living cells, producing fluorescent products that can be read quantitatively with fluorescent plates. This fluorescence signal is proportional to the number of viable cells, and thus loss of cell signal corresponding to exposure to Cdk inhibitor correlates with loss of viability. This assay can distinguish cell cycle arrest from loss of viability, where the cells may still be viable, and is therefore well suited for evaluating Cdk inhibitors. In such assays, potent cytotoxic compounds can result in a significant loss of cell viability.

Determination of cellular IC in human colorectal cancer cell line, HCT-116, Normal human fibroblasts, IMR90₅₀. Protein regulated IC₅₀Also in HCT-116.

The results of this viability assay are listed in Table 3 (HCT-116 (viability/protein regulated) and IMR-90). Other compounds of the invention were tested for HCT-116 cell viability IC50 (. mu.M, non-protein regulated) in Table 6.

Similar cell viability assays were performed for other cell lines as above. Other compounds of the invention were found to have an IC50(μ M) of: (i) compound a 37: HCT-116(<50nM), HCT-116 protein regulated (<500nM), a2780(<10nM), IMR90(<50 nM); (ii) compound B16: HCT-116(<10nM), HCT-116 protein regulated (<500nM), A2780(<10nM), IMR90(<100 nM). Calcein AM viability assay protocol.

Recovering HCT-116 or IMR90 cells from the plates by trypsinization, inoculating 1,000 or 4,000 cells into 24-well dishes at 37 deg.C and 5% CO₂The mixture was incubated overnight. HCT-116 cells were cultured in RPMI-1640, 10% FCS, while IMR90 cells were cultured in α -minimal essential medium, 10% FCS. After overnight incubation to allow adhesion, the media in each well was aspirated and media containing test compounds at concentrations of 0 to 250nM, spanning a total of 7 doses, was added. The plate was again placed in the incubator and incubated for a further 72 hours (3 days). The medium used for determining protein-regulated IC50 was RPMI-1640, 10% FCS plus 1mg/ml acid alpha-glycoprotein (Sigma G-9885) and 45mg/ml human serum albumin (SigmaA 3782). After 72 hours incubation with test compound, cells were washed twice with 1 × PBS, taking special care to remove all remaining buffer.

A50. mu.g aliquot of calcein (molecular Probe catalog No. C3100) was dissolved in 50. mu.l DMSO to prepare a 5. mu.M solution of calcein AM. After calcein was completely dissolved (10 min at room temperature), it was diluted to 10ml PBS. calcein/PBS (0.5ml) was added to each well. Plates were incubated for 75 minutes at 37 ℃ (protected from light) and fluorescence signals were read on a fluorescence plate reader (excitation 485/20, emission 530/25).

Measurement 5: arrested cell assay

Cyclin-dependent kinase (Cdk) activity is required to facilitate the progression of cells through different phases of the cell division cycle. In culture, normal non-transformed cell proliferation requires the presence of growth factors, which are removed by serum removal, resulting in loss of Cdk activity, with the result that when the cells enter the resting phase G₀When it is time, the cell cycle is exited. Thus, from a mechanistic point of view, but not limited by theory, withThe potency of Cdk inhibitors in arresting normal cells should be significantly reduced compared to their transformed counterparts.

The results of viability assays performed on arrested normal cells (IMR90) and arrested tumor cells (HT-116) with certain compounds of the invention are set forth in Table 4 below. FIG. 4 shows that the inhibitory activity of Compound A37 on the viability of arrested normal cells (IMR90) and tumor cells (HT-116) is increased. Compound a37 was found to have an IC50<50nM for arrested HCT-116 cells and an IC50>10 μ M for arrested IMR90 cells. Compound B16 demonstrated an IC50<50nM for arrested HCT-116 cells and an IC50>10 μ M for arrested IMR90 cells.

Evaluation of Compound potency by serum starvation-arrested HCT-116 and IMR90 cells

For each test compound concentration, HCT-116 cells were plated in triplicate in RPMI 1640 medium containing 10% fetal bovine serum, 24-well dishes at 200 or 2,000 cells/well, 5% CO at 37 deg.C₂The mixture was incubated overnight. The medium was removed from the plates containing 2,000 cells/well, the cells were washed once with serum-free medium, and 1m1 serum-free medium was added to the cells. Plates containing cells in the presence of serum and cells without serum were incubated for an additional 6 days.

For each compound concentration tested, IMR90 cells were plated in triplicate in MEM-alpha medium containing 10% fetal bovine serum, in 24-well dishes at 2,000 or 20,000 cell/well densities at 37 ℃ with 5% CO₂The mixture was incubated overnight. The medium was removed from 20,000 cells/well dish, the cells were washed once with serum-free medium, and serum-free medium was added to the cells. Plates containing cells in the presence of serum and cells without serum were incubated for an additional 3 days.

Evaluation of cell cycle arrest of HCT-116 and IMR90 cells by serum starvation

To ensure that the cells did break away from the cell cycle after serum removal, the percentage of BrdU positive cells representing those processes that passed S phase was determined in each experiment. For this experiment, cell viability was simultaneously assessed using the fluorogenic substrate SNARF-1 of an intracellular esterase active only in living cells. BrdU binding and SNARF-1 division were assessed together by flow cytometry, providing an assessment of the viability of arrested cells on a single cell basis. For this analysis, cells were stained with SNARF-1 as follows, and then the BrdU binding assay described above was prepared.

HCT-116 and IMR90 cells were seeded in serum-containing medium (RPMI-1640 or MEM-. alpha.with 10% FCS, respectively) in T25 flasks at the following densities. After 24 hours of growth, the medium was removed, the cells were washed and serum-free medium was added.

HCT-116+ FCS 5,000 cells

HCT-116-FCS 100,000 cells

IMR90+ FCS 100,000 cells

IMR90-FCS 200,000 cells

IMR90 cells were grown for a further 3 days, HCT-116 cells were grown for a further 6 days, and then pulsed with BrdU. A50. mu.g aliquot of SNARF-1 (molecular Probe catalog number C1272) was dissolved in 50. mu.l DMSO for 10 minutes at room temperature, and then diluted to 10ml of PBS. SNARF-1 was further diluted to 1:64,000, and then 200. mu.l was added to each cell tube in which cells had been cultured in the presence or absence of serum and pulsed with BrdU for 20 hours. Cells were incubated at 37 ℃ for 30 minutes and then washed with 3ml PBS.

These cells were then fixed in preparation for measuring BrdU binding as described above. The percentage of live (FL-2) and BrdU positive (FL-1) cells was determined on a FACScan flow cytometer.

Evaluation of viability of arrested HCT-116 and IMR90 cells following exposure to Compounds of the invention

5% CO at 37 ℃ in the presence of the Compound₂Next, cells were incubated for 72 hours (3 days) as follows, and the potency of the compounds on cycling and arrested cells was determined. Exposure of cycle and arrested HCT-116 cells and cycle IMR90 cells to in the 5 to 250nM range6 dose series. For arrested normal cells, the dose range was increased to 50nM to 25 μ M, compensating for the expected decrease in activity.

The effect on cell viability upon exposure to compound for 72 hours was evaluated using the calcein AM assay. Calcein AM is a fluorogenic substrate for intracellular esterases which are active only in living cells. This substrate division thus provides a measure of viability which is proportional to the number of cells.

Calcein AM stock was prepared by dissolving 50 μ g aliquots (molecular probe catalog No. C3100) in 50 μ l dmso. The tube was incubated at room temperature for about 10 minutes to ensure complete solubilization of calcein. The final solution was prepared by diluting calcein into 10ml PBS, which was protected from light.

The medium in the cells was aspirated, the cells were then washed twice with 1ml of PBS, and the PBS in the cells was completely removed by aspiration. 0.5ml of calcein/PBS solution was pipetted into each well. Plates were incubated at 37 ℃ for 75 minutes (protected from light) and read on fluorescent plate readout (excitation 485/20, emission 530/25).

Measurement 6: biochemical kinase inhibition assay

Enzyme: cdc 2/cyclin B was purchased from the market. Cdk 2/his-cyclin E_{Short term}Expressed with Sf9 cells. Cdk 2/cyclin A, Cdk 4/cyclin D1 and Cdk 6/cyclin D2, expressed in Sf9 cells. Protein kinase a (catalytic subunit, from bovine heart) and protein kinase C (mixed isozyme from rat brain) were purchased from the market.

Substrate: histone H1 was purchased from the market. GST-Rb is a glutathione S-transferase fused N-terminally to residues 379-928 of the Rb protein.

And (3) determination: measurement of [ gamma-³²P]Adenosine triphosphate radioactivity was used to determine Cdc 2/cyclin B activity. Cdc 2/cyclin B kinase and histone H1 were purchased from the market. The final assay contained 50mM Tris.HCl, 10mM MgCl₂1mM dithiothreitol, 50mu.M adenosine triphosphate, 2. mu. Ci³²P, 10% dimethylsulfoxide (from compound), pH7.5, 20. mu.g histone H1, 6U enzyme, 50. mu.L volume. The compound was added at each concentration of 1nM to 10. mu.M. The reaction was started by adding the enzyme, and was carried out at 30 ℃ for 20min, followed by addition of 20. mu.L of a reaction terminator (237mM disodium ethylenediaminetetraacetate, 105mM adenosine triphosphate, pH8.0) to terminate the reaction. Protein was precipitated by adding 35. mu.L of 70% (w/v) trichloroacetic acid, and the precipitate was captured on a 96-well glass fiber filter plate (Millipore, Inc.) and wetted with 25% (w/v) trichloroacetic acid. The filters were washed 10 times with 25% (w/v) trichloroacetic acid, 100. mu.l of scintillant (Microscint 20, packard Instruments) were added and binding was determined by scintillation counting³²The amount of P. Relative activity was determined by dividing the amount of bound radioactivity in the presence of compound by the amount of bound radioactivity in control experiments with DMSO alone and without compound. Before calculation, the background radioactivity measured in the 50mM EDTA-free compound-containing experiment was subtracted from all results. The 50% inhibitory concentration (IC50) of compound was determined by substituting the data into a standard equation:

P＝min+(max-min)(1/(1+(IC50/[I])^s)) (1)

where P-1-relative activity is relative inhibition, [ I ] is the compound concentration, max and min are the maximum and minimum relative inhibition (1 and 0), respectively, and s is the so-called Hill slope.

Cdk 2/cyclin E, Cdk 2/cyclin A, Cdk 4/cyclin D1 and Cdk 6/cyclin D2 activities were determined using a glutathione agarose capture assay. The enzyme is expressed in Sf9 insect cells as a heterodimer, and the substrate (GST-Rb) is glutathione-S-transferase fused to residues Rb retinoblastoma protein 379-928 expressed in E.coli (E.coli). The assay contained 50mM Tris.HCl, 10mM MgCl₂1mM dithiothreitol, 50. mu.M adenosine triphosphate, 2. mu. Ci [ gamma-³³P]Adenosine triphosphate, 10% dimethyl sulfoxide (from compound), pH7.5, 40. mu.g GST-Rb and enzyme, 100. mu.L volume. The compound was added at each concentration of 1nM to 10. mu.M. The reaction was allowed to proceed at 30 ℃ for 15min, and 70. mu.L of a reaction stop solution (237mM disodium ethylenediaminetetraacetate, 105mM adenosine triphosphate, p)H8.0) to stop the reaction. GST-Rb was captured by allowing it to bind to glutathione agarose beads (Amersham) for 110min, and the suspension was filtered through a glass fiber filter. The retained beads were washed 5 times with phosphate buffered saline containing 0.3% (w/v) Tween-20, 100. mu.l of scintillant was added, and binding was determined by scintillation counting³³The amount of P. Relative activity was determined by dividing the amount of bound radioactivity in the presence of compound by the amount of bound radioactivity in control experiments with DMSO alone and without compound. Before calculation, the background radioactivity measured in the experiment containing 50mM disodium EDTA without compound was subtracted from all results. 50% Inhibitory Concentration (IC) of a compound was determined by substituting data into equation (1)₅₀)。

Protein kinase C and protein kinase a were determined by TCA precipitation assay using histone H1 as a substrate. For protein kinase A, the final assay contained 50mM Tris, 10mM MgCl₂1mM dithiothreitol, pH7.5, 12. mu.M adenosine triphosphate, 10% (v/v) dimethylsulfoxide (from compound), 20. mu.g histone H1, 2. mu. Ci [ gamma-³²P]Adenosine triphosphate, 0.2U protein kinase A, 100. mu.l of the assay solution. The protein kinase C determination solution contains 50mM Tris and 10mM MgCl₂1mM dithiothreitol, 0.8mM CaCl₂pH7.5, 5. mu.M adenosine triphosphate, 10% (v/v) dimethylsulfoxide (from compound), 20. mu.g histone H1, 2. mu. Ci [ gamma-³²P]Adenosine triphosphate, 0.01U protein kinase C, 50. mu.l of the assay solution. The assay was started by adding the enzyme, allowing the reaction to proceed at 30 ℃ for 10min, and then stopping the reaction by adding 237mM disodium EDTA, 105mM adenosine triphosphate, and pH8.0 in 0.4 volume. 0.5 vol 75% (w/v) trichloroacetic acid was added to precipitate the protein from the termination reaction, which was captured by filtration through a 96-well glass fiber filtration unit (Millipore). The filter was washed 10 times with 25% (w/v) trichloroacetic acid, 100. mu.l of scintillator was added, and bound [ 2], [ 2] was measured by scintillation counting³²P]The amount of phosphate ester. 50% Inhibitory Concentration (IC) of a compound was determined by substituting data into equation (1)₅₀)。

The results of the above measurements are shown in tables 5 and 6.

Measurement 7: xenograft tumor model

A medicine is provided. The compounds of the invention are synthesized and formulated with biocompatible carriers for intravenous administration. Obtaining CPT-11(Pharmacia) drug, prepared with 5% dextrose-water (D5W). All formulations were made fresh weekly and the injection volume was adjusted to body weight (0.2ml/20g mouse).

Mice/chow. Female nu/nu mice from Charles River were housed in quiet mini-cages, provided with free access and irradiated standard Rodent chow (Purina Pico-Lab Rodent Diet 20).

The Maximum Tolerated Dose (MTD) was determined. 8 week old mice were paired and grouped into groups of 5-8 animals and a preliminary toxicity study was performed with unknown test compounds. Animals were treated with test compound daily for 10 consecutive days by intravenous injection and weighed twice weekly. Mice were frequently examined for clinical signs due to any drug related adverse reactions. Acceptable toxicity of anticancer drugs in mice is defined by NCI as: the average group weight reduction is no more than 20%, and the toxic mortality of the treated animals is no more than 10%.

Standard protocol. To make a single 1mm³Tumor fragments (tumor brie) were implanted s.c. into athymic nude mice (6-7 weeks old males or females) or 5-10X 106 tissue culture derived cells were implanted into synadrome. Animals were initially monitored twice weekly for tumor growth, then when the implant approached about 100mm³At the predetermined volume, monitoring was daily. When tumors grew to a calculated weight of 62-221mg, animals were paired and organized into appropriate experimental treatment groups (8-10 animals/group) and treatment with test compounds was initiated. The positive control group was administered as the past control group. Tumor weights were calculated and body weights were weighed twice weekly and animals were frequently observed for adverse drug reactions. The protocol requires that any animal with a tumor mass of 1000mg die immediately.

Tumors were measured by measuring their length and width with digital calipers. Tumor weight was estimated using the formula:

tumor weight (mg) ═ w² x l)/2

Where w is the tumor width and l is the tumor length in mm. These values may also be in units of volume (mm)³) And (4) showing.

Experimental treatment may result in Partial Regression (PR) or Complete Regression (CR) of the tumor. PR is where the tumor size is 50% or less than the initial (day 1) size but greater than 0.0mg for three consecutive days during the study, and CR occurs when there is no measurable tumor mass for three consecutive days. Cure is defined as an animal whose tumor shrinks to 0mg and remains in that state until the end of the experiment.

Log Cell Kill (LCK) is a calculation that determines the percentage of tumor cells killed after initial treatment and can be used as a quantitative measure of potency:

log Cell Kill (LCK) ═ T-C)/(3.32) (Td)

Where T is the average time required for the treated mice to reach 1000mg, C is the average time for the control tumor size to reach 1000mg, Td is the time estimated to double the tumor in exponential growth phase using linear regression analysis of the control tumor semilog growth curve, and 3.32 is the fold increase required for the population to increase 1-log10 units. Each LCK unit represents 1-log10 units of cell kill (e.g., 90% kill at 1LCK, 99% kill at 2LCK, etc.). We believe that when the LCK values of the compounds are >1, which corresponds to > 90% tumor cell killing, they are significantly active.

Tumor Growth Inhibition (TGI) is a calculation that describes the amount of a compound that inhibits tumor growth over a period of time. It is expressed as:

％TGI＝100(1-T/C)

where T is the average tumor size of the compound-treated group on the indicated day, and C is the average tumor size of the vehicle control group on the same day.

Toxic death is defined as death resulting from compound treatment and not from a disease state. If the tumor size does not reach 1000mg after the final compound treatment, the animal dies within 1 week, and death due to toxicity is considered. There were non-tumor related death records after this point, but not considered toxic deaths.

Tumor regression is defined by the following convention: regression was defined as fraction (PR) if tumor weight was reduced to < 50% of initial weight (<50 mg). Regression was defined as Complete (CR) if tumor weight decreased to less than measurable weight during the experiment. Cure is defined as animals without tumor at the end of the observation period.

As a result: figure 6 shows the results obtained for several compounds of the invention in a HCT116 xenograft tumor model. Figure 6 shows the results obtained for compound a37 in an a2780 xenograft tumor model. Figure 7 shows the results obtained for compound a37 in the PC3 xenograft tumor model. Figure 8 shows the results obtained for compound B16 in an a2780 xenograft tumor model.

Measurement 8: measuring the affinity between a target molecule and a compound

To demonstrate the suitability of a particular compound for the uses set forth herein, it may be useful to characterize the binding properties of such a compound with its known binding partners (if any). This should not be taken as limiting the scope of the invention.

The affinity of the compounds for their respective binding partners can be determined, e.g. using BIACORE^TMAssay system (Biacore AB, Uppsala, SE). Other systems that produce similar quantitative results, such as those developed by Affinity Sensors (Cambridge, UK), will be apparent to those skilled in the art.

In one representative method, compound R is assayed for binding to its known binding partner CDK 2/cyclin E. The assay was performed at 22 ℃ on a BIACORE 2000 SPR-Biosensor in electrophoresis buffer containing 20mM HEPES (pH7.4), 150mM NaCl, 1mM DTT and 0.005% Tween 20 (protein grade, Calbiochem). A10. mu.M solution of Compound R at pH8.0 was coupled by amide couplingThe linker chemistry was coupled to the dextran surface of a CM5 sensor protein chip (research grade). To characterize the binding of compound R to proteins such as CDK 2/cyclin E, the purified protein fraction was diluted in running buffer to give 9 different protein concentrations, then each concentration was passed successively over the sensor protein surface for 5min, followed by running buffer over the surface at the same flow rate over 5 min. The association and dissociation of the CDK 2/cyclin E complex on the CM 5-compound R-loaded chip was measured at a flow rate of 30 μ l/min. After each experiment, two consecutive injections of 3M hydrochloric acid were performed before loading the next sampleThe chip was regenerated (20 seconds, 30. mu.l/min).

Data were analyzed using Bioevaluation software version 3.1 (Biacore AB, Uppsala, SE). Curves were normalized to the start of injection, and background obtained with control surfaces. The association and dissociation rates were determined separately, or all using the Langmuir 1:1 binding model. The following equation was used to calculate affinity (K)_D)：

K_DAssociation of k dissociation/k

Similar operations can be performed as above with other target proteins such as Cdk9, Cdk4, etc. For example, inhibitors of Cdk9 are effective in the treatment or prevention of HIV and/or AIDS.

FIG. 9 shows an example of the results obtained with CDK 2/cyclin E bound to a CM 5-compound R-loaded chip. K calculated from these data_DEqual to 8, 0+/-2, 8 nM.

Measurement 9: antiviral activity

The activity of certain compounds of the invention was evaluated by measuring the potency of these compounds on acutely infected cells in low-passage (low passage), clinically isolated HIV-1 ROJO-infected Peripheral Blood Mononuclear Cells (PBMCs). The therapeutic index of these compounds can be estimated using these normal human cells. Fresh PBMCs from both donors were pooled and stimulated with PHA-P for 48-72 hours. The cells are then cultured in the presence of IL-2 to maintain cell division by mitogenic signaling. The virus was added at a multiplicity of infection of 0.1. After infection, cells were cultured for 7 days, and then evaluated for potency. Viral replication was measured by the level of reverse transcriptase activity in the supernatant and cytotoxicity was determined using the MTS assay. The results of two assays for anti-HIV potency and cytotoxicity of these compounds are listed in table 7.

All references, patents, and publications cited herein are hereby incorporated by reference in their entirety.

Table 1.

Compound concentration range for assay 1.

Concentration of the Compound

0

5nM

10nM

25nM

50nM

100nM

250nM

Table 2.

Results of certain compounds of the invention in the BrdU binding assay described above.

TABLE 3

The results of certain compounds of the invention in the following in vitro cell activity assays described above: viability and clonogenic cell viability assays were performed with HCT-116 cells, viability assays with IMR90 cells, and measurement of the activity of two anti-NCI cell lines (mean-curve MID value and IC50 for the anti-doxorubicin-resistant cell line).

Table 4.

Results of certain compounds of the invention in the above described arrested cell assay (IC50, nM).

Table 5.

Results of certain compounds of the invention in the above biochemical inhibition assay (IC50, μ M)

Compound (I)	Cdk 2/cyclin E	Cdk 2/cyclin A	Cdk 4/cyclin D	Cdc 2/cyclin B	Cdk 6/cyclin D2	PKA	PKC	c-Abl
Compound (I)	Cdk 2/cyclin E	Cdk 2/cyclin A	Cdk 4/cyclin D	Cdc 2/cyclin B	Cdk 6/cyclin D2	PKA	PKC	c-Abl	A	<0.1	<0.1	<1	<1	<1
B	<0.01	<0.1	<10	<0.1					A	<0.1	<0.1	<1	<1	<1
B	<0.01	<0.1	<10	<0.1					C	<0.1	<0.1	<1	<1		>10	>10	>10
D	<0.1	<0.1	<1	<1					C	<0.1	<0.1	<1	<1		>10	>10	>10
D	<0.1	<0.1	<1	<1					E	<0.01	<0.01	<0.01	<0.1	<0.01	<10	<10	<10
F	<0.1	<0.1	<0.01	<0.1	<0.01				E	<0.01	<0.01	<0.01	<0.1	<0.01	<10	<10	<10
F	<0.1	<0.1	<0.01	<0.1	<0.01				G	<0.1	<0.1	<0.01	<0.1		>10		>10
H	<0.1	<0.1	<0.1	<0.1					G	<0.1	<0.1	<0.01	<0.1		>10		>10
H	<0.1	<0.1	<0.1	<0.1					I	<0.1	<0.1	<0.01	<0.1
J	<0.1								I	<0.1	<0.1	<0.01	<0.1
J	<0.1								K	<0.1
L	<0.1		<0.1	<0.1					K	<0.1
L	<0.1		<0.1	<0.1					M	<0.1
N	<0.1			<0.01					M	<0.1
N	<0.1			<0.01					O	<0.1
P	<0.1								O	<0.1
P	<0.1								Q	<0.01	<0.1	<0.01	<0.1	<0.1

Table 6.

Results of other compounds in the above biochemical inhibition and HCT-116 viability assay (non-protein regulation).

TABLE 7

The antiviral activity of certain compounds of the present invention results. IC 50: 50% inhibition of viral replication as measured by reverse transcriptase levels in the supernatant; TC 50: 50% cytotoxicity (MTS); TI: TC50/IC 50.

Compound (I)	IC50(μM)	TC50(μM)	TI
Compound (I)	IC50(μM)	TC50(μM)	TI	A32	<0.01	<0.1	>10
A61	<0.01	<0.1	>10	A32	<0.01	<0.1	>10
A61	<0.01	<0.1	>10	A64	<0.01	<0.1	>10

C3	<0.01	<0.1	>10
C3	<0.01	<0.1	>10	C4	<0.1	<0.1	>1
AZT	<0.01	>1.0	>100	C4	<0.1	<0.1	>1

TABLE A

TABLE B

Watch C

Table D

Other compounds of the invention that result from the selection of appropriate characteristics from the possible characteristics in the table below. Compound a77 was produced, for example, from the following selection: non-morpholino-aryl-OCH₂(CO) -piperazine-CH₃。

Levorotatory cycloaryl or ring substituent N dextrorotatory substituent

Heteroaryl character of the substituent

CH₃Morpholino aryl OCH₂NHM alkyl

Isopropyl piperazine thiophene OCH₂(CO) NMM alkoxy

CH₃CH₂O(CO)CH₂ SO₂Morpholino alcohols

No OCH₂(CO)OCH₂Piperazine substituted amines

Piperidine acid

Pyrazole esters

Pyrrolidine CH₂CH₂OCH₃

CH₂CH₂OH

CH₂NH₂

CH₂NHCH₂CH₂CH₃

CH₂NHCH₃

CH₂NHCHCH₃CH₃

CH₃

CHCH₃CH₃

COOCH₂CH₃

Is free of

TABLE E

Other compounds of the invention that result from the selection of appropriate characteristics from the possible characteristics in the table below. Compound B3 was produced, for example, from the following selection: non-morpholino-aryl-CH₂-piperazine-CH₂CH₂OH。

Levorotatory cycloaryl or ring substituent N dextrorotatory substituent

Heteroaryl character of the substituent

CH₃Morpholino aryl CH₂NHM alkyl

Isopropylpiperazine thiophene CH₂CH₂NMM alkoxyBase of

CH₃CH₂O(CO)CH₂ CH₂CH₂CH₂Morpholino alcohols

Without CH₂CH₂CH₂CH₂Piperazine substituted amines

Piperidine acid

Pyrazole esters

Pyrrolidine CH₂CH₂OCH₃

CH₂CH₂OH

CH₂NH₂

CH₂NHCH₂CH₂CH₃

CH₂NHCH₃

CH₂NHCHCH₃CH₃

CH₃

CHCH₃CH₃

COOCH₂CH₃

Substituted or unsubstituted aryl

Carbocyclic ring of family

Substituted or unsubstituted aryl

Group heterocyclic ring

Is free of

Claims

1. A compound having the structure of formula I:

2. the compound of claim 1, wherein the pharmaceutically acceptable salt form has the structure:

3. a pharmaceutical composition comprising a pharmaceutically acceptable excipient and a compound of any one of claims 1-2.

4. Use of a compound according to any one of claims 1-2 in the manufacture of a medicament for the treatment of a hyperproliferative disorder.

5. Use of a compound according to any one of claims 1-2 in the manufacture of a medicament for inhibiting cell proliferation.

6. Use of a compound according to any one of claims 1-2 in the manufacture of a medicament for the treatment of a viral infection.

7. The use of claim 6, wherein the viral infection is caused by Human Immunodeficiency Virus (HIV).

8. Use of a compound of any one of claims 1-2, or a composition comprising a therapeutically effective amount of said compound, in the manufacture of a medicament for inhibiting a cyclin dependent kinase in a host in need of such treatment.

9. Use of a compound of any one of claims 1-2, or a composition comprising a therapeutically effective amount of said compound, in the manufacture of a medicament for treating a condition associated with a cyclin dependent kinase in a host in need of such treatment.