AU779980B2

AU779980B2 - New stilbenes with vascular damaging activity

Info

Publication number: AU779980B2
Application number: AU64581/00A
Authority: AU
Inventors: Peter David Davis
Original assignee: Angiogene Pharmaceuticals Ltd
Current assignee: Angiogene Pharmaceuticals Ltd
Priority date: 1999-02-16
Filing date: 2000-08-09
Publication date: 2005-02-24
Anticipated expiration: 2020-02-15
Also published as: AU6458100A

Description

WO 01/12579 PCT/GB00/03067 NEW STILBENES WITH VASCULAR DAMAGING ACTIVITY Formation of new vasculature by angiogenesis is a key pathological feature of several diseases (J Folkman, New England Journal of Medicine 333, 1757-1763 (1995)). For example, for a solid tumour to grow it must develop its own blood supply upon which it depends critically for the provision of oxygen and nutrients; if this blood supply is mechanically shut off the tumour undergoes necrotic death. Neovascularisation is also a clinical feature of skin lesions in psoriasis, of the invasive pannus in the joints of rheumatoid arthritis patients and of atherosclerotic plaques. Retinal neovascularisation is pathological in macular degeneration and in diabetic retinopathy. In all these diseases reversal of neovascularisation by damaging the newly-formed vascular endothelium is expected to have a beneficial therapeutic effect.

Compounds able to damage neovasculature have advantages in the treatment of disease. For example, attacking tumour vasculature has several important advantages over a direct attack on the tumour. In particular the endothelial cells of tumour vasculature are more genetically stable than those of the tumour itself and are therefore less likely to become resistant to the damaging agent. Thus a major problem in conventional anti-tumour chemotherapy, that of drug resistance, is circumvented by this approach. Furthermore, since the endothelial cells of the tumour vasculature, unlike the tumour cells themselves, are similar between different solid tumour types, vascular damaging agents are able to attack a wide range of tumour types.

A number of tubulin-binding agents including the stilbenes combretastatin Al, combretastatin A4 J. Chaplin et al.,British J. Cancer 27, S86-S88 (1996))and combretastatin A4 phosphate Chaplin et al., Anticancer Research 19, 189-196, (1999)) are known to selectively damage neovasculature of solid tumours in animal models. While there are reports of the activity of other analogues of combretastatin A4 in tubulin binding assays, in cytotoxicity assays and in tumour models there have been no reports of the vascular damaging activities of analogues. Since the activity of WO 01/12579 PCT/GB00/03067 2 tubulin-binding compounds against in vitro assays are poor predictors of selective vascular damaging activity and activity of such compounds in vivo can also be mediated by direct antimitotic effects on the tumour itself, no prediction can be made of the selective vascular damaging activity of known or novel analogues of the combretastatins from published reports. Thus compounds which have the advantages of a selective anti-vascular mechanism given above, rather than acting through a direct effect on the tumour tissue itself, are not apparent.

We have found a series of novel cis-stilbenes with vascular damaging activity. These compounds specifically damage newly-formed vascular endothelium, especially that associated with solid tumours, without affecting the normal, established vascular endothelium of the host species. Such compounds are of use in the prophylaxis and treatment of cancers involving solid tumours and in other diseases where there is inappropriate formation of new vasculature such as diabetic retinopathy, psoriasis, rheumatoid arthritis, macular degeneration and the formation of atherosclerotic plaques.

Known vascular-damaging stilbenes, combretastatin Al, combretastatin A4 and combretastatin A4 phosphate have a 4-methoxy group in the ring. The compounds of the invention lack a 4-methoxy group in the ring corresponding to the ring of combretastatin A4. Several studies suggest that substituting alternative groups for the 4-methoxy group in the B-ring of combretastatin A4 would considerably reduce biological activity: In J. Med. Chem 1991, 34, 2579-2588, Cushman et al. state, regarding analogues of combretastatin A4: "the presence of a 4-methoxy group in the B-ring plays a very important role for this compound to be highly cytotoxic". Replacement of the 4methoxy group with chlorine, for example, gave compounds that were three to four orders of magnitude less potent against a panel of five different cell lines.

WO 01/12579 PCTIGB00/03067 3 In J. Med. Chem. 1998, 41, 3022-3032 Ohsumi et al. disclose anilino analogues of combretastatin A4 in which the replacement of the B-ring 4-methoxy group by either a methyl group or a chlorine atom gave a reduction in biological potency of 8.5-fold and 13.5-fold respectively.

Similarly in Brit. J. Cancer 1995, 71, 705-711 Woods el al. disclose analogues of combretastatin with reduced potency. For example the 4-methyl compound shows to 36-fold reduction in potency against four cell lines compared to the 4-methoxy compound.

It cannot be anticipated from the above studies that compounds in which the B-ring 4methoxy group is replaced would retain anti-vascular activity. It is particularly unexpected that replacing the B-ring methoxy group of combretastatin A4 would result in a compound with similar potency as a vascular damaging agent.

Thus according to one aspect of the invention we provide a compound of formula RRO OH

R

1 0 OH Wherein:

R',R

2 and R 3 are each independently alkyl, R' is alkyl, haloalkyl, alkenyl, alkynyl, alkylthio, alkylsulphinyl, alkylsulphonyl or halo, R' is hydrogen, alkoxy, alkyl, alkylthio, hydroxy or halo, and the pharmaceutically acceptable salts, solvates, hydrates and prodrugs thereof.

According to a second aspect of the invention, there is provided (Z)-l-(3-hydroxyl- 4-methylphenyl)-2-(3,4,5-trimethoxyphenyl) ethene.

According to a third aspect of the invention, there is provided a cis-stilbene of formula: R

R

1 0 OX R20 OR 3

R

4 wherein:

R

2 and R 3 are each independently alkyl,

R

4 is alkyl, haloalkyl, alkenyl, alkynyl, alkylthio, alkylsulphinyl, alkylsulphonyl or halo,

R

5 is hydrogen, alkoxy, alkyl, alkylthio, hydroxy or halo; and X is a group which can be removed in vivo by hydrolysis.

According to a fourth aspect of the invention, there is provided (Z)-2-methyl-5-[2- (3,4,5-trimethoxyphenyl)ethenyl]phenyl dihydrogen phosphate.

According to a fifth aspect of the invention, there is provided a composition for use S 15 in the treatment of neovascularisation which composition contains an effective amount of Sthe cis-stilbene dihydrogen phosphate according to the fourth aspect of the invention and a pharmaceutically acceptable excipient.

S

According to a sixth aspect of the invention, there is provided a composition for use in the treatment of neovascularisation which composition contains an effective amount of the cis-stilbene according to the first or second aspect of the invention or a cis-stilbene prodrug according to the third or fourth aspect of the invention and a pharmaceutically acceptable excipient.

According to a seventh aspect of the invention, there is provided use of a cisstilbene according to the first or second aspect of the invention or a cis-stilbene prodrug 25 according to the third or fourth aspect of the invention in the manufacture of a

S

medicament for the treatment of a disease involving neovascularisation.

According to a seventh aspect of the invention, there is provided A method for treating neovascularisation, wherein said method comprises administering a therapeutically effective amount of the cis-stilbene according to the first or second aspect of the invention or a cis-stilbene prodrug according to the third or fourth aspect of the invention.

According to an eighth aspect of the invention, there is provided a method for treating a disease involving neovascularisation, wherein said method comprises (R:\LIBFF]9971 administering a therapeutically effective amount of the cis-stilbene according to the first or second aspect of the invention or a cis-stilbene prodrug according to the third or fourth aspect of the invention.

According to a ninth aspect of the invention, there is provided use of a cis-stilbene s according to the first or second aspect of the invention or a cis-stilbene prodrug according to the third or fourth aspect of the invention for the manufacture of a medicament for treating neovascularisation According to a tenth aspect of the invention, there is provided use of a cis-stilbene according to the first or second aspect of the invention or a cis-stilbene prodrug according to the third or fourth aspect of the invention for the manufacture of a medicament for treating a disease involving neovascularisation.

As used herein the term "alkyl", alone or in combinations, means a straight or branched-chain alkyl group containing from one to seven, preferably a maximum of four, carbon atoms such as methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, t-butyl and pentyl.

Examples of alkoxy groups are methoxy, ethoxy, propoxy, isopropoxy, butoxy and tbutoxy.

The term "halogen" means fluorine, chlorine, bromine or iodine.

An alkenyl group may be for example an olefinic group containing from two to seven carbon atoms for examples methylene, ethylene, n-propylene, i-propylene, nbutylene, i-butylene, s-butylene and t-butylene. An alkynyl group may be for example an ethynyl, propynyl or butynyl group.

Where one or more functional groups in compounds of formula are sufficiently basic or acidic the formation of salts is possible. Suitable salts include pharmaceutically acceptable salts for example acid addition salts including hydrochlorides, hydrobromides, 25 phosphates, sulphates, hydrogen sulphates, alkylsulphonates, arylsulphonates, acetates, benzoates, citrates, maleates, fumarates, succinates, lactates and tartrates, salts derived from inorganic bases including alkali metal salts such as sodium or potassium salts, alkaline earth metal salts such as magnesium or calcium salts, and salts derived from organic amines such as morpholine, piperidine or dimethylamine salts.

Prodrugs of the invention are compounds which upon administration to a mammal produce compounds of formula Such prodrugs can be for example converted within the mammal by hydrolysis. Prodrugs are preferably ester derivates of the phenolic hydroxy group contained in compounds of formula such as, for example, phosphate esters, carboxylate esters, sulphate esters and carbonates.

[R:\LIBFF]997 4b Preferred compounds of the invention are those of formula I in which R 2 and R 3 are all methyl, and prodrugs thereof.

R:LIB FF]99715 .doc:ANB WO 01/12579 PCTIGB00/03067 Further preferred compounds of the invention are those of formula 1 in which R 2 and R 3 are all methyl and R' is hydrogen and prodrugs thereof Still further preferred compounds of the invention are those of formula 1 in which R',

R

2 and R 3 are all methyl, R 5 is hydrogen and R 4 is alkyl or halo and prodrugs thereof Preferred prodrugs of the invention are phosphate esters. Particularly preferred prodrugs of the invention are dihydrogen phosphate esters.

Specifically preferred compounds of the invention are: (Z)-1-(3-hydroxy-4-methylphenyl)-2-(3,4,5-trimethoxyphenyl)ethene (Z)-2-methyl-S-[2-(3,4.5-trimethoxyphenyl)ethenyl]phenyl dihydrogen phosphate Compounds of the invention can be prepared by any process known to a person skilled in the art. Compounds of formulae can be prepared by a number of processes as generally described hereinbelow and more specifically in the Examples hereinafter. In the general preparations described below it may be necessary to employ protecting groups which are then removed during the final stages of the synthesis. The appropriate use of such protecting groups and processes for their removal will be readily apparent to those skilled in the art. In the following process description, the symbols R 2

R

4 and R

S

when used in the formulae depicted are to be understood to represent those groups described above in relation to formula unless otherwise indicated In one general example compounds of formula can be prepared by Wittig olefin synthesis involving reaction of a phosphonium salt of formula with a strong base, for example an alkyllithium such as n-butyllithium or t-butyllithium or a metal hydride such as sodium hydride in a solvent such as an ether solvent for example diethyl ether WO 01/12579 PCT/GB00/03067 6 or tetrahydrofuran or in a solvent such as a hydrocarbon solvent for example toluene at a temperature of between about -100 0 C to about 30 0 C followed by treatment with an aldehyde of formula in which R 6 is a protecting group, to give an intermediate of formula The synthesis of compounds of formula is then completed by removal of the group R 6 Suitable protecting groups R 6 include trialkylsilyl, for example tbutyldimethylsilyl, and allyl. Where R 6 is a trialkylsilyl group it may be removed, for example, by treatment with a quaternary ammonium fluoride such as tetrabutylammonium fluoride in an ether solvent such as tetrahydrofuran at a temperature of about -30 0 C to about 40 0 C conveniently at or near ambient temperature. Where R 6 is an allyl group it may be removed for example by treatment with a palladium(0) complex such as tetrakis(triphenylphosphine)Pd(0) in a solvent such as a chlorinated solvent, for example dichloromethane, at a temperature of about 0 C to about 40°C conveniently at or near ambient temperature, in the presence of an allyl scavenger such as morpholine.

Br Ph. 3

HO

R

s

R

S

R

RO OR 6

R

O

O OR s RO /OH R3O OR 3 RO OR' R* RO OR 3 (1) Aldehydes of formula can be prepared by any process known to a person skilled in the art. In one general example an aldehyde of formula can be prepared from an alcohol of formula by oxidation with a suitable oxidising agent. Suitable oxidising agents include the Dess-Martin reagent and manganese dioxide. Alcohols of formula can be prepared by application of standard methods of organic synthesis including the selective protection of phenols of formula Where the protecting group R 6 is a trialkylsilyl group, for example t-butyldimethylsilyl, alcohols of formula may be prepared, for example, by treatment of a phenol of formula with a strong base, for example an alkyllithium such as n-butyllithium or t-butyllithium or a metal hydride such as sodium hydride in a solvent such as an ether solvent for example diethyl ether or tetrahydrofuran or in a solvent such as a hydrocarbon solvent for example toluene at a WO 01/12579 PCT/GB00/03067 7 temperature of between about -100 0 C to about 40 0 C followed by treatment with tertbutylchlorodimethylsilane.

Phenols of formula are either known or may be prepared from known compounds using standard methods of organic synthesis.

HOH H 2 OH HO s O.Rs

R!

/OH R ORS (3) Compounds of formula may also be prepared from other compounds of formula by chemical modification. Examples of such chemical modifications that may be applied are standard alkylation, halogenation, oxidation and coupling reactions. These reactions may be used to add new substituents or to modify existing substituents.

Prodrugs of compounds of formula can be prepared by any process known to a person skilled in the art. Processes for the preparation of prodrugs of compounds of formula 1 include standard acylation, sulphation and phosphorylation reactions. In one general example dihydrogen phosphate esters of compounds of formula can be prepared by treatment of the corresponding di-t-butylphosphate esters with an acid, for example hydrochloric acid or trifluoroacetic acid, in a solvent such as a chlorinated solvent, for example dichloromethane, at a temperature of from about -20 0 C to about 0 C, conveniently at room temperature.

Compounds according to the invention are able to destroy tumour vasculature and vasculature that has been newly formed while leaving unaffected normal, mature vasculature. The ability of the compounds to act in this way may be determined by the tests described hereinafter.

WO 01/12579 PCT/GB00/03067 8 The compounds according to the invention are thus of particular use in the prophylaxis and treatment of cancers involving solid tumours and in the prophylaxis and treatment of diseases where inappropriate angiogenesis occurs such as diabetic retinopathy, psoriasis, rheumatoid arthritis, atherosclerosis and macular degeneration.

The compounds of the invention may be administered as a sole therapy or in combination with other treatments. Thus the invention includes compositions for the treatment of neovascularisation which compositions contain an effective amount of a cis-stilbene or prodrugs thereof as hereinbefore defined. The invention also includes the use in the preparation of a composition for the treatment of neovascularisation of a cis-stilbene or prodrugs therofas hereinbefore defined. For the treatment of solid tumours compounds of the invention may be administered in combination with radiotherapy or in combination with other anti-tumour substances for example those selected from mitotic inhibitors, for example vinblastine, vincristine, vinorelbine, paclitaxel and docetaxel; platinum derivatives for example cisplatin and carboplatin; alkylating agents, for example melphalan, chlorambucil, busulphan, ifosfamide and cyclophosphamide; antimetabolites, for example methotrexate, 5-fluorouracil, cytosine arabinoside, gemcitabine and hydroxyurea; antitumour antibiotics for example bleomycin, doxorubicin, daunomycin, epirubicin, idarubicin, mitomycin-C, dactinomycin and mithramycin enzymes, for example aspariginase; topoisomerase inhibitors for example etoposide,teniposide, topotecan and irinotecan; thymidylate synthase inhibitors for example raltitrexed; biological response modifiers for example interferon; antibodies for example edrecolomab and trastuzumab; anti-hormones for example tamoxifen, toremifene, raloxifene, droloxifene, iodoxyfene, anastrozole, letrazole, vorazole ,exemestane, flutamide, nilutamide and bicalutamide; anti-growth factor compounds for example EGFr tyrosine kinase inhibitors VEGFr kinase inhibitors and PDGFr tyrosine kinase inhibitors; and anti-angiogenesis agents such as angiostatin, endostatin and thalidomide. Such combination treatment may involve simultaneous or sequential application of the individual components of the treatment.

WO 01/12579 PCT/GB00/03067 9 For the prophylaxis and treatment of disease the compounds according to the invention may be administered as pharmaceutical compositions selected with regard to the intended route of administration and standard pharmaceutical practice. Such pharmaceutical compositions may take a form suitable for oral, buccal, nasal, topical, rectal or parenteral administration and may be prepared in a conventional manner using conventional excipients. For example for oral administration the pharmaceutical compositions may take the form of tablets or capsules. For nasal administration or administration by inhalation the compounds may be conveniently delivered as a powder or in solution. Topical administration may be as an ointment or cream and rectal administration may be as a suppository. For parenteral injection (including intravenous, subcutaneous, intramuscular, intravascular or infusion) the composition may take the form of, for example, a sterile solution, suspension or emulsion.

The dose of a compound of the invention required for the prophylaxis or treatment of a particular condition will vary depending on the compound chosen, the route of administration, the form and severity of the condition and whether the compound is to be administered alone or in combination with another drug. Thus the precise dose will be determined by the administering physician but in general daily dosages may be in the range 0.001 to 100mg/kg preferably 0.1 to BIOLOGICAL ACTIVITY The following test was used to demonstrate the activity of compounds according to the invention.

Activity against tumour vasculature measured by fluorescent dye.

The following experiment further demonstrates the ability of the compounds to damage tumour vasculature.

Tumour functional vascular volume in CaNT tumour-bearing mice was measured using the fluorescent dye Hoechst 33342 according to the method of Smith et al (Brit J Cancer 57, 247-253, 1988). At least three animals were used in control and treated WO 01/12579 PCT/GB00/03067 groups. The fluorescent dye was dissolved in saline at 6.25 mg/ml and injected intravenously at 10 mg/kg 24 hours after intraperitoneal drug treatment. One minute later, animals were killed and tumours excised and frozen; 10 um sections were cut at 3 different levels and observed under UV illumination using an Olympus microscope equipped with epifluorescence. Blood vessels were identified by their fluorescent outlines and vascular volume was quantified using a point scoring system based on that described by Chalkley, (J Natl Cancer Inst, 4, 47-53, 1943). All estimates were based on counting a minimum of 100 fields from sections cut at the 3 different levels.

Examples of the activity of compounds of the invention in this test are given in the table: Compound of Example Dose (mg/kg) Reduction in Functional Vascular Volume 1 50 88 3 50 27 50 The following non-limiting Examples illustrate the invention: EXAMPLE I 1-(3-hvdroxv-4-methylphenvl)-2-(3.4. A solution of 1-(3-tert-butyldimethylsilyloxy-4-methylphenyl)-2-(3,4,5trimethoxyphenyl)ethene (491mg) in anhydrous tetrahydrofuran (10ml) at room temperature was treated slowly with a 1. 1M solution of tetrabutylammonium fluoride in tetrahydrofuran (I.1ml). After 30 minutes crushed ice (5ml) and diethylether WO 01/12579 PCT/GB00/03067 11 were added and the aqueous phase extracted with diethylether (5 portions of The combined extracts were washed with water (3 portions of 10ml) and brine dried (MgSO4) and concentrated under reduced pressure to give a solid.

Recrystallisation from ethyl acetate/hexane gave the title compound (208mg) as a white solid m.p. 123-125 0 C. nmr: 8H (500MHz, d6-DMSO) 2.07 3H), 3.57 (s, 6H), 3.62 3H), 6.40 J 12Hz, 1H), 6.46 J 12 Hz, IH), 6.56 2H), 6.61 (dd, J 8Hz, 2Hz, 1H), 6.76 J= 1.7Hz, 1H), 6.98 J 8Hz, 1H), 9.21 (s 1H).

The 1-(3-tert-butyldimethylsilyloxy-4-methylphenyl)-2-(3,4,5-trimethoxyphenyl)ethene used as starting material in the above preparation was prepared as follows: A suspension of 3,4,5-trimethoxybenzyltriphenylphosphonium bromide (848mg) in dry tetrahydrofuran (50ml) at -78 0 C was treated dropwise with n-butyllithium (0.9ml of a 1.8M solution in hexane) and the mixture allowed to warm to -40 0 C and stir for lh.

The mixture was recooled to -78°C and a solution of 3-tert-butyldimethylsilyloxy-4methylbenzaldehyde (390mg) in tetrahydrofuran (40ml) added slowly. After a further 2h the mixture was allowed to warm to room temperature before being poured into ice water (20ml). The aqueous phase was extracted with diethylether (5 portions of and the combined extracts were washed with water (3 portions of 20ml) and brine (2 portions of 20ml), dried (MgSO4) and concentrated under reduced pressure to give an oil. Purification by chromatography on silica gel, eluting with petroleum ether ethyl acetate (90:10) gave 1-(3-tert-butyldimethylsilyloxy-4-methylphenyl)-2-(3,4,5trimethoxyphenyl)ethene (456mg) as a red oil.

The 3-lert-butyldimethylsilyloxy-4-methylbenzaldehyde used as starting material in the above preparation was prepared as follows: A solution ofDess-Martin periodinane (187mg) in dichloromethane (4ml) was treated slowly with a solution of 3-t-er-butyldimethylsilyloxy-4-methylbenzyl alcohol (100mg) in dichloromethane (4ml) and the mixture stirred for Ih at room temperature.

Diethylether (10ml) was added followed by aqueous sodium thiosulphate solution (10ml) and the mixture stirred for 15 minutes. The aqueous phase was extracted with diethylether (5 portions of 20ml) and the combined extracts were washed with aqueous WO 01/12579 PCT/GB00/03067 12 sodium thiosulphate solution (3 portions of 10ml), water (3 portions of 10ml) and brine (2 portions of 10mi), dried (MgSO4) and concentrated under reduced pressure to give a yellow solid. Purification by chromatography on silica gel, eluting with petroleum ether diethyl ether (50:50) gave 3-tert-butyldimethylsilyloxy-4methylbenzaldehyde The 3-tert-butyldimethylsilyloxy-4-methylbenzyl alcohol used as starting material in the above preparation was prepared as follows: A solution of 3-hydroxy-4-methylbenzyl alcohol (275mg) in dry tetrahydrofuran (15ml) at -78°C was treated slowly with n-butyllithium (1.2ml of a 1.8M solution in hexane) and the mixture stirred for 15minutes before being allowed to warm to room temperature and stir for a further 30minutes. A solution of tertbutylchlorodimethylsilane (287mg) in tertrahydrofuran (10ml) was added and the mixture stirred for 16h. Water (20ml) was added and the mixture extracted with diethylether (5 portions of 20ml) and the combined extracts were washed with water (2 portions of 10ml) and brine (20ml), dried (MgSO4) and concentrated under reduced pressure. Purification by chromatography on silica gel, eluting with petroleum ether diethyl ether (50:50) gave 3-tert--butyldimethylsilyloxy-4-methylbenzyl alcohol (390mg).

EXAMPLE 2 (Z)-2-methvl-5-[2-(3.4.5-trimethoxyphenyl)ethenyl]phenvl dihydrogen phosphate Trifluoroacetic acid (0.22mL, 2.95mmol) was added dropwise to a stirred solution of (Z)-2-methyl-5-[2-(3,4,5-trimethoxyphenyl)ethenyl]phenyl di-tertbutyl phosphate (401mg, 0.82mmol) and dichloromethane (16mL). The mixture was stirred at room temperature overnight. Solvent was removed in vacuo, and the residue azeotroped four times with toluene. The colourless oil was triturated with ether to give the title compound as a white solid (181mg, 58%) m.p. 109-113°C. nmr: 6H (500MHz, d6- DMSO) 2.39 3H), 3.81 6H), 3.87 3H), 6.69 J=12Hz, IH), 6.74 (d, WO 0 1112579 PCT/GBO0103067 13 Jl12Hz, 6.78 2H), 7.07 J=8Hz, 1H), 7.28 J=8Hz, 1H), 7.49 1H!), (bs, 2H).

(Z)-2-methyl-S-[2-(3 5-trimethoxyphenyl)ethenyl~phenyl di-teributyl phosphate was prepared as follows: Di-tert-butylphosphoramidite (498mg, 2.O0mmol) in dichloromethane (1 m.L) was added to a solution of (Z)-1-(3-hydroxy-4-methylphenyl)-2-( 3 4 5 trimethoxyphenyl)ethene (300mg, 1 .O0mmol), IH-tetrazole (I182mg, 2.6Ommol) in dichloromethane (3 mL) under nitrogen. After 2h, magnesium monoperoxyphthalate hexahydrate (1.24g, 2.O0mmol) was added in portions. After stirring for a further 2h, the reaction mixture was partitioned between ethyl acetate and water; the aqueous phase was extracted (ethyl acetate x2), the combined organic extracts were washed (water x2, brine x dried (MgSO 4 and concentrated in vacuc. Flash chromatography, eluting with 33% ethyl acetate/hexane, gave (Z')-2-methyl-5-[2- (3,4,5-trimethoxyphelyl)ethenyl]phenyI di-iertbutyl phosphate as a yellow oil (40 1mg, 82%).

EXAMPLE 3 I-(4-fluoro-3-hydroxvphenyl)-2-(3 .4 This compound was isolated directly from the Wittig reaction between 3,4,5trimethoxybenzyltriphenylphosphoflium bromide and 3 -tert-butyldimethylsilyloxy- 4 fluorobenzaldehyde (340mg) performed in an analogous manner to that of Example 1.

There was obtained the title compound (80mg) as a colourless oil. nmr:- (300N4iHz, d6- DMSQ) 3.59 6H), 3.63 6.46 1=12Hz, 6.48 J=12Hz, 6.54 2H), 6.68 (in, IH), 6.90 (dd, J=8.8, 2.1Hz, 1H), 7.06 (dd, J=1 1.4, 8.414z, IR), 9.80 I H).

The following compounds were prepared in an analogous manner to that of Example I1: WO 0 1/12579 PTGOI36 PCT/GBOO/03067 14 EXAMPLE 4 I (4-chloro-3-hydroxyphenfl)-2-(3.4.5-trimethoxyphenl)ethele From I-(3.ierti-butydimethylsilyloxy-4-chlorophenyl)- 2 3 4

,S-

trimethoxyphenyl)ethene (240mg) there was obtained the title compound (12 1mg) as a colourless oil. nmr: (300M1-z, d6-DMSO) 3.59 6H), 3.63 3H), 6.49 (in, 2H), 6.54 2H), 6.71 (dd, J=8.2, 0.9Hz, 6.93 J=0.9Hz, IR), 7.25 3=8.2Hz, 1H), 10. 11 (bs, I H).m/e 320 EXAMPLE -(4-et hyl-3 -hydroxyphenfl-2-(3.4,S-t rimet hoxyphenyI)ethefle From -lwri-butyldimethylsilyloxy-4-ethylphenyl)-2-(3 trimethoxyphenyl)ethene (926mg) there was obtained the title compound (208mg) as a white solid m.p. 105-107'C, nmr: 5H (300MfHz, CDCI3) 1.02 J=7.6Hz, 2.6 (q, 2H) 3.7 6H), 3.8 3H), 4.6 (bs, IH), 6.4 J 12Hz, 18), 6.5 J= 12 Hz, I1H), 6.5 2H), 6.7 IH), 6. 8 J= 7.6Hz, I1H), 7. 0 J3 7.6H4z, 18H).

Claims

1. A cis-stilbene of formula O H R1O OH R2d OR 3 R4 (1) wherein: R 2 and R 3 are each independently alkyl, R 4 is alkyl, haloalkyl, alkenyl, alkynyl, alkylthio, alkylsulphinyl, alkylsulphonyl or halo, R 5 is hydrogen, alkoxy, alkyl, alkylthio, hydroxy or halo, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

2. A cis-stilbene according to claim 1 wherein R 2 and R 3 are all methyl.

3. A cis-stilbene according to claim 2 wherein R 5 is hydrogen and R 4 is alkyl or halo.

4. (Z)-1-(3-hydroxyl-4-methylphenyl)-2-(3,4,5-trimethoxyphenyl) ethene.

5. A cis-stilbene of formula: halo, wherein: R 2 and R 3 are each independently alkyl, R 4 is alkyl, haloalkyl, alkenyl, alkynyl, alkylthio, alkylsulphinyl, alkylsulphonyl or R 5 is hydrogen, alkoxy, alkyl, alkylthio, hydroxy or halo; and X is a group which can be removed in vivo by hydrolysis.

6. A cis-stilbene prodrug according to claim 5 wherein: R 2 and R 3 are all methyl.

7. A cis-stilbene prodrug according to claim 5 or claim 6 wherein: R 5 is hydrogen and R 4 is alkyl or halo.

8. A cis-stilbene prodrug according to claim 7 wherein R 4 is methyl.

9. A cis-stilbene prodrug according to claim 5, 6, 7 or 8, which is a carboxylic phosphate ester, sulphate ester or carbonate. A cis-stilbene prodrug according to claim 9, which is a phosphate ester. ester, [R:\LIBFF]99715.doc:ANB

11. A cis-stilbene prodrug according to claim 9 or 10, which is a dihydrogen phosphate ester.

12. (Z)-2-methyl-5-[2-(3,4,5-trimethoxyphenyl)ethenyl]phenyl dihydrogen phosphate.

13. A composition for use in the treatment of neovascularisation which composition contains an effective amount of the cis-stilbene dihydrogen phosphate according to claim 12 and a pharmaceutically acceptable excipient.

14. A composition for use in the treatment of neovascularisation which composition contains an effective amount of the cis-stilbene according to any of claims 1 to 4 or a cis-stilbene prodrug according to any one of claims 5 to 12 and a pharmaceutically acceptable excipient. A composition as claimed in claim 13 or 14 in combination with at least one further anti-tumour substance.

16. A use of a cis-stilbene as claimed in any of claims 1 to 4 or a cis-stilbene prodrug as claimed in any one of claims 5 to 12 in the manufacture of a medicament for the treatment of a disease involving neovascularisation.

17. A use according to claim 16, wherein the disease is cancer involving a solid tumour.

18. A use according to claim 17, wherein said medicament is for administration in 20 combination with radiotherapy or another anti-tumour substance. *oo0

19. A use according to claim 16, wherein the disease is diabetic retinopathy, psoriasis, rheumatoid arthritis, atherosclerosis or macular degeneration.

20. A use according to any one of claims 16 to 19, wherein the cis-stilbene prodrug is (Z)-2-methyl-5-[2-(3,4,5-trimethoxyphenyl)ethenyl]phenyl dihydrogen *00 25 phosphate.

21. A use according to any one of claims 16 to 20, substantially as herein described.

22. A composition according to claim 13, 14 or 15, substantially as herein described.

23. A cis-stilbene according to any one of claims 1 to 4, substantially as herein described.

24. A cis-stilbene prodrug according to any one of claims 5 to 12, substantially as herein described. [R:\LIBFF]99715.doc:ANB 17 A method for treating neovascularisation, wherein said method comprises administering a therapeutically effective amount of the cis-stilbene of any one of claims 1 to 4 or 23 or a cis-stilbene prodrug of any one of claims 5 to 12 or 24.

26. A method for treating a disease involving neovascularisation, wherein said method comprises administering a therapeutically effective amount of the cis-stilbene of any one of claims 1 to 4 or 23 or a cis-stilbene prodrug of any one of claims 5 to 12 or 24.

27. Use of a cis-stilbene of any one of claims 1 to 4 or 23 or a cis-stilbene prodrug of any one of claims 5 to 12 or 24 for the manufacture of a medicament for treating neovascularisation

28. Use of a cis-stilbene of any one of claims 1 to 4 or 23 or a cis-stilbene prodrug of any one of claims 5 to 12 or 24 for the manufacture of a medicament for treating a disease involving neovascularisation. Dated 25 November, 2004 Angiogene Pharmaceuticals Ltd Patent Attorneys for the Applicant/Nominated Person SPRUSON FERGUSON C.. o0r 0 C [R:\LIBFF]9971