IE20010784A1

IE20010784A1 - N-benzylindole-3-acetic acid derivatives

Info

Publication number: IE20010784A1
Application number: IE20010784A
Authority: IE
Inventors: Martin Michael Clynes
Original assignee: Univ Dublin City
Priority date: 2000-08-24
Filing date: 2001-08-24
Publication date: 2003-04-16
Also published as: WO2002020478A1; AU2001282410A1

Abstract

A family of compounds, N-Benzylindole-3-acetic acid derivatives, are active as inhibitors of multiple drug resistance protein (MRP) while having low direct toxicity, low COX -1 inhibitory activity and, in some cases, having useful activities such as COX -2 inhibition. The agents are useful in combination with MRP substrate drugs in the treatment of, for example, drug resistant cancer or tumours likely to develop drug resistant cancers because they can be given at higher concentration while being less toxic and having less side effects than known MRP inhibitors. The agents may also be more beneficial in the treatment of diseases which are dependent on MRP-1 activity or where MRP-1 activity reduces the effectiveness of existing therapies.

Description

The invention relates to N-benzylindole-3-acetic acid derivatives for use in the prophylaxis and/or treatment of drug resistant cancer.

Background art During the last few decades, new cytotoxic agents have been developed that can improve the treatment outcome of many cancers, including solid tumours. Among those drugs, the natural product isolates and their derivatives - including anthracydines, vinca alkaloids, epipodophyllotoxins, and taxanes - have proved to be effective in inducing remissions and cures in many malignancies 1,z.

The development of drug resistance is considered one of the most significant obstacles to the effective treatment of cancer 3’4. Elucidation of the mechanisms determining inherent or chemotherapy-induced resistance in human tumours to many cancer agents is of great interest to researchers and of great importance to patients.

Resistance to drugs may be due to one or more general mechanisms including, changes in drug distribution in the body (for example modifications of blood flow), changes in cellular permeability to the drug in question, modifications in target mechanisms (e.g. modifications of the topoisomerase enzyme), adaptive changes in the metabolism of the drug, changes in the downstream effectors of drug action (e.g. inhibition of apoptosis mechanism in cancer treatment), sequestration of drug into areas of the cell where it cannot affect the cellular target or active efflux of the drug from the target cell5.

DUBL01/C -2In the field of cancer treatment, several different efflux mechanisms have now been characterised which are thought to reduce the active intracellular concentration of drug within the cancer cell, thereby allowing the cell to survive in the presence of these drugs and continue as a growing, viable cancer4.

The first such mechanism which was discovered and characterised was resistance associated with the over-expression of a membrane bound protein pump, termed P-glycoprotein (P-gp) also known as Pl70 6’7,8. This pump utilises the energy of ATP to actively efflux a variety of agents including a broad spectrum of cancer chemotherapeutic drugs 9. The net effect of this efflux mechanism is to reduce the time a cell is exposed to the agent and to reduce its maximum intracellular concentration. Since most cytotoxic cancer therapies are given at or near the maximum tolerable dose, and have little selectivity for cancer cells, the dose given cannot be increased to compensate without becoming toxic for the patient. P-gp can pump, a wide variety of structurally dissimilar agents including many of the more effective, first-line anti-cancer drugs such as doxorubicin and vinca alkaloids 10. If cells already express a high level of P-gp activity they will be intrinsically resistant to the cytotoxic effects of these agents. Likewise, if P-gp expression is induced or a P-gp-overexpressing subpopulation is selected, in a tumour by initial cytotoxic exposure, all of the other substrate agents will be less effective if given subsequently10. Because P-gp was the earliest multidrug resistance (MDR) mechanism to be investigated clinically, a significant amount of information has amassed on the clinical impact of P-gp inhibition in different cancers and with different combinations of drugs. To date almost all trials of P-gp modulators have failed to show significant clinical benefit for reasons which included toxicity associated with the inhibitors, toxicity from prolonged inhibition of P-gp, intolerable side effects of the inhibitors and unfavourable pharmacokinetic interactions between the P-gp substrate drugs and P-gp inhibitors11.

DUBL01/C ΙΕϋΐΟΉ -3More recently a new family of cancer-related efflux proteins was discovered 12. The first member of this family was a 190 KDa glycoprotein termed multidrug resistance protein (MRP) (also known as Multidrug resistance-related Protein & Multidrug Resistance-associated protein)13,14 but it is now clear that this protein (more recently termed MRP-1) 12 is one of at least 6 proteins (MRP 1-6) which share an identifiable level of homology and which actively efflux drugs across cellular lipid membranes 63. As with P-gp, the MRP proteins are also ATPdependent efflux pumps. MRP-1, the most studied member of the family, has similar (but not identical) substrate specificities to P-gp but the two proteins only share a 30 % similarity in protein sequence 12. It is accepted in the literature that MRP substrates include the anthracyclines (e.g. doxorubicin or epiruhicin), epipodophyllotoxins (e.g.etoposide), vinca alkaloids (especially vincristine) methotrexate and taxanes (especially taxol) 15,16.

MRP-1 is over-expressed in a number of multidrug-resistant cell lines, but, more interestingly, it now appears that it may be over-expressed, ahead of the overexpression of P-170, in a variety of human cancers, including breast carcinoma 17,18. MRP is also present in many normal cells but over-expression in tumour cells appears to be associated with poor prognostic outcome in several forms of cancer and chemotherapeutic treatment19. The role of MRP expression in drug resistance remains to be fully explored, although evidence suggests a correlation with prognosis or impact of chemotherapy in neuroblastoma20, retinoblastoma 2I, primary breast carcinoma 22, relapsed acute leukemia 23, acute myeloid leukemia 24, malignant melanoma 25, and non-small cell lung cancer 26. As investigations of the role of MRP in cancer progress, it is likely that it will be recognised as an important factor in many other forms of cancer.

Inhibition of MRP-1 has been suggested as a mechanism to improve the cancer therapy with MRP-1 substrate drugs. To date no clinical trials of specific MRP-1 inhibitors have been initiated but several agents have been suggested as viable, DUBL01/C -4specific inhibitors of MRP for clinical use27.

Several groups have demonstrated that MRP-1 can be inhibited by clinically relevant concentrations of NSAID agents such as indomethacin28,29,3°. NSAIDs are currently used to treat a variety of inflammatory conditions where their therapeutic activity is associated with the ability to inhibit the enzyme cyclooxygenase (COX) in the body. Two forms of cyclooxygenase have been described, a constitutively expressed variant COX-1, which is largely responsible for cytoprotective mechanisms, such as those operating in the stomach lining, and COX-2, which is an inducible form, primarily responsible for a variety of inflammatory reactions 31,32. COX-2 activity has also been associated with progression of certain forms of colon cancers and specific inhibitors of COX-2 have received approval for use in preventing the emergence of cancer in patients genetically predisposed to develop colon cancer as young adults (Familial Adenomatous Polyposis)33,34,35. The NSAIDs currently in common clinical use generally have little specificity and inhibit both forms of COX. This means that prolonged use, which is often required with chronic immunological conditions such as arthritis, is commonly associated with negative effects such as gastrointestinal intolerance, gastro-iniesrinal ulceration and reduced renal blood flow (all associated with COX-1 inhibition). Agents such as indomethacin are also quite toxic directly and this limits the maximum dose which can be given acutely36.

Earlier work 27 has established that a number of NSAIDs, including indomethacin and sulindac, have the ability to potentiate the toxicity of a range of MRP-substrate anticancer drugs such as doxorubicin, vincristine and VP-16, especially in MRP- expressing cell lines such as DLKP and A549 while this effect was not evident in P-gp expressing cell lines such as DLKPA. Significantly, the activity in enhancing the toxicity of the anticancer drugs does not appear to be directly related to cyclooxygenase inhibition as a number of DUBLO1/C IE ο 1 ο I 8 4 -5NSAIDs which are known to act as COX inhibitors do not display this effect. Two other groups have reported similar effects with indomethacin in other MRP-expressing cell lines.28,29 Indomethacin was developed by Shen and co-workers in the Merck, Sharp and Dohme Research Laboratories in the early 1960’s37,3S. Significantly the lead compound was l-henzyl-5-mefhoxy-2-methylindole-3-acetamide. When the side chain was modified to a carboxylic acid, improved anti-inflammatory activity resulted. In an exhaustive study, Shen and co-workers prepared a further three hundred and fifty analogues to determine the structure-activity relationship.

The result of this study was four candidates that went forward for clinical evaluation and of these MK-615, later to receive the generic name indomethacin, proved to be the most effective anti-inflammatory agent in human subjects.

Indomethacin, in common with most other NSAIDs, has been shown to inhibit both COX-1 and COX-2 isoforms; the gastrotoxicity associated with this class of compounds is believed to be due to COX-1 inhibition and accordingly extensive searching for selective COX-2 inhibitors has been undertaken in recent years 39,4°.

Indeed the indomethacin derivative in which the benzoyl group is replaced by a 4-bromobenzyl group has been reported as a highly selective COX-2 inhibitor 41. However, some NSAIDs also inhibit other enzymes, including phospholipase A2 (PLA2). In 1978, Kaplan and coworkers found that indomethacin inhibits PLA2 from rabbit polymorphonuclear leukocytes, 42 while more recent reports have studied the inhibitory abilities of other NSAIDs 43. Development of selective inhibitors of human PLA2, based on zV-benzyl derivatives of indomethacin, has attracted considerable attention recently, including work by Kreft and coworkers44 at Wyeth-Ayerst and Shevitz, Bach and co-workers 45,46’4/’48 at Lilly.

DUBL01/C -6A number of studies have considered the lipoxygenase and cycloxygenaseinhibitory properties of NS AID s, including indomethacin, aspirin and sulindac, and their effects on tumour growth and promotion 49,50.

It is clear that an efficient inhibitor of MRP-1 with little or no side effects would be highly valuable therapeutically. Of the agents currently available, MRPinhibitory NSAIDs represent the most likely agents to provide therapeutically useful MRP inhibition. However, the MRP-inhibitory NSAIDS, particularly indomethacin, have side effects, particularly gastrointestinal pathology, which would likely limit their clinical and commercial potential. This invention is directed towards providing inhibitors of MRP which provide for a similar level of MRP inhibition while having less toxicity and associated side effects than known MRP inhibitors.

Statements of Invention According to the invention there is provided a compound of the formula RO I DUBL01/C -7- IEOH*78* wherein R1 = Me, Et R= CrC6 alkyl which may be substituted or unsubstituted Y = OHorNH2 wherein X is H, Cl, Br, F with the provisos that: (i) when X in the 3 position then X =F (ii) when X is in the 4 position then X = Cl, Br, or F, and pharmacologically acceptable salts thereof, in the preparation of a medicament for the prophylaxis and/or treatment of drug resistance cancer.

Preferably in the compound of formula I the CrC6 alkyl are substituted with one or more of the same or different of: hydroxy, carboxy, phosphonic groups.

Most preferably the compound of Formula I has a carboxylic acid or a phosphonic acid functional group.

Preferably the pharmacologically acceptable salt of a compound of Formula I is an alkali metal salt selected from the group comprising sodium, potassium and lithium or an alkali earth metal salt selected from the group comprising magnesium and calcium.

A preferred embodiment of the invention provides use of a compound of the formula DUBL01/C co2h II wherein X is F, Cl or Br and pharmacologically acceptable salts thereof, in the preparation of a medicament for the prophylaxis and/or treatment of drug resistance cancer.

Compounds of formula II have been shown in particular to increase the anticancer effect of anti-cancer drugs such as adriamycm and to inhibit the transport of MRP-1 substrate drugs from cells. The compounds have also been found to be efficient inhibitors of COX-2. These compounds by virtue of their low toxicity and low side effects have major therapeutic potential in the prophylaxis and/or treatment of drug resistant cancer.

A further preferred embodiment of the invention provides use of a compound of formula DUBL01/C ΙΕΟ 1 07 8 4 wherein X is Me or Et and pharmacologically acceptable salts thereof, in the preparation of a medicament for the prophylaxis and/or treatment of drug resistance cancer.

Compounds of formula III have been found in particular to increase the effect of anti-cancer drugs such as adriamycin and taxol. Because the compounds have low toxicity and low side effects the compounds have major therapeutic potential in the prophylaxis and/or treatment of drug resistant cancer.

One embodiment of the invention provides use of a compound of formula I, II or TTT for the prophylaxis and/or treatment of multiple drug resistance related disease Another embodiment of the invention provides use of a compound of Fonnnla L II or ΠΤ for the prophylaxis and/or treatment of leishmaniasis.

One embodiment of the invention provides for the use of a compound of formula I, Π or ΠΙ with an identifiable marker, preferably the identifiable marker is a radiolabelled marker.

A further embodiment of the invention provides for the use of compounds of formula I, II or III for identifying tumours which are mutidrug resistant.

The invention also provides a pharmaceutical composition comprising a compound of formula I, II or III as hereinbefore described.

DUBL01/C -10Preferably in the compound of formula I the Ci-C6 alkyl are substituted with one or more of the same or different of hydroxy, carboxy, phosphonic groups. Most preferably the compound of formula I has a carboxylic acid or a phosphonic acid functional group. Preferably the compound of formula I comprises a pharmacologically acceptable salt preferably an alkali metal salt selected from the group comprising sodium, potassium and lithium or an alkali earth metal salt selected from the group comprising magnesium and calcium.

Preferably in the pharmaceutical composition of the invention the MRP substrate drug is a MRP-1 substrate drug.

Most preferably the substrate drug is an anti-cancer drug selected from the group comprising an anthracycline, a vinca alkaloid, an epipodophyllotoxin or a taxane. Ideally the anti-cancer drug is selected from any one or more of anthracycline, adriamycin, daunorubicin, epirubicin, vinca alkaloid, vincristine, epipodophyllotoxin etoposide (VP-16), teniposide or taxol.

The pharmaceutical composition of the invenrion may be in the form of an emulsion, liposome, patch, powder and/ or complex.

Preferably the composition comprises an ingestable carrier in which case the composition is in the form of a tablet, capsule, syrup or powder.

In one embodiment of the invention the pharamceutical composition is in combination with an agent which is a substrate for multidrug resistance related protein for simultaneous, sequential or separate use.

Preferably the pharmaceutical composition comprises an adjuvant such as an anti-emetic, an anti-inflammatory agent or a cancer chemotherapy medicament.

DUBL01/C ¢010784 -11Most preferably the pharmaceutical composition of the invention is in a form for oral, intravenous, intramuscular, intraperitoneal, intradermal, intravesicular and/or rectal administration.

The invention provides a compound of formula I or pharmacologically acceptable salts thereof and an identificable marker, preferably die identifiable marker is a radiolabelled marker. These compounds are potentially therapeutically valuable in identifying whether tumours are mutidrug resistant or not.

The invention also provides a compound of the formula wherein X is 3-F.

The invention further provides a compound of the formula X DUBL01/C wherein X is F, Cl, Me or MeS.

The invention also provides a compound selected from l-(4-fluorobenzyl)-5-methoxy-2-methylindole-3-acetic acid; l-(3-fluorobenzyl)-5-methoxy-2-methyhndole-3-acetic acid; and l-(4-chlorobenzyl)-5-methoxy-2-methylindole-3-acetic acid.

The invention further provides a compound selected from l-(4-methylthiohenzyl)-5-methoxy-2-methylindole-3-acetic acid; l-(4-methylbenzyl)-5-methoxy-2-methylindole-3-acetic acid; l-(3-chlorobenzyl)-5-methoxy-2-methylindole-3-acetic acid; -(2-chlorobenzyl)-5-methoxy-2-methyIindole-3-acetic acid; l-(4-Chlorobenzyl)-5-methoxyindole-3-acetic acid; and l-{4-Chlorohenzyl)-2-methoxyindole-3-acetic acid; The invention fsrtiserprovides a compound selected from 3-Ccaibomeahoxy)-2-ethyl-l-(phenylmethyl)-5-methoxy indole; 3-(hydroxymethyl)-2-methyl-l-(phenylniethyl)-5-methoxy indole; 3-(hydroxymethyl)-2-ethyl-l-(phenylmethyl)-5-methoxy indole; 3-(cyanomethyl)-2-methyl-l-(phenylmethyl)-5-methoxy indole; and 3-(cyanomethyl)-2-ethyl-1 -(phenylmethyl)-5-methoxy indole.

The invention also provides a pharmaceutical composition comprising a compound of the invention. Preferably the composition comprises a compound of the invention and an MRP substrate drug.

DUBL01/C -13The invention further provides use of a compound of formula I, II or III in the preparation of a medicament for the prophylaxis and/or treatment of drug resistant cancer, multiple drug resistance related disease or leishmaniasis.

The compound may be present in the medicament at an amount suitable to produce a concentration in the bloodstream of from 20ng/ml to 100pg/ml most preferably from lgg /ml to 20gg/ml.

The compounds of the invention are likely to be less toxic than NSAID-based inhibitors and can also have less side effects on other enzymes including COX-1 or have specific effects on potentially damaging enzymes such as COX-2. This is likely to make these compounds more useful as they are likely to have better specificity and less toxicity and side effects.

DUBL01/C -14|E 0 Α ϋ 7 8 4 Detailed Description The invention will be more clearly understood from the following description thereof given by way of example only.

We have found a family of compounds, N-Benzylindole-3-acetic acid derivatives, which are active as inhibitors of MRP while having less direct toxicity, less COX -1 inhibitory activity and, in some cases, having useful activities such as COX-2 inhibition, in comparison to other known MRP inhibitors. A number of these compounds are known per se, however none have previously been described as having MRP inhibitory activity48'60,61.

These compounds appear to be more useful in combination with MRP-1 substrate drugs in the treatment of, for example, drug resistant cancer or tumours likely to develop drug resistant cancers because they can be given at higher concentration (producing better inhibition of MRP-1) while being less toxic and having less side effects than other known MRP inhibitors. These compounds have been found to potentiate the toxicity of MRP substrate anti-cancer drugs. The compounds may also be more beneficial in the treatment of diseases which are dependent on MRP-1 activity or where MRP-1 activity reduces the effectiveness of existing therapies.

We have also found that combinations of these compounds are useful in increasing the potency of substrates for MRP and display less toxicity and side effects than existing agents.

In experimental biological research, these compounds may also be useful as they allow scientific investigators to look at the effect of inhibition of MRP-1 activity in combination with or in the absence of other specific effects (depending on the exact agent chosen) such as COX activity.

DUBL01/C MRP inhibitors of the present invention may be used with radiolabelled markers, and the like, attached, to identify whether tumours are multidrug resistant or not allowing the treatment regime to be altered accordingly.

Synthesis of N-benzylindomethacin derivatives A series of TVbenzyl derivatives compounds 4, 5, 7-11, 13-20, were prepared by benzylation of commercially available indole-3-acetic acid and its derivative compounds 30, 31 and 32 as illustrated in Schemes 1-3. Martinelli and coworkers 51 have reported a practical synthetic route to compound 29 employing a Nenitzescu reaction as the key step, and a modification of this approach was employed in this work for the synthesis of each of the compounds 26-29 48 as summarised m Scheme 4.

CO2H H i, NaH (2.4 eq.) DMF, 0°C, 0.5 h .4-XC6H4CH2Br or i-XOcHzCHjCI 0°C, 1 h Br 10 23% MeO 11 47% Scheme 1 DUBLOl/C ¢0 A Q7 8 4 -16MeO ΌΟ2Η i, NaH (2.4 eq.) DMF, 0°C, 0.5 h ZEZ Me ii, XC6H4CH2Br or XC6H4CH2CI 0°C, 1 h H 13 38% 3-CI 16 46% Cl 4 41% 2-CI 17 35% Br 7 20% 3-F 18 43% F 14 29% MeS 5 17% Me 15 12% Scheme 2 R1- H i, NaH (2.4 eq.) DMF, 0°C, 0.5 h ii, 4-C!C6H4CH2CI 0°C, 1 h MeO H R1 R2 MeO H 31 H Me 32 H 19 28% Me 20 36% Scheme 3 DUBL01/C £ ο 1 01 8 4 Me 26 80% Scheme 4 DUBL01/C -18General procedure for benzvlations To a slurry of sodium hydride (60% dispersion in mineral oil) in dry DMF under nitrogen at 0°C was added the indole-3-acetic acid derivative in one portion. Stirring at 0°C was continued for 30 min. The appropriate benzyl halide was added in one portion and stirring continued for 1 hr. The reaction was quenched by pouring into ice-water, the resultant mixture acidified with 10% hydrochloric acid and the crude product obtained by filtration.

Example 1: l-(4-Chlorobenzyl)-5-methoxy-2-methylindole-3-acetic acid Compound 4 The procedure followed was as detailed above, using 5-methoxy-2methylindole-3-acetic acid (219.2 mg, 1 mmol), para-chlorobenzyl chloride (193.2 mg, 1.2 mmol, 1.2 eq.), sodium hydride (60% dispersion in mineral oil; 96.0 mg, 2.4 mmol, 2.4 eq.) and DMF (5 mL). Recrystallisation of the crude product (ether / ethanol) afforded the title compound as a pale yellow solid (141.1 mg, 41.1%). m.p. 185-7°C; UmaxCKBrj/an-1 3200-2500 (OH), 1706 (C=O); iRNMR (270 MHz, CDCI3) δ 6.74-7.25 (m. E_ & C6H4), 5.22 (s, 2 H, N-CH2), 3.84 (s, 3 H, CH3O), 3.72 (s, 2 H, C2%CO2H), 2.29 (s, 3 H, CH3); l^CNMR (67.8 MHz, CDCI3) δ 177.3 (C=O), 154.4 (C5), 136.3 (p-Ph), 135.1, 133.1, 131.5, 128.1 (4 X qC), 129.0, 127.3 (σ- & m-Ph), 111.2, 109.8 (C4 & C6), 103.8 (C3), 100.5 (C7), 55.9 (CH3O), 46.2 (N-CH2), 30.4 (CH2CO2H), 10.4 (CH3).

DUBL01/C 19. ΙΕΟ 107 84 Example 2: l-(4r-Bromobenzyl)-5-methoxy-2-methylindole-3-acetic acid Compound 7 The procedure followed was as detailed above, using 5-methoxy-2methylindole-3-acetic acid (328.8 mg, 1.5 mmol), pa/a-bromobenzyl bromide (281.2 mg, 1.35 mmol, 0.9 eq.), which was added dropwise via syringe as a solution in DMF (1 mL), sodium hydride (60% dispersion in mineral oil; 144.0 mg, 3.6 mmol, 2.4 eq.) and DMF (4 mL). Flash column chromatography (chloroform / methanol 15:1) afforded the tide compound as a pale yellow solid (119.6 mg, 20.5%). m.p. 191-2°C; OmaxiKBrj/cm'1 3300-2500 (OH), 1700 (C=O); 1HNMR (270 MHz, CDCI3) δ 6.77-7.35 (m, 7 H, indole-H & C6H4), 5.20 (s, 2 H, N-CH2), 3.84 (s, 3 H, CH3O), 3.72 (s, 2 H, CH2CO2H), 2.29 (s, 3 H, CH3); 13CNMR (67.8 MHz, CDCI3) δ 177.7 (C=O), 154.4 (C5), 136.8, 135.0, 132.3, 128.1 (4 X qC), 132.5, 127.7 (o- & m-Ph), 121.1 (p-Ph), 111.1, 109.7 (C4 & C6), 104.0 (C3), 100.5 (C7), 55.9 (CH3O), 46.2 (N-CH2), 30.5 (CH2CO2H), 10.3 (CH3); m/z387, 389 (M+[79/81Br], 1 %), 239(25), 121(68), 78(94), 63(100); (Found: C, 59.0; H, 5.0; N, 3.9. Ci9HigNBrO3 requires C, 58.8; H, 4.7; N, 3.6%).

Example 3: l-(4-Fluorobenzyl)-5-methoxy-2-methylindole-3-acetic acid Compound 14 The procedure followed was as detailed above, using: 5-methoxy-2methylindole-3-acetic acid (328.8 mg, 1.5 mmol), //ara-fluorobenzyl bromide (187 pL, 1.5 mmol, 1.0 eq.), which was added dropwise via syringe, sodium hydride (60% dispersion in mineral oil; 144.0 mg, 3.6 mmol, 2.4 eq.) and DMF DUBL01/C 8F 0 t 0 7 8 4 -20- ilk= (5 mL). Flash column chromatography (chloroform / methanol 20:1) afforded the title compound as a white solid (140.8 mg, 28.7%). m.p. 167-9°C; UmaxiKBrj/cm-1 3300-2500 (OH), 1686 (C=O); iHNMR (270 MHz, CDCI3) 56.74-7.07 (m, 7 H, indole-H & C6H4), 5.21 (s, 2 H, N-CH2), 3.83 (s, 3 H, CH3O), 3.71 (s, 2 H, CH2CO2H), 2.28 (s, 3 H, CH3); 13CNMR (67.8 MHz, CDCI3) 5177.1 (C=O), 162.1 (d, JcF 246, C4'), 154.4 (C5), 135.0, 133.5, 131.5, 128.1 (4 X qC), 127.6 (d, VcF 8.7, C2' & C6’), 115.7 (d, 2/CF 22.4, C3' & C5'), 111.1, 109.8 (C4 & C6), 103.8 (C3), 100.5 (C7), 55.8 (CH3O), 46.2 (N-CH2), 30.4 (CH2CO2H), 10.4 (CH3); m/z 327(M+, 2%), 283(5), 205(19), 109(17), 84(73), 49(100); (Found: C, 69.9; H, 5.6; N, 4.0; F, 5.7.

C19H18NFO3 requires C, 69.7; H, 5.5; N, 4.3; F, 5.8%).

Example 4: l-(3-Fluorobenzyl)-5-methoxy-2-methylindole-3-acetic acid 15 Compound 18 The procedure followed was as detailed above, using 5-methoxy-2Tnetbyhndnie-3-aceric acid (548.1 mg, 2.5 mmol), mera-fluorobenzyl bromide (363 uL, 3 mmol, 1.2 eq.), sodium hydride (60% dispersion in mineral oil; 240.0 mg, 6 mmol, 2.4 eq.) and DMF (5 mL). Recrystallisation (dichloromethane / hexane) afforded the tide compound as a white solid (350.2 mg, 42.8%).

M.p. 182-3°C; UmaxiKBrj/cm'1 3300-2200 (OH), 1702 (C=O); iHNMR (270 MHz, CDCI3) 56.61-7.26 (m, 7 H, indole-H & C6H4), 5.25 (s, 2 H, N-CH2), 3.85 (s, 3 H, CH3O), 3.73 (s, 2 H, CL72CO2H), 2.30 (s, 3 H, CH3); 13CNMR (67.8 MHz, CDCI3) 5176.4 (C=O), 163.2 (d, JcF 246, C3’), 154.4 (C5), 140.5 (d, 3/CF 7.5, Cl'), 135.0, 131.5, 128.1 (3 X qC), 130.4 (d, VcF 7.5, C5’), 121.5 DUBL01/C (d, VcF 2.5, C6'), 114.3, 113.0 (2 d, 2/CF 21.2, 2/cp 21.2, C2' & C4'), 111.2, 109.7 (C4 & C6), 103.9 (C3), 100.5 (C7), 55.9 (CH3O), 46.4 (N-CH2), 30.3 (CH2CO2H), 10.4 (CH3); m/z 327(M+, 84%), 282(100), 117(88), 109(77); (Found; C, 70.1; H, 5.5; N, 4.3; F, 6.1. C19H18NFO3 requires C, 69.7; H, 5.5; N, 4.3; F, 5.8%).

Comparative Examples The following N-benzyl derivatives, compounds 5, 13, 15-17, 19 and 20 were found to be inactive in inhibiting MRP even though they have similar chemical structures to the active compounds of examples 1 to 4.

Example 5: 1-(4-Methylthiobenzyl)-5-methoxy-2-methylindole-3-acetic acid Compound 5 The procedure followed was 2s detailed above, using 5-methoxy-2methyIindole-3-acetic add (438.5 mg, 2 mmol), pars-methylthiobenzyl chloride (414.4 mg, 2.4 mmol, 1.2 eq.), which was added dropwise as a solution in DMF (1 mL), sodium hydride (60% dispersion in mineral oil; 192.0 mg, 4.8 mmol, 2.4 eq.) and DMF (5 mL). Flash column chromatography (chloroform / methanol 20:1) afforded a dark orange solid that was further purified by recrystallisation (dichloromethane/ hexane), giving the title compound as an off-white solid (119.2 mg, 16.8%). m.p. 159-60°C (Lit.52 155-6.5°C); max(KBr)/cm-l 3300-2500 (OH), 1709 (C=O); 1HNMR (270 MHz, CDCI3) 66.74-7.16 (m, 7 H, indole-H & C6H4), 5.22 (s, 2 H, N-CH2), 3.84 (s, 3 H, CH3O), 3.72 (s, 2 H, C//2CO2H), 2.42 (s, 3 DUBL01/C H, CH3S), 2.30 (s, 3 H, CH3); 13CNMR (67.8 MHz, CDCI3) 6176.9 (C=O), 154.4 (C5), 137.5, 135.1, 131.6, 128.1 (4XqC), 134.8 (p-Ph), 127.2, 126.5 (o-& m-Ph), 111.1, 109.9 (C4 & C6), 103.6 (C3), 100.5 (C7), 56.0 (CH3O), 46.4 (NCH2), 30.4 (CH2CO2H), 15.9 (CH3S), 10.4 (CH3); m/z 355(M+, 17%), 311(12), 137(100); (Found: C, 67.5; H, 6.2; N, 4.2; S, 8.8. C20H21NSO3 requires C, 67.6; H, 6.0; N, 3.9; S, 9.0%).

Example 6: l-(3-Chlorobenzyl)-5-methoxy-2-methylindole-3-acetic acid Compound 16 The procedure followed was as detailed above, using 5-methoxy-2methylindole-3-acetic acid (219.2 mg, 1 mmol), mefa-chlorobenzyl chloride (148 gL, 1.2 mmol, 1.2 eq.), sodium hydride (60% dispersion in mineral oil; 96.0 mg, 2.4 mmol, 2.4 eq.) and DMF (5 mL). Recrystallisation of the crude product (dichloromethane / hexane) afforded the title compound as a white solid (157.1 mg, 45.7%). m.p. 177-9°C; UmaxfKBrycnr1 3200-2500 (OH), 1710 (C=O); iHNMR (270 MHz, CDCI3) 56.74-7.22 (m, 7 H, indole-H & C0H4), 5.23 (s, 2 H, N-CHt). 3.85 (s, 3 H, CH3O), 3.73 (s, 2 H, CH2CO2H), 2.28 (s, 3 H, CH3); 13CNMR (67.8 MHz, CDCI3) 5176.8 (C=O), 154.4 (C5), 139.9 (C3'), 135.0, 134.8, 131.5, 128.1 (4X qC), 130.1, 127.6, 126.1, 124.1 (C2', C4’, C5’& C6'), 111.2, 109.8 (C4 & C6), 103.9 (C3), 100.5 (C7), 55.9 (CH3O), 46.3 (N-CH2), 30.3 (CH2CO2H), .4 (CH3); m/z 343(M+[3 3 Cl], 1%), 299(12), 125(16), 84(68), 43(100); (Found: C, 65.9; H, 5.4; N, 4.0. C19H18NCIO3 requires C, 66.4; H, 5.3; N, 4.1%).

DUBLOI/C IF 0 A 01 8 4 - 23 Example 7: l-(2-Chlorohenzyl}-5-methoxy-2-methylindole-3-acetic acid Compound 17 The procedure followed was as detailed above, using 5-methoxy-2methylindole-3-acetic acid (219.2 mg, 1 mmol), ort&o-chlorobenzyl chloride (147 pL, 1.2 mmol, 1.2 eq.), sodium hydride (60% dispersion in mineral oil; 96.0 mg, 2.4 mmol, 2.4 eq.) and DMF (5 mL). Recrystallisation of the crude product (dichloromethane/ hexane) afforded the title compound as a white solid (119.2 mg, 34.7%).

M.p. 193-6°C; umax(KBr)/cm-l 3300-2400 (OH), 1702 (C=O); *HNMR (270 MHz, CDCI3) 86.98-7.41 (m, 5 H, C6H4 & 4-H), 6.77 (dd, /8.6, 2.4, 1 H, 6H), 6.23 (d, /7.6, 1 Η, 7-H), 5.32 (s, 2 H, N-CH2), 3.85 (s, 3 H, CH3O), 3.76 (s, 2 H, CJ/2CO2H), 2.28 (s, 3 H, CH3); l^CNMR (67.8 MHz, CDCI3) 8176.7 (C=O), 154.6 (C5), 135.2 (C2'), 131.9, 131.6, 128.2 (3 X qC), 129.4, 128.7, 127.4, 127.0 (C3\ C4', C5' & C6’), 111.3, 109.7 (C4 & C6), 103.9 (C3), 100.6 (C7), 55.9 (CH3O), 44.6 (N-CH2), 30.4 (CH2CO2H), 10.2 (CH3); m/z 343(M-[35ci]. 1%), 299(8), 125(19), 84(75), 49(100).

Example 8: 1—Benzyi-5-methoxy—2-mefhylindole-3—acetic acid Compound 13 The procedure followed was as detailed above, using 5-methoxy-2methylindole-3-acetic acid (328.8 mg, 1.5 mmol), benzyl chloride (207 pL, 1.8 mmol, 12 eq.), sodium hydride (60% dispersion in mineral oil; 144.0 mg, 3.6 mmol, 2.4 eq.) and DMF (4 mL). Flash column chromatography (chloroform/ methanol 20:1) afforded the title compound as a pale yellow solid (175.0 mg, 37.7%).

DUBL01/C m.p. 169-73°C; Omax/KBrj/cm'1 3300-2500 (OH), 1700 (C=O); 7HNMR (270 MHz, CDCI3) δό.73-7.25 (m, 8 H, indole-H & Ph), 5.22 (s, 2 H, N-CH2), 3.81 (s, 3 H, CH3O), 3.69 (s, 2 H, C7%CO2H), 2.26 (s, 3 H, CH3); 13CNMR (67.8 MHz, CDCI3) 6177.6 (C=O), 154.3 (C5), 137.8, 153.2, 131.7, 128.1 (4 X qC), 127.3 OPh), 128.8, 125.9 (o- & m-Ph), 111.0, 109.9 (C4 & C6), 103.7 (C3), 100.4 (C7), 55.9 (CH3O), 46.7 (N-CH2), 30.5 (CH2CO2H), 10.4 (CH3); m/z 309(M+, 82%), 264(70), 173(37), 158(38), 91(100); (Found: C, 73.4; H, 6.15; N, 4.2. C19H19NO3 requires C, 73.8; H, 6.2; N, 4.5%).

Example 9: l-(L-Chlorobenzyl)-5-methoxyindole-3-acetic acid Compound 19 The procedure followed was as detailed above, using 5-methoxyindole-3-acetic acid (410.4 mg, 2 mmol), paza-chlorobenzyl chloride (386.5 mg, 2.4 mmol, 1.2 eq.), sodium hydride (60% dispersion in mineral oil; 192.0 mg, 4.8 mmol, 2.4 eq.) and DMF (5 mL). Flash column chromatography (chloroform/ methanol 20:1) afforded a pale yellow solid that was further purified by recrystallisation (dicfaloromethane/ hexane), giving the tide compound as a white solid (185.6 mg. 28,1%). m.p. 145-6°C; UmaxiKB^/cm'l 3300-2400 (OH), 1706 (C=O); 7HNMR (270 MHz, CDCI3) 86.81-7.27 (m, 8 H, indole-H & C6H4), 5.20 (s, 2 H, N-CH2), 3.84 (s, 3 H, CH3O), 3.77 (s, 2 H, C//2CO2H); 13CNMR (67.8 MHz, CDCI3) 8177.3 (C=O), 154.3 (C5), 135.9 OPh), 133.5, 131.7, 128.3 (3 X qC), 128.7, 128.1 (o— & m-Ph), 127.8 (C2), 112.6, 110.6 (C4 & C6), 106.6 (C3), 100.9 (C7), 55.9 (CH3O), 49.6 (N-CH2), 31.0 (CH2CO2H); m/z 329, 331(M+[35/37Cl], 14%), 284(12), 127(19), 125(60), 69(82), 41(100); (Found: C, 65.9; H, 5.2; N, 4.3; Cl, 11.0. C18H16NCIO3 requires C, 65.55; H, 4.9; N, 4.25; Cl, 10.75%).

DUBL01/C -25- ΙΕ 0 1 0 7 8 4 Example 10: l-Benzylindole-3-acetic acid Compound 8 To a slurry of sodium hydride (60% dispersion in mineral oil; 0.55 g, 13.8 mmol, 2.4 eq.) in dry DMF (10 mL) under nitrogen at 0°C was added indole-3-acetic acid (1.00 g, 5.71 mmol) in one portion. Stirring at 0°C was continued for 30 min. Benzyl bromide (0.82 mL, 6.84 mmol, 1.2 eq.) was added in one portion and stirring continued for 1 hr. The reaction was quenched by pouring into icewater (circa lOOmL), the resultant mixture acidified with 10% hydrochloric acid and the crude product obtained by filtration. Recrystallisation (ether/ethanol) afforded the title compound as a white solid (0.4459 g, 29.5%). m.p. 153-4°C (Lit.53 148°C); nmax (KBr)/cm'l 3000-2500 (OH), 1702 (C=O); iHNMR (270 MHz, CDCI3) 67.10-7.63 (m, 10 H, indole-H & Ph), 5.27 (s, 2 H, N-CH2), 3.80 (s, 2 H, CB2CO2H); l^CNMR (67.8 MHz, CDCI3) 6177.8 (C=O), 137.3 (PPh), 136.6 (C7a), 128.8, 126.9 (o-&m-Ph), 127.9 (C3a), 127.6, 127.3 (C2 & p-Ph), 122.1 (C5), 119.6, 119.1 (C4 & C6), 109.8 (C7), 106.9 (C3), 50.0 (N-CH2), 31.0 (CH2CO2H); m/z265(M+ 8%), 149(12), 91(28), 84(66). 49(100); (Found; C, 76.7; H, 6.0; N, 5.4. C17H15NO2 requires C, 77.0; H, 5.7: N, 5.3%).

Example 11: l-(4-Chlorobenzyl)indole-3-acetic acid Compound 9 The procedure followed was as detailed above, using para-chlorobenzyl chloride (1.10 g, 6.84 mmol, 1.2 eq.). Upon acidification, the crude product was extracted with dichloromethane (50 mL), the organic layer washed with water (3 X 50 mL) and brine (50 mL), dried (MgSC>4), evaporated and concentrated in vacuo. Recrystallisation (ether/ethanol) afforded the title compound as a pale yellow solid (0.6925 g, 40.5%).

DUBL01/C -26- SEQ 10? 84 m.p. 146-8°C; OmayfKBrVcnr1 3300-2500 (OH), 1713 (C=O); iHNMR (270 MHz, CDCI3) 67.00-7.63 (m, 9 H, indole-H & C6H4), 5.23 (s, 2 H, CH2), 3.80 (s, 2 H, C#2CO2H); 13CNMR (67.8 MHz, CDCI3) 6177.6 (C=O), 136.5 (A Ph), 135.9 (C7a), 133.5 (p-Ph), 129.0, 128.4 (a- & m-Fti), 127.9 (C3a), 127.1 (C2), 122.3 (C5), 119.8, 119.2 (C4 & C6), 109.7 (C7), 107.2 (C3), 49.4 (N-CH2), 31.0 (6H2CO2H); m/z 299, 301(M+[35/37Cl], 32%), 255(31), 125(100); (Found: C, 67.8; H, 4.9; N, 4.65; Cl, 11.4. C17H14NCIO2 requires C, 68.1; H, 4.7; N, 4.7; Cl, 11.8%).

Example 12: l-(4~Bromobenzyl)indole~3-acetic acid Compound 10 The procedure followed was as detailed above, using /wa-bromobenzyl bromide (1.71 g, 6.84 mmol, 1.2 eq.). Recrystallisation of the crude product (ether/ ethanol) afforded the title compound as a pale yellow solid (0.4493 g, 22.9%). m.p. 147-9°C; Vm^HKBD/an-l 3000-2500 (OH), 1710 (C=O); ^HNMR (270 MHz, CDCI3) δό.85-7.63 (m, 9 H. indole-H & C6H4), 5.21 (s, 2 H, CH2), 3,78 (s, 2 H, C/^COpHb 15CNMR (67.8 MHz, CDCI3) 6177.6 (C=O), 136.4 (C7a), 131.9 (m-Ph), 1285 (o-Ph), [APh not seen], 128.0 (C3a), 127.1 (C2), 122.3 (C5), 121.6 (p-Ph), 119.7, 119.2 (C4 & C6), 109.7 (C7), 107.2 (C3), 49.5 (NCH2), 30.9 (CH2CO2H); m/z 343, 345(M+[79/81Br], 30%), 299(30), 224(60), 125(100); (Found: C, 59.0; H, 4.3; N, 3.8; Br, 23.1. Ci7Hi4NBrO2 requires C, 59.3; H, 4.1; N, 4.1; Br, 23.2%).

DUBL01/C tgi % Example 13: l-(4-Methoxybenzyl)indole-3-acetic acid Compound 11 The procedure followed was as detailed above, using para-methoxybenzyl bromide (0.93 mL, 6.84 mmol, 1.2 eq.). Recrystallisation of the crude product (ether / ethanol) afforded the title compound as a pale yellow solid (0.7957 g, 47.2%). m.p. 133-5°C; umax(KBr)/cm-l 3300-2200 (OH), 1712 (C=O); iHNMR (270 MHz, CDCI3) 56.79-7.62 (m, 9 H, indole-H & C6H4), 5.20 (s, 2 H, N-CH2), 3.78 (s, 2 H, CJT2CO2H), 3.76 (s, 3 H, CH3O); 13CNMR (67.8 MHz, CDCI3) 5 177.6 (C=O), 159.1 (p-Ph), 136.5 (C7a), 129.3 (APh), 128.3 (α-Ph), 127.9 (C3a), 127.1 (C2), 122.0 (C5), 119.5, 119.0 (C4 & C6), 114.2 (zn-Ph), 109.8 (C7), 106.7 (C3), 55.3 (CH3O), 49.5 (N-CH2), 31.0 (CH2CO2H); Wz295(M+, 10%), 251(17), 121(100); (Found: C, 72.7; H, 6.2; N, 4.4. C18H17NO3 requires C, 73.2; H, 5.8; N, 4.7%).

Example 14: 1-(4-Me±yibenzyI)-5-methoxy-2-msrhphnijoic-3—acetic acid Compound 15 The procedure followed was as detailed above, using 5-messaxy-2-methylindole3-acetic acid (400 mg, 1.82 mmol), para-methylbenzyl bromide (405 mg, 2.19 mmol, 1.2 eq), sodium hydride (60% dispersion in mineral oil: 175.2 mg, 4.38 mmol, 2.4 eq) and DMF (5 mL). Recrystallisation of the crude product (methanol) afforded the title compound as a white solid (70mg, 12%) m.p. 189-90°C; UmaxtKBrj/cm1 3300-2500 (OH), 1698 (C=O); ’H NMR (270 MHz, CDCb) δ 7.32-6.74 (m, 7 H, indole-H & C6H4), 5.22 (s, 2 H, N-CH2), 3.84 (s, 3 H, CH3O), 3.72 (s, 2 H, C/%CO2H), 2.30 (s, 3 H, CH3), 2.28 (s, 3 H, CHa); DUBL01/C IE 5 1 0 7 8 4 - 28 13C NMR (67.8 MHz, CDC13) δ 177.8 (C=O), 154.2 (C5), 136.9, 135.2, 134.7, 131.6 (4 x qC), 129.4, 125.9 (o & m-Ph), 127.9 (p-Ph), 111.0, 110.0 (C4 & C6), 103.3 (C3), 100.2 (C7), 55.9 (CH3O), 46.6 (N-CH2), 30.5 (CH2CO2H), 21.0 (CH3), 10.4 (CH3); m/z 323(M+, 74%), 278(67), 105(100); (Found : C, 73.91 ; H, 6.51; N, 4.17; C20H21NO3 requires C, 74.28; H, 6.39; N, 4.33%).

Example 15: l-(4-Chlorobenzyl)-2-methylindole-3-acetic acid Compound 20 The procedure followed was as detailed above, using 2-methylindole-3-acetic acid (378.4 mg, 2 mmol), para-chlorobenzyl chloride (386.5 mg, 2.4 mmol, l. 2 eq.), sodium hydride (60% dispersion in mineral oil; 192.0 mg, 4.8 mmol, 2.4 eq.) and DMF (10 mL). Flash column chromatography (chloroform/ methanol 20:1) afforded a pale yellow solid that was further purified by recrystallisation (dichloromethane/ hexane), giving the title compound as a white solid (229.2 mg, 36.5%). m. p. 182-4°C; umax(KBr)/cm-l 3300-2200 (OH), 1706 (C=O); ^HNMR (270 MHz, CDCI3) 86.86-7.58 (m, 8 H, indole-H & C6H4), 526 (s, 2 H, N-CH2), 3.76 (s, 2 H, C25CO2H), 231 (s, 3 H, CH3); 13CNMR (67.8 MHz, CDCI3) 8177.0 (C=O), 136.3 (p-Ph), 134.4, 1333, 127.7 (3 X qC), 129.0, 127.4 (o- & m-Ph), 121.5 (C5), 119.8, 118.2 (C4 & C6), 109.1 (C7), 104.2 (C3), 46.1 (NCH2), 30.3 (CH2CO2H), 10.3 (CH3); m/z 313, 315(M+[35/37Cl], 14%), 268(20), 127(36), 125(100); (Found: C, 69.1; H, 5.5; N, 4.6; Cl, 11.3. C18H16NCIO2 requires C, 68.9; H, 5.1; N, 4.5; CI, 11.3%).

DUBL01/C -29Synthesis of precursors for 26-29 The synthesis of compounds 26-29 required the production of a number of precursors. The synthetic route for these intermediates (46-63) is described below.

Example 16: Methyl 3-(phenylmethyl)amino-2-pentenoate Compound 47 A solution of methyl propionylacetate (12.6 mL, 0.1 mmol), benzylamine (11.4 mL, 0.105 mmol, 1.05 eq.) and /Moluenesulphonic acid monohydrate (0.95 g, 5 mmol, 5 mol%) in toluene (50 mL) was refluxed under Dean-Stark conditions under nitrogen for 3 hr. The reaction solution was cooled in an ice-bath to ~10°C, the fine white crystalline precipiate that formed was removed by filtration, and the filtrate evaporated and concentrated in vacuo affording the title compound as a clear orange liquid (21.1 g, 95.7%) that was used without subsequent purification. lH NMR (270 MHz, CDCI3) 08.93 (s, 1 H, NH), 7.18-7.37 (m, 5 H, C6H5), 4.56 (s, H, C=CH-C=O), 4.43 (d. J 6Λ 2 H. N-CH2), 3.64 (s, 3 H, OCH3), 2.23 (q, J15, H, CH2CH3), 1.12 (L J 7A, CH2CW3): 13CNMR (67.8 MHz, CDCI3) 6171/2 (C=O), 167.0 (C3), 138.8 G-PbL 128.7. 126.9, (o- & nz-Ph), 127.4 (σ-Ph), 81.0 (C2), 49.9 (N-CH2), 25.1 (CH2CH3,, 12.1 (CH2CH3).

Example 17: Methyl 3-(phenrimethyl)amino-2-butenoate Compound 46 The procedure followed was as detailed above, using methyl acetoacetate (10.8 mL, 0.1 mol). The title compound was obtained as a clear orange liquid (20.1 g, 97.8%) that was used without subsequent purification.

DUBLOl/C IE 0 1 0 7 8 4. -301HNMR (270 MHz, CDC13) 68.93 (s, 1 H, NH), 7.13-7.38 (m, 5 H, C6H5), 4.54 (s, 1 H, C=CH-C=O), 4.42 (d, J 6.2, 2 H, N-CH2), 3.63 (s, 3 H, OCH3), 1.91 (s, 3 H, CH3); 13CNMR (67.8 MHz, CDCI3) 6170.9 (C=0), 161.9 (C3), 138.7 (i-Ph), 128.8, 126.7, (o-&m-Ph), 127.4 (p-Ph), 82.9 (C2), 50.0 (N-CH2), 19.3 (CH3).

Example 18: 3-(Carbomethoxy)-2-ethyl-l-(phenylmethyl)-5-hydroxyindole Compound 49 To a solution of benzoquinone (10.17 g, 93.9 mmol, 1.38 eq.) in nitromethane (40 mL) at r.t. under nitrogen was added a solution of methyl 3(phenylmethyl)amino-2-pentenoate (15.00 g, 68.1 mmol) in nitromethane (20 mL) dropwise over 30 min. Stirring at r.t. was continued for 48 hr. The reaction mixture was cooled to 0°C in an ice-bath, filtered, washed with cold nitromethane (~50 mL) affording the title compound as a pale brown solid (11.32 g, 53.7%) after drying in vacuo that was used without subsequent purification. However, an analytical sample was obtained thus: a portion of the product (2.89 g) was suspended in 1,2-dichloroethane (10 mL) and the mixture refluxed for 30 mm. Filtration of the hot mixture afforded a pale brown solid (1-68 g). m.p. 191—3°C (Lit 51 194-5°C); vmax (KBr)/cm'l 3281, 1646, 1142; iRNMR (270 MHz, CDCI3) 66.72-7.67 (m, 8 H, indole-H & C6H5), 5.34 (s, 2 H. N-CHt), 3.93 (s, 3 H, CO2CH3), 3.14 (q, J 7.5, 2 H, Ctf2CH3), 1.18 (t, J Ί.4, 3 H. CH2CH3); 13CNMR (67.8 MHz, CDCI3) 6166.3, (C=O), 155.6 (C5), 151.4, 136.6, 131.5, 127.9 (4 x qC), 128.9, 125.8 (o- & w-Ph), 127.7 (p_Ph), 111.7, 110.7 (C4 & C6), 106.6 (C7), 103.1 (C3), 50.8 (CO2CH3), 46.7 (N-CH2), 19.3 (CH2CH3), 13.8 (CH2CH3).

DUBL01/C -si- 8E 0 1 07 8 4 Example 19: 3-(Carbomethoxy)-2-methyl-l-(phenylmethyl)-5-hydroxyindole Compound 48 The procedure followed was as detailed above, using: a solution of benzoquinone (14.54 g, 0.134 mol, 1.38 eq.) in nitromethane (50 mL) and a solution of methyl 3-(phenylmethyl)amino-2-butenoate (20.00 g, 0.974 mol) in nitromethane (25 mL). Stirring at r.t. under nitrogen was continued for 66 hr. The title compound was obtained as a pale brown solid (13.73 g, 47.7%). A portion (2.50 g) was refluxed in 1,2-dichloroethane (5 mL) for 1 hr and the resultant mixture filtered whilst hot to give an analytical sample (0.87 g) as a pale brown solid. m.p. 191-3°C; vmax (CHClal/cm-1 3016, 1676,1118; iHNMR (270 MHz, DMSOd6) 68.99 (s, 1 H, OH), 6.63-7.42 (m, 8 H, indole-H & C6H5), 5.43 (s, 2 Η, NCH2), 3.81 (s, 3 H, CO2CH3), 2.65 (s, 3 H, CH3); 13CNMR (67.8 MHz, DMSOd6) 0165.4, (C=O), 152.8 (C5), 144.9, 137.2, 136.5, 130.3 (4x qC), 128.6, 126.1 (o& zra-Ph), 127.2 (p-Ph), 111.6, 110.7 (C4 & C6), 105.4 (C7), 102.5 (C3), 50.3 (CO2CH3), 45.8 (N-CH2), 11.8 (CH3). -nnri· 3-(Carbomeffioxy)-2-ethyUi-(phcnyirrieifayi)-5-meihcncyindoie To a mixture of 3-(carhomethoxy)-2-ethyl-l-(phenyhnethyl)-5-hydroxymdole (11.31 g, 36.56 mmol) and tetrabutylammonium bromide (1.179 g, 3.66 mmol, 10 moi%) in water (100 mL) was added a 50% aq. solution of sodium hydroxide (35 ml.) and methyl iodide (6.83 mL, 0.110 mol, 3.0 eq.). This heterogeneous mixture was brought to reflux, resulting in a dark brown solution, and reflux continued for 30 min. Once cooled to r.t., the reaction mixture was extracted with ethyl acetate (3 x 85 mL), the combined extracts washed with brine (150 mL), dried (MgSO4), evaporated and concentrated in vacuo to a pale brown DUBL01/C -32- ΙΕΟ I 07 84 solid (10.82 g). Recrystallisation of the crude product (zso-propanol) afforded the title compound as colourless crystals (7.160 g, 60.6%). m.p. 100-1°C (Lit.51 102-3°C); vmax (CHCl3)/cm-l 2950,1689,1480, 1462,1439, 1120; iHNMR (270 MHz, DMSO-^) 06.78-7.53 (m, 8 H, indole-H & C6H5), 5.51 (s, 2 H, N-CH2), 3.84 (s, 3 H, CO2CH3), 3.79 (s, 3 H, CH3O), 3.11 (q, J 7.4, 2 H, C7/2CH3), 1.06 (t, /7.4, 3 H, CH2CH3).

Example 21: 3-(Carbomethoxy)-2-methyl-l-(phenylmethyl)-5-methoxyindole Compound 50 The procedure followed was as detailed above, using: 3-(carbomethoxy)-2methyl-l-(phenylmethyl)-5-hydroxy-indole (12.99 g, 43.98 mmol), tetrabutylammonium bromide (1.418 g, 4.40 mmol, 10 mol%), water (125 mL), 50% aq. sodium hydroxide solution (42 mL) and methyl iodide (8.21 mL, 0.132 mol, 3.0 eq.). The crude product was obtained as a pale brown solid that upon rw-ryatallisatinn (zso-propanol) afforded the title compound as beige crystals (7.775 g. 572%). nup. 127-8°C; VTOax (CHCl3)/cm'7 3007. 2950. 1688, 1482, 1415, 1119; 1HNMR (270 MHz, DMSO-/6) 66.78-7.54 (m. 8 H. indole-H & C6H5), 5.48 (s, 2 Η, NCH2), 3.84 (s, 3 H, CO2CH3), 3.79 is. 3 H. CH3O), 2.67 (s, 3 H, CH3); 73CNMR (67.8 MHz, DMSO-/6) 6165.4, (C=O). 1552 (C5), 145.2, 137.1, 131.1, 126.9 (4 x qC), 128.7, 126.0 (0-& m-Ph), 127.3 (p-Ph), 111.3, 111.1 (C4 & C6), 103.3 (C7), 103.0 (C3), 55.3 (CH3O), 50.5 (CO2CH3), 45.9 (N-CH2), 11.8 (CH3).

DUBL01/C -33Example 22: 3-(Hydroxyrnethyl)-2-ethyl-l-(phenylmethyl)-5-methoxyindole Compound 53 To a slurry of lithium aluminium hydride (2.46 g, 64.9 mmol, 3.0 eq.) in THF (50 mL) at r.t. under nitrogen was added a solution of 3-(carbomethoxy)-2ethyl-l-(phenylmethyl)-5-methoxyindole (7.00 g, 21.6 mmol) in THF (15 mL) dropwise and stirring continued for 17 hr. The reaction was quenched by the sequential addition of water (2.5 mL), 15% aq. sodium hydroxide solution (2.5 mL) and water (7.5 mL). The resultant mixture was stirred at r.t. for 30 min during which the grey slurry was transformed to a fine white solid and a clear yellow solution. This mixture was filtered, the filtrate dried (MgSO4), evaporated and concentrated in vacuo to give the title compound (4.24 g, 66.4%) without further purification as a yellow oil that solidified upon standing. m.p. 69-72°C (Lit.51 69-70°C); vmax (CHC13)/0^1 3008, 1485, 1454, 1158; iHNMR (270 MHz, DMSO-cfc) δό.63-7.30 (m, indole-H & C6H5), 5.37 (s, 2 H, N-CH2), 4.61 (s, 3 H, CH2OH; accidental equivalence), 3.76 (s, 3 H, CH3O), 2.74 (q, J 7.5, 2 H, CH2CH3), 1-05 (t, J 7.6, 3 H. CH2C//3).

Example 23: 3-{HydroxymetbyI}-2-methyl-l-(phenylmethyl)-5-methoxyindole Compound 52 The procedure followed was as detailed above, using a slurry of lithium aluminium hydride (2.85 g, 75.2 mmol, 3.0 eq.) in THF (50 mL) and a solution of 3-(carbomethoxy)-2-methyl-l-(phenylmethyl)-5-methoxyindole (7.75 g, 25.1 mmol) in THF (30 mL). Work-up in a similar fashion afforded the title compound as a yellow solid (4.45 g, 63.0%) without further purification.

DUBL01/C Vmax (KBr)/cm-l 3500-2700 (OH), 2923, 1447, 1147; iHNMR (270 MHz, DMSOd$ 66.61-7.30 (m, 8 H, indole-H & C6H5), 5.33 (s, 2 H, N-CH2), 4.60 (s, 3 H, CH2OH; accidental equivalence), 3.75 (s, 3 H, CH3O), 2.31 (s, 3 H, CH3); 13CNMR (67.8 MHz, DMSO^) 6153.4 (C5), 138.6, 134.9, 131.2, 128.0 (4 X qC), 128.5, 126.2 (o- & zn-Ph), 127.0 (p-Ph), 111.9 (C3), 109.9 (C4 & C6), 100.6 (C7), 55.3 (CH3O), 53.9 (CH2OH), 45.8 (N-CH2), 10.0 (CH3).

Example 24: 3-(Cyanomethyl)-2-ethyl-l-(phenylmethyl)-5-methoxyindole Compound 55 To a solution of boron trifluoride etherate (4.06 mL, 32.1 mmol, 3.0 eq.) and cyanotrimethylsilane (5.70 mL, 42.7 mmol, 4.0 eq.) in dichloromethane (65 mL) at 0°C under nitrogen in a 3-necked RB-flask fitted with a thermometer and a dropping funnel was added a solution of 3-(hydroxymethyl)-2-ethyl-l-(phenylmethyl)-5-methoxyindole (3.156 g, 10.7 mmol) in dichloromethane (15 mL) dropwise at such a rate that the internal temperature was maintained at <7°C. Stirring at 0°C was continued for 1 hr. The reaction was quenched by the addition of sat. aq. sodium bicarbonate solution (10 mL) and stirring at r.t. continued for 45 mm. The organic layer was separated, washed with 1 M hydrochloric acid (25 mL), sat. aq. sodium bicarbonate solution (25 mL) and brine (25 mL), dried (MgSO4), evaporated and concentrated in vacuo to a brown oil (2.194 g). Elution of the crude product through a silica gel plug with » dichloromethane (500 mL) afforded the title compound, after evaporation and concentration in vacuo, as a light amber semi-solid (2.319 g, 71.3%).

VmaxiCHClsVcm-1 3012, 2249, 1486, 1454, 1157; iHNMR (270 MHz, DMSO- DUBL01/C - 35 - IE Ο 1 0 7 8 4 CH2CN), 3.76 (s, 3 H, CH3O), 2.78 (q, J 7.6, 2 H, CH2CH3), 1.05 (t, J 7.4, 3 H, CH2C/73).

Example 25: 3-(Cyanomethyl)-2-methyl-l-(phenyl-methyl)-5-methoxy indole Compound 54 The procedure followed was as detailed above, using a solution of boron trifluoride etherate (5.84 mL, 46.1 mmol, 3.0 eq.) and cyano-tri-methylsilane (8.19 mL, 61.4 mmol, 4.0 eq.) in dichloro-methane (90 mL) and a solution of 3(hydroxymethyl)-2-methyl-l-(phenyl-methyl)-5-methoxy-indole (4.32 g, 15.4 mmol) in dichloro-methane (25 mL). Work-up in a similar fashion afforded the crude product as a dark red semi-solid (4.266 g) that, upon elution through a silica gel plug with dichloro-methane (700 mL), gave the title compound as a light yellow semi-solid (3.233 g, 72.5%).

'VmaxtCHClaVcnr1 3004, 2249,1487, 1454, 1157; *HNMR (270 MHz, DMSO-CNMR (67.8 MHz, DMSOd6) S154.1 (C5), 138.5, 135.4, 131.4, 127.2 (4 x qC), 128.9, 126.4 (o- & w-Ph), 127.4 (p-Ph), 119.6 (CN), 110.9, 110.8 (C4 & C6), 100.3 (C7), [C3 not seen], 55.7 (CH3O), 46.2 (N-CH2), 12.5 (CH2CN), 10.2 (CH3).

Example 26; 3-(Amidomethyl)-2-ethyl-l-(phenyl-methyl)-5-methoxy-indole Compound 57 To a solution of 3-(cyanomethyl)-2-ethyl-l-(phenylmethyl)-5-methoxy-indole (2.307 g, 7.58 mmol) in ferf-butanol (30 mL) was added powdered potassium hydroxide (2.127 g, 37.9 mmol, 5.0 eq.) and the mixture refluxed under nitrogen DUBL01/C for 1 hr. Once cooled to r.t., the reaction mixture was partitioned between ethyl acetate (75 mL) and brine (75 mL). The organic layer was separated, washed with brine (75 mL), dried (MgSO4), evaporated and concentrated in vacuo. Flash column chromatography (dichloromethane / ethyl acetate 1:2) afforded the title compound as a pale yellow solid (1.680 g, 66.2%).

'VmaxiCHClsVcm-1 3514, 3398, 3008, 1671 (C=O), 1485, 1154; iHNMR (270 MHz, DMSO-J6) 66.63-7.30 (m, 10 H, indole-H, C6H5 & ΝΉ2), 5.36 (s, 2 Η, NCH2), 3.74 (s, 3 H, CH3O), 3.44 (s, 2 H, 3-CH2), 2.73 (q, J 7.4, 2 H, C/Z2CH3), 1.04 (t, J 7.4, 3 H, CH2CH3).

Compound 27: 3-(Amidomethyl)-2-methyl-l-(phenyl-methyl)-5-methoxyindole Compound 56 The procedure followed was as detailed above, using: 3-(cyanomethyl)-2methyl-l-(phenylmethyl)-5-methoxyindole (3.113 g, 10.7 mmol), powdered potassium hydroxide (3.01 g, 53.6 mmol, 5.0 eq.) and iezi-butanol (40 mL). Work-up in a similar fashion gave the crude product as a yellow solid (2.956 g).

Recrystallisation (ethyl acetate) afforded the product as colourless crystals (1.139 g). Concentration of the mother liquor and subsequent recrystallisation (hexane / dichloromethane) afforded the product as pale yellow crystals (0.725 g). Concentration of the mother liquor and subsequent flash column chromatography (ethyl acetate then ethyl acetate / methanol 20:1) afforded the product as a pale yellow solid (0.463 g). Combination of the three materials afforded the title compound (2.327 g, 70.4 %). vmax (CH Cl 3) /cm’1 3514, 3398, 3003, 1671 (C=O), 1486, 1454, 1154; iHNMR (270 MHz, DMSCMi) 66.64-7.30 (m, 10 H, indole-H, C6H5 & NHz), 5.33 (s, 2 H, DUBL01/C N-CH2), 3.75 (s, 3 H, CH3O), 3.45 (s, 2 H, 3-CH2), 2.29 (s, 3 H, CH3); *3CNMR (67.8 MHz, DMS0- Example 28: 3-(Amidomethyl)-2-ethyl-l-(phenyl-methyl)-5-hydroxy indole Compound 59 To a solution of 3-(amidomethyl)-2-ethyl-l-(phenyl-methyl)—5-methoxyindole (1.622 g, 5.03 mmol) in dichloro-methane (35 mL) at 0°C under nitrogen was added boron tribromide (1.0 M solution in dichloromethane; 15.1 mL, 15.1 mmol, 3.0 eq.) dropwise. Stirring at r.t. was continued for 3 hr. The reaction solution was diluted with dichloromethane (50 mL) and quenched with water (75 mL). The organic layer was separated, washed with sat. aq. ammonium chloride solution (75 mL), brine (75 mL), dried (Na2SC>4), evaporated and concentrated in vacuo affording the tide compound as a pale brown solid (1.551 g, 98.2%) without further purification. vmax(CHCl3)/cm-l 3500-3100 (OH), 3512, 3396, 3006, 1668 (C=O), 1482, 1454, 1153; iHNMR (270 MHz, DMSO-ifg) 68.61 (br s, 1 H, OH), 6.49-7.30 (m, 10 H, indole-H, C6H5 & NH2), 5.31 (s, 2 H, N-CH2), 3.39 (s, 2 H, 3-CH2), 2.70 (q, J 7.4, 2 H, C/72CH3), 1.03 (t, 77.4, 3 H, CH2CZ/3).

DUBL01/C .38. ΙΕΟ 1 0 7 8 4 Example 29: 3-(Amidomethyl)-2-methyl-l-(phenyl-methyl)-5-hydroxy-indole Compound 58 The procedure followed was as detailed above, using: 3-(amidomethyl)-2methyl-l-(phenyl-methyl)-5-methoxy-indole (2.32 g, 7.52 mmol) in dichloromethane (25 mL) and boron tribromide (1.0 M solution in dichloromethane; 22.6 mL, 22.6 mmol, 3,0 eq.). Work-up in a similar fashion (a further portion of dichloromethane, ~125 mL, was added prior to the aqueous washes to effect complete dissolution of the crude product) afforded the title compound as a brown solid (1.941 g, 87.7%) without further purification.

Vmax(CHCl3)/cm-l 3600-3100 (OH), 3513, 3398, 3008, 1669 (C=O), 1483, 1454, 1153; iHNMR (270 MHz, DMSO-^6) 68.59 (br s, 1 H, OH), 6.50-7.31 (m, 10 H, indole-H, C6H5 & NH2), 5.29 (s, 2 H, N-CH2), 3.37 (s, 2 H, 3-CH2), 2.26 (s, 3 H, CH3); l^CNMR (67.8 MHz, DMSO-<) 6172.8 (C=O), 150.6 (C5), 138.8, 134.4, 130.6 (3 x qC, 1 qC not seen), 128.5, 126.2 (o- & m-Ph), 126.9 fy-Ph), 110.2, 109.5 (C4 & C6), 105.1 (C3), 102.7 (C7), 45.8 (N-CH2), 31.7 (3-CH2), 10.2 (CH3).

Example 30: Ethyl 4[[3-(amidomeihyl)-2-ethyl-l-(phenylmethyl)-indol-5yl]oxy]butanoate Compound 61 To a solution of 3-(amidomethyl)-2-ethyl-l-(phenyl-methyl)-5-hydroxy-indole (894 mg, 2.90 mmol) in DMF (5 mL) at r.t. under nitrogen was added sodium hydride (60% dispersion in mineral oil; 139 mg, 3.48 mmol, 1.2 eq.) in one portion. Stirring at r.t. was continued for 1 hr. Ethyl 4-bromobutyrate (415 pL, 2.90 mmol) was added in one portion and stirring at r.t. was continued for 2 hr. The reaction was quenched with water (10 mL) and extracted with dichloromethane (15 mL). The organic layer was separated, the aqueous layer extracted DUBLOI/C .a,- ΙΕΟ 10/64 with dichloromethane (10 mL), the combined organics washed with water (3 x 25 mL) and brine (25 mL), dried (MgSO4), evaporated and concentrated in vacuo to a brown oil. Flash column chromatography (dichloromethane / isopropanol 20:1, 10:1 then 5:1) afforded the title compound as a beige solid (600.5 mg, 49.0%; 57.6% based on recovered starting material) and unreacted stenting material was recovered (133.3 mg). iHNMR (270 MHz, DMSO-/6) 66.62-7.30 (m, 10 H, indole-H, C6H5 & NH2), 5.35 (s, 2 H, N-CH2), 4.07 (q, J 7.1, 2 H, CH3CH2OC=O), 3.96 (t, J 6.2, 2 Η, γCH2), 3.43 (s, 2 H, 3-CH2), 2.73 (q, J 7.4, 2 H, 2-C//2CH3), 2.47 (t, J 7.0, 2 Η, aCH2), 1.97 (qn, J 6.8, 2 H, |3-CH2), 1.18 (t, J 7.2, 3 H, Ctf3CH2OC=O), 1.03 (t, J 7.4, 3 H, 2-C/73CH2).

Example 31: Ethyl 4-[[3-(amidomethyl)-2-methyl-l-(phenylmethyl)-indol-5yl]oxy]butanoate Compound 60 The procedure followed was as detailed above, using: 3—(amidomethyl)-2methyl-l-(pheiiyl-methyI)-5-hydroxy-mdole (950 mg. 3_23 mmol) in DMF (10 mL), sodium hydride (60% dispersion in mineral oil: 155 mg, 3.87 mmol, 1.2 eq.) and ethyl 4-bromobutyrate (415 pL, 323 mnsoi). Work-up in a similar fashion gave the crude product as a brown oil. Flash column chromatography (dichloromethane / iso-propanol 20:1, 10:1 then 5:1) afforded the title compound as a beige solid (506.5 mg, 38.4%; 47.3% based on recovered starting material) and unreacted starting material was recovered (178.6 mg).

VmaxCTCfy/cm-1 3514, 3399, 3002, 1727 (ester C=O), 1672 (amide C=O), 1485, 1156; iHNMR (270 MHz, DMSCM5) 66.67-7.35 (m, 10 H, indole-H, C6H5 & NH2), 5.38 (s, 2 H, N-CH2), 4.12 (q, J 7.1, 2 H, CH3C#2OC=O), 4.01 (t, /6.3, 2 DUBL01/C -40H, 7-CH2), 3.47 (s, 2 H, 3-CH2), 2.52 (t, J 7.3, 2 H, cc-CH2), 2.34 (s, 3 H, CH3), 2.02 (qn, J 6.8, 2 H, |3-CH2), 1.22 (t, 7 7.3, 3 H, Ctf3CH2OC=O); 13CNMR (67.8 MHz, DMSO-76) δ 172.7, 172.5 (2 x 0=0), 152.4 (C5), 138.6, 134.8, 131.2, 128.1 (4 x qC), 128.4, 126.1 (0- & w-Ph), 126.9 (p-Ph), 110.1, 109.7 (C4 & C6), 105.7 (C3), 101.9 (C7), 67.0 (y-CH2), 59.7 (CH3CH2OC=O), 45.8 (N-CHfy), 31.5 (3CH2), 30.2 (cc-CH2), 24.5 (|3-CH2), 14.0 (CH3CH2OC=O), 10.2 (CH3).

Example 32: Dimethyl [3-[[3-(amidomethyl)-2-methyl-l-(phenylmethyl)indol-5-yl]oxy]-propyl]phosphonate Compound 62 The procedure followed was as detailed above, using 3-(amidomethyl)-2methyl-l-(phenylmethyl)-5-hydroxyindole (1.103 g, 3.75 mmol) in DMF (10 mL), sodium hydride (60% dispersion in mineral oil; 180 mg, 4.50 mmol, 1.2 eq.) and dimethyl (3-bromopropyl)phosphonate (0.866 g, 3.75 mmol). Work-up in a similar fashion gave the crude product as a light brown oil. Flash column chromatography (dichloromethane / iso-propanol 9:1 then 9:2) afforded the title compound as an off-white solid (0.964 g, 57.9%). vmax(CHCl3)/cnrl 3515, 3399, 3002, 1671 (C=O), 1485, 1156, 1039; iHNMR (270 MHz, DMSO-76) 66.71-7.37 (m, 10 H, indole-H, C6H5 & NH2), 5.40 (s, 2 H, N-CH2), 4.05 (t, 75.5, 2 H, y-Ofy), 3.68 (d, 37Ph 15.1, 6 H, (CH3O)2P=O), 3.48 (s, 2 H, 3-CH2), 2.35 (s, 3 H, CH3), 1.90-2.06 (m, 4 H, a- & |3-CH2); 13CNMR (67.8 MHz, DMSO-76) 6174.2 (C=O), 153.9 (C5), 140.1, 136.4, 132.9, 129.7 (4 x qC), 130.0, 127.8 (o- & m-Ph), 128.5 (p-Ph), 111.8, 111.3 (C4 & C6), 107.3 (C3), 103.7 (C7), 69.3 (d, 37cp 16.2, γ-ΟΗ2), 53.5 (d, 2yCP 6.2, (CH3O)2P=O), 47.4 (NCH2), 33.1 (3-CH2), 23.9 (d, 27CP 3.7, |3-CH2), 22.0 (d, Vqp 139, a-CH2), 11.7 (CH3).

DUBL01/C ΙΕΟ 1 07 8 4 Example 33: Dimethyl [3-[[3-(amidomethyl)-2-ethyl-l-(phenylmethyl)indol-5-yl]oxy]-propyl]phosphonate Compound 63 The procedure followed was as detailed above, using 3-(amidomethyl)-2-ethyll-(phenylmethyl)-5-hydroxyindole (2.513 g, 8.15 mmol) in DMF (20 mL), sodium hydride (60% dispersion in mineral oil; 0.391 g, 9.78 mmol, 1.2 eq.) and dimethyl (3-bromopropyl)phosphonate (1.883 g, 8.15 mmol). Work-up in a similar fashion gave the crude product as a light brown solid. A portion (100 mg) of this material was purified by flash column chromatography (dichloromethane / iso-propanol 7:1 then 5:1) affording an analytically pure sample (63.8 mg, overall yield 64.8%). 7HNMR (270 MHz, DMSO-/6) 66.65-7.31 (m, 10 H, indole-H, C6H5 &NH2), 5.35 (s, 2 H, N-CH2), 4.00 (t, J 5.7, 2 H, y-CH2), 3.64 (d, 3/pH 14.8, 6 H, (CH3O)2P=O), 3.44 (s, 2 H, 3-CH2), 2.73 (q, J 7.4, 2 H, 2-C//2CH3), 1.80-2.01 (m, 4 H, a- & (3-CH2), 1.04 (t, J 7.4, 3 H, 2-CH2CZ/3); 13CNMR (67.8 MHz, DMSO-/6) 6172.7 (C=O), 152.4 2/CP 6.2, (CH3O)2P=O), 45.9 (N-CH?), 31.4 (3-CH2), 22.3 (d, 2/CP 4.9, β-CHz), 20.2 (d, Vcp 139, cc-CH2), 17.3 (2CH2CH3), 14.4 (2-CH2CH3).

Example 34: 4-[[3-(Amidomethyl)-2-methyl-l-(phenylmethyl)-indol-5-yl] oxy] butanoic acid Compound 26 To a solution of ethyl 4-[[3-(amidomethyl)-2-methyl-l-(phenylmethyl)-indol5-yl]oxy]butanoate (495.9 mg, 1.21 mmol) in ethanol (40 mL) at r.t. was added a 1 M aq. solution of sodium hydroxide (3 mL). Stirring at r.t. was continued for 19 hr. The reaction mixture was evaporated to dryness, water (10 mL) added DUBL01/C and the resultant mixture acidified with 10% hydrochloric acid (~1 mT.) The crude product was obtained by filtration, stirred with boiling ethyl acetate (50 mL) for 5 min, cooled to ~5°C and filtered to give, after drying (50°C / 1.0 mm Hg, 3 hr), the title compound as an off-white solid (368.0 mg, 79.7%). m.p. 232-4°C (Lit. 48 218-21°C); umax(KBr)/cm-l 3548 & 3345 (NH), 3600-2200 (OH), 2962, 2917, 1729 (carboxyl C=O), 1641 (amide C=O), 1578, 1473, 1161, 1038; 1HNMR (270 MHz, DMSO^) 86.65-7.33 (m, 10 H, indole-H, C6H5 & NH2), 5.35 (s, 2 H, N-CH2), 3.97 (t, 76.3, 2 H, y-CH2), 3.44 (s, 2 H, 3-CH2), 2.40 (t, 7 7.2, 2 H, a-CH2), 2.31 (s, 3 H, CH3), 1.90-2.01 (m, 2 Η, β-ΟΗ2); 13CNMR (67.8 MHz, DMSO-76) 6174.1, 172.6 (2 X C=O), 152.5 (C5), 138.5, 134.7, 131.3, 128.1 (4 X qC), 128.4, 126.1 (o- & zra-Ph), 126.9 (p-Ph), 110.2, 109.7 (C4 & C6), 105.7 (C3), 101.9 (C7), 67.2 (y-CH2), 45.8 (N-CH2), 31.5 (3-CH2), 30.4 (0C-CH2), 24.6 (β-ΟΗ2), 10.1 (ΟΗβ).

Example 35: 4~[[3-(Amidomethyl)-2-ethyl-l-(phenylmethyl)-indol-5yl]oxy]-butanoic add Compound 27 To a solution of ethyl 4-[[3-(amidomethyl)-2-ethyl-l-(phenylmethyi)-indol-5yl]-oxy]-butanoate (403.9 mg, 0.956 mmol) in ethanol (15 mL) at r.t. was added a 1 M aq. solution of sodium hydroxide (2 mL). Stirring at r.t. was continued for 2 hr. The reaction mixture was evaporated to dryness, water (10 mL) added and the resultant mixture acidified with 10% hydrochloric acid (~1 mL). The crude product was extracted with ethyl acetate (~200 mL), the organic layer separated, washed with brine (50 mL), dried (MgSOzj), evaporated and concentrated in vacuo. This material was swirled with ether / methanol (3-4 mL / 6 drops), allowed to settle, the liquor carefully removed by pipette, and the DUBL01/C .43. ΙΕ 0 1 0 7 8 4 residual solid dried in vacuo to give die tide compound as an off-white solid (254.2 mg, 67.4%). m.p. 196-8°C (Lit.48 196-9°C); UmaxiKBrj/cm-1 3548 (NH), 3600-2200 (OH), 2965, 1715 (carboxyl C=O), 1644 (amide C=O), 1487, 1044; iHNMR (270 MHz, DMSO-ob) 66.63-7.35 (m, 10 H, indole-H, C0H5 & NH2), 5.35 (s, 2 H, N-CH2), 3.96 (t, /6.3, 2 H, γ-ΟΗ2), 3.44 (s, 2 H, 3-CH2), 2.73 (q /7.4, 2 H, C7/2CH3), 2.40 (t, /7.2, 2 H, 0C-CH2), 1.91-2.03 (m, 2 Η, β-ΟΗ2), 1.04 (t, / 7.4, 3 H, CH2C7/3); 13CNMR (67.8 MHz, DMSO-^) 5174.1, 172.7 (2 X C=O), 152.5 (C5), 140.4, 138.7, 131.2, 128.2 (4 X qC), 128.4, 125.9 (o- & mPh), 126.8 (p-Ph), 110.4, 110.0 (C4 & C6), 105.2 (C3), 102.0 (C7), 67.1 (yCH2), 45.8 (N-CH2), 31.4 (3-CH2), 30.2 (a-CH2), 24.5 (β-ΟΗ2), 17.3 (LH2CH3), 14.4 (CH2CH3); (Found: C, 69.9; H, 6.9; N, 7.4. C23H26N2O4 requires C, 70.0; H, 6.6; N, 7.1%).

Example 36: [3-[[3-(Amidomeihyl)-2-methyLT-(phenylmeihyI)-indol-5-yl] oxy]-propyl]-phosphonic acid Compound 28 To a solution of sodium iodide (2.55 g, 10.0 mmol, 9.0 eq.) in acetonitrile (13 mL) at r.t. under nitrogen in a 20 mL narrow, straight-walled flask (a new B19 cone sealed at the end by a glassblower is ideal) was added chlorotrimethylsilane (2.16 mL, 17.0 mmol, 9.0 eq.) in one portion. A fine white precipitate fonned immediately and was allowed to settle over 15 min affording a clear yellow solution of iodotrimethylsilane. To a solution of dimethyl [3-[[3(amidomethyl)-2-methyl-l-(phenylmethyl)-indol-5-yl]oxy]-propyl]-phosphonate (842 mg, 1.89 mmol) in dichloromethane (20 mL) at r.t. under nitrogen was added a portion of the iodotrimethylsilane solution (5 mL, 3.0 eq.) in one portion. Stirring at r.t. was continued for 1.5 hr. The reaction contents were DUBL01/C -44evaporated to dryness and the residue stirred with methanol (10 mL) for 10 min, resulting in a white suspension. Evaporation and concentration in vacuo afforded the crude product. Recrystallisation (ethyl acetate / acetonitrile / acetic acid / water 21:7:7:9) afforded the title compound as a white solid (531 mg, 67.3%). m.p. 201-3°C (Lit.48 201-3°C); nmax(KBr)/cm-l 3600-2400 (OH), 3455 (N-H), 2928, 1655 (C=O), 1489, 1154; iHNMR (270 MHz, DMSO-afc) 66.66-7.32 (m, 10 H, indole-H, C6H5 & NH2), 5.34 (s, 2 H, N-CH2), 4.01 (t, J6.5, 2 Η, γCH2), 3.44 (s, 2 H, 3-0¾). 2.31 (s, 3 H, CH3), 1.83-2.01, 1.64-1.76 (2 m, 4 H, ce- & β-ΟΗ2); 13CNMR (100.54 MHz, DMSO-dfe) 6174.9 (C=O), 153.1 (C5), 138.8, 135.9, 131.9, 128.6 (4 X qC), 129.1, 126.7 (0- & m-Ph), 127.7 (p-Ph), 111.0, 110.5 (C4 & C6), 105.6 (C3), 102.6 (C7), 69.0 (d, 3JcP 17.7, 7-0¾). 46.5 (N-CH2), 31.9 (3-CH2), 24.4 (d, Vcp 137, 0C-CH2), 23.3 (s, 2/CP 0, β0¾). 10.6 (CH3); 3lPNMR (161.83 MHz, DMSO-^fc, phosphoric acid external reference) 6275; (Found: C, 60.3; H, 5.9; N, 6.3. C21H25N2PO5 requires C, 60.6; H, 6.05; N, 6.7%).

Example 37: [3-[[3-(Amidomethyl)-2-ethyl-l-(phenylmethyl)-indol-5-yl]oxypropyl]phosphonic acid Compound 29 The procedure followed was as detailed above, using sodium iodide (5.86 g, 39.1 mmol, 6.0 eq.), chlorotrimethylsilane (4.96 mL, 39.1 mmol, 6.0 eq.) and acetonitrile (35 mL). A portion of the iodotrimethylsilane solution (20 mL, 3.0 eq.) was added to a solution of dimethyl [3-[[3-(amidomethyl)-2-ethyl-l(phenylmethyl)-indol-5-yl]oxy]propyl]phosphonate (3.793 g, 6.51 mmol) in dichloromethane (50 mL) in one portion and stirring at r.t. under nitrogen DUBL01/C ΙΕΟ 1 O7 8 4 -45continued for 1.5 hr. Work-up in a similar fashion gave the crude product as an orange solid. Recrystallisation (ethyl acetate / acetonitrile / acetic acid / water 21:7:7:9) afforded the title compound as a pale brown solid (0.970 g, 34.6%). m.p. 190-2°C (Lit. 51 194-6°C); umax(KBr)/cm-l 3600-2400 (OH), 3457 (N5 H), 2361, 1654 (C=O), 1487, 1154; iHNMR (270 MHz, DMSO-cfc) 66.64-7.38 (m, 10 H, indole-H, C6H5 & NH2), 5.35 (s, 2 H, N-CH2), 4.00 (t, 76.3, 2 Η,γCH2), 3.44 (s, 2 H, 3-CH2), 2.73 (q, 77.4, 2 H, 2-C7/2CH3), 1.63-1.78, 1.872.00 (2 m, 4 Η, α- & β-ΟΗ2), 1.04 (t, 77.4, 3 H, 2-CH2CL73); 13CNMR (67.8 MHz, DMSO-76) 6172.8 (C=O), 152.5 (C5), 140.4, 138.7, 131.2, 128.2 (4 X qC), 128.4, 125.9 (o- & m-Ph), 126.8 (p-Ph), 110.4, 110.0 (C4 & C6), 105.2 (C3), 102.3 (C7), 68.2 (d, VCP 16.1, y-CH2), 45.8 (N-CH2), 31.4 (3-CH2), 24.1 (d, l7cp 145, a-CH2), 23.1 (s, 2/CP Ο, β-ΟΗ2), 17.3 (2-CH2CH3), 14.4 (2-CH2CH3); (Found: C, 61.6; H, 6.3; N, 6.4. C22H27N2PO5 requires C, 61.4; H, 6.3; N, 6.5%).

DUBLOl/C -46Pharmacological Tests Example A To assess the ability of these agent to increase the effect of MRP-1-substrate anticancer drugs, toxicity assays were performed which compared the effect of the drug adriamycin (doxorubicin) in the absence and presence of an inhibitor.

The test compounds were prepared as described previously.

Method The DLKP cell line used in these examples was derived from a lung cancer patient and developed by researchers in the National Cell and Tissue Culture Centre, Dublin City University. The CORL23 (R) lung cell line was a gift of Dr.P.Twentyman, MRC Clinical Oncology and Radiotherapeutics unit, Hills Road, Cambridge CB2 2QH, U.K.

Indomethacin and all other named chemicals used in this example were supplied by the Sigma Chemical Company Ltd. Fancy Road, Dorset, BH12 4QH, England- Media for all assays was provided by GibcoBRL, Life Technologies Ltd, 3 Fountain Drive, Inchinnan Business Park, Paisley PA4 9RF Scotland. The test compounds were synthesised in our own laboratories.

DLKP cell line Cells were trypsinised from the flask in the exponential phase of growth. Cell suspensions containing lxl04 cells/ml were prepared in cell culture medium. Volumes of ΙΟΟμΙ of this cell suspension were added into 96-well plates using a multichannel pipette. Plates were agitated gently in order to ensure even DUBL01/C dispersion of cells over a given well. Cells were then incubated overnight at 37°C in an atmosphere containing 5% CO2.

Cytotoxic drug dilutions and test compound dilutions were prepared at 4X their final concentration in media. Volumes of 50uL of the drug dilution and 50pL of the test compound dilution were then added to each relevant well so that a total final volume of 200pL was present in each well. All potential toxicity-enhancing agents were dissolved in DMSO, ethanol or media. Stock solutions were prepared at approximately 15mg/10ml media, filter sterilised with a 0.22pm filter and then used to prepare all subsequent dilutions. Solvent control experiments showed that no toxicity enhancement effects were due to the presence of DMSO or ethanol.

Cells were incubated for a further 6 days at 37°C in an atmosphere containing 5% CO2. At this point the control wells would have reached approximately 8090% confluency.

Cell number was assessed using the acid phosphatase assay. Each well on the plate was washed with lOOuI PBS. This was then removed and ΙΟΟμΙ of freshly prepared phosphatase substrate (lOmM p-nitrophenol phosphate in O.lM sodium, 0.1% triton X-100, pH 5.5) was added to each well.

The plates were then incubated in the dark at 37°C for 2 hours and the enzymatic reaction was stopped by the addition of 50μ1 of IN NaOH. The plate was read in a dual beam plate reader at 405nm with a reference wavelength of 620nm.

Statistical analysis of the data was performed as follows. The results obtained from the analysis of data using the fractional method were confirmed using a DUBLOI/C IF 01 01 8 4 - 48 - 1 computer package for multiple drug effect analysis, "Dose-Effect Analysis with Microcomputers", 54,55. The program provides combination index (CI) values which are a quantitative measure of drug interaction in terms of an additive (CI = 1), synergistic (CI < 1) or antagonistic (CI > 1) effect for a given endpoint of the assay used. The toxicity values associated with the highest concentration of the compound combined with adriamycin were used to produce the CI values found in the tables.

Results In the results given below the % cell survival in the drug-free control is 100%. All results are the cell survival means ± the standard deviation (SD) of that mean from a minimum of three experiments. The derivative concentrations used in all cases are non-toxic, i.e. there is no expectation of significantly increasing cell death by combining the two drugs. Indomethacin, a known potentiator of toxicity in MRP-1-expressing cell lines, is used as a positive control. The Combination Index values quoted are a statistical measure of the toxic synergism (or lack of) between the compound and adriamycin. The smaller the value the greater the synergism evident. Values of 1 or greater indicate only additive toxiciiy or antagonism of the toxic effect of adriamycin.

DUBL01/C -49- ΙΕΟ 1018 4 Table 1.1 DLKP, Adriamycm and Indomethacin, Compound 4, Test Sample % Cell Survival S.D. Adr. lOng/ml 58.6 1.7 Indo (2.5pg/ml) 94.1 4.4 Indo + Adr 24.5 1.1 Indo (1.25pg/ml) 97.7 7.4 Indo + Adr 30.7 1.7 Indo (0.625pg/ml) 95.1 2.1 Indo + Adr 36.3 1.0 Combination Index Value 0.557 Cpd. 4 (5 pg/ml) 93.8 0.9 Cpd. 4 + Adr 20.3 1.8 Cpd. 4 (2.5ug/ml) 98.6 0.8 Cpd. 4 -h Adr 31.5 2.8 Cpd. 4 (I25ug/ml) 101.0 2.3 Cpd. 4 * Adr 40.3 13.5 Combination Index Value 0.483 DUBL01/C -so- £O1°78* Table 1.2 DLKP, Adriamycin and Compounds 7 & 14 Test Sample % Cell Survival S.D. Adr. lOng/ml 44.9 5.5 Cpd. 7 (10pg/ml)) 99.2 2.5 Cpd. 7 + Adr 10.3 1.2 Cpd. 7 (5pg/ml) 104.2 2.7 Cpd. 7 + Adr 12.3 1.8 Cpd. 7 (2.5pg/ml) 103.3 3,4 Cpd. 7 + Adr 18.4 2.7 Combination Index Value 0.437 Cpd. 14 (lOug/ml) 93.1 6.7 Cpd. 14 +Adr 18.6 0.5 Cpd. 14 (5pg/ml) 93.0 7.7 Cpd. 14 +Adr 243 2.0 Cpd. 14 (2.5ug/ml) 94.5 33 Cpd. 14 + Adr 30.4 3.7 Combination Index Value 0.563 DUBL01/C Table 1.3 DLKP P32, Adriamycin and Compound 18 Test Sample % Cell Survival S.D Adr. lOng/ml 57.6 1.1 Cpd. 18 (15pg/ml) 93.6 1.8 Cpd. 18 + Adr 13.4 2.0 Cpd. 18 (7.5pg/ml) 98.0 1.9 Cpd. 18 + Adr 24.0 0.8 Cpd. 18 (3.75pg/ml) 98.1 1.8 Cpd. 18 + Adr 55.7 0.7 Combination Index Value 0.340 Table 1.4 DLKP, Adriamycin and Compound 27 Test Sample % Cell Survival S.D. Adr. lOng/ml 41.28 9.48 Cpd. 27 (50pg/ml) 97.8 3.1 Cpd. 27 + Adr 13.2 2.9 Cpd. 27 (25pg/ml) 99.1 1.4 Cpd. 27 + Adr 15.7 3.8 Cpd. 27 (12.5pg/ml) 99.5 2.1 Cpd. 27 + Adr 23.2 8.5 Combination Index Value 0.397 DUBLOl/C Table 1.5 IED 1 07 8 4 DLKP, Adriamycin and Compound 26 Test Sample % Cell Survival S.D. Adr. lOng/ml 57.6 1.1 Cpd. 26 (20μg/ml) 99.9 0.5 Cpd. 26 + Adr 26.9 0.1 Cpd. 26 (10pg/ml) 100.3 0.0 Cpd. 26 + Adr 38.9 1.3 Cpd. 26 (5pg/ml) 100.6 0.3 Cpd. 26 + Adr 49.0 3.0 Combination Index Value 0.551 Unexpectedly, compounds with very similar structures did not produce synergistic toxicity in these cells.

DUBL01/C -53- IE 0 1 0 7 8 4 Table 1.6 DLKP, Adriamycin and Compound 5 Test Sample % Cell Survival S.D Adr. lOng/ml 52.5 4.0 Cpd. 5 (5 pg/ml) 99.0 7.7 Cpd. 5 + Adr 42.2 3.3 Cpd. 5 (2.5pg/ml) 100.3 0.9 Cpd. 5 + Adr 47.9 5.3 Cpd. 5 (1.25pg/ml) 99.3 3.1 Cpd. 5 + Adr 50.0 2.7 Combination Index Value 1.000 DUBLOI/C -54Table 1.7 DLKP, Adriamycin and Compounds 16 & 17 Test Sample % Cell Survival S.D Adr. lOng/ml 44.9 5.5 Cpd. 16 (lOpg/ml) 99.5 0.5 Cpd. 16 + Adr 39.4 5.4 Cpd. 16 (5 pg/ml) 98.8 3.1 Cpd. 16 + Adr 42.7 1.1 Cpd. 16 (2.5pg/ml) 100.7 3.5 Cpd. 16 + Adr 41.8 1.3 Combination Index Value 1.035 Cpd. 17 (5pg/ml) 96.7 4.2 Cpd. 17+Adr 34.1 2.8 Cpd. 17 (2.5pg/ml) 98.9. 4.1 Cpd. 17 +Adr 38.0 15 Cpd. 17 (l-25pg/ml) 99.0 45 Cpd. 17+Adr 41.0 1.0 Combination Index Value 1.000 DUBLOl/C Table 1.8 -55IE o 1 0 7 8 4 DLKP, Adriamycin and Compound 29 Test Sample % Cell Survival S.D. Adr. lOng/ml 57.6 1.1 Cpd. 29 (50pg/ml) 100.4 0.1 Cpd. 29 + Adr 49.3 3.5 Cpd. 29 (25pg/ml) 92.0 3.1 Cpd. 29 + Adr 60.6 3.5 Cpd. 29 (12.5pg/ml) 100.9 0.1 Cpd. 29 + Adr 63.3 5.1 Combination Index Value 1.035 DLKP cell line These results indicate that compounds 4, 7, 14, 18, 26 and 27 are all capable of increasing the anti-cancer effect of adriamycin (CI values «1). The mechanism of this effect is through a blocking of fhe action of the drug resistance pump MRP-1, which is active in this cell line and makes it resistant to a number of anti-cancer drugs including adriamycin. Compounds with very similar structures (13, 5, 16, 17, and 29) do not possess this ability (CI values > 1).

DUBL01/C -56COR L23 R cell line Experiments were also performed in the MRP-1 resistant cell line COR L23 R. Again a synergistic increase in toxicity was seen when adriamycin was combined with a compound, 27, from the active class (CI <1) and no synergy was evident with a compound with a very closely related structure but from the inactive group, 28 (CI =1).

Table 1.9 CORL23(R), Adriamycin and Compound 27 Test Sample % Cell Survival S.D Adr. 125 ng/ml 74.6 2.8 Indo (2.5 pg/ml) 102.3 3.7 Indo + Adr 31.2 7.5 Indo (1.25pg/ml) 102.0 3.6 Indo +Adr 47.0 5.1 Indo (0.625pg/ml) 99.1 7.6 Indo +Adr 602 6.5 Combination Index Value 0.779 l , Cpd. 27 (50pg/ml) 102.2 4.4 Cpd. 27 + Adr 18.5 5.2 Cpd. 27 (25pg/ml) 98.3 7.6 Cpd. 27 + Adr 48.8 2.9 Cpd. 27 (12.5pg/ml) 105.0 4.6 Cpd. 27 +Adr 63.7 4.7 Combination Index Value 0.813 DUBL01/C -57Table 1.10 CORL23 (R) Adriamycin and Compound 28 Test Sample % Cell Survival S.D Adr. 250ng/ml 50.8 2.9 Cpd. 28 (25pg/ml) 103.2 6.4 Cpd. 28 + Adr 46.9 2.6 Cpd. 28 (12.5pg/ml) 96.4 3.1 Cpd. 28 + Adr 47.9 2.4 Cpd. 28 (6.25pg/ml) 103.3 6.4 Cpd. 28 + Adr 51.1 4.3 Combination Index Value 1.000 Combinations with taxol F.TpeTrments were also undertaken with active and inactive inhibitors in combination with taxol. Table 1.11 illustrates that a synergistic increase in the toxidty of taxol was evident in the MRP-1 resistant cell line COR L23 R when this anti cancer drug was combined with Compound 27. Table 1.12 illustrates that this effect was not evident when the structurally similar agent compound 28 was combined with taxol.

DUBL01/C IE Ο 107 8 4 Table 1.11 CORL23(R) Taxol and Compound 27 Test Sample % Cell Survival S.D. Taxol. 7.0ng/ml 42.1 1.7 Cpd. 27 (50pg/ml) 94.6 0.2 Cpd. 27 + Taxol 17.6 0.2 Cpd. 27 (25pg/ml) 98.5 2.7 Cpd. 27 + Taxol 23.7 1.3 Cpd. 27 (12.5pg/ml) 100.4 0.6 Cpd. 27 + Taxol 23.4 0.2 Combination Index Value 0.352 Table 1.12 I CORL23(R) Taxol and Compound 28 Test Sample % Cell Survival S.D. Taxol. 7. Ong/ml 39.9 4.9 Cpd. 28 (25pg/ml) 99.2 5.1 Cpd. 28 + Taxol 34.4 5.4 Cpd. 28 (12.5pg/ml) 100.7 2.4 Cpd. 28 + Taxol 35.7 5.6 Cpd. 28 (6.25pg/ml) 101.1 1.9 Cpd. 28 + Taxol 39.7 2.8 Combination Index Value 1.365 DUBL01/C Example B To further explore the effect of this group of compounds/agents. Experiments were undertaken to examine the effect of a positive and negative compound on the direct action of MRP-1 using a purified preparation of membrane containing MRP-1 protein. These MRP-1 containing vesicles (Inside Out Vesicles, IOVs) are prepared in such a way as to actively transport MRP-1 substrates, such as radiolabelled Leukotriene C4 into the membrane, where the activity of the pump can be measured by quantifying the total radioactivity of the vesicles over time. Agents which inhibit the action of MRP-1 reduce the amount of radiation accumulated into the vesicles.

The test compounds were prepared as described previously.

Method Preparation of Vesicles The isolation of IOVs from the HL60 ADR cells was performed as described by Ishikawa etal., (1994)56.

HL60 ADR cells (donated by Dr. Melvin Centre, Kansas State University, Manhattan KS 66506, USA) were cultured (in RPMI media supplemented with 10% serum) to a high number of cells in spinner flasks at 37°C and placed on a spinner apparatus.

Approximately 7x10s cells were pelleted at 5,000r.p.m. (l,200g) for 10 minutes at 4°C in a Sorvall refrigerated centrifuge. The supernatant was decanted and the pellets resuspended in 50 ml of ice cold PBS. The combined pellets were then transferred to a 50 ml tube and spun at 4,000 rpm. for 5 minutes. The resulting cell pellet was then resuspended in 230mls hypotonic buffer with the following components.

DUBL01/C Buffer constituent Preparation instructions 0.5mM Sodium phosphate (pH 7.0) 30 mg NaP in 500ml UHP O.lmM EGTA (Ethylene Glycol-bis(P-aminoethyl Ether) Ν,Ν,Ν’,Ν’-Tetraacetic acid) 19.2 mg EGTA added to NaP solution O.lmM PMSF lOOmM stock prepared in EtOH The PMSF was added to the buffer immediately before use.

Cells were lysed by gentle agitation at 4 °C for 1.5 hours. The cell lysate was then centrifuged at 28,000 rpm. (100,000g) for 35 minutes at 4° C with a Beckman SW28 rotar in a Beckman XL-80 ultracentrifuge. The resulting pellets were then resuspended in 10ml of hypotonic buffer and then homogenised for 15 minutes at 4° C with a Braun Potter S886 homogeniser.

The homogenised cell extract was diluted to a final volume of 20ml with incubation buffer which contained the following Buffer constituent Preparation instruction lOmM TRIS -HCi (pH 7.4) 1.211 gTRIS in IL UHP water 250mM Sucrose 42.79g Sucrose in 500 ml 10 mM TRIS-HC1 (pH 7.4) The crude membrane fraction was layered over 38% sucrose/lOmM TRIS-HC1 pH 7.4, (38g sucrose in 100ml lOmM TRIS HCl pH 7.4) and centrifuged at 28,000 r.p.m (100,000g) for 35 minutes at 4°C with a SW28 rotor. A volume of 10ml crude membrane fraction was layered over 28.5ml 38% sucrose/lOmM TRIS HCl pH 7.4. The interface was marked to specify the location of the DUBLOl/C .6,. 1E0UH4 plasma membrane band which developed after the sucrose centrifugation step.

A thin white band became localised at the interface after centrifugation and this was removed with a Pasteur pipette and diluted to a total volume of 20ml with incubation buffer. The suspension was then centrifuged at 38,200 rpm (100,000g) for 35 minutes at 4 °C using a Beckman 70.1 rotor.

The pellets were then resuspended in 0.2ml incubation buffer. Vesicles were formed by passing resuspended pellets through a 27-gauge needle 20 times using a lml syringe.

A protein assay was then performed and the IOV preparation was then diluted to a concentration of 5mg protein /ml with incubation buffer. Volumes of 50μ1Σ IOVs were then frozen at -80° C.

Protein assay Protein levels were determined using the Bio-Rad protein assay kit (Bio-Rad, 5000006) as follows.

A 2mg/ml bovine serum albumin (BSA) solution (Sigma, A9543) was prepared freshly in lysis buffer. A protein standard curve was prepared from the BSA stock with dilutions made in lysis buffer. The Bio-Rad reagent was diluted 1:5 in UHP water and filtered through Whatman paper before use. Α 20μ1 volume of protein standard dilution or sample was added to lml of diluted dye reagent and the mixture vortexed. After 5 minutes incubation, absorbance was assessed at 570nm. The concentration of the protein samples was determined from the plot of the absorbance at 570nm versus concentration of the protein standard.

Transport assays with IOVs.

Transport assay with IOVs were performed as described by Ishikawa et al., (1994). The protocol used in these assays is as follows: DUBLOI/C ,62. IEO 1 0?8 4 Prior to performing the assay, filters (Millipore, GSWP-02500) were soaked in the incubation buffer for 1 hour at 4°C. Once soaked, the filters were applied to the filter apparatus (Millipore, 12-25 Sampling Manifold) and vacuum was applied to the system. An eppendorf thermomixer (Eppendorf, 5436) was allowed to equilibrate at 37°C and once at temperature, the ATP, AMP, creatine kinase and IOV solutions were thawed rapidly at 37°C. After thawing, solutions were immediately placed on ice.

The ATP/creatine phosphate/MgCl2/10mM TRIS-HC1 (pH 7.4) solution was prepared as follows Buffer constituent Preparation instructions MgCl2 6H20 203.3mg in 30ml Incubation buffer ATP (Disodium salt) 6.05 mg ATP in 3 ml MgCl2 6H20 solution Creatine phosphate 32.7 mg in 3ml ATP solution Stock volumes of200pL were maintained frozen at -80°C until required.

For the AMP solution, 4.99mg AMP (Sigma, A1752) was substituted for the ATP (Sigma A7699). Once prepared as in Table 2.11.1 above, 100pL volumes were frozen at -80° C.

The creatine kinase solution (2mg/ml), (Sigma, C5755) was prepared in incubation buffer and 50pL aliquots frozen at -80 °C.

An eppendorf was placed in the thermomixer and the following added sequentially: 60pL incubation buffer, 30 pL ATP, 5pL creatine kinase, 5ul [3H]LTC4 (DuPont NEN, NET-1018, 0.01 mCi/ml) and lOpL IOVs. After every DUBL01/C sequential addition the thermomixer was adjusted to half speed mixing to allow agitation of the various components of the mixture.

Aliquots of 20pL were removed at appropriate time-points and added in to lml of ice cold incubation buffer. These were then washed through the filter apparatus. The eppendorf tube was washed out with lml of ice cold incubation buffer. The filter was finally washed with 2 ml of ice cold incubation buffer. Filters were removed and placed in 8ml scintillation cocktail (ICN, 882475) in a scintillation vial. After allowing 12 hours for the filters to fully disolve, the vials were counted for [3H] content using a Beckman LS-6500 scintilation counter using a 1 minute count time.

For an AMP negative control, the above procedure was repeated with ATP replaced by AMP. For a total negative control, neither ATP nor AMP were included but were instead replaced with 30pL incubation buffer.

For assessment of a compounds ability to inhibit LTC4 transport ability, the compound of interest was dissolved in incubation buffer at the desired concentration. 5pL of this was added to an eppendorf in the thermomixer. 55uL of incubation buffer was then added. The standard volumes of ATP, AMP, creatine kinase, LTC4 and IOV were then added to a total final volume of 110pL.

DUBL01/C ·64’ ΙΕΟ 1 07 84 Results Table 2.1 Compound Molarity of compound in test solution Average % Inhibition S.D. Indomethacin 46. OmM 83.1 10.4 Cpd. 4 47.6mM 57.7 4.7 Cpd. 14 50.ImM 67.1 13.5 Cpd. 27 41.8mM 85.0 9.0 Cpd. 7 47.2mM 66.9 7.7 Cpd. 28 39.8mM 0.0 0.8 Cpd. 16 50.ImM 26.8 5.2 Cpd. 29 38.2mM 22.5 5.6 Relative ATP-dependent rates shown are expressed as a percentage of untreated control, taken as 100%, by subtracting the rate in the presence of AMP, which was used as the blank. Data given are from a minimum of three assay repeats.

These results indicate that the active compounds are all good inhibitors of MRP1 transport as compared to the positive control inhibitor, indomethacin. Compounds 16, 28, & 29 which have very similar structures possess little or no MRP-1 inhibitory activity.

DUBL01/C -65- if ο 1 07 8 4.

Example C To further investigate the mechanism of action of the inhibitors discovered, experiments were undertaken to quantitate the effect of one of the compounds on the MRP-l-associated efflux of adriamycin from DLKP cells. Cells were exposed to adriamycin for 2 hours which loaded the cells with the anti-cancer drug. When re-fed with fresh, non-adriamycin containing media, the drug is effluxed from the cells by the MRP-1 pump, leading to a steady decline in the cellular level of adriamycin. Agents which block this efflux lead to less reduction in the overall cellular concentration of adriamycin.

The test compounds were prepared as described previously.

Method DLKP cells were seeded into 75cm2 flasks (Costar, 3375) at 0.5xl06 cells per flask. Cells were incubated for 48 hours, after which time medium was removed and fresh medium containing adriamycin (ΙΟμτη), indomethacin or test compound (27.95um), or combination of both adriamycin and indomethacin/compound of interest, was added. Flasks were incubated at 37°C for a period of two hours.

After this two hour incubation, the media was removed from all flasks and replaced with fresh media, or media containing indomethacin/test compound or adriamycin, as the experiment required. The flasks were returned to the 37°C incubator. At relevant time points the media was removed from the flasks and the flasks were washed twice with PBS. Cells were then trypsinised and counted. Pellets were then washed with PBS and frozen at -20°C.

DUBL01/C ft Lk 7f When required for HPLC analysis, the frozen pellets were thawed, resuspended in ΙΟΟμΙ UHP water and added to glass tubes (test samples). Untreated cells were resuspended in 800μ1 UHP and ΙΟΟμΙ of this were placed in to 8 glass tubes. These were die adriamycin control tubes and were labelled as follows: 50pg/ml, ^g/ml, 5pg/ml, 2μg/ml, Ιμβ/πύ, 0^g/ml, 0.25μg/ml, 0μg/ml adriamycin.

A volume of ΙΟΟμΙ 33% aqueous silver nitrate (Sigma, S6506) was then added to the pellets (all tubes), followed by mixing for 5 minutes. A quantity of 300μ1 of the internal standard (daunorubicin, 6ug/ml in 50mls methanol) was then added to all tubes. 1.3ml HPLC grade acetonitrile (Labscan) was added to the test sample tubes only. 300μ1 HPLC grade acetonitrile was added to the adriamycin control tubes, lml of appropriate concentration of adriamycin was then added to the adriamycin control tubes. All tubes were maintained at 4°C for 1 hour. This was followed by centrifugation at 4000rpm for 15 minutes. 1.1ml of the supernatant was removed and added to HPLC autosampler vials. All solvent was then removed under a stream of nitrogen gas. The remaining solids were resuspended in 50μΤ of HPLC mobile phase.

The HPLC mobile phase was prepared as follows: 64ml of O.lM phosphoric acid (Sigma, P6560) was added to 488 UHP water. The pH was then adjusted to 2.3 with IN potassium hydroxide (Sigma P6310). A volume of 248 ml acetonitrile was added finally and the completed mobile phase allowed to degas at 4°C overnight.

DUBLOl/C .67. IEO1Q7 84 The samples for analysis were automatically injected in to the HPLC system (Beckman System Gold 507 autosampler, 125 pump and 166 detector). Mobile phase flow rate was set at 0.5ml per minute with a total run time of 16 minutes. The column used for HPLC analysis of adriamycin in DLKP was a Cw reversed phase Prodigy 5μιη particle size ODS-3 column (Phenomenex, U.K.). Absorbance was monitored at 253nm.

A standard curve of adriamycin peak area /daunorubicin internal standard versus adriamycin concentration was used to quantify the levels of adriamycin present in the samples. Results were finally reported as the content of adriamycin per million cells.

Results Table 3.1 Time Treatment Adriamycin content ug per million cells S.D. TO Adriamycin alone 1.41 0.03 T5 Adriamycin alone 0.57 0.05 TO Adriamycin + Indomethadn 1.31 0.09 T5 (+) Indomethacin 1.04 0.10 T5 (-) Indomethacin 0.70 0.10 TO Adriamycin + Cpd. 18 1.30 0.07 T5 (+) Cpd. 18 1.08 0.04 T5 (-)Cpd. 18 0.60 0.13 TO Adriamycin + Cpd. 28 1.19 0.10 T5 (+) Cpd. 28 0.53 0.05 T5 (-) Cpd. 28 0.40 0.05 DUBL01/C Adriamycin levels in DLKP cells treated with adriamycin alone versus DLKP cells treated with adriamycin and indomethacin / indomethacin analogues combined. Data shown are the average of three separate determinations.

TO: Time point immediately after initial 2 hour loading period T5: Time point 5 hours after initial 2 hour loading period (+) Indomethacin/analogue: Flasks re-fed with either indomethacin or test compound (28 um) after initial 2 hour loading period.

(-) Indomethacin/test compound: Flasks re-fed with media only after initial 2 hour loading period.

These results show that after 5 hours the adriamycin content of the cells reduces from 1.42 pg/million cells to 0.57 pg /million cells due to the effects of MRP-1 actively pumping the anti-cancer drug out of the cells. Where the MRP inhibitor, indomethacin, is maintained for the duration of the experiment, the adriamycin content only falls to 1.04 pg /million cells. Removal of indomethacin after the 2 hour loading period leads to greater efflux of the adriamycin leading to a cellular adriamycin content of 0.70 pg.'million cells. Similar results are seen with the test inhibitor Compound 18. Using this agent the adriamycin content of the cells drops to 1.08 pg /million cells indicating that this drug has potent activity as an MRP-1 inhibitor. Removal of Compound 18 after the 2 hour period leads to a cellular adriamycin content of 0.60 pg/million cells. The inactive compound, 28, does not maintain the cellular content of adriamycin and levels drop to from 1.19 pg/million cells to 0.53 pg/million cells in the presence of this agent.

DUBL01/C *69' τε* gi R 7 8 4 Example D Indomethacin has inherent toxicity to cells and this may limit any future application to the field of MRP-resistance circumvention. Experiments were undertaken to examine the toxicity of these agents by measuring the highest concentration of agent which did not produce a toxic effect in the cells (highest non-toxic concentration).

The test compounds were prepared as described previously.

Method Cells in the exponential phase of growth were harvested by trypsinisation. Cell suspensions containing 1x104 cells/ml were prepared in cell culture medium. Volumes of ΙΟΟμΙ of these cell suspensions were added in to 96 well plates (Costar, 3599) using a multichannel pipette. Plates were agitated gently in order to ensure even dispersion of cells over a given well. Cells were then incubated overnight at 37°C in an atmosphere containing 5% CO2.

Test compound dilutions were prepared at 2X their final concentration in cell culture medium. Volumes of the drug dilutions (IOOpL) were then added to each well using a multichannel pipette. Plates were then mixed gently as above. The cells were incubated for a further 6 days at 37°C and 5% CO2. At this point the control wells would have reached approximately 80-90% confluency.

Assessment of cell survival in the presence of drug was determined by acid phosphatase assay (see example 1). The concentration of drug which caused 50% cell kill (IC50 of the drug) was determined from a plot of the % survival (relative to the control cells) versus cytotoxic drug concentration.

DUBL01 ZC Results -70,E n 1 0 7 8 * Table 4.1 The table below charts the highest non-toxic concentrations for all of the 5 compounds tested compared with indomethacin.

Compound Highest non-toxic concentration pg/ml Molar cone. (mM) Indomethacin 2.5 0.007 Cpd. 4 5.0 0.015 Cpd. 16 10.0 0.029 Cpd. 17 5.0 0.015 Cpd. 7 10.0 0.026 Cpd. 14 10.0 0.031 Cpd. 13 10.0 0.032 Cpd. 20 5.0 0.016 Cpd. 19 5.0 0.015 Cpd. 5 5.0 0.013 Cpd. 27 50.0 0.130 CpcL26 20.0 0.054 Cpd. 28 25.0 0.062 Cpd. 29 50.0 0.119 Cpd. 18 15.0 0.046 The non-toxic concentration of each compound was determined using data from three separate experiments.

The results indicate that both the active and in-active test compounds are all less toxic than indomethacin. This suggests that any of these agents would be safer to administer than indomethacin.

DUBLOI/C -71Exatnple E Cyclooxygenase-1 (COX-1) inhibition. COX-1 is a constitutive enzyme and is thought to be associated with the important positive aspects of prostaglandins (cytoprotection and maintenance of renal blood flow). Indomethacin is a potent inhibitor of COX-1 and as a result has a number of side effects associated with its use. Less inhibition of COX-1 is likely to be associated with fewer of these side effects.

The test compounds were prepared as described previously.

Method The COX -1 assay was based on that used by Boopathy et al.57 and Piazza et al.5*. 100ml of lOOmM TRIS HCl (Sigma T-1378) (pH 7.4 - 8) was prepared (1.211gin lOOmlsUHP).

The following compounds were weighed out and added to the lOOmM TRIS HCL 0.05mM Glutathione 15.37mgs 0.625μΜ Haemoglobin 4.03mgs 0.5mM Hydroquinone (Sigma H-9003) 5.5lmgs 1.25mM CaCl2 18.3mgs The reaction mixture for the COX-1 assay was prepared as follows: 250units of Prostaglandin H syntheses 1 (COX-1) (Cayman Chemicals) lOOpm Arachidonic acid (Cayman Chemicals, 90010) TRIS-HC1 + components (as prepared above) - added to make a final volume of lml in the reaction mixture.

DUBLOI/C OE R 1 Ο 1 8 ίί -72- — 4' · =- · ^ (Note : 250 units of COX-1 enzyme was used in the assay as gave the optimum level of activity when incubated with arachidonic acid.) The reaction mixture was incubated at 37°C for 20 minutes and then terminated by the addition of 0.2ml of 100% (w/v) trichloroacetic acid in lm- HC1 (91.7ml H2O + 8.3ml Cone HC1).

After thorough mixing, 0.2ml of 1% (w/v) thiobarbituric acid solution (in IM NaOH) was added and the mixture was heated in a boiling water bath for 20 min. After cooling to room temperature and brief centrifugation, the enzyme activity was measured by the thiobarbituric acid colour reaction of malonaldehyde formed in the reaction and determined by a spectrophotometer at 530nm.

To assess the ability of the test compounds to inhibit COX-1, 12μβ/πύ of the compounds were added to the reaction mixture, the volume brought up to 1 ml with TRIS HCL + components and the COX-1 assay carried out as above.

The enzymatic activity of COX-1 was assessed by its ability to act on the substrate arachidonic acid and form malonaldehyde. The greater the optical density reading the greater the activity of COX-1 in the reaction mixture. Indomethacin was used as the positive control. The test compounds were added to the reaction mixture at the same concentration as indomethacin and their abilities to inhibit COX-1 were compared.

The controls used in the experiment were: Arachidonic negative control.

Indomethacin negative control DMSO control.

DUBL01/C -73- ΙΕΟΙΟΙδί In the control reaction mixture arachidonic acid or enzyme was added to the reaction mixture after the addition the trichloroacetic acid.

Results The table below outlines the level of COX-1 inhibition associated with the test compounds as compared to the inhibition seen with inhibition. All agents are used at the same concentration of 12 ug/ml.

Table 5.1 Compound (12μβ/πύ) Molar cone. of compound in assay Average % Inhibition of COX-1 % S.D. Indomethacin 0.033 60.9 12.2 Cpd. 4 0.035 16.3 12.4 Cpd. 7 0.030 24.5 5.8 Cpd. 14 0.037 26.6 9.9 Cpd. 27 .031 0.1 7.4 Cpd. 18 €.057 0.8 3.6 Cpd. 26 0.032 22.1 8.2 Cpd. 29 0.030 1.7 8.1 Cpd. 28 0.037 -7.4 2.8 Cpd. 13 0.038 -1.0 7.4 No compound (Control) N/A 0.0 0.0 With DMSO (no compound) N/A -1.3 9.0 The results are the average of a minimum of three assay repeats. Data is expressed as % inhibition relative to an untreated control. Negative values indicate an increased activity of the enzyme in the presence of the compound.

DUBL01/C -74These results indicate that all of the compounds tested (both active and inactive) have less COX-1 inhibitor activity and are therefore likely to have less side effects in the clinical setting.

Example F Cyclooxygenase-2 (COX-2) is associated with the negative aspects of inflammatory conditions. Specific inhibition of this enzyme is thought to be therapeutically useful in the treatment of chronic inflammatory conditions and may also have a beneficial role to play in the chemopreventation of certain form of colon cancer. Inhibition of COX-2, therefore, would be expected to provide additional useful effects in treatment using any of the positive compounds outlined.

The test compounds were prepared as described previously.

Methods COX-2-inhibitory activity was measured using an indirect method. It was previously shown by Asano ef al.fr, that COX-2 is the constitutive and dominant isoform in un-stimulated and stimulated cultured human lung epithelial cells. A lung adenocarcinoma cell line, A549 was used as a source of COX-2. This cell line was purchased from the American Type Culture Collection, 10801 University Boulevard Manassas, VA 20110-2209, USA. Stimulation of this cell line with interleukin 1β (IL-Ιβ) induces production of prostaglandin E2 (PGE2) from arachidonic acid by COX-2. The amount of PGE2 produced is quantified by ELISA. Inhibitors of COX-2 reduce the production of PGE2.

DUBLOl/C Cells were seeded at high density (2.5 x 10 5 cells per well) in 6 well plates (Falcon, 3046).

Plates were then incubated overnight in serum containing media. The media 5 was then removed and cells washed twice with DME. The compounds of interest (such as indomethacin and IL-Ιβ) were then added to the cells at a concentration appropriate for the cell density present. Control wells were treated with media only.

After 24 hours the media was removed from the wells, placed into appropriately labelled eppendorf tubes and stored at -80°C.

Samples were analysed using a PgE2 enzyme immunoassay kit (Cayman Chemical, 514010). Concentrations of PgE2 present in the samples were determined from a standard curve of absorbance at 405 nm versus PgE2 concentration.

DUBL01/C -76Table6.1 Treatment % Inhibition of production of PgE2 by COX- 2 S.D. Cell control 0.0 0.0 IL-1B (lOmg/ml) 0.0 0.0 Indomethacin (lOnM) + IL-IB 86.1 5.9 Cpd. 4 (lOnM) + IL-1B 78.1 11.1 Cpd. 7 (lOnM) + IL-1B 44.1 12.2 Cpd. 14 (lOnM) + IL-1B 40.1 18.5 Cpd. 27 (lOnM) + IL-1B 14.7 15.0 Cpd. 18 (lOnM) + IL-1B 13.6 10.6 Cpd. 26 (lOnM) + IL-1B 24.2 8.1 Cpd. 29 (lOnM) + IL-1B 0.2 0.3 Cpd. 28 (lOnM) + IL-1B 48.0 0.8 Results are represented as means i S.D. for duphcare determinations carried out 5 on three separate occasions. Inhibition is expressed £s a percentage of untreated control (IL-Ιβ (10mg/mI)), taken as 100%.

These results indicate that indomethacin is also a potent inhibitor of COX-2 activity. Activity varies among the all of compounds tested (both positive and negative) with Compound 4 having COX-2-inhibitory activity near to that of indomethacin.

The invention is not limited to the embodiments hereinbefore described which may be varied in detail.

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DUBLOI/C

Claims

1. -(4-CfrloTobenzyi)-2-methoxyindole-3-acetic acid. 31. A compound selected from

1. Use of a compound of the formula I

2. Use of a compound as claimed in claim 1 wherein the C r C 6 alkyl are substituted with one or more of the same or different of: hydroxy, carboxy, phosphonic groups. 3. -(cyanomethyl)-2-ethyl-1 -(phenylmethyl)-5-methoxy indole. 32. A pharmaceutical composition comprising a compound as claimed in 3-(cyanomethyl)-2-methyl-l-(phenylmethyl)-5~methoxy indole; and DUBLOI/C 3-(hydroxymethyl)-2-ethyl-l -(phenylmethyl)-5-methoxy indole; 3-(hydroxymethyl)-2-methyl-l-(phenylmethyl)-5-methoxy indole; 3-(carbomethoxy)-2-ethyl-l-(phenylmethyl)-5-methoxy indole;

3. Use of a compound as claimed in claim 1 or 2 which has a carboxylic acid or a phosphonic acid functional group.

4. Use of a compound as claimed in claim 1 wherein the pharmacologically 10 acceptable salt is an alkali metal salt selected from the group comprising sodium, potassium and lithium or an alkali earth metal salt selected from the group comprising magnesium and calcium. 5. Claim 27 to 31. 33. A pharmaceutical composition comprising a compound as claimed in claim 27 to 32 and an MRP substrate drug. 5 R= C r C 6 alkyl which may be substituted or unsubstituted Y = OH or NH 2 wherein X is H, Cl, Br, F with the provisos fflar fi) when X in the 3 position then X =F (h) when X is in the 4 position then X = Cl, Br, or F, or pharmacologically acceptable salts thereof, and a identifiable marker. 26. A compound as claimed in claim 25 wherein the identifiable marker is a radiolabelled marker. 27. A compound of the formula DUBLOI/C -88ΊΠΞ’ ΐ Ί £ wherein X is 3-F. 28. A compound of the formula wherein X is F, Cl, Me or MeS. DUBL01/C ΙΕ0 1 0 7 8 4 29. A compound selected from l-(4-fluorobenzyl)-5-methoxy-2-methylindole-3-acetic acid; l-(3-fluorobenzyl)-5-methoxy-2-methylindole-3-acetic acid; and l-(4-chlorobenzyl)-5-methoxy-2-methylindole-3-acetic acid; 30. A compound selected from l-(4-methylthiobenzyl)-5-methoxy-2-methylindole-3-acetic acid; l-(4-methylbenzyl)-5-methoxy-2-methylindole-3-acetic acid; l-(3-chIorobenzyl)-5-methoxy-2-methylmdole-3-acetic acid; l-(2-chlorobenzyl)-5-methoxy-2-methyiindole-3-acetic acid; l-(4-Chlorobenzyl)-5-methoxyindole-3-acetic acid; and

5. Use of a compound of the formula 15 Π wherein X is F, Cl or Br and pharmacologically acceptable salts thereof, in the preparation of a medicament for the prophylaxis and/or treatment of drug resistance cancer. DUBL01/C Hfc· ή·· τ 7 @ & -84- ? '· - · 5 wherein R 1 = Me, Et R= Ci-C 6 alkyl which may be substituted or unsubstituted Y = OHorNH 2 10 wherein X is H, CL, Br, F wah the provisos that (i) when X in the 3 position then X =F (ii) when X is in the 4 position then X = Cl, Br, or F, and pharmacologically acceptable salts thereof, in the preparation of a medicament for the prophylaxis and/or treatment of drug resistance cancer. DUBLOI/C 83 8E 0 ii y. ί 4

6. Use of a compound of formula wherein X is Me or Et and pharmacologically acceptable salts thereof, in the preparation of a medicament for the prophylaxis and/or treatment of drug resistance cancer.

7. Use of a compound as claimed in any of claims 1 to 6 for the prophylaxis and/or treatment of multiple drug resistance related disease.

8. Use of a compound as daimed m any of claims 1 to 6 for the prophylaxis and/or rreannent of kshmamaas

9. Use of a compound as claimed in any of claims 1 to 6 with an identifiable marker. 10. Use as claimed in claim 9 wherein the identifiable marker is a radiolabelled marker. 11. Use of compounds as claimed in any of claims 1 to 6 for identifying tumours which are mutidrug resistant. DUBL01/C -85- IE01Q784 12. A pharmaceutical composition comprising a compound of the formula II and III or pharmacologically acceptable salts thereof and a multidrug resistance related protein (MRP) substrate drug. 5 13. A pharmaceutical composition comprising a compound of the formula I or pharmacologically acceptable salts thereof and a multidrug resistance related protein (MRP) substrate drug. 14. A pharmaceutical composition as claimed in claim 13 wherein in the 10 compound of formula I the C r C 6 alkyl are substituted with one or more of the same or different of: hydroxy, carboxy, phosphonic groups. 15 15. A pharmaceutical composition as claimed in claim 13 or 14 wherein the compound of formula I has a carboxylic acid or a phosphonic acid functional group. 16. A pharmaceutical composmcn as in any of claims 12 to 15 wherein the compound comprises a pharmacologically acceptable salt preferably an aTkafi metal salt selected from the group comprising sodium, potassium and lithium or an alkali earth metal salt selected from the group comprising magnesium and calcium. 25 17. A pharmaceutical composition as claimed in any of claims 12 to 16 wherein the MRP substrate drug is a MRP-1 substrate drug. 18. A pharmaceutical composition as claimed in 12 to 17 wherein the substrate drug is an anti-cancer drug selected from the group comprising an anthracycline, a vinca alkaloid, an epipodophyllotoxin or a taxane. DUBL01/C 19. A pharmaceutical composition as claimed in any of claims 12 to 18 wherein the anti-cancer drug is selected from any one or more of anthracycline adriamycin, daunorubicin, epirubicin, vinca alkaloid vincristine, epipodophyllotoxin etoposide (VP-16), teniposide or taxol. 20. A pharmaceutical composition as claimed in any of claims 12 to 19 in the form of an emulsion, liposome, patch, powder and/or complex. 21. A pharmaceutical composition as claimed in claim 20 wherein the 10 composition comprises an ingestable carrier in the form of a tablet, capsule, syrup or powder. 22. A pharmaceutical composition as claimed in any of claims 12 to 21 in combination with an agent which is a substrate for multidrug resistance 15 related protein for simultaneous, sequential or separate use. 23. A pharmaceutical composition as claimed in any of claims 12 to 22 which comprises an adjuvant such as an anti-emetic, an antiinfiammarnry agent or a cancer chemotherapy medicament, 'in 24. A pharmaceuiical composition as claimed in any of claims 12 to 23 in a form for oral, intravenous, intramuscular, intraperitoneal, intradermal, intravesicular and/or rectal administration. DUBL01/C -8725. A compound of the formula RO I wherein R 1 = Me, Et

10. 34. Use of a compound as claimed in any of claims 27 to 33 in the preparation of a medicament for the prophylaxis and/or treatment of drug resistance cancer, multiple drug resistance related disease or leishmaniasis.