ZA200601715B

ZA200601715B - Pyrimidothiophene compounds

Info

Publication number: ZA200601715B
Application number: ZA200601715A
Authority: ZA
Inventors: Dymock Brian; Fromont Christofe; Barril-Alonso Xavier; Drysdale Martin James; Jordan Allan
Original assignee: Vernalis Cambridge Ltd; Cancer Rec Tech Ltd; Cancer Res Inst
Priority date: 2003-08-29
Filing date: 2006-02-27
Publication date: 2007-05-30
Also published as: CN1842532A; CN100455586C; GB0320300D0

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 HSPS0 in various critical oncogenic pathways and the discovery that certain natural products with anticancer activity are targeting this molecular chaperone, the fascinating new concept has been developed that inhibiting HSP function may be useful 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 ane 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, HSPS0 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 HSPQ0 family in humans consists of four genes, namely the cytosolic HSP90a and HSP90B 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 TRAP1 (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 HSPS0 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, 2000Db).

Known HSPS0 Inhibitors

The first class of HSP90 inhibitors to be discovered was the benzoquinone ansamycin class, which includes the compounds herbimycin A and geldanamycin. They were shown to reverse the malignant phenotype of fibroblasts transformed by the v-Src oncogene (Uehara et al., 1985), and subsequently to exhibit potent antitumour activity in both in vitro (Schulte et al., 1998) and in vivo animal models (Supko et al., 1995).

Immunoprecipitation and affinity matrix studies have shown that the major mechanism of action of geldanamycin involves binding to HSP90 (Whitesell et al., 1994; Schulte and Neckers, 1998). Moreover, X-ray crystallographic studies have shown that geldanamycin competes at the ATP binding site and inhibits the intrinsic ATPase activity of HSP90 (Prodromou et al., 1997,

Panaretou et al., 1998). This in turn prevents the formation of mature multimeric HSP90 complexes capable of chaperoning client proteins. As a result, the client proteins are targeted for degradation via the ubiquitin proteasome pathway. 17-Allylamino, 17-demethoxygeldanamycin (17AAG) retains the property of HSP90 inhibition resulting in client protein depletion and antitumour activity in cell culture and xenograft models (Schulte et al, 1998; Kelland et al, 1999), but has significantly less hepatotoxicity than geldanamycin (Page et al, 1997). 17AAG is currently being evaluated in

Phase | clinical trials.

Radicicol is a macrocyclic antibiotic shown to reverse the malignant phenotype of v-Src and v-Ha-Ras transformed fibroblasts (Kwon et al, 1992;

Zhao et al, 1995). It was shown to degrade a number of signalling proteins as a consequence of HSP90 inhibition (Schulte et al., 1998). X-ray crystallographic data confirmed that radicicol also binds to the N terminal domain of HSPQ0 and inhibits the intrinsic ATPase activity (Roe et al., 1998).

Radicicol lacks antitumour activity in vivo due to the unstable chemical nature of the compound.

Coumarin antibiotics are known to bind to bacterial DNA gyrase at an ATP binding site homologous to that of the HSP90. The coumarin, novobiocin, was shown to bind to the carboxy terminus of HSPOO, i.e., at a different site to that occupied by the benzoquinone ansamycins and radicicol which bind at the N-terminus (Marcu et al., 2000b). However, this still resulted in inhibition of HSPS0 function and degradation of a number of HSP90-chaperoned signalling proteins (Marcu et al., 2000a). Geldanamcyin cannot bind HSPS0 subsequent to novobiocin; this suggests that some interaction between the N and C terminal domains must exist and is consistent with the view that both sites are important for HSP90 chaperone properties.

A purine-based HSP90 inhibitor, PU3, has been shown to result in the degradation of signalling molecules, including ERBB2, and to cause cell cycle arrest and differentiation in breast cancer cells (Chiosis et al., 2001).

Patent publications WO 2004/050087 and WO 2004/056782 relate to known classes pyrazole derivatives which are HSP90 inhibitors.

HSP90 as a Therapeutic Target

Due to its involvement in regulating a number of signalling pathways that are crucially important in driving the phenotype of a tumour, and the discovery that certain bioactive natural products exert their effects via HSP90 activity, the molecular chaperone HSP90 is currently being assessed as a new target for anticancer drug development (Neckers et al., 1999).

The predominant mechanism of action of geldanamycin, 17AAG, and radicicol involves binding to HSPS0 at the ATP binding site located in the N-terminal domain of the protein, leading to inhibition of the intrinsic ATPase activity of

HSP90 (see, e.g., Prodromou et al., 1997; Stebbins et al., 1997; Panaretou et al., 1998). inhibition of HSP90 ATPase activity prevents recruitment of co-chaperones and encourages the formation of a type of HSP90 heterocomplex from which these client proteins are targeted for degradation via the ubiquitin proteasome pathway (see, e.g., Neckers et al., 1999; Kelland et al., 1999).

Treatment with HSPQ0 inhibitors leads to selective degradation of important proteins involved in cell proliferation, cell cycle regulation and apoptosis, processes which are fundamentally important in cancer.

Inhibition of HSPYO function has been shown to cause selective degradation of important signalling proteins involved in cell proliferation, cell cycle regulation and apoptosis, processes which are fundamentally important and which are commonly deregulated in cancer (see, e.g., Hostein et al., 2001).

An attractive rationale for developing drugs against this target for use in the clinic is that by simultaneously depleting proteins associated with the transformed phenotype, one may obtain a strong antitumour effect and achieve a therapeutic advantage against cancer versus normal cells. These events downstream of HSP90 inhibition are believed to be responsible for the antitumour activity of HSP90 inhibitors in cell culture and animal models (see, e.g., Schulte et al., 1998; Kelland et al., 1999).

Brief description of the invention

The present invention relates to the use of a class of substituted thieno[2,3- d]pyrimidine compounds (referred to herein as pyrimidothiophenes) as HSP90 inhibitors, for example for inhibition of cancer cell proliferation. A core pyrimidothiophene ring with aromatic substitution on one ring carbon atom are principle characterising features of the compounds with which the invention is concerned.

Detailed description of the invention

In one broad aspect the present invention provides the use of a compound of formula (1), or a salt, N-oxide, hydrate, or solvate thereof in the preparation of a composition for inhibition of HSPYO activity in vitro or in vivo:

NH, oy

CY y v

Ry R, wherein

R, is a group of formula (lA): (AMA) (2)-(AI®)s-Q (IA) wherein

Ar! is an optionally substituted aryl or heteroaryl radical,

Alk! and Alk? are optionally substituted divalent C+-C3 alkylene or C»-C; alkenylene radicals, m, p, r and s are independently 0 or 1,

Z is —O-, -S-, -(C=0)-, -(C=8)-, -802-, -C(=0)0-, -C(=O)NR*-, -C(=S)NRA-, -SO,NR™-, -NRAC(=0)-, -NRSO>- or ~NRA- wherein R* is hydrogen or C-Cs alkyl, and

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

Rs is hydrogen, an optional substituent, or an optionally substituted (C4-

Ce)alkyl, aryl or heteroaryl radical; and

R, is a carboxylic ester, carboxamide or sulfonamide group.

In another broad aspect, the invention provides a method of treatment of diseasess which are responsive to inhibition of HSP90 activity in mammals, : which method comprises administering to the mammal an amount of a compound as defined in claim 1 effective to inhibit said HSPO0 activity.

The in vivo use, and method, of the invention is 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 | 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 Hsp70 in the heart and brain; scrapie/CJD, Huntingdon’s or Alzheimer’s disease. Use for the treatment of cancer is especially indicated.

The publications WO 01/62233, Transition Metal Chemistry Vol. 19, 1994, pages 335-339, Journal of Heterocyclic Chemistry Vol. 30, 1993, pages 1065-

1072, and Synthesis No. 5, 1983, pages 402-404, disclose specific compounds falling within formula (I) above, or relate to compound classes which encompass some compounds of formula (I). However, the majority of the compounds of formula (1) with which the above broad aspects of the invention are concerned are believed novel in their own right. The invention includes such novel compounds, and in particular compounds of formula (1), and salts, N-oxides, hydrates, or solvates thereof:

NH, oy

Be, 0

Rs R, wherein

R2 is a group of formula (IA):

CAMA )p-@)-(AK?)-Q (1A) wherein

Ar' is an optionally substituted aryl or heteroaryl radical,

Alk! and Alk? are optionally substituted divalent C4-C; alkylene or C.-C alkenylene radicals, m, p, r and s are independently 0 or 1,

Z is —O-, -S-, -(C=0)-, (C=S)-, -SOs-, -C(=0)0-, -C(=O)NR"-, -C(=8)NR"-, -SO:NR*-, -NR*C(=0)-, -NR*SO, or -NR"- wherein R* is hydrogen or C4-Cs alkyl, and

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

R; is hydrogen, an optional substituent, or an optionally substituted (C4-

Ce)alkyl, aryl or heteroaryl radical; and

R, is a carboxylic ester, carboxamide or sulfonamide group,

PROVIDED THAT (i) R3 is not —NH; or (ii) when R4 is ~-COOCH3 and Rs is hydrogen then R; is not ethylamino, diethylamino, phenylamino or

-N(Ph)(C,Hs) wherein Ph is phenyl.

As used herein: the term "carboxyl group” refers to a group of formula -COOH; the term "carboxy! ester group” refers to a group of formula -COOR, wherein R is a radical actually or notionally derived from the hydroxyl compound ROH; and the term "carboxamide group” refers to a group of formula ~-CONRRb, wherein -NRgRy, is an amino (including cyclic amino) group actually or notionally derived from ammonia or the amine HNR;Re. the term "sulfonamide group" refers to a group of formula -SO:NR2Ry, wherein -NRgRy, is an amino (including cyclic amino) group actually or notionally derived from ammonia or the amine HNR;R;

As used herein, the term “(C4-Cp)alkyl” wherein a and b are integers refers to a straight or branched chain alkyl radical having from a to b carbon atoms.

Thus when a is 1 and b is 6, for example, the term includes methyl, ethyl, n- propyl, isopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, n-pentyl and n-hexyl.

As used herein the term "divalent (C,-Cp)alkylene radical" wherein a and b are integers refers to a saturated hydrocarbon chain having from a to b carbon atoms and two unsatisfied valences.

As used herein the term “(C,-Cp)alkenyl“ wherein a and b are integers refers to a straight or branched chain alkenyl moiety having from a to b carbon atoms having at least one double bond of either E or Z stereochemistry where applicable. The term includes, for example, vinyl, allyl, 1- and 2-butenyl and 2-methyl-2-propenyl.

As used herein the term "divalent (Ca-Co)alkenylene radical” refers to a hydrocarbon chain having from a to b carbon atoms, at least one double bond, and two unsatisfied valences.

As used herein the term "cycloalkyl" refers to a saturated carbocyclic radical having from 3-8 carbon atoms and includes, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl.

As used herein the term "cycloalkenyl" refers to a carbocyclic radical having from 3-8 carbon atoms containing at least one double bond, and includes, for example, cyclopentenyl, cyclohexenyl, cycloheptenyl and cyclooctenyl.

As used herein the term "aryl" refers to a mono-, bi- or tri-cyclic carbocyclic aromatic radical. Wustrative of such radicals are phenyl, biphenyl and napthyl.

As used herein the term "carbocyclic" refers to a cyclic radical whose ring atoms are all carbon, and includes monocyclic aryl, cycloalkyl, and cycloalkenyl radicals.

As used herein the term "heteroaryl" refers to a mono-, bi- or tri-cyclic aromatic radical containing one or more heteroatoms selected from S, N and

O. lllustrative of such radicals are thienyl, benzthienyl, furyl, benzfuryl, pyrrolyl, imidazolyl, benzimidazolyl, thiazolyl, benzthiazolyl, isothiazolyl, benzisothiazolyl, pyrazolyl, oxazolyl, benzoxazolyl, isoxazolyl, benzisoxazolyl, isothiazolyl, triazolyl, benztriazolyl, thiadiazolyl, oxadiazolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, indolyl and indazolyl.

As used herein the unqualified term "heterocyclyl" or "heterocyclic" includes "heteroaryl" as defined above, and in particular refers to a mono-, bi- or tri- eyclic non-aromatic radical containing one or more heteroatoms selected from 8, N and O, and to groups consisting of a monocyclic non-aromatic radical containing one or more such heteroatoms which is covalently linked to another such radical or to a monocyclic carbocyclic radical. Illustrative of such radicals are pyrrolyl, furanyl, thienyl, piperidinyl, imidazolyl, oxazolyl,

isoxazolyl, thiazolyl, thiadiazolyl, pyrazolyl, pyridinyl, pyrrolidinyl, pyrimidinyl, morpholinyl, piperazinyl, indolyl, morpholinyl, benzfuranyl, pyranyl, isoxazolyl, benzimidazolyl, methylenedioxyphenyl, ethylenedioxyphenyl, maleimido and succinimido groups.

Unless otherwise specified in the context in which it occurs, the term "substituted" as applied to any moiety herein means substituted with at least one substituent, for example selected from (C4-Ceg)alkyl, (C1-Cs)alkoxy, hydroxy, hydroxy(C+-Ce)alkyl, mercapto, mercapto(C4-Ce)alkyl, (C1- ~~ Ce)alkylthio, halo (including fluoro and chloro), trifluoromethyl, trifluoromethoxy, nitro, nitrile (-CN), oxo, phenyl, -COOH, -COOR"®, -CORA,

SORA, -CONH, -S02NH,, -CONHR?, -SO.NHR?, -CONR"R®, -SO,NR"R®, -NHa, -NHR?, -NR*R, -OCONH,, -OCONHR" , -OCONR?R®, -NHCOR?, -NHCOOR", -NRECOORA, -NHS0,0R”, -NR®SO,0R”, -NHCONH;, -NRACONH,, -NHCONHR® -NRACONHRE, -NHCONR*R® or -NR*CONR*R® wherein R* and R® are independently a (C4-Cg)alkyl group. An “optional substituent” may be one of the foregoing substituent groups.

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 concemed 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 Ror S stereochemistry at each chiral axis. The invention includes all such enantiomers and diasterecisomers and mixtures thereof

So-called ‘pro-drugs’ of the compounds of formula (i) are also within the scope of the invention. Thus certain derivatives of compounds of formula (1) which may have little or no pharmacological activity themselves can, when administered into or onto the body, be converted into compounds of formula (1) having the desired activity, for example, by hydrolytic cleavage. Such derivatives are referred to as ‘prodrugs’. Further information on the use of prodrugs may be found in Pro-drugs as Novel Delivery Systems, Vol. 14, ACS

Symposium Series (T. Higuchi and W. Stella) and Bioreversible Carriers in

Drug Design, Pergamon Press, 1987 (ed. E. B. Roche, American

Pharmaceutical Association).

Prodrugs in accordance with the invention can, for example, be produced by replacing appropriate functionalities present in the compounds of formula (1 with certain moieties known to those skilled in the art as ‘pro-moieties’ as described, for example, in Design of Prodrugs by H. Bundgaard (Elsevier, 1985).

Also included within the scope of the invention are metabolites of compounds of formula (1), that is, compounds formed in vivo upon administration of the drug. Some examples of metabolites include

(i) where the compound of formula (1) contains a methyl group, an hydroxymethyl derivative thereof (-CH3 -> -CH-OH): (ii) where the compound of formula (1) contains an alkoxy group, an hydroxy derivative thereof (-OR -> -OH); (iii) where the compound of formula (I) contains a tertiary amino group, a secondary amino derivative thereof (-NR'R? -> -NHR" or -NHR?); (iv) where the compound of formula (I) contains a secondary amino group, a primary derivative thereof (-NHR'-> -NH); (v) where the compound of formula (I) contains a phenyl moiety, a phenol derivative thereof (-Ph -> -PhOH); and (vi) where the compound of formula (I) contains an amide group, a carboxylic acid derivative thereof (-CONH, -> COOH).

The radical Ro

As stated, R; is a group of formula (IA): (AMm-(AKYe-@2)-(Ak)-Q (IA) wherein in any compatible combination Ar! is an optionally substituted aryl or heteroaryl radical, Alk' and Alk? are optionally substituted divalent C+-Cs alkylene or C,-C; alkenylene radicals, m, p, r and s are independently 0 or 1,

Z is —=O-, -S-, -(C=0)-, -(C=S)-, -SO,-, -C(=0)0-, -C(=0)NR"- , -C(=S)NR*-, -SO,NRA-, -NRAC(=0)-, -NRAS0,- or -NR*- wherein R* is hydrogen or C4-Cg alkyl, and Q is hydrogen or an optionally substituted carbocyclic or heterocyclic radical

When present in the radical Rg,

Ar’ may be, for example, a phenyl, cyclohexyl, pyridyl, morpholino, piperidiny! or piperazinyl ring. Presently it is preferred that Ar’, when present, by a phenyl ring;

Alk' and Al? may be, for example, optionally substituted divalent radicals selected from ~CHa, CH2CHz- or —-CH=CH-. Optional substituents in Alk! and Alk? include, for example mono- or di(C+-

Csalkyl)amino and C4-Csalkoxy; and

Z may be, for example, —O- or -NH-; and Q is hydrogen.

In a simple subclass of compounds with which the invention is concemed, m is 1 and each of p, rand s is 0, and Q is hydrogen, so that Rz is optionally substituted ary! or heteroaryl. In such cases, Re may be, for example, optionally substituted phenyl, 2- or 3-thienyl, 2- or 3- furanyl, 2-, 3- or 4- pyridinyl, morpholinyl, or piperidinyl. Currently preferred are compounds wherein R; is optionally substituted phenyl, for example where the optional ; substituents are selected from methyl, ethyl, n- or isopropyl, vinyl, allyl, methoxy, ethoxy, n-propyloxy, benzyloxy, allyloxy, cyanomethoxy chloro, bromo, cyano, formyl, methyl-, ethyl-, or n-propyl-carbonyloxy, methyl- or ethylaminocarbonyl. More complex substituent groups which may be present in the R, ring include those (i) of formula -O(CH2)nZ" whereinnis 1,2 0r3 and Z! is a primary, secondary, tertiary or cyclic amino group, or a Cs-

Cealkoxy group; or (ii) of formula —(Al®)mZ' wherein Alk® is a divalent straight or branched chain (C4-C3) alkylene, mis 0 or 1, and Z' is a primary, secondary, tertiary or cyclic amino group, or a C4-Cgalkoxy group. Preferred substitution positions in the phenyl ring are positions 2, 4 and 5.

In other simple structures, m is 1, p, r and s are again each 0, and Q may be an optionally substituted carbocyclic or heterocyclic ring, for example phenyl, cyclohexyl, pyridyl, morpholino, piperidinyl, or piperazinyl ring. In such cases,

Q is a direct substituent in the optionally substituted Ar’ ring.

In more complex structures with which the invention is concerned, one or more of m, p, r and s may be 1, and Q may be hydrogen or an optionally substituted carbocyclic or heterocyclic ring. For example, p and/or s may be 1 and r may be 0, so that Q is linked to Ar! by an alkylene or alkenylene radical,

for example a C4-Cj3 alkylene radical, which is optionally substituted. in other cases each of p, r, and s may be 1, in which cases, Q is linked to Ar' by an alkylene or alkenylene radical which is interrupted by the hetero atom- containing Z radical. In still other cases, p and s may be 0 and r may be 1, in which case Q is linked to Ar via the hetero atom-containing Z radical.

Specific examples of Rz groups usable in compounds of the invention include those present in the compounds of the Examples herein.

The optional substituent Ry

Rj is hydrogen or an optional substituent, as defined above. Presently it is preferred that Rs be hydrogen.

The group Rs

When Ry is a carboxamide or sulfonamide group, examples include those of formula —CONR®(AIk),.R* or -SO,NR®(Ak),R* wherein

Alk is a divalent alkylene, alkenylene or alkynylene radical, for example a —CHy-, -CH,CH2-, -CH,CH,CHy-, ~CH,CH=CH-, or -CH,CCCH,- radical, and the Alk radical may be optionally substituted, nisQort,

R® is hydrogen or a C4-Cs alkyl or C»-Cs alkenyl group, for example methyl, ethyl, n- or iso-propyl, or allyl, Currently it is preferred that R® be hydrogen.

RA is an optional substituent such as hydroxyl, amino (including mono- and di-(C4-Cs)alkylamino), carbamoyl (-C(=0)NH,), -SO,0H, trifluoromethyl; or optionally substituted carbocyclic, for example cyclopropyl, cyclopentyl, cyclohexyl, phenyl optionally substituted by hydroxyl, amino, fluoro, chloro, bromo, 3,4 methylenedioxy, sulfamoy! (-SO2NH3), -SO,0H, methoxy, methylsulfonyl, trifuoromethyl,; or heterocyclyl, for example pyridyl, furyl, thienyl, diazolyl, N-piperazinyl,

pyrrolyl, tetrahydrofuranyl, thiazolyl, 1-aza-bicyclo[2,2,2]octanyl, or N- morpholinyl any of which heterocyclic rings may be substituted, for example on a ring nitrogen by (C4-Cz)alkyl, or R* and R® taken together with the nitrogen to which they are attached form an N-heterocyclic ring which may optionally contain one or more additional hetero atoms selected from O, S and N, and which may optionally be substituted on one or more ring C or N atoms, examples of such N-heterocyclic rings including morpholino, piperidinyl, piperazinyl and N-phenylpiperazinyl.

Presently it is preferred that Rs be a corboxamide group.

When Ry is a carboxylic ester group, examples include those of formula -COOR® wherein RC is a C;-Cs alkyl or C,-Cs alkeny! group, for example methyl, ethyl, n- or iso-propyl, or allyl; or an optionally substituted aryl or heteroaryl group, for example optionally substituted phenyl, pyridyl or thiazolyl; or an optionally substituted aryl(C4-Cg alkyl)- or heteroaryl(C+-Cs alkyl)- group such as benzyl or pyridylmethyl; or an optionally substituted cycloalkyl group such as cyclopentyl or cyclohexyl.

Specific examples of R4 groups usable in compounds of the invention include those present in the compounds of the Examples herein.

A preferred subclass of the compounds with which the invention is concerned has formula (11)

Ris

Riz

Rio (if) 2

XX

HNT NT °S A wherein

Ais a secondary amino group

Rio is H, Cl, Br, or CHa;

R11 is hydrogen, CI, Br, CN, methyl, ethyl, n- or iso-propyl, vinyl or allyl;

R42 is (i) a radical of formula -O(CHj)»Z' whereinn is 1,2or3 and Z'is a primary, secondary, tertiary or cyclic amino group, or a C4-Cgalkoxy group; or (ii) a radical of formula —(Alk®)»Z" wherein Alk® is a divalent straight or branched chain (C4-C3) alkylene, mis 0 or 1, and Z' is a primary, secondary, tertiary or cyclic amino group, or a C4-Cgalkoxy group. In this subclass of compounds (ll) it is preferred that A is a secondary C4-Csalkylamino group, for example wherein the C4-Cgalkyl substituent is selected from methyl, ethyl, and n- and iso-propyl, and Ryz is (i) a radical of formula -O(CH2).Z" wherein nis 1, 2or3and Z'is di(C4-Czalkyl)amino or C4-Csaikoxy, for example wherein the

C4-Csalkyl component(s) is/are selected from methyl, ethyl, and n- and iso- propyl.

Specific compounds with which the invention is concerned include those of the Examples, particularly those exemplified compounds which have structure (i) above.

There are multiple synthetic strategies for the synthesis of the compounds (1) with which the present invention is concerned, but all rely on known chemistry, known to the synthetic organic chemist. Thus, compounds according to formula (I) can be synthesised according to procedures described in the standard literature and are well-known to the one skilled in the art. Typical literature sources are “Advanced organic chemistry’, 4"

Edition (Wiley), J March, “Comprehensive Organic Transformation”, 2"

Edition (Wiley), R.C. Larock , “Handbook of Heterocyclic Chemistry’, 2™

Edition (Pergamon), A.R. Katritzky), review articles such as found in “Synthesis”, “Acc. Chem. Res.” , “Chem. Rev”, or primary literature sources identified by standard literature searches online or from secondary sources such as “Chemical Abstracts” or “Beilstein”. Such literature methods include those of the preparative Examples herein, and methods analogous thereto.

For example the following general reaction scheme can be employed:

NH, 1 Pi

A HS OR! NEN e.g. Suzuki NN

Cy CL oY coupling NC or Cl heat — or chloro =.

OR? (cyclisation) Rr? Rt displacement g° Re

Starting material are either available commercially or can be made according to literature methods. Subsequent reactions may be carried out on R? R3or

R* to prepare additional compounds of formula (1)

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);

Immunosupression such as in transplantation (Bijimakers, 2000 and Yorgin, 2000); Anti-inflammatory diseases (Bucci, 2000) such as Rheumatoid arthritis,

Asthma, MS, Type | 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, heurogenerative disorders such as scrapie/CJD, Huntingdon's and Alzheimer's (Sittler, 2001; Trazelt, 1995 and

Winkihofer, 2001)".

Accordingly, the invention also includes: (i) A pharmaceutical or veterinary composition comprising a compound of formula (I) above, together with a pharmaceutically or veterinarily acceptable carrier. (ii) The use of a compound a compound of formula (I) above in the preparation of a composition for composition for inhibition of HSP90 activity in vitro or in vivo. (iii). A method of treatment of diseases or conditions which are responsive to inhibition of HSP9O0 activity in mammals which method comprises administering to the mammal an amount of a compound of formula (1) above effective to inhibit said HSP90 activity.

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.

Example 1 2-Amino-4-phenyl-thieno[2,3-d]pyrimidine-6-carboxylic acid ethyl ester

NT J hey

HN" NT TS $

Step 1 2-Amino-4-chloro-thieno[2,3-d]pyrimidine-6-carboxylic acid ethyl ester

Cl

NT Oo [ons

HN™ NT 7S $

To a stirred mixture of 2-amino-4,6-dichloro-5-formyl-pyrimidine (1 eq.) and potassium carbonate (1 eq.) in acetonitrile at ambient temperature was added ethyl-2-mercaptoacetate (0.95 eq.) and the mixture stirred at ambient temperature for three hours, followed by heating at 80°C for one hour. After cooling, the mixture was concentrated to dryness in vacuo. Column chromtaography on silica, eluting with ethyl acetate and hexanes, gave example 1 as a yellow powder.

LC-MS retention time: 2.371 minutes, [M+H]* 258.0

Step 2 (Suzuki Reaction): 2-Amino-4-phenyl-thieno[2,3-d]pyrimidine-6-carboxylic acid ethyl ester ay

HNN S $

A solution of example 1 step 1 (1 eq.), phenyl boronic acid (1.2 eq) and sodium carbonate (1.2 eq) in 1,4-dioxane and water (3.5:1) were degassed by bubbling though nitrogen gas for 5 mins. Pd(PPhs)s (0.05 eq.) was added and the mixture heated in a Personal Chemistry Synthesiser microwave at 150 °C for 10 minutes. After cooling and concentration in vacuo, perparative HPLC gave example 2 as a white powder.

LC-MS retention time: 2.545 minutes, [M+H]" 300.10

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

Example 2: 2-Amino-4-(4-trifluoromethyl-phenyl)-thieno[2,3-d]pyrimidine-6- carboxylic acid ethyl ester

FOF

NTS 0

Q

HNT ONT 7S $ prepared as for Example 1.

LC-MS retention time: 2.768 minutes, [M+H]" 368.1

'H NMR (400MHz, d6-DMSO): § = 1.07 (3H, t, J=7.1Hz), 4.09 (2H, q, J = 7.1Hz), 7.25 (2H, br s), 7.68 (1H, s), 7.76 (2H, d, J = 8.0Hz), 7.85 (2H, d, J = 8.0Hz).

This compound had activity ‘B’ in the fluorescence polarization assay described below.

The following compounds in Table 1 were synthesised and tested in the fluorescence polarization assay described below. Suzuki reactions were carried out as in Example 1 Step2. Reductive amination reactions were carried out as for Example 33, as follows: 2-Amino-4-(4-piperidin-1-ylmethyl-phenyl)-thieno[2,3-d]pyrimidine-6- carboxylic acid ethyl ester (Example 33 in Table 1) ; = ©.

HNT NSO ne REN

Pyrrolidine (5 equiv) was added to a suspension of 2-amino-4-(4-formyl- phenyl)-thieno[2,3-d]pyrimidine-8-carboxylic acid ethyl ester (1 equiv) in methanol. Reaction mixture was heated to reflux for 3.5 hours then cooled to room temperature. Sodium borohydride (3 equiv) was added and stirred for 10 mins. The mixture was concentrated in vacuo then partitioned between ethyl acetate and water. The phases were separated and the organic layer was washed with brine, dried and evaporated to a yellow oil. The crude product was purified by preparative HPLC.

LC-MS retention time: 1.803 minutes, [M+H]" 383.

Chloride displacement reactions were carried out as for Example 22 as follows: 2-Amino-4-benzylamino-thieno[2,3-djpyrimidine-6-carboxylic acid methyl ester (Example 22 in Table 1)

Cl > $ —

HN NTS 0 oe E

Won s ©

The 2-amino-4-chloro-thieno[2,3-d]pyrimidine-6-carboxylic acid methyl ester (100mg, 0.39mmol), benzylamine (100u!) in 4mL of THF are submitted to MW irradiation at 110°C for 35 mins. The reaction was cooled to room temperature and worked up (acidic) and purified using standard conditions for a neutral compound.

LC-MS: RT = 2.391mins; MS m/z = 329 (M+1).

Note: Intensity and reaction time depends on reactivity of amine. For example, for less reactive amines (such as N-methyl aniline), suitable reaction conditions are MW 160°C for 30 mins and 0.5mL of amine.

The fourth column of Table 1 states the activity of the compound in the fluorescence polarization assay described below.

Table 1

Hsp90 FP ;

C50 Comment

NH, i.

ZZ

3 joqeal 348 A Suzuki

Cl 0 [eo] 7

NH,

Ft na 4 opal 436 A Suzuki . -

F ANE

M

NH,

NS

(~

BEN EEE o — lo] 0 = poy 328 A Suzuki

HN NTS $

H ? reductive 7 oF amination on 7 383 pk

NZ o/ made by “DD

HSN S Y Suzuki reaction)

JR

NSN : 2 : oR 344 A Suzuki

AN = © Q a —

NH,

Cl No lz _ 8 334 A Suzuki 0] /°

NH,

NS l~ 5 318 Suzuki

F le} /° 11 2 314 A Suzuki

HAN s $

0 12 ‘ 330 Suzuki

TOI

HAN? s $

NH, ® 13 ated 340 Suzuki dw ) gd — a 14 N™ =r 0 334 Suzuki

Mo

HNTONT TS $

NNT 314 A Suzuki

Pp

HNT NTS $

NH, 0 16 Ps 325 A Suzuki 7 ~ 0

N Io

NH, 9 17 og. 325 Suzuki

A

NH,

Oo 18 oC 306 Suzuki

S )

Jd — 19 JL Ww, 316 chloride 08 Ye displacement

HNN s ©

~~ bal © 9) chloride i» A 342 dso

Pp n

HN” NPS 0 splaceme pe 2 1 o 30 1 chioride

N I i displacement

HNN tS] 0] o— displacement

N™S

A

H LN N S [o] 23 “Nn : 329 chloride o— displacement .

NT

A

HNT TN S [o]

WA

24 o/ 321 chloride

N =r displacement

HN S [v] hioride nd 405 © ® N Se dl displacement

HAN? Ss [o] hloride 26 be 343 ©

NANA o— displacement

Pp

HN NTS 0 lo} g . 27 § H_ 301 . crore

A > A isplacemen

HNN TS ©

HN ¢ chloride 28 yo 315 displacement ae Se : chloride 29 HN / 357 displacement ry

HAN s © reductive

N

S amination on 4-aldehyd 383 aldehyde (made by j® A o-/ Suzuki

HN NS 0 reaction)

[0] NTN

Cy FZ

N S chloride 31 Ho 364 chlo o displacement )

NH,

ZZ

32 8 339 A Suzuki

NZ 0 a

OO) reductive amination on 4-aldehyd 33 397 eee (made by

NZ TY o-/ Suzuki

HS § © reaction)

NH,

NN lL 34 S 328 A

Q

OH reduction of 4-aldehyde 330 A (made by x > A ol Suzuki

HN" NTS © reaction)

N-N

HO (7 36 363 chloride

HN displacement _/ [oe Xy

HN ONTTS ©

H

N-N [ 2 37 361 chloride

HN displacement _/ a) [0] joss

HN NTS © =N,

Ps 38 HN 355 chloride 00 / displacement

HAN S [¢] jg! 39 HN H J 355 chloride [oa displacement

HN NTS © 0

NH, 40 343 Suzuki

N= 0.

I A 4

Sh SB 'S; reductive amination on 41 309 4-aldehyde (made by hy TN o Suzuki

HNN TS reaction)

Example 42.

2-Amino-4-phenyl-thieno[2,3-d]pyrimidine-6-carboxylic acid amide a.

HNN SNH,

The compound of example 1 was suspended in concentrated ammonium hydroxide and heated in a Personal Chemistry Synthesiser microwave at 140 °C for 20 minutes. Concentration in vacuo gave example 42 as a white solid.

LC-MS retention time: 1.824 minutes, [M+H]" 271.10

Example 43 2-Amino-4-phenyl-thieno[2,3-d]pyrimidine-6-carboxylic acid ethylamide

Ng

HN NTS 0

Step 1 2-Amino-4-phenyl-thieno[2,3-d]pyrimidine-6-carboxylic acid

N™ OH hay

HNT NTS 0

Sodium Hydroxide (0.66 g; 16.5 mmol) was added to a suspension of 2- amino-4-phenyl-thieno[2,3-d]pyrimidine-6-carboxylic acid ethyl ester (Example 1) (1.00 g; 3.34 mmol) in ethanol (20 ml) and water (2 ml). The mixture was heated at reflux for 1 hour (affording a homogeneous pale-yellow solution)

and allowed to cool to ambient temperature. Solvents were removed in vacuo and the solid residue was dissolved in water (30 mL) and cooled with ice- water bath. The mixture was stirred and adjusted to pH 1-2 by drop-wise addition of concentrated Hydrochloric acid. The resulting precipitate was filtered, washed with water, then ethanol and finally diethyl ether. The off- white product was dried in vacuo to afford 2-amino-4-phenyl-thienof2,3- d]pyrimidine-6-carboxylic acid as a colourless solid (0.784 g; 87%).

LC/MS RT = 1.845 min; m/z = 272 (M+H)+

Step 2 2-Amino-4-phenyl-thieno[2,3-d]pyrimidine-6-carboxylic acid ethylamide

H ry

HN" NT 78 0

O-(7-Azabenzotriazol-1-yl)-N,N,N’,N'-tetramethyluronium hexafluorophosphate (0.380 g, 1.0 mmol) was added to 2-amino-4-phenyl- thieno[2,3-d]pyrimidine-6-carboxylic acid (0.187 g, 0.69 mmol). This mixture was suspended in dimethylformamide (DMF) (5.0 ml) and diisopropylethylamine (0.696 ml; 4.0 mmol) added to afford a yellow solution.

Diethylamine hydrochloride (0.122 g; 5.0 mmol) was added and the reaction mixture was heated for ten minutes at 100 °C in a sealed vial in a microwave synthesiser. DMF was removed in vacuo and the residue was partitioned between ethyl acetate (30 mi) and water (30 ml). The phases were separated and the organic phase was washed with saturated sodium chloride solution and dried over sodium sulphate. Mixture was filtered and the filtrate solvents were removed in vacuo to leave a yellow solid which was adsorbed onto silica gel and purified by flash chromatography on silica gel (20 g), eluting with a solvent gradient of 15 to 50% ethyl acetate in hexane. This affords 2-amino-4- phenyl-thieno([2,3-d]pyrimidine-6-carboxylic acid ethylamide as a pale yellow solid (0.051 g; 25%).

LC/MS RT = 2.08 min; m/z = 299 (M+H)+ 1H NMR (400MHz, d6 DMSO) § 1.11 (t, 3H), 3.26 (m, 2H), 7.12 (s, 2H), 7.61 (m, 3H), 7.86 (m, 2H), 8.03 {(s, 1H), 8.71 (t, 1H).

The compound of Example 43 had activity ‘A’ in the fluorescence polarization assay described below.

The following compounds (Table 2) were made by the method of Example 43 from the corresponding ester (Table 1) and the appropriate amine.

The final column of Table 2 states the activity of the compound in the fluorescence polarization assay described below.

Table 2

Hsp90 FP

Example Structure MH+

IC50 44 NA H) 342 A

HN Ss ©

NOI NAN

HNN SB

[0]

QR

46 SA H) 384

HAN s 0 47 N 313 A yg \ “\

HNTSNTTS Ty : 48 2 NN 327

Ho s

49 ‘ay 375

HAN s 0° YD 50 2 389

NTS A N

Ha s © 51 oy 376

NT \ N ==N > Lar =

HS $s ©

OH

53 ry 329 A

HNP s © 54 ay 73 357 A

HAS S$ s 55 2a ~~ 381

NSN § © 56 313 A

Die

HNN

57 365 A

NZ NP N hey ™

HNT NTS (3

' 58 ND adh TI

HAN 5 0 59 oy 327

NZ N

Ir

H 347 hy

HNTSNTTS 0

NH,

NS

ZZ

61 fo qey 31 | A 0

HN

_J 62 ~ 339 A

HNN SO

LJ

63 ' Ly gn 300

HAN S [0]

WJ

64 oe 300

To

HNN s 0 65 bs 314

Sen

HAN s ©

®

N

» 398

TO

HN Se

Low 209 | A

ALS

HN NSO

1

Te i}

HAN 5 9 0 70 0 341

DR

HN s ©

PU

HN" NTS 0 72 ry OH 315 A

I

HNP? s ©

Hp SNS

Example 74

_N N NH, (GE a ELIS Sa Le

A PY A 2 s O

HN" "N° 70 HN NTS HN" °N

H 2 H { 2,5-Diamino-4-phenyl-thieno[2,3-d]pyrimidine-6-carboxylic acid ethyl ester

NH,

NTS Oo a Lg

HN” ON” 7S $

Step 1 2-Amino-6-0xo-4-phenyl-1,6-dihydro-pyrimidine-5-carbonitrile

ZN ne ~

HN 0

H

Benzaldehyde (15g, 141.3mmol, 1eq), guanidine carbonate (25.47g, 141.3mmol, 1eq), ethyl cyanoacetate (15.99¢, 141.3mmol, 1eq) and anhydrous sodium acetate (11.59g, 141.3mmol, 1eq) were added to 300ml anhydrous pyridine and refluxed for 4 hours. The reaction was then cooled to room temperature and the solvent was removed under reduced pressure. The brown residue was triturated with 400 ml aqueous acetic acid (30%) and filtered off. The yellow solid was then triturated with 300ml diethyl ether and filtered off to yield 2-amino-6-o0x0-4-phenyi-1,6-dihydro-pyrimidine-5- carbonitrile as an off-white solid.

Yield: 14.469 (48%)

LCMS retention time = 1.34min, m/z calcd for C11HgN4O 213.22 (M + H), found 213.1

Step 2 2-Amino-4-phenyl-6-thioxo-1,6-dihydro-pyrimidine-5-carbonitrile

ZN

N°

HA S

H

2-Amino-6-oxo-4-phenyl-1,6-dihydro-pyrimidine-5-carbonitrile (0.200g, 0.942mmol, 1eq) and phosphorous pentasulfide (0.838g, 3.770mmol, 4eq) were dissolved in 5ml pyridine. The reaction was heated under reflux for 2 hours, cooled to room temperature and poured onto 100ml! water. The mixture was boiled for 1 hour, cooled to room temperature and extracted with dichloromethane. The combined organic extracts were washed with saturated brine and dried over anhydrous sodium sulphate. The solvent was removed in vacuo and the orange residue was triturated with diethyl ether to give 2- amino-4-phenyl-6-thioxo-1,6-dihydro-pyrimidine-5-carbonitrile as a yellow solid.

Yield: 0.118g (55%)

LCMS retention time = 1.94min, m/z calcd for C411HgN4S 229.29 (M + H), found 229.1

Step 3 2,5-Diamino-4-phenyl-thieno[2,3-d]pyrimidine-6-carboxylic acid ethyl ester

NH, 0]

NT

Ip:

HN" NTS ¢

Sodium (0.010g, 0.438mmol, 1eq) was dissolved in 4ml anhydrous ethanol under nitrogen. 2-Amino-4-phenyl-6-thioxo-1,6-dihydro-pyrimidine-5-

carbonitrile (0.100g, 0.438mmol, 1eq) was added and the reaction was stirred at room temperature for 1 hour. 2-Bromoethylacetate (0.073g, 0.438mmol, 1eq) was added. The reaction was stirred for further 30 minutes at room temperature. Then sodium (0.010g, 0.438mmol, 1eq) dissolved in 1m! anhydrous ethanol was added. The reaction was then refluxed for 5 hours.

The reaction was cooled to room temperature and quenched with water. The precipitate was filtered off and triturated with diethyl ether to yield 2,5-diamino- 4-phenyl-thieno[2,3-d]pyrimidine-6-carboxylic acid ethyl ester as a yellow solid.

Yield: 0.059g (43%)

LCMS retention time = 2.42min, m/z calcd for C4sH1sN4O2S 315.38 (M + H), found 315.1 "TH NMR (DMSO-ds, 2.50) 8 1.19 (t, 3H, J= 7.1), 4.13 (q, 2H, J = 7.1), 5.79 (bs, 2H), 7.29 (bs, 2H), 7.50-7.56 (m, 5H)

This compound had activity B in the fluorescence polarisation assay described below.

Example 75 2-Amino-5-methyl-4-phenyl-thieno[2,3-d]pyrimidine-6-carboxylic acid amide (8)

N ’d hay

HNT NT 7S NH,

Step 1 5-Amino-4-benzoyl-3-methyl-thiophene-2-carboxylic acid ethyl ester

Lu

X

0 0 \ J C

HN

Prepared by literature method

Bryan P. McKibben, Craig H. Cartwright, Arlindo L. Castelhano Tetrahedron

Lett. 1099, 44, 5471

Step 2 2-Amino-5-methyl-4-phenyl-thieno[2,3-d]pyrimidine-6-carboxylic acid amide oO

N a hay

HN ONT TS NH,

Guanidine carbonate was added to a solution of 5-amino-4-benzoyl-3- methylthiophene-2-carboxylic acid ethyl ester, and the suspension heated, 1750C, for ~3hrs. under a nitrogen atmosphere. The suspension was allowed to cool and water added. The mixture was extracted with ethyl acetate, extracts were washed and dried. Solution was concentrated and the residue purified by chromatography eluting with mixtures of ethyl acetate and hexane.

LC retention time 2.17 minutes [M+H}+ 285.1 (Run time 3.75mins)

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

Additional examples prepared by methods similar to those described above are listed in Table 3. The fourth column of Table 3 states the activity of the compound in the fluorescence polarization assay described below.

Claims

PCT/GB2004/003641 C 119 Claims:

1. The use of a compound of formula (1), 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: N” N A (h R, R, wherein R; is a group of formula (IA): (AMA) 2)-(AK?)-Q (IA) wherein Ar' is an optionally substituted aryl or heteroaryl radical, Alk® and AIK? are optionally substituted divalent C41-Cs alkylene or C,-Caalkenylene radicals, m, p, r and s are independently O or 1, Z is —O-, -S-, «(C=0)-, (C=S)-, -SO02-, -C(=0)0-, -C(=O)NR*- _C(=S)NRA-, -SO,NR*-, -NR*C(=0)-, -NR*SO- or ~NR"- wherein R” is hydrogen or C4-Cs alkyl, and Q is hydrogen or an optionally substituted carbocyclic or heterocyclic radical; R3 is hydrogen, an optional substituent, or an optionally substituted (C- Ce)alkyl, aryl or heteroaryl radical; and R, is a carboxylic ester, carboxamide or sulfonamide group.

2. The use as claimed in claim 1 for immunosuppression or the treatment of viral disease, inflammatory diseases such as rheumatoid arthritis, asthma, multiple sclerosis, Type | diabetes, lupus, psoriasis and inflammatory bowel AMENDED SHEET

PCT/GB2004/003641 ¢® 120 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 Hsp70 in the heart and brain: scrapie/CJD, Huntingdon's or Alzheimer's disease.

3. The use as claimed in claim 1 for the treatment of cancer.

4. The use as claimed in any of the preceding claims wherein, in the compound (I), mis 1, each of p, rand s is 0, and Q is hydrogen.

5. The use as claimed in any of the preceding claims, wherein, in the compound (1), Ry is optionally substituted phenyl, 2- or 3-thienyl, 2- or 3- furanyl, 2-, 3- or 4-pyridinyl, morpholinyl, or piperidinyl.

6. The use as claimed in claim 5 wherein, in the compound (1), Rois phenyl, optionally substituted by a one or more substituents selected from methyl, ethyl, n- or isopropyl, vinyl, allyl, methoxy, ethoxy, n-propyloxy, benzyloxy, allyloxy, cyanomethoxy chloro, bromo, cyano, formyl, methyl-, ethyl-, or n-propyl-carbonyloxy, methyl- or ethylaminocarbonyl, and substituents of formula -O(CH2)n2' wherein nis 1, 2 or 3 and Z' is a primary, secondary, tertiary or cyclic amino group, or a C,-Cealkoxy group; or of formula «(Alk®)mZ" wherein Alk® is a divalent straight or branched chain (Cs- Cs) alkylene, mis O or 1, and Z' is a primary, secondary, tertiary or cyclic amino group, or a C4-Cealkoxy group.

7. The use as claimed in claim 6 wherein optional substituents are in the 2- and/or 4- and/or 5-position of the phenyl ring.

8. The use as claimed in any of the preceding claims, wherein, in the compound (1), mis 1,and p, rand s are 0, and Q is an optionally substituted carbocyclic or heterocyclic ring. AMENDED SHEET

PCT/GB2004/003641 ® 121

9. The use as claimed in any of the preceding claims, wherein, in the compound (1), Ar is a phenyl, cyclohexyl, pyridyl, morpholino, piperidinyl, or piperazinyl ring.

10. The use as claimed in any of the preceding claims, wherein, in the compound (l),wherein Rz is hydrogen.

11. The use as claimed in any of the preceding claims, wherein, in the compound (1), Rs is a carboxamide group of formula —CONRB(AIK),R? or a sulphonamide group of formula —SO,NRE(AIK),R? wherein Alk is an optionally substituted divalent alkylene, alkenylene or alkynylene radical, nisOor1, RB is hydrogen or a C1-Cs alkyl or C2-Ce alkenyl group, R” is hydroxy or optionally substituted carbocyclic or heterocyclyl, or R* and R® taken together with the nitrogen to which they are attached form an N-heterocyclic ring which may optionally contain one or more additional hetero atoms selected from O, S and N, and which may optionally be substituted on one or more ring C or N atoms.

12. The use as claimed in claim 11 wherein Alk is optionally substituted —CH.-, —CH,CH,-, -CH2CH>CHz-, — CH,CH=CH-, or -CH>CCCH_-, RB is hydrogen or methyl, ethyl, n- or iso-propyl, or allyl, AMENDED SHEET

PCT/GB2004/003641 ® 122 R” is hydroxy or optionally substituted phenyl, 3.4 methylenedioxyphenyl, pyridyl, furyl, thienyl, N-piperazinyl, or N- morpholinyl, or R* and R® taken together with the nitrogen to which they are attached form an N-heterocyclic ring which may optionally contain one or more additional hetero atoms selected from O, S and N, and which may optionally be substituted on one or more ring C or N atoms.

13. The use as claimed in any of claims 1 to 10, wherein, in the compound (I), Rs is a carboxylic ester group of formula _COORC wherein RC is a C1-Cs alkyl or C2-Cg alkenyl group, or an optionally substituted ary! or heteroaryl group, or an optionally substituted aryl(C4-Cg alkyl)- or heteroaryl(C4-Cs alkyl)- group or an optionally substituted cycloalkyl group.

14. The use as claimed in any of claims 1 to 10, wherein, in the compound (1), Ry is a carboxylic ester group of formula -COORC wherein RC is optionally substituted methyl, ethyl, n- or iso-propyl, allyl, phenyl, pyridyl, thiazolyl, benzyl, pyridylmethyl, cyclopentyl or cyclohexyl.

15. The use as claimed in any of claims 1 to 3, wherein the compound (1) has formula (11): Ri Riz Rio (I) KI J HN NTS A wherein A is a secondary amino group; AMENDED SHEET

PCT/GB2004/003641 C 123 Rip is H, Cl, Br, or CHj3; R11 is hydrogen, Cl, Br, CN, methyl, ethyl, n- or iso-propyl, vinyl or allyl;

Rs. is (i) a radical of formula -O(CHz)sZ' wherein nis 1, 2 or 3 and Z'isa primary, secondary, tertiary or cyclic amino group, or a C,-Cealkoxy group; or (ii) a radical of formula (Alk®)mZ" wherein Alk® is a divalent straight or branched chain (C1-C3) alkylene, mis 0 or 1, and Z' is a primary, secondary, tertiary or cyclic amino group, or a C4-Cgalkoxy group.

16. The use as claimed in claim 15 wherein, in the compound (In, Ais a secondary C4-Cealkylamino group.

17. The use as claimed in claim 15 or claim 16 wherein, in the compound (II), Riz is (i) a radical of formula -O(CHz)sZ' wherein nis 1,2 or 3 and Z'is di(C4-Caalkyl)amino or C4-Cjalkoxy.

18. A compound of formula (I), or a salt, N-oxide, hydrate, or solvate thereof: Nid h EN ( De, Ry R, wherein R, is a group of formula (IA): (AM m~AK YZ) (AK?) Q (IA) wherein Ar' is an optionally substituted aryl or heteroaryl radical, AMENDED SHEET

PCT/GB2004/003641 ® 124 Alk' and Alk? are optionally substituted divalent C;-C; alkylene or Cz Cs alkenylene radicals, m, p, r and s are independently 0 or 1, Z is —O-, -S-, -(C=0)-, (C=S)-, -SO,-, -C(=0)0O-, -C(=O)NR*- -C(=S)NR*-, -SO,NR*-, -NRAC(=0)-, -NR*SO, or —-NR"- wherein RA is hydrogen or C,4-Cg alkyl, and Q is hydrogen or an optionally substituted carbocyclic or heterocyclic radical; Rj; is hydrogen, an optional substituent, or an optionally substituted (C4-Cg)alkyl, aryl or heteroaryl radical; and R, is a carboxylic ester, carboxamide or sulfonamide group, PROVIDED THAT (i) Rs is not —=NH; and (ii) when R, is -COOCH; and Rs is hydrogen then R; is not —NH,, ethylamino, diethylamino, phenylamino or — N(Ph)(C.Hs) wherein Ph is phenyl, and (iii) when Ry is —CONH; and R; is hydrogen then R, is not —NH,. 19 A compound as claimed in claim 18 wherein R; is hydrogen.

20. A compound as claimed in claim 18 or claim 19 wherein m is 1,eachofp,r and s is 0, and Q is hydrogen.

21. A compound as claimed in claim 20 wherein R; is optionally substituted phenyl, 2- or 3-thienyl, 2- or 3- furanyl, 2-, 3- or 4-pyridinyl, morpholinyl, or piperidinyl.

22. A compound as claimed in claim 20 wherein R; is phenyl, optionally substituted by methyl, ethyl, n- or isopropyl, vinyl, allyl, methoxy, ethoxy, n-propyloxy, benzyloxy, allyloxy, cyanomethoxy chloro, bromo, cyano, formyl, methyl-, ethyl-, or n-propyl-carbonyloxy, methyl- or ethylaminocarbonyl,.

23. A compound as claimed in claim 22 wherein optional substituents are in the 2- and/or 4- and/or 5-position of the phenyl ring. AMENDED SHEET

PCT/GB2004/003641

24. A compound as claimed in claim 18 or claim 19 wherein mis 1, and p, r and s are 0, and Q is an optionally substituted carbocyclic or heterocyclic ring.

25. A compound as claimed in claim 18 or claim 19 wherein mis 1 and at least one of p, rand sis 1.

26. A compound as claimed in any of claims 18 to 25 wherein Ar'is an optionally substituted phenyl ring.

27. A compound as claimed in claim 18 or claim 19 wherein mis 0.

28. A compound as claimed in claim 25 or claim 26 wherein Alk' when present is optionally substituted —CH,, CH,CH;- or -CH=CH-; Alk? when present is optionally substituted —CHz, CH,CH,- or -CH=CH-; Z when present is —O- or -NH-; and Q is hydrogen.

29. A compound as claimed in claim 28 wherein Z and Alk? are present, and Alk? is substituted by di(C1-Csalkyl)amino or C4-Cgalkoxy.

30. A compound as claimed in any of claims 18 to 29 wherein Rs is a carboxamide group of formula ~CONRB(Alk),R* or a sulphonamide group of formula —SO,NRB(AIk),R* wherein Alk is a n optionally substituted divalent alkylene, alkenylene or alkynylene radical, nis 0 or 1, RB is hydrogen or a C4-Ce alkyl or C.-C alkenyl group, R” is hydroxy or optionally substituted carbocyclic, for example hydroxy and/or chloro-substituted phenyl and 3,4 methylenedioxyphenyl;, or AMENDED SHEET

PCT/GB2004/003641 heterocyclyl, for example pyridyl, furyl, thienyl, N-piperazinyi, or N- morpholinyl any of which heterocyclic rings may be substituted, or R® and R® taken together with the nitrogen to which they are attached form an N-heterocyclic ring which may optionally contain one or more additional hetero atoms selected from O, S and N, and which may optionally be substituted on one or more ring C or N atoms.

31. A compound as claimed in claim 30 wherein Alk is optionally substituted —CH2-, =CH2CHz-, —CH,CH.CH32-, — CH,CH=CH-, or -CH,CCCH>-, R® is hydrogen or methyl, ethyl, n- or iso-propyl, or allyl, R” is hydroxy or optionally substituted phenyl, 3,4 methylenedioxyphenyl, pyridyl, furyl, thienyl, N-piperazinyl, or N- morpholinyl, or R* and R® taken together with the nitrogen to which they are attached form an N-heterocyclic ring which may optionally contain one or more additional hetero atoms selected from O, S and N, and which may optionally be substituted on one or more ring C or N atoms.

32. A compound as claimed in claim 30 or claim 31 wherein Ry is a carboxamide group.

33. A compound as claimed in any of claims 18 to 29 wherein Ry is a carboxylic ester group of formula -COORC wherein RC is a C4-Cs alkyl or Cz- Cs alkenyl group, or an optionally substituted aryl or heteroaryl! group, or an optionally substituted aryl(C4-Ce alkyl)- or heteroaryl(C4-Cs alkyl)- group or an optionally substituted cycloalkyl group. AMENDED SHEET

PCT/GB2004/003641 <9 127

34. A compound as claimed in any of claims 18 to 29 wherein Rsis a carboxylic ester group of formula -COORC wherein RC is optionally substituted methyl, ethyl, n- or iso-propyl, allyl, phenyl, pyridyl, thiazolyl, benzyl, pyridyimethyl, cyclopentyl or cyclohexyl.

35. A compound as claimed in claim 18 having formula (if): Ri Ri, Rio (1) JO Na HN ONT °S A wherein A is a secondary amino group Rio is H, Cl, Br, or CHjs; R14 is hydrogen, Cl, Br, CN, methyl, ethyl, n- or iso-propyl, vinyl or allyl; Riz is (i) a radical of formula -O(CH2)nZ' wherein nis 1, 2 or 3 and Z'isa primary, secondary, tertiary or cyclic amino group, or a Cs-Cealkoxy group; or (ii) a radical of formula «(Alk*)nZ' wherein Alk® is a divalent straight or branched chain (C4-C3) alkylene, mis 0 or 1, and Z' is a primary, secondary, tertiary or cyclic amino group, or a C4-Cealkoxy group.

36. A compound as claimed in claim 35 wherein Ais a secondary Cs- Cealkylamino group. AMENDED SHEET

PCT/GB2004/003641 < 128

37. A compound as claimed in claim 35 or claim 36 wherein Ry2 is (i) a radical of formula -O(CH;).Z' wherein nis 1, 2 or 3 and Z' is di(Cy- Csalkyl)amino or C4-Csalkoxy.

38. A compound as claimed in claim 37 which is the subject of any of the Examples herein except Example 74.

39. A pharmaceutical or veterinary composition comprising a compound as claimed in any of claims 18 to 38, together with one or more pharmaceutically or veterinarily acceptable carriers and/or excipients.

40. Use according to any one of claims 1 to 17, substantially as herein described with reference to and as illustrated in any of the examples.

41. A compound according to any one of claims 18 to 38, substantially as herein described with reference to and as illustrated in any of the examples.

42. A composition according to claim 39, substantially as herein described with reference to and as illustrated in any of the examples. AMENDED SHEET