WO2006090094A1

WO2006090094A1 - Pyrimidothiophene compounds for use as hsp90 inhibitors

Info

Publication number: WO2006090094A1
Application number: PCT/GB2005/000736
Authority: WO
Inventors: Paul Andrew Brough; Xavier Barril-Alonso; Martin James Drysdale
Original assignee: Vernalis R & D Ltd
Priority date: 2005-02-28
Filing date: 2005-02-28
Publication date: 2006-08-31

Abstract

Certain specific compounds of formula (I) are inhibitors of HSP90 activity in vitro or in vivo, and of use in the treatment of inter alia, cancer: formula (I) wherein R2 is a group of formula -(Ar1)m-(Alk1)P-(Z)r-(Alk2)S-Q wherein Ar1 is an optionally substituted aryl or heteroaryl radical, Alk1 and Alk2 are optionally substituted divalent C1-C3 alkylene or C2-C3 alkenylene radicals, m, p, r and s are independently 0 or 1, Z is -0-, -S-, -(C=0)-, -(C=S)-, -SO2-, -C(=0)O-, -C(=O)NRA- , -C(=S)NRA-, -SO2NRA-, -NRAC(=0)-, -NRASO2- or -NRA-wherein RA is hydrogen or C1-C6 alkyl, and Q is hydrogen or an optionally substituted carbocyclic or heterocyclic radical; R3 is hydrogen, an optional substituent, or an optionally substituted (C1-C6)alkyl, aryl or heteroaryl radical; and R4 is a carboxylic ester, carboxamide or sulfonamide group.

Description

Pyrimidothiophene Compounds

This invention relates to substituted bicyclic thieno[2,3-d]pyrimidine (herein referred to as 'pyrimidothiophene') compounds having HSP90 inhibitory activity, to the use of such compounds in medicine, in relation to diseases which are responsive to inhibition of HSP90 activity such as cancers, and to pharmaceutical compositions containing such compounds.

Background to the invention

Molecular chaperones maintain the appropriate folding and conformation of proteins and are crucial in regulating the balance between protein synthesis and degradation. They have been shown to be important in regulating many important cellular functions, such as cell proliferation and apoptosis (Jolly and Morimoto, 2000; Smith et al., 1998; Smith, 2001).

Heat Shock Proteins (HSPs)

Exposure of cells to a number of environmental stresses, including heat shock, alcohols, heavy metals and oxidative stress, results in the cellular accumulation of a number of chaperones, commonly known as heat shock proteins (HSPs). Induction of HSPs protects the cell against the initial stress insult, enhances recovery and leads to maintenance of a stress tolerant state. It has also become clear, however, that certain HSPs may also play a major molecular chaperone role under normal, stress-free conditions by regulating the correct folding, degradation, localization and function of a growing list of important cellular proteins.

A number of multigene families of HSPs exist, with individual gene products varying in cellular expression, function and localization. They are classified according to molecular weight, e.g., HSP70, HSP90, and HSP27. Several diseases in humans can be acquired as a result of protein misfolding (reviewed in Tytell et al., 2001 ; Smith et al., 1998). Hence the development of therapies which disrupt the molecular chaperone machinery may prove to be beneficial. In some conditions (e.g., Alzheimer's disease, prion diseases and Huntington's disease), misfolded proteins can cause protein aggregation resulting in neurodegenerative disorders. Also, misfolded proteins may result in loss of wild type protein function, leading to deregulated molecular and physiological functions in the cell.

HSPs have also been implicated in cancer. For example, there is evidence of differential expression of HSPs which may relate to the stage of tumour progression (Martin et al., 2000; Conroy et al., 1996; Kawanishi et al., 1999; Jameel et al., 1992; Hoang et al., 2000; Lebeau et al., 1991 ). As a result of the involvement of HSP90 in various critical oncogenic pathways and the discovery that certain natural products with anticancer activity are targeting this molecular chaperone, inhibition of HSP function is a mechanism for drug intervention in the treatment of cancer. The first molecular chaperone inhibitor is currently undergoing clinical trials.

HSP90

HSP90 constitutes about 1-2% of total cellular protein, and is usually present in the cell as a dimer in association with one of a number of other proteins (see, e.g., Pratt, 1997). It is essential for cell viability and it exhibits dual chaperone functions (Young et al., 2001 ). It plays a key role in the cellular stress response by interacting with many proteins after their native conformation has been altered by various environmental stresses, such as heat shock, ensuring adequate protein folding and preventing non-specific aggregation (Smith et al., 1998). In addition, recent results suggest that HSP90 may also play a role in buffering against the effects of mutation, presumably by correcting the inappropriate folding of mutant proteins (Rutherford and Lindquist, 1998). However, HSP90 also has an important regulatory role. Under normal physiological conditions, together with its endoplasmic reticulum homologue GRP94, HSP90 plays a housekeeping role in the cell, maintaining the conformational stability and maturation of several key client proteins. These can be subdivided into three groups: (a) steroid hormone receptors, (b) Ser/Thr or tyrosine kinases (e.g., ERBB2, RAF-1 , CDK4, and LCK), and (c) a collection of apparently unrelated proteins, e.g., mutant p53 and the catalytic subunit of telomerase hTERT. All of these proteins play key regulatory roles in many physiological and biochemical processes in the cell. New HSP90 client proteins are continuously being identified.

The highly conserved HSP90 family in humans consists of four genes, namely the cytosolic HSP90α and HSP90β isoforms (Hickey et al., 1989), GRP94 in the endoplasmic reticulum (Argon et al., 1999) and HSP75/TRAP1 in the mitochondrial matrix (Felts et al., 2000). It is thought that all the family members have a similar mode of action, but bind to different client proteins depending on their localization within the cell. For example, ERBB2 is known to be a specific client protein of GRP94 (Argon et al., 1999) and type 1 tumour necrosis factor receptor (TNFR1 ) and RB have both been shown to be clients of TRAPI (Song et al., 1995; Chen et al., 1996).

HSP90 participates in a series of complex interactions with a range of client and regulatory proteins (Smith, 2001). Although the precise molecular details remain to be elucidated, biochemical and X-ray crystallographic studies (Prodromou et al., 1997; Stebbins et al., 1997) carried out over the last few years have provided increasingly detailed insights into the chaperone function of HSP90.

Following earlier controversy on this issue, it is now clear that HSP90 is an ATP-dependent molecular chaperone (Prodromou et al, 1997), with dimerization of the nucleotide binding domains being essential for ATP hydrolysis, which is in turn essential for chaperone function (Prodromou et al, 2000a). Binding of ATP results in the formation of a toroidal dimer structure in which the N terminal domains are brought into closer contact with each other resulting in a conformational switch known as the 'clamp mechanism' (Prodromou and Pearl, 2000b.

Our copending international application PCT/GB2004/003641 relates to the use of a compound of formula (I), or a salt, N-oxide, hydrate, or solvate thereof in the preparation of a composition for inhibition of HSP90 activity in vitro or in vivo:

wherein

R₂ is a group of formula (IA):

-(Ar¹)_m-(AlkV(Z)_r(Alk²)s-Q ('A) wherein

Ar¹ is an optionally substituted aryl or heteroaryl radical,

AIk¹ and AIk² are optionally substituted divalent CrC₃ alkylene or C-₂-C₃ alkenylene radicals, m, p, r and s are independently 0 or 1 ,

Z is -O-, -S-, -(C=O)-, -(C=S)-, -SO₂-, -C(=O)O-, -C(=O)NR^A- ,

-C(=S)NR^A-, -SO₂NR^A-, -NR^AC(=O)-, -NR^ASO₂- or -NR^A- wherein R^A is hydrogen or C1-C-₆ alkyl, and

Q is hydrogen or an optionally substituted carbocyclic or heterocyclic radical;

R₃ is hydrogen, an optional substituent, or an optionally substituted (C₁- Cβjalkyl, aryl or heteroaryl radical; and

R₄ is a carboxylic ester, carboxamide or sulfonamide group.

Description of the Invention

The present invention relates to certain compounds of formula (I) above which are inhibitors of HSP90 activity and have the utility disclosed in PCT/GB2004/003641 , said compounds being selected from the group consisting of 2-amino-4-[2,4-dichloro-5-(3-morpholin-4-yl-propoxy)-phenyl]- thieno[2,3-d]pyrimidine-6-carboxylic acid ethylamide,

2-amino-4-[2,4-dichloro-5-(2-methoxy-ethoxy)-phenyl]-thieno[2,3- d]pyrimidine-6-carboxylic acid isopropylamide,

2-amino-4-[2,4-dichloro-5-(2-diethylamino-ethoxy)-phenyl]-thieno[2,3- d]pyrimidine-6-carboxylic acid (2,2,2-trifluoro-ethyl)-amide,

2-amino-4-[2,4-dichloro-5-(3-dimethylamino-propoxy)-phenyl]- thieno[2,3-d]pyrimidine-6-carboxylic acid (2,2,2-trifluoro-ethyl)-amide,

2-amino-4-[2,4-dichloro-5-(2-pyrrolidin-1-yl-ethoxy)-phenyl]-thieno[2,3- d]pyrimidine-6-carboxylic acid cyclopropylamide,

2-amino-4-[2,4-dichloro-5-(2-hydroxy-ethoxy)-phenyl]-thieno[2,3- d]pyrimidine-6-carboxylic acid cyclopropylamide,

2-amino-4-[2,4-dichloro-5-(3-piperidin-1-yl-propoxy)-phenyl]-thieno[2,3- d]pyrimidine-6-carboxylic acid ethylamide,

2-amino-4-{2,4-dichloro-5-[2-(1-methyl-pyrrolidin-2-yl)-ethoxy]-phenyl}- thieno[2,3-d]pyrimidine-6-carboxylic acid ethylamide,

2-amino-4-[2,4-dichloro-5-(1-methyl-piperidin-4-yloxy)-phenyl]- thieno[2,3-d]pyrimidine-6-carboxylic acid ethylamide,

2-amino-4-[2,4-dichloro-5-(1-ethyl-pyrrolidin-3-yloxy)-phenyl]- thieno[2,3-d]pyrimidine-6-carboxylic acid ethylamide, and

2-amino-4-[2,4-dichloro-5-(3-diethylamino-propoxy)-phenyl]-thieno[2,3- d]pyrimidine-6-carboxylic acid ethylamide, and salts, N-oxides, hydrates, and solvates thereof.

In other aspects, the invention provides

(i) a method of treatment of diseases which are responsive to inhibition of HSP90 activity in mammals, which method comprises administering to the mammal an amount of a compound selected from the above- defined group effective to inhibit said HSP90 activity;

(ii) the medicinal use of a compound selected from the above-defined group;

(iv) the use of a compound selected from the above-defined group for inhibition of HSP90 activity in vitro or in vivo;

(iv) the use of a compound selected from the above-defined group in the preparation of a composition for the treatment of diseases in which HSP90 activity is implicated; and

(v) a pharmaceutical composition comprising a compound selected from the above-defined group and a pharmaceutically acceptable carrier.

As used herein the term "salt" includes base addition, acid addition and quaternary salts. Compounds of the invention which are acidic can form salts, including pharmaceutically or veterinarily acceptable salts, with bases such as alkali metal hydroxides, e.g. sodium and potassium hydroxides; alkaline earth metal hydroxides e.g. calcium, barium and magnesium hydroxides; with organic bases e.g. N-ethyl piperidine, dibenzylamine and the like. Those compounds (I) which are basic can form salts, including pharmaceutically or veterinarily acceptable salts with inorganic acids, e.g. with hydrohalic acids such as hydrochloric or hydrobromic acids, sulphuric acid, nitric acid or phosphoric acid and the like, and with organic acids e.g. with acetic, tartaric, succinic, fumaric, maleic, malic, salicylic, citric, methanesulphonic and p- toluene sulphonic acids and the like.

For a review on suitable salts, see Handbook of Pharmaceutical Salts: Properties, Selection, and Use by Stahl and Wermuth (Wiley-VCH, Weinheim, Germany, 2002).

The term 'solvate' is used herein to describe a molecular complex comprising the compound of the invention and a stoichiometric amount of one or more pharmaceutically acceptable solvent molecules, for example, ethanol. The term 'hydrate' is employed when said solvent is water.

Compounds with which the invention is concerned which may exist in one or more stereoisomeric form, because of the presence of asymmetric atoms or rotational restrictions, can exist as a number of stereoisomers with R or S stereochemistry at each chiral centre or as atropisomeres with R or S stereochemistry at each chiral axis. The invention includes all such enantiomers and diastereoisomers and mixtures thereof.

The compounds of the invention are inhibitors of HSP90 and are useful in the treatment of diseases which are responsive to inhibition of HSP90 activity such as cancers; viral diseases such as Hepatitis C (HCV) (Waxman, 2002); lmmunosupression such as in transplantation (Bijlmakers, 2000 and Yorgin, 2000); Anti-inflammatory diseases (Bucci, 2000) such as Rheumatoid arthritis, Asthma, MS, Type I Diabetes, Lupus, Psoriasis and Inflammatory Bowel Disease; Cystic fibrosis (Fuller, 2000); Angiogenesis-related diseases (Hur, 2002 and Kurebayashi, 2001): diabetic retinopathy, haemangiomas, psoriasis, endometriosis and tumour angiogenesis. Also an Hsp90 inhibitor of the invention may protect normal cells against chemotherapy-induced toxicity and be useful in diseases where failure to undergo apoptosis is an underlying factor. Such an Hsp90 inhibitor may also be useful in diseases where the induction of a cell stress or heat shock protein response could be beneficial, for example, protection from hypoxia-ischemic injury due to elevation of Hsp70 in the heart (Hutter, 1996 and Trost, 1998) and brain (Plumier, 1997 and Rajder, 2000). An Hsp90 inhibitor - induced increase in Hsp70 levels could also be useful in diseases where protein misfolding or aggregation is a major causal factor, for example, neurogenerative disorders such as scrapie/CJD, Huntingdon's and Alzheimer's (Sittler, 2001 ; Trazelt, 1995 and Winklhofer, 2001 )".

Specifically the use and method of the invention are applicable to the treatment of diseases in which HSP90 activity is implicated, including use for immunosuppression or the treatment of viral disease, inflammatory diseases such as rheumatoid arthritis, asthma, multiple sclerosis, Type I diabetes, lupus, psoriasis and inflammatory bowel disease; cystic fibrosis angiogenesis- related disease such as diabetic retinopathy, haemangiomas, and endometriosis; or for protection of normal cells against chemotherapy-induced toxicity; or diseases where failure to undergo apoptosis is an underlying factor; or protection from hypoxia-ischemic injury due to elevation of Hsp90 in the heart and brain; scrapie/CJD, Huntingdon's or Alzheimer's disease. Use for the treatment of cancer is especially indicated.

It will be understood that the specific dose level for any particular patient will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, route of administration, rate of excretion, drug combination and the causative mechanism and severity of the particular disease undergoing therapy. In general, a suitable dose for orally administrable formulations will usually be in the range of 0.1 to 3000 mg, once, twice or three times per day, or the equivalent daily amount administered by infusion or other routes. However, optimum dose levels and frequency of dosing will be determined by clinical trials as is conventional in the art.

The compounds with which the invention is concerned may be prepared for administration by any route consistent with their pharmacokinetic properties. The orally administrable compositions may be in the form of tablets, capsules, powders, granules, lozenges, liquid or gel preparations, such as oral, topical, or sterile parenteral solutions or suspensions. Tablets and capsules for oral administration may be in unit dose presentation form, and may contain conventional excipients such as binding agents, for example syrup, acacia, gelatin, sorbitol, tragacanth, or polyvinyl-pyrrolidone; fillers for example lactose, sugar, maize-starch, calcium phosphate, sorbitol or glycine; tabletting lubricant, for example magnesium stearate, talc, polyethylene glycol or silica; disintegrants for example potato starch, or acceptable wetting agents such as sodium lauryl sulphate. The tablets may be coated according to methods well known in normal pharmaceutical practice. Oral liquid preparations may be in the form of, for example, aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, or may be presented as a dry product for reconstitution with water or other suitable vehicle before use. Such liquid preparations may contain conventional additives such as suspending agents, for example sorbitol, syrup, methyl cellulose, glucose syrup, gelatin hydrogenated edible fats; emulsifying agents, for example lecithin, sorbitan monooleate, or acacia; non-aqueous vehicles (which may include edible oils), for example almond oil, fractionated coconut oil, oily esters such as glycerine, propylene glycol, or ethyl alcohol; preservatives, for example methyl or propyl p-hydroxybenzoate or sorbic acid, and if desired conventional flavouring or colouring agents.

For topical application to the skin, the drug may be made up into a cream, lotion or ointment. Cream or ointment formulations which may be used for the drug are conventional formulations well known in the art, for example as described in standard textbooks of pharmaceutics such as the British Pharmacopoeia.

The active ingredient may also be administered parenterally in a sterile medium. Depending on the vehicle and concentration used, the drug can either be suspended or dissolved in the vehicle. Advantageously, adjuvants such as a local anaesthetic, preservative and buffering agents can be dissolved in the vehicle. The following examples illustrate the preparation and activities of specific compounds of the invention.

Scheme 1 is a general scheme for the synthesis of the compounds of the invention.

Ig 1 h

Scheme 1

Suitable reagents, solvents, methodology and reaction temperatures for the synthetic procedures of scheme 1 will be known to those skilled in the art of organic chemistry. Examples of synthetic procedures used to synthesize compound 1e (steps A to D) are given in example 1. Step E is an amide forming reaction which may be performed by direct reaction of a primary amine with 1 e (as described in example 1 ). Alternatively 1e may be converted to the intermediate carboxylic acid 1 i followed by amide formation (as described in example 2). There are many methods known to the organic chemist for the transformation of carboxylic acid functional groups to amides. For example the intermediate carboxylic acid 1 i can activated with O-(7-

Azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate and diisopropylethylamine and mixed with a primary or secondary amine in THF or DMF at rt to 100 ⁰C for 1-16 hours.

Step F is the selective removal of a benzyl protecting group (Greene and Wutts 1991 ). Step G is the alkylation of the phenol 1g which may (for example) be accomplished by reaction of 1g with a primary or secondary alkyl halide in the presence of a base, for example cesium carbonate or potassium carbonate in a suitable solvent, for example THF or DMF with heating to 140 °C 1-16 hours. Alternatively, phenol 1g can be reacted with a primary or secondary alcohol under Mitsunobu conditions (Organic Reactions, Beak et a/, vol. 42, 1992).

General Procedures

All reagents obtained from commercial sources were used without further purification. Anhydrous solvents were obtained from commercial sources and used without further drying. Flash chromatography was performed with prepacked silica gel cartridges (Strata SI-1 ; 61 A, Phenomenex, Cheshire UK or IST Flash II, 54A, Argonaut, Hengoed, UK). Thin layer chromatography was conducted with 5 x 10 cm plates coated with Merck Type 60 F₂54 silica gel.

The compounds of the present invention were characterized by LC/MS using a Hewlett Packard 1100 series LC/MSD linked to quadripole detector (ionization mode: electron spray positive; column: Phenomenex Luna 3u C18(2) 30 x 4.6 mm; Buffer A prepared by dissolving 1.93g ammonium acetate in 2.5 L HPLC grade H₂O and adding 2 ml_ formic acid. Buffer B prepared by adding 132 ml_ buffer A to 2.5 L of HPLC grade acetonitrile and adding 2 mL formic acid; elution gradient 95:5 to 5:95 buffer A : buffer B over 3.75 minutes. Flow rate = 2.0 mL/min)

Nuclear magnetic resonance (NMR) analysis was performed with a Brucker DPX-400 MHz NMR spectrometer. The spectral reference was the known chemical shift of the solvent. Proton NMR data is reported as follows: chemical shift (δ) in ppm, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, p = pentet, m = multiplet, dd = doublet of doublet, br = broad), coupling constant, integration.

2-Amino-4-chloro-thieno[2,3-d]pyrimidine-6-carboxylic acid ethyl ester

This compound was used in the preparation of the compounds of the invention by reacting with compound 1d of scheme 1 (step D) to afford compound 1e. It may be synthesized (for example) as described:

To a stirred mixture of 2-amino-4,6-dichloro-5-formyl-pyrimidine (available from Bionet Research Intermediates, UK) (5.Og 1 eq.) and potassium carbonate (9.0 g; 2.5 equiv.) in acetonitrile (160ml) at ambient temperature was added ethyl-2-mercaptoacetate (2.86 ml; 1.0 equiv.). The resulting mixture was stirred at reflux for three hours. After cooling, the solvents were removed in vacuo and the residue partitioned between ethyl acetate and water, the phases separated and the organic phase washed with saturated aqueous sodium chloride solution. Phases were separated and the organic phase was dried over Na₂SO₄ then filtered and filtrate solvents removed in vacuo. The crude product was purified by column chromatography on silica gel, eluting with ethyl acetate and hexanes, to afford 2-Amino-4-chloro- thieno[2,3-d]pyrimidine-6-carboxylic acid ethyl ester as a yellow powder (60%).

LC-MS: RT = 2.371 minutes, m/z = 258.0 [M+H]⁺ (Total run time = 3.5 minutes)

Example 1

2-Amino-4-[2,4-dichloro-5-(3-morpholin-4-yl-propoxy)-phenyl]-thieno[2,3- d]pyrimidine-6-carboxylic acid ethylamide

Step i

1-Benzyloxy-2,4-dichloro-5-nitro-benzene

Potassium carbonate (12g, 87mmol) was added to a solution of 2,4-dichloro- 5-nitrophenol (Lancaster Synthesis, Morecambe, Lancashire, UK) (15.6g, 75mmol) in acetone. Benzyl bromide (9ml, 76mmol) was added and the suspension heated at 75⁰C (oil bath temperature) for ~3hrs. The resulting suspension was allowed to cool and water (500ml) was added, the mixture was extracted with dichloromethane (2x200ml). The combined extracts were washed with aqueous sodium hydroxide (150ml, 2M), water (2x200ml) and saturated aqueous sodium chloride solution (150ml). The solution was dried over anhydrous sodium sulphate and concentrated to a pale yellow solid (21.5g, 96%) Rf 0.73 CH₂Cl₂ (SiO₂)

LC retention time 2.915min [M+H]⁺ no ionisation (run time 3.75 min)

Step 2

5-Benzyloxy-2,4-dichloro-phenylamine

Iron powder (21 g, 376mmol) was added to a suspension 1-Benzyloxy-2,4- dichloro-5-nitro-benzene (21.5g, 72mmol) in acetic acid (300ml) / water (150ml) and the mixture was heated at 85⁰C (oil bath temperature) for ~90mins. The resulting suspension was filtered. The filtrate was allowed to cool, water (750ml) was added and the mixture extracted with dichloromethane (3x150ml). The combined extracts were washed with aqueous sodium hydroxide (300ml, 2M), water (2x500ml) and saturated aqueous sodium chloride solution (200ml). The solution was dried over anhydrous sodium sulphate filtered and the filtrate solvents removed in vacuo to afford product as a pale brown solid (18.6g, 96%) Rf 0.57 CH₂CI₂ (SiO₂)

LC retention time 2.792min [M+H]⁺ 270 /268 (run time 3.75 min)

Step 3

1-Benzyloxy-2,4-dichloro-5-iodo-benzene

Hydrochloric acid (60ml, 6M) was added to a solution of the 5-Benzyloxy-2,4- dichloro-phenylamine (16.2g, 60mmol) in acetic acid (240ml) and the resulting suspension cooled (ice/water/salt). Aqueous sodium nitrite (4.8g, 69.5mmol in 40ml) was added slowly (keeping the temperature <5°C). On complete addition the resulting solution was stirred for ~30mins. The resulting solution was poured into a solution of potassium iodide (2Og, 120mmol) and iodine (4g, 16mmol) in water (200ml), and the mixture stirred for ~90mins. Water (800ml) was added and the mixture extracted with dichloromethane (3x250ml). The combined extracts were washed with aqueous sodium thiosulphate solution (2x150ml, 10%), aqueous sodium hydroxide (250ml, 2M), water (2x250ml) and saturated aqueous sodium chloride solution (200ml). The solution was dried over anhydrous sodium sulphate and concentrated to a pale brown oil, solidified on standing. (20.6g, 90%) R_f 0.82 CH₂CI₂ (SiO₂)

LC retention time 3.084min [M+H]⁺ no ionisation (run time 3.75 min)

Step 4

2-Amino-4-(5-benzyloxy-2,4-dichIoro-phenyl)-thieno[2,3-d]pyrimidine-6- carboxylic acid ethyl ester

Potassium acetate (16g, 163mmol) was added to a solution of 1-Benzyloxy- 2,4-dichloro-5-iodo-benzene (20.6g, 54mmol) and jb/s(pinacolato)diboron (14.5g, 57mmol) in DMF (50ml), under a nitrogen atmosphere. Palladium acetate (450mg, cat.) was added and the mixture heated, oil bath temperature 90⁰C, for ~18hrs. The resulting solution was concentrated, and the residue taken up in ethyl acetate (200ml), the solution was washed with water

(3x200ml) and saturated aqueous sodium chloride solution (150ml). The solution was dried over anhydrous sodium sulphate and concentrated to a pale brown gum.

The residue was taken up in 1 ,4-dioxan (160ml) and 2-Amino-4-chloro- thieno[2,3-d]pyrimidine-6-carboxylic acid ethyl ester (12.85g, 50mmol) and aqueous potassium phosphate (40ml, 2M) added, under a nitrogen atmosphere. Dichloro bis(triphenylphosphine) palladium(ll) (cat.) was added and the mixture heated, oil bath temperature 100⁰C, for ~3hrs. The mixture was allowed to cool and ethyl acetate (400ml) added. The mixture was washed with saturated aqueous sodium chloride solution (100ml). The solution was dried over anhydrous sodium sulphate and concentrated to a pale yellow solid. Solids were washed with diethyl ether/ hexane (1 :1), to give an off-white solid. Dried in vacuo (40⁰C). 10.7g (45%) R_f 0.13 EtO Ac/Hex (1 :3) (SiO₂)

LC retention time 2.972min [M+H]⁺ 476/474 (run time 3.75 min)

Step 5

2-Amino-4-(2,4-dichloro-5-hydroxy-phenyl)-thieno[2,3-d]pyrimidine-6- carboxylic acid ethyl amide

A suspension of 2-Amino-4-(5-benzyloxy-2,4-dichlorophenyI)-thieno[2,3- d]pyrimidine-6-carboxylic acid ethyl ester in methanolic ethylamine (~2M) was heated, at -75⁰C, for ~18hrs. The resulting solution was concentrated and the residue triturated with diethyl ether/ hexane to give a pale brown powder. LC retention time 2.654 minutes [M+H]⁺ 475.1/ 473.1 (Run time 3.75mins)

Boron trichloride solution (1 M in dichloromethane) was added to a suspension of 2-Amino-4-(5-benzyloxy-2,4-dichlorophenyl)-thieno[2,3-d]pyrimidine-6- carboxylic acid ethyl amide in dichloromethane, at -78⁰C under a nitrogen atmosphere. The suspension was stirred for ~3hrs at room temperature. The suspension was cooled in ice and methanol added, the resulting mixture was stirred for ~1hr. and concentrated to a yellow green solid. The solids were suspended in aqueous sodium acetate (10%) and extracted with ethyl acetate. The extracts were washed with water and saturated aqueous sodium chloride solution. The solution was dried over anhydrous sodium sulphate and concentrated to a pale brown solid, washed with hexane dried in vacuo.

LC retention time 2.180 minutes [M+H]⁺ 385/383 (Run time 3.75mins)

Step 6

4-(3-Hydroxylpropyl) morpholine (0.043 ml; 0.31 mmol) was added to a stirred solution of 2-amino-4-(2,4-dichIoro-5-hydroxy-phenyl)-thieno[2,3-d]pyrimidine- 6-carboxylic acid ethylamide (0.100 g; 0.26 mmol) in anhydrous THF (30 ml) under N₂. Triphenylphosphine (0.102 g; 0.39 mmol) was added followed by diisopropyl azodicarboxylate (0.077 ml; 0.39 mmol). The reaction mixture was stirred at room temperature for 18 hour. The reaction mixture was diluted with ethyl acetate (50 ml) and then washed with water (50ml) followed by saturated aqueous sodium hydrogen carbonate solution (50 ml) and saturated aqueous sodium chloride solution (50 ml). The organic phase was dried over anhydrous sodium sulphate, filtered and the filtrate solvents removed in vacuo to afford a brown oil. The crude product was purified by flash chromatography on silica gel (10g Flash Si cartridge) eluting with 2:1 ethyl acetate/ hexanes followed by 5% methanol/ ethyl acetate. This affords product as a yellow foam (0.047 g; 35%).

LC retention time 1.789 minutes [M+H]⁺ 512/510 (Run time 3.75mins)

¹H NMR (400MHz, d6-DMSO): δ = 1.07 (3H, t, J = 7.2Hz), 1.90 (2H₁ m, J = 6.6Hz), 2.33 (4H, br s), 2.41 (2H, t, J = 6.9), 3.21 (2H, m, J = 7.2Hz), 3.51 (4H, t, 6.2), 7.27 (2H, br s), 7.35 (1 H, s), 7.57 (1 H, s), 7.83 (1 H, s), 8.55 (1 H, t, J = 5.6).

This compound had activity A in the fluorescence polarization assay described below.

Example 2

2-Amino-4-[2,4-dichloro-5-(2-methoxy-ethoxy)-phenyl]-thieno[2,3- d]pyrimidine-6-carboxylic acid isopropylamide

Stepi

2-Amino-4-(5-benzyloxy-2,4-dichloro-phenyl)-thieno[2,3-d]pyrimidine-6- carboxylic acid

Sodium hydroxide (0.190 g; 4.75 mmol) was added to 2-Amino-4-(5- benzyloxy-2,4-dichIoro-phenyl)-thieno[2,3-d]pyrimidine-6-carboxylic acid ethyl ester (step 4 example 1 ). Ethanol (25 ml) was added followed by water (2.5 ml) and the reaction mixture was heated to reflux for 1 hour. The reaction mixture was allowed to cool and solvents were removed in vacuo. The resulting residue was dissolved in water and stirred in ice-water bath and the neutralized by the drop-wise addition of 37% (aq) hydrochloric acid solution. The reaction mixture was freeze dried to afford product as a yellow powder (containing 2 equivalents of NaCI) 1.33 g; 100%.

LC retention time 2.579 minutes [M+H]⁺ 448/446 (Run time 3.75mins)

Step 2

2-Amino-4-(5-benzyloxy-2,4-dichloro-phenyl)-thieno[2,3-d]pyrimidine-6- carboxylic acid isopropylamide

O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (1.176 g; 3.07 mmol) was added to 2-Amino-4-(5-benzyloxy-2,4-dichloro- phenyl)-thieno[2,3-d]pyrimidine-6-carboxylic acid.2NaCI (1.33 g; 2.38 mmol) then DMF (25 ml) was added to afford a turbid brown solution, lsopropylamine (1.01 ml; 11.9 mmol) was added and reaction mixture was heated at 60 ⁰C (oil bath temperature) for 18 hours. Reaction mixture was allowed to cool to room temperature and DMF was removed in vacuo. The residue was partitioned between ethyl acetate (200 ml) and water (200 ml). The phases were separated and the organic phase was washed with saturated aqueous sodium chloride solution (200 ml) the dried over anhydrous sodium sulphate, filtered and the filtrate solvents removed in vacuo to afford a yellow solid. The crude product was purified by flash chromatography on silica gel (5Og IST Flash Si cartridge) eluting with a solvent gradient of 20 to 50% ethyl acetate in hexane. This affords product as a colourless solid (0.612 g; 53%)

LC retention time 2.756 minutes [M+H]⁺ 489/487 (Run time 3.75mins)

Step 3

2-Amino-4-(2,4-dichloro-5-hydroxy-phenyl)-thieno[2,3-d]pyrimidine-6- carboxylic acid isopropylamide

Prepared as for step 5 example 1 from 2-Amino-4-(5-benzyloxy-2,4-dichloro- phenyl)-thieno[2,3-d]pyrimidine-6-carboxylic acid isopropylamide (0.594 g). Product was purified by flash chromatography on silica gel (2Og IST Flash Si cartridge) eluting with a solvent gradient of 20 to 100% ethyl acetate in hexane. This affords product as a colourless solid (0.350 g; 72%)

LC retention time 2.353 minutes [M+H]⁺ 399/397 (Run time 3.75mins)

Step 4 2-Amino-4-[2,4-dichloro-5-(2-methoxy-ethoxy)-phenyl]-thieno[2,3- d]pyrimidine-6-carboxylic acid isopropylamide

Cesium carbonate (0.255 g; 0.78 mmol) was added to a stirred solution of 2- amino-4-(2,4-dichloro-5-hydroxy-phenyl)-thieno[2,3-d]pyrimidine-6-carboxylic acid isopropylamide (0.155 g; 0.39 mmol) in DMF. 2-Bromoethyl methyl ether (0.044 ml; 0.47 mmol) was then added and the reaction mixture heated to 140 °C (oil bath temperature) for 1.5 hours. The reaction mixture was allowed to cool to room temperature and DMF was removed in vacuo. The residue was partitioned between ethyl acetate (100 ml) and water (100 ml). The phases were separated and the organic phase was washed with saturated aqueous sodium chloride solution (100 ml) then dried over anhydrous sodium sulphate, filtered and the filtrate solvents removed in vacuo to afford a brown. The crude product was purified by flash chromatography on silica gel (10g Flash Si cartridge) eluting with 30% ethyl acetate in hexanes followed by 50% ethyl acetate in hexanes. This affords product as a pale yellow solid (0.294 g; 17%) LC retention time 2.502 minutes [M+H]⁺ 457/454 (Run time 3.75mins)

¹H NMR (400MHz, d6-DMSO): δ = 1.11 (6H, d, J = 6.6Hz), 3.32 (3H, s), 3.67 (2H, t, J = 4.5Hz), 4.01 (1 H, m, J = 6.02), 4.22 (2H, t, J = 4.5Hz), 7.26 (2H, br s), 7.38 (1 H, s), 7.61 (1 H, s), 7.84 (1 H, s), 8.34 (1 H, d, J = 7.7).

Example 3

2-Amino-4-[2,4-dfchloro-5-(2-diethylamino-ethoxy)-phenyl]-thieno[2,3- d]pyrimidine-6-carboxylic acid (2,2,2-trifluoro-ethyl)-amide

Step i

2-Amino-4-(2,4-dichloro-5-hydroxy-phenyl)-thieno[2,3-d]pyrimidine-6- carboxylic acid (2,2,2-trifluoro-ethyl)-amide

This compound was synthesized by following scheme 1 and example 2 (steps 1 , 2, 3) using 2,2,2-trifuoroethylamine for the amide forming reaction of example 2, step 2. The product was obtained as a cream-coloured solid.

LC retention time 2.393 minutes; [M+H]⁺ 439/437 (Run time 3.75mins)

Step 2

2-Amino-4-[2,4-dichloro-5-(2-diethylamino-ethoxy)-phenyl]-thieno[2,3- d]pyrimidine-6-carboxylic acid (2,2,2-trifluoro-ethyl)-amide

This compound was prepared as for step 4 example 2 using 2-bromo~N,N- diethylamine hydrobromide for the alkylation. The crude product was purified by flash chromatography on silica gel (Phenomenex Strata SI-1) eluting with a mixture of 1 N ammonia in methanol solution : ethyl acetate (1 : 10). This afforded product as a colourless solid.

LC retention time 2.032 minutes [M+H]⁺ 538/536 (Run time 3.75mins)

Example 4

2-Amino-4-[2,4-dichloro-5-(3-dimethylamino-propoxy)-phenyl]-thieno[2,3- d]pyrimidine-6-carboxylic acid (2,2,2-tπfluoro-ethyl)-amide

This compound was synthesized by following methods described for scheme 1 and reacting 2-Amino-4-(2,4-dichloro-5-hydroxy-phenyl)-thieno[2,3- d]pyrimidine-6-carboxylic acid (2,2,2-trifluoro-ethyl)-amide (step 1 example 3) with 3-dimethylamino-1-propanol using method of step 6 example 1.

The crude product was purified by flash chromatography on silica gel, eluting with 10% methanol in dichloromethane. This afforded product as a colourless solid.

LC retention time 1.947 minutes [M+H]⁺ 524/521 (Run time 3.75mins) This compound had activity A in the fluorescence polarization assay described below.

Example 5

Z-Amino^-^^-dichloro-S^-pyrrolidin-i-yl-ethoxyJ-phenyll-thieno^.S- d]pyrimidine-6-carboxylic acid cyclopropylamide

Step i

2-Amino-4-(2,4-dichloro-5-hydroxy-phenyl)-thieno[2,3-d]pyrimidine-6- carboxylic acid cyclopropylamide

This compound was synthesized by following the methods described for scheme 1 and example 2 (steps 1 , 2, 3) using cyclopropylamine for the amide forming reaction (example 2, step 2). The product was obtained as a pale yellow coloured solid following trituration with diethyl ether.

LC retention time 2.201 minutes [M+H]⁺ 397/395 (Run time 3.75mins)

Step 2 2-Amino-4-[2,4-dichloro-5-(2-pyrrolidin-1-yl-ethoxy)-phenyl]-thieno[2,3- d]pyrimidine-6-carboxylic acid cyclopropylamide

This compound was prepared using the methods of example 2 step 4 using 2- Amino-4-(2,4-dichloro-5-hydroxy-phenyl)-thieno[2,3-d]pyrimidine-6-carboxylic acid cyclopropylamide and 1-(2-chloroethyl)pyrrolidine for the alkylation. The crude product was purified by flash chromatography on silica gel (Phenomenex Strata SI-1 ) eluting with a mixture of 1 N ammonia in methanol solution : ethyl acetate (1 : 10). This afforded product as a light-brown solid.

LC retention time 1.870 minutes [M+H]⁺ 494/492 (Run time 3.75mins)

Example 6

2-Amino-4-[2,4-dichloro-5-(2-hydroxy-ethoxy)-phenyl]-thieno[2,3- d]pyrimidine-6-carboxylic acid cyclopropylamide

This compound was prepared using the methods of example 5 using -Amino-

4-(2,4-dichloro-5-hydroxy-phenyl)-thieno[2,3-d]pyrimidine-6-carboxylic acid cyclopropylamide (step 1 example 5) and 2-bromoethanol. The crude product was purified by flash chromatography on silica gel (Phenomenex Strata SI-1 ) eluting with ethyl acetate to afford product as a colourless solid.

LC retention time 2.138 minutes [M+H]⁺ 441/439 (Run time 3.75mins)

Example 7

2-Amino-4-[2,4-dichloro-5-(3-piperidin-1-yl-propoxy)-phenyl]-thieno[2,3- d]pyrimidine-6-carboxyIic acid βthylamide

This compound was prepared following the methods of scheme 1 using 1-(3- chloropropyl)-piperidine and 2-Amino-4-(2,4-dichloro-5-hydroxy-phenyl)- thieno[2,3-d]pyrimidine-6-carboxylic acid ethyl amide (step 5 example 1). The crude product was purified by flash chromatography on silica gel, eluting with 10% methanol in dichloromethane.

LC retention time 1.880 minutes [M+H]⁺ 510/508 (Run time 3.75mins)

Example 8 2-Amino-4-{2,4-dichloro-5-[2-(1-methyl-pyrrolidin-2-yl)-ethoxy]-phenyl}- thieno[2,3-d]pyrimidine-6-carboxylic acid ethylamide

This compound was prepared following the methods of scheme 1 and example 2 using 2-(2-chloroethyl)-1-methylpyrrolidine and 2-Amino-4-(2,4- dichloro-δ-hydroxy-phenyO-thienoP.S-dJpyrimidine-δ-carboxylic acid ethyl amide (step 5 example 1 ) for the alkylation. The crude product was purified by flash chromatography on silica gel, eluting with 10% methanol in dichloromethane.

LC retention time 1.843 minutes [M+H]⁺ 496/494 (Run time 3.75mins)

Example 9

2-Amino-4-[2,4-dichloro-5-(1-methyl-piperidin-4-yloxy)-phenyl]- thieno[2,3-d]pyrimidine-6-carboxylic acid ethylamide

This compound was prepared following the methods of scheme 1 using 4- chloro-1 -methylpiperidine and 2-Amino-4-(2,4-dichloro-5-hydroxy-phenyl)- thieno[2,3-d]pyrimidine-6-carboxylic acid ethyl amide (step 5 example 1 ). The crude product was purified by flash chromatography on silica gel eluting with 10-15% methanol in dichloromethane.

LC retention time 1.846 minutes [M+H]⁺ 480/482 (Run time 3.75mins)

Example 10

2-Amino-4-[2,4-dichloro-5-(1-ethyl-pyrrolidin-3-yloxy)-phenyl]-thieno[2,3- d]pyrimidine-6-carboxylic acid ethylamide

This compound was prepared following the methods of scheme 1 and using 1- ethyl-2-pyrrolidinol (racemic) and 2-Amino-4-(2,4-dichloro-5-hydroxy-phenyl)- thieno[2,3-d]pyrimidine-6-carboxylic acid ethyl amide (step 5 example 1 ). The crude product was purified by flash chromatography on silica gel eluting with 10% methanol in dichloromethane to afford product as a pale yellow solid.

LC retention time 1.831 minutes [M+H]⁺ 480/482 (Run time 3.75mins)

Example 11

2-Amino-4-[2,4-dichIoro-5-(3-dlethyIamino-propoxy)-phenyl]-thieno[2,3- d]pyrimidine-6-carboxylic acid ethylamide

This compound was prepared following the methods of scheme 1 and using 1- ethyl-2-pyrrolidinol and 2-Amino-4-(2,4-dichloro-5-hydroxy-phenyl)-thieno[2,3- d]pyrimidine-6-carboxy!ic acid ethyl amide (step 5 example 1). The crude product was purified by flash chromatography on silica gel eluting with 10% methanol in dichloromethane to afford product as a pale yellow solid.

LC retention time 2.202 minutes [M+H]⁺ 498/496 (Run time 3.75mins)

Fluorescence Polarization Assay

Fluorescence polarization {also known as fluorescence anisotropy} measures the rotation of a fluorescing species in solution, where the larger molecule the more polarized the fluorescence emission. When the fluorophore is excited with polarized light, the emitted light is also polarized. The molecular size is proportional to the polarization of the fluorescence emission. The fluoroscein-labelled probe - VER00051001 -FAM -

binds to HSP90 { full-length human, full-length yeast or N-terminal domain HSP90 } and the anisotropy {rotation of the probe:protein complex} is measured.

Test compound is added to the assay plate, left to equilibrate and the anisotropy measured again. Any change in anisotropy is due to competitive binding of compound to HSP90, thereby releasing probe.

Materials

Chemicals are of the highest purity commercially available and all aqueous solutions are made up in AR water.

1 ) Costar 96-well black assay plate #3915

2) Assay buffer of (a)100mM Tris pH7.4; (b) 2OmM KCI; (c) 6mM MgCI₂. Stored at room temperature.

3) BSA (bovine serum albumen) 10 mg/ml (New England Biolabs # B9001S)

4) 20 mM probe in 100 % DMSO stock concentration. Stored in the dark at RT. Working concentration is 200 nM diluted in AR water and stored at 4 ⁰C. Final concentration in assay 80 nM.

5) E. coli expressed human full-length HSP90 protein, purified >95% (see, e.g., Panaretou et al., 1998) and stored in 50μL aliquots at -8O⁰C .

Protocol 1 ) Add 100μMx buffer to wells 11A and 12A (=FP BLNK)

2) Prepare assay mix - all reagents are kept on ice with a lid on the bucket as the probe is light-sensitive.

i. Final Cone"

• 1x Hsp90 FP Buffer 10 ml 1x

• BSA 10mg/ml (NEB) 5.0 μl 5 μg/ml

• Probe 200μM 4.0 μl 80 nM

• Human full-length Hsp90 6.25 μl 200 nM

3) Aliquot 100μl assay mix to all other wells

4) Seal plate and leave in dark at room temp for 20 minutes to equilibrate

Compound Dilution Plate - 1 x 3 dilution series

1) In a clear 96-well v-bottom plate - {# VWR 007/008/257} add 10 μl 100% DMSO to wells B1 to H11

2) To wells A1 to A11 add 17.5μl 100% DMSO

3) Add 2.5 μl cpd to A1. This gives 2.5 mM {50x} stock cpd - assuming cpds 20 mM.

4) Repeat for wells A2 to A10. Control in columns 11 and 12.

5) Transfer 5 μl from row A to row B- not column 12. Mix well.

6) Transfer 5 μl from row B to row C. Mix well.

7) Repeat to row G.

8) Do not add any compound to row H - this is the 0 row.

9) This produces a 1x3 dilution series from 50 μM to 0.07 μM. 1O)In well B12 prepare 20 μl of 100 μM standard compound.

11 )After first incubation the assay plate is read on a Fusion™ α-FP plate reader (Packard BioScience, Pangbourne, Berkshire, UK).

12)After the first read, 2 μl of diluted compound is added to each well for columns 1 to 10. In column 11 {provides standard curve} only add compound B11 - H11. Add 2 μl of 10OmM standard cpd to wells B12 - H12 {is positive control } 13)The Z' factor is calculated from zero controls and positive wells. It typically gives a value of 0.7 - 0.9.

The compounds tested in the above assay were assigned to one of two activity ranges, namely A = <10μM; B = >10μM, and those assignments are reported above.

A growth inhibition assay was also employed for the evaluation of candidate HSP90 inhibitors:

Assessment of cytotoxicity by Sulforhodamine B (SRB) assay: calculation of 50% inhibitory concentration (ICso).

Day i

1 ) Determine cell number by haemocytometer.

2) Using an 8 channel multipipettor, add 160μl of the cell suspension (3600 cells/well or 2 x 10⁴ cells/ml) to each well of a 96-well microtitre plate.

3) Incubate overnight at 37⁰C in a CO₂ incubator.

Day 2

4) Stock solutions of drugs are prepared, and serial dilutions of each drug are performed in medium to give final concentrations in wells.

5) Using a multipipettor, 40μl of drug (at 5x final concentration) is added to quadruplicate wells.

6) Control wells are at either side of the 96 well plates, where 40μl of medium is added.

7) Incubate plates in CO₂ incubator for 4 days (48 hours).

Day 6 8) Tip off medium into sink and immerse plate slowly into 10% ice cold trichloroacetic acid (TCA). Leave for about 30mins on ice.

9) Wash plates three times in tap water by immersing the plates into baths of tap water and tipping it off.

10)Dry in incubator.

11 )Add 100μl of 0.4% SRB in 1 %acetic acid to each well (except the last row

(right hand)of the 96 well plate, this is the 0% control, ie no drug, no stain.

The first row will be the 100% control with no drug, but with stain). Leave for 15 mins.

12)Wash off unbound SRB stain with four washes of 1 % acetic acid. 13)Dry plates in incubator. 14)Solubilise SRB using 100μl of 1OmM Tris base and put plates on plate shaker for 5 mins. 15)Determine absorbance at 540nm using a plate reader. Calculate mean absorbance for quadruplicate wells and express as a percentage of value for control, untreated wells. 16)Plot % absorbance values versus log drug concentration and determine the IC₅₀.

By way of illustration, the compound of Example 2 gave an IC50 in the 'A¹ range (<50uM) for the SRB growth arrest assay.

REFERENCES

A number of publications are cited above in order to more fully describe and disclose the invention and the state of the art to which the invention pertains. Full citations for these references are provided below. Each of these references is incorporated herein by reference in its entirety into the present disclosure.

Argon Y and Simen BB. 1999 "Grp94, an ER chaperone with protein and peptide binding properties", Semin. Cell Dev. Biol., Vol. 10, pp. 495-

505. Conroy SE and Latchman DS. 1996 "Do heat shock proteins have a role in breast cancer?", Brit. J. Cancer, Vol. 74, pp. 717-721. Felts SJ, Owen BAL, Nguyen P, Trepel J, Donner DB and Toft DO. 2000 "The

HSP90-related protein TRAP1 is a mitochondrial protein with distinct functional properties", J. Biol. Chem.. Vol. 5, pp. 3305-3312. Hickey E, Brandon SE, Smale G, Lloyd D and Weber LA. 1999 "Sequence and regulation of a gene encoding a human 89-kilodalton heat shock protein", MoI. Cell. Biol., Vol. 9, pp. 2615-2626. Hoang AT, Huang J, Rudra-Gonguly N, Zheng J, Powell WC, Rabindron SK,

Wu C and Roy-Burman P. 2000 "A novel association between the human heat shock transcription factor I (HSF1) and prostate adenocarcinoma, Am. J. Pathol., Vol. 156, pp. 857-864. Jolly C and Morimoto Rl. 2000 "Role of the heat shock response and molecular chaperones in oncogenesis and cell death", J. Natl. Cancer

Inst, Vol. 92, pp. 1564-1572. Kawanishi K, Shiozaki H, Doki Y, Sakita I, lnoue M, Yano M, Tsujinata T,

Shamma A and Monden M. 1999 "Prognostic significance of heat shock proteins 27 and 70 in patients with squamous cell carcinoma of the esophagus", Cancer. Vol. 85, pp. 1649-1657. Lebeau J, Le Cholony C, Prosperi MT and Goubin G. 1991 "Constitutive overexpression of 89 kDa heat shock protein gene in the HBL100 mammary cell line converted to a tumorigenic phenotype by the EJ/T24

Harvey-ras oncogene", Oncogene, Vol. 6, pp. 1125-1132. Martin KJ, Kritzman BM, Price LM, Koh B, Kwan CP, Zhang X, MacKay A,

OΗare MJ, Kaelin CM, Mutter GL, Pardee AB and Sager R. 2000

"Linking gene expression patterns to therapeutic groups in breast cancer", Cancer Res., Vol. 60, pp. 2232-2238. Panaretou B, Prodromou C, Roe SM, O'Brien R, Ladbury JE, Piper PW and

Pearl LH. 1998 "ATP binding and hydrolysis are essential to the function of the HSP90 molecular chaperone in vivo", EMBO J., Vol. 17, pp. 4829-4836. Pratt WB. 1997 "The role of the HSP90-based chaperone system in signal transduction by nuclear receptors and receptors signalling via MAP kinase", Annu. Rev. Pharmacol. Toxicol.. Vol. 37, pp. 297-326. Prodromou C and Pearl LH. 2000a "Structure and in vivo function of HSP90",

Curr. Qpin. Struct. Biol., Vol. 10, pp. 46-51. Prodromou C, Roe SM, O'Brien R, Ladbury JE, Piper PW and Pearl LH. 1997

"Identification and structural characterization of the ATP/ADP-binding site in the HSP90 molecular chaperone", CeH, Vol. 90, pp. 65-75. Prodromou C, Panaretou B, Chohan S, Siligardi G, O'Brien R, Ladbury JE, Roe SM, Piper PW and Pearl LH. 2000b "The ATPase cycle of HSP90 drives a molecular 'clamp' via transient dimerization of the N-terminal domains", EMBO J., Vol. 19, pp. 4383-4392. Rutherford SL and Lindquist S. 1998 "HSP90 as a capacitor for morphological evolution. Nature, Vol. 396, pp. 336-342. Smith DF. 2001 "Chaperones in signal transduction", in: Molecular chaperones in the cell (P Lund, ed.; Oxford University Press, Oxford and NY), pp. 165-178. Smith DF, Whitesell L and Katsanis E. 1998 "Molecular chaperones: Biology and prospects for pharmacological intervention", Pharmacological

Reviews, Vol. 50, pp. 493-513. Song HY, Dunbar JD, Zhang YX, Guo D and Donner DB. 1995 "Identification of a protein with homology to hsp90 that binds the type 1 tumour necrosis factor receptor", J. Biol. Chem., Vol. 270, pp. 3574-3581. Tytell M and Hooper PL. 2001 "Heat shock proteins: new keys to the development of cytoprotective therapies", Emerging Therapeutic Targets, Vol. 5, pp. 267-287.

Young JC, Moarefi I and Haiti FU. 2001 "HSP90: a specialized but essential protein-folding tool", J. Cell. Biol., Vol. 154, pp. 267-273.

Claims

Claims:

1. A compound selected from the group consisting of

2-amino-4-[2,4-dichloro-5-(3-morpholin~4-yl-propoxy)-phenyl]- thieno[2,3-d]pyrimidine-6-carboxylic acid ethylamide,

2-amino-4-{2,4-dichIoro-5-[2-(1-methyl-pyrrolidin-2-yl)-ethoxy]-phenyl}- thieno[2,3-d]pyrimidine-6-carboxylic acid ethylamide,

2-amino-4-[2,4-dichloro-5-(1-ethyl-pyrrolidin-3-yloxy)-phenyl]- thieno[2,3-d]pyrimidine-6-carboxylic acid ethylamide, and 2-amino-4-[2,4-dichloro-5-(3-diethylamino-propoxy)-phenyl]-thieno[2,3- d]pyrimidine-6-carboxylic acid ethylamide,

and salts, N-oxides, hydrates, and solvates thereof.

2. A method of treatment of diseases which are responsive to inhibition of HSP90 activity in mammals, which method comprises administering to the mammal an amount of a compound as claimed in claim 1 effective to inhibit said HSP90 activity.

3. The medicinal use of a compound as claimed in claim 1.

4. The use of a compound as claimed in claim 1 for inhibition of HSP90 activity in vitro or in vivo.

5. The use of a compound as claimed in claim 1 in the preparation of a composition for the treatment of diseases in which HSP90 activity is implicated; and

6. A pharmaceutical composition comprising a compound as claimed in claim 1 and a pharmaceutically acceptable carrier.