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Definitions

• This invention relates to substituted bicyclic pyrrolopyrimidine 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.
• Hsps heat shock proteins
• Hsps 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).
• therapies which disrupt the molecular chaperone machinery may prove to be beneficial.
• misfolded proteins can cause protein aggregation resulting in neurodegenerative disorders.
• 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).
• Hsp90 in various critical oncogenic pathways and the discovery that certain natural products with anticancer activity are targeting this molecular chaperone suggests that inhibiting the function of Hsp90 may be useful in the treatment of cancer.
• the first in class natural product 17AAG is currently in Phase Il clinical trials.
• Hsp90 constitutes about 1-2% of total cellular protein. In cells, it forms dynamic multi- protein complexes with a wide variety of accessory proteins (referred to as co- chaperones) which appear responsible for regulating the chaperone function. It is essential for cell viability and it exhibits dual chaperone functions (Young et al., 2001).
• co- chaperones accessory proteins
• Hsp90 forms a core component of the cellular stress response by interacting with many proteins after their native conformation has been altered.
• Environmental stresses such as heat shock, heavy metals or alcohol, generate localised protein unfolding.
• Hsp90 (in concert with other chaperones) binds these unfolded proteins allowing adequate refolding and preventing non-specific aggregation (Smith et al., 1998).
• 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).
• 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 many client proteins. These can be subdivided into three groups: (a) steroid hormone receptors (e.g.
• Hsp90 is responsible for stabilising and activating mutated kinases where the wild type kinase is not an Hsp90 client (for an example see the B-Raf story published in da Rocha Dias et al., 2005). All of these proteins play key regulatory roles in many physiological and biochemical processes in the cell. New client proteins of Hsp90 are being constantly identified; see http://www.picard.ch/downloads/Hsp90interactors.pdf for the most up to date list.
• Hsp90 The highly conserved Hsp90 family in humans consists of four genes, namely the cytosolic Hsp90 ⁇ and Hsp90 ⁇ 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). Apart from the differences in sub-cellular localisation, very little is known about the differences in function between Hsp90 ⁇ / ⁇ , GRP94 and TRAP1.
• Initial reports suggesting that certain client proteins were chaperoned by a specific Hsp90 e.g. Her2 by Grp94 alone
• 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.
• Hsp90 is an ATP-dependent molecular chaperone (Prodromou et al, 1997), with dimerisation 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). This conformational switching is, in part, regulated by the various co-chaperones associated with Hsp90 (Siligardi et al., 2004).
• 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). lmmunoprecipitation 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).
• 17-Allylamino, 17-demethoxygeldanamycin 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 has been shown to be much more active on tumour cells than its affinity for purified Hsp90 would suggest.
• tumour cells but not non-tumourigenic cells
• 17AAG contains a high-affinity conformation of Hsp90 to which 17AAG binds more tightly, and confers tumour selectivity on Hsp90 inhibitors (Kamal et al., 2003). 17AAG is currently being evaluated in Phase Il 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 Hsp90 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 Hsp90, 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).
• Geldanamcyin cannot bind Hsp90 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 Her2, and to cause cell cycle arrest and differentiation in breast cancer cells (Chiosis et al., 2001). Recent studies have identified other purine- based compounds with activity against Her2 and activity in cell growth inhibition assays (Dymock et al 2004; Kasibhatla et al 2003; Llauger et al 2005).
• Patent publications WO 2004/050087, WO 2004/056782, WO 2004/072051 , WO 2004/096212, WO 2005/000300, WO 2005/021552, WO 2005/034950 relate to Hsp90 inhibitors.
• Hsp90 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).
• geldanamycin, 17AAG, and radicicol The predominant mechanism of action of geldanamycin, 17AAG, and radicicol involves binding to Hsp90 at the ATP binding site located in the N-terminal domain of the protein, leading to inhibition of the intrinsic ATPase activity of Hsp90 (Prodromou et al., 1997; Stebbins et al., 1997; Panaretou et al., 1998).
• Hsp90 ATPase activity by 17AAG induces the loss of p23 from the chaperone-client protein complex interrupting the chaperone cycle. This leads to the formation of a Hsp90-client protein complex that targets these client proteins for degradation via the ubiquitin proteasome pathway (Neckers et al., 1999; Whitesell & Lindquist, 2005). Treatment with Hsp90 inhibitors leads to selective degradation of important proteins (for example Her2, Akt, estrogen receptor and CDK4) involved in cell proliferation, cell cycle regulation and apoptosis, processes which are fundamentally important in cancer.
• important proteins for example Her2, Akt, estrogen receptor and CDK4
• Hsp90 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 (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 (Schulte et al., 1998; Kelland et al., 1999).
• Hsp90 inhibitors therefore resensitise strains which have become resistant to, for example, azole antifungal agents (e.g. fluconazole) as well as newer agents such as echinocandins.
• azole antifungal agents e.g. fluconazole
• newer agents such as echinocandins.
• the present invention provides a compound of formula (I), or a pharmaceutically acceptable salt thereof salt thereof:
• R 1 is hydrogen, fluoro, chloro, bromo, or a radical of formula ((1A):
• X is -O-, -S- -S(O)-, -SO 2 -, or -NH-,
• AIk 1 and AIk 2 are optionally substituted divalent C 1 -C 3 alkylene or C 2 -C 3 alkenylene radicals, m, n and p are independently O or 1 , and
• Q is hydrogen or an optionally substituted carbocyclic or heterocyclic radical
• R 2 is a radical of formula (IB):
• Ar 1 is an optionally substituted aryl or heteroaryl radical
• AIk 1 , AIk 2 , Z, and Q are as defined in relation to formula (IA), and p, q, r and s are independently O or 1; and
• the invention provides 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:
• the invention also provides 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 defined in claim 1 effective to inhibit said HSP90 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, drug resistant fungal infection (since HSP90 inhibitors are able to resensitise strains which have become resistant to, for example, azole antifungal agents (e.g.
• fluconazole as well as newer agents such as echinocandins
• 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 Hsp70 in the heart and brain; scrapie/CJD, Huntingdon's or Alzheimer's disease.
• Use for the treatment of cancer is especially indicated.
• (C a -C b )alkyl wherein a and b are integers refers to a straight or branched chain alkyl radical having from a to b carbon atoms.
• a 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.
• divalent (C a -C b )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.
• (C a -C b )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.
• divalent (C a -C b )alkenylene radical refers to a hydrocarbon chain having from a to b carbon atoms, at least one double bond, and two unsatisfied valences.
• cycloalkyl refers to a saturated carbocyclic radical having from 3-8 carbon atoms and includes, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl.
• 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.
• aryl refers to a mono-, bi- or tri-cyclic carbocyclic aromatic radical, and includes aromatic monocyclic or bicyclic carbocyclic radicals fused to a non aromatic carbocyclic or heterocyclic ring.
• aromatic monocyclic or bicyclic carbocyclic radicals fused to a non aromatic carbocyclic or heterocyclic ring are phenyl, biphenyl and napthyl, and radicals of the formula:
• ring A (i) is optionally substituted, (ii) has 5 or 6 ring members including the carbons of the phenyl ring to which it is fused, and (iii) has at least one heteroatom O, S or N hetero atom as a ring member.
• Carbocyclic refers to a cyclic radical whose ring atoms are all carbon, and includes aryl, cycloalkyl, and cycloalkenyl radicals.
• heteroaryl refers to a mono-, bi- or tri-cyclic aromatic radical containing one or more heteroatoms selected from S, N and O.
• Illustrative 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.
• heterocyclyl or “heterocyclic” includes “heteroaryl” as defined above, and in particular refers to a mono-, bi- or tri-cyclic non-aromatic radical containing one or more heteroatoms selected from S, 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.
• 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.
• substituted as applied to any moiety herein means substituted with at least one substituent, for example selected from (CrC 6 )alkyl, (C r C 6 )alkoxy (including methylenedioxy and ethylenedioxy substitution on adjacent carbon atoms of a carbocyclic or heterocyclic ring), hydroxy, hydroxy(d-C 6 )alkyl, mercapto, mercaptoCCVCeJalkyl, (C r C 6 )alkylthio, monocyclic carbocyclic of 3-6 ring carbon atoms, monocyclic heterocyclic of 5 or 6 ring atoms, halo (including fluoro and chloro), trifluoromethyl, trifluoromethoxy, nitro, nitrile (-CN), oxo, -COOH, -COOR A , -COR A , -S0 2 R A , -CONH 2 ,
• the optional substituent contains an alkyl radical
• that alkyl radical may be substituted by a monocyclic carbocyclic group of 3-6 ring carbon atoms, or a monocyclic heterocyclic group of 5 or 6 ring atoms.
• the optional substituent is or comprises a monocyclic carbocyclic group of 3-6 ring carbon atoms, or a monocyclic heterocyclic group of 5 or 6 ring atoms, that ring may itself be substituted by any of the non-cyclic optional substituents listed above.
• An "optional substituent” may be one of the substituent groups encompassed in the above description.
• 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.
• 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.
• hydrohalic acids such as hydrochloric or hydrobromic acids, sulphuric acid, nitric acid or phosphoric acid and the like
• organic acids e.g. with acetic, tartaric, succinic, fumaric, maleic, malic, salicylic, citric, methanesulphonic and p-toluene sulphonic acids and the like.
• hydrates and solvates are of course merely specific physico-chemical forms of the active compounds of the invention and therefore form part of the invention.
• Any unqualified reference herein to a compound which falls within formula (I) is to be construed as a reference to that compound, irrespective of whether it is or is not in the form of a hydrate or solvate.
• 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.
• N-oxides may be oxidesed to form N-oxides.
• Such N-oxides substantially retain the HSP90 inhibitory activity of the parent compounds, and are thus form part of the invention.
• Any unqualified reference herein to a compound which falls within formula (I) is to be construed as a reference to that compound, irrespective of whether it is or is not in the form of an N-oxide.
• 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.
• So-called 'pro-drugs' of the compounds of formula (I) are also within the scope of the invention.
• certain derivatives of compounds of formula (I) which may have little or no pharmacological activity themselves can, when administered into or onto the body, be converted into compounds of formula (I) 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 (I) 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).
• metabolites of compounds of formula (I), that is, compounds formed in vivo upon administration of the drug are also included within the scope of the invention.
• Some examples of metabolites include
• R 1 is a radical of formula (IA):
• X may be -0-, -S- -S(O)-, -SO 2 -, or -NH-. At present -O- and -S- are preferred;
• m and n are both 0.
• m is 1 and n is 0.
• m is 0 and n is 1;
• Q may be hydrogen or an optionally substituted carbocyclic or heterocyclic radical.
• carbocyclic radicals Q include phenyl, cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.
• heterocyclic radicals Q include heteroaryl radicals such as pyridyl, thienyl and furanyl, and non-aromatic heterocyclic radicals such as piperidinyl, piperazinyl, tetrahydropyrrole, and morpholinyl.
• AIk 1 and AIk 2 are unsubstituted.
• Q when carbocyclic or heterocyclic may be unsubstituted, but when substituted, optional substituents may be selected from, for example, methyl, ethyl, n- or isopropyl, vinyl, allyl, methoxy, ethoxy, n-propyloxy, isopropyloxy, benzyloxy, allyloxy, cyanomethoxy, fluoro, chloro, bromo, cyano, oxo, formyl, methyl-, ethyl-, or n-propyl-carbonyloxy, methyl- or ethylaminocarbonyl, and substituents of formula -O(CH 2 ) a Z 1 wherein a is 1, 2 or 3 and Z 1 is a primary, secondary, tertiary or cyclic amino group, or a C 1 - C 6 alkoxy group; or of formula
• R 1 substituent has the formula -O(CH 2 ) n Z 1 or -S(CH 2 ) ⁇ Z 1 wherein n is 1 , 2 or 3 and Z 1 is a primary, secondary, tertiary or cyclic amino group, the latter being optionally substituted, or a C r C 6 alkoxy group.
• R 1 include hydrogen, methoxy, ethoxy, methylthio, ethylthio, hydroxyeththylthio, methylamino, diethylaminomethylthio, methylaminocarbonylmethylthio, and groups of formula (A) -(H):
• R 2 is a radical of formula (IB): -(Ar 1 ) p -(Alk 1 ) q -(Z) r -(Alk 2 ) s -Q.
• Ar 1 is an optionally substituted aryl or heteroaryl radical, for example phenyl, 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.
• Ar 1 is optionally substituted phenyl.
• Z when present, is -0-, or -NH-;
• Q may be, for example a phenyl, cyclohexyl, pyridyl, morpholino, piperidinyl, or piperazinyl ring, such as optionally substituted phenyl, 2- or 3-thienyl, 2- or 3- furanyl, 2-, 3- or 4-pyridinyl, morpholinyl, or piperidinyl.
• p is 1 , each of q, r and s is 0, and Q is hydrogen.
• p is 1 , and q, r and s are 0, and Q is an optionally substituted carbocyclic or heterocyclic ring.
• p, q, r and s are each 1 , and Q is hydrogen.
• R 2 is phenyl, optionally substituted by one or more substituents selected from methyl, trifuoromethyl, ethyl, n- or isopropyl, vinyl, allyl, methoxy, trifuoromethoxy, ethoxy, methylenedioxy, ethylenedioxy, n-propyloxy, benzyloxy, allyloxy, cyanomethoxy, fluoro, chloro, bromo, cyano, formyl, methyl-, ethyl-, or n-propyl-carbonyloxy, methyl- or ethylaminocarbonyl, and substituents of formula - O(CH 2 ) n Z 1 wherein n is 1 , 2 or 3 and Z 1 is a primary, secondary, tertiary or cyclic amino group, or a C r C 6 alkoxy group; or of formula -(Alk 3 ) m
• R 3 is cyano (-CN).
• R 1 is (a) C 1 -C 6 alkylthio or C 1 -C 6 alkoxy in either of which one or more hydrogen atoms are optionally replaced by fluorine atoms, or (b) a substituent of formula -O(CH 2 ) n Z 1 or -S(CH 2 ) n Z 1 wherein n is 1 , 2 or 3 and Z 1 is a primary, secondary, tertiary or cyclic amino group the latter being optionally substituted.
• R 10 is H, Cl, Br, or -CH 3 ;
• R 11 is hydrogen, Cl, Br, CN, methyl, ethyl, n- or iso-propyl, methoxy, ethoxy, vinyl or allyl;
• R 12 is (i) a radical of formula -O(CH 2 ) n Z 1 or -S(CH 2 ) n Z 1 wherein n is 1 , 2 or 3 and Z 1 is (i) a primary, secondary, tertiary or cyclic amino group, or a C- ⁇ -C 6 alkoxy group; or (ii) a radical of formula -(Alk 3 ) m Z 1 wherein AIk 3 is a divalent straight or branched chain (C 1 -C 3 ) alkylene, m is 0 or 1 , and Z 1 is a primary, secondary, tertiary or cyclic amino group, or a C r C 6 alkoxy group.
• 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.
• HCV Hepatitis C
• lmmunosupression such as in transplantation (Bijlmakers, 2000 and Yorgin, 2000)
• Anti-inflammatory diseases (Bucci, 2000) such
• 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).
• 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)".
• the invention also includes:
• a pharmaceutical or veterinary composition comprising a compound of formula (I) above, together with a pharmaceutically or veterinarily acceptable carrier.
• a method of treatment of diseases or conditions which are responsive to inhibition of HSP90 activity in mammals which method comprises administering to the mammal an amount of a compound of formula (I) above effective to inhibit said HSP90 activity.
• a suitable dose for orally administrabie 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.
• 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 administrabie 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 polyvinylpyrrolidone; 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.
• 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.
• the drug can either be suspended or dissolved in the vehicle.
• adjuvants such as a local anaesthetic, preservative and buffering agents can be dissolved in the vehicle.
• Compounds of the invention may be administered together with other classes opf pharmaceutically active drugs.
• combination therapy with two or more different classes of anticancer agent is a recognised and widespread practice.
• the present compounds may be used in such combination therapy, particularly where the other drug(s) have a mode of action different from HSP90 inhibition.
• RT Retention Times
• HPLC purifications were performed on a Waters FractionLynx MS Autopurification system with a Gemini ® 5 ⁇ M C18(2), 100 mm * 20 mm i.d. column from Phenomenex, running at a flow rate of 20 mL min "1 with UV diode array detection (210 - 400 nm) and mass-directed collection. Gradients used for each compound are shown in Table 1.
• Solvent B 95% v/v HPLC grade acetonitrile + 5% v/v Solvent A + 0.08% v/v formic acid.
• Solvent B 95% v/v HPLC grade acetonitrile + 5% v/v Solvent A + 0.08% v/v ammonia solution.
• the mass spectrometer was a Waters Micromass ZQ2000 spectrometer operating in positive or negative ion electrospray ionisation modes, with a molecular weight scan range of 150 to 1000.
• Table 1 Preparative HPLC gradients
• step 2 To a solution of 4-(2,4-dimethyl-phenyl)-2-methylsulfanyl-7-(2-trimethylsilanyl- ethoxymethyl)-7H-pyrrolo[2,3-d]pyrimidine (step 2) (100mg, 0.25 mmol) in CH 2 CI 2 (3 ml) at O 0 C was added dropwise a solution of N-Bromosuccinimide in CH 2 CI 2 (45 mg, 0.25 mmol). After 5 minutes the reaction was allowed to warm to ambient temperature. The solution was evaporated in vacuo and the residue was partitioned between EtOAc (2 x 20 ml) and sat. aqueous sodium thiosulfate solution ( 20 ml).
• This compound had activity 'A' in the fluorescence polarization assay described below.
• the title compound was made by way of the method of example 1 step 5 (TBAF mediated SEM deprotection).
• the crude product was purified by flash chromatography on silica gel, eluting with ethyl acetate and hexane mixture to afford an off white solid.
• This compound had activity 'A' in the fluorescence polarization assay described below.
• step 3 with tetra butylammonium fluoride using the method outlined in example 1 step
• the title compound was prepared by treating 4-(2,4-dimethyl-phenyl)-5-methyl-2- methylsulfanyl-7-(2-trimethylsilanyl-ethoxymethyl)-7H-pyrrolo[2,3-d]pyrimidine with tetrabutylammonium fluoride using the method outlined in example 1 step 5. Purification was by flash chromatography on silica gel eluting with ethyl acetate / hexane mixture; followed by trituration with diethyl ether to afford title compound as a colourless solid.
• This compound had activity 'B' in the fluorescence polarization assay described below.
• the title compound was prepared by the route outlined in scheme 2 and by way of the methods of example 2, using 3-hydroxyphenyl boronic acid and 4-chloro-2- methylsulfanyl-7-(2-trimethylsilanyl-ethoxymethyl)-7H-pyrrolo[2,3-d]pyrimidine-5- carbonitrile in the appropriate step (cross coupling).
• the title compound was prepared by reacting 4-[(3-(2-Diethylamino ⁇ ethoxy)-phenyl]-2- methylsulfanyl-7-(2-trimethylsilanyl-ethoxymethyl)-7H-pyrrolo[2,3-d]pyrimidine-5- carbonitrille with tetrabutylammonium fluoride and ethylene diamine in THF, using the method outlined in example 1 step 5. Purification was by flash chromatography on silica gel eluting with gradient 1 % triethylamine in dichloromethane to 1 % triethylamine; 15% methanol; 84% dichloromethane to afford title compound as a pale yellow solid.
• 6-amino-5-(2,2-diethoxy-ethyl)-2-mercapto-pyrimiclin-4-ol pyrimidine 3.Og (11.6 mmol) [prepared as detailed in Davoll. J., J. Chem. Soc. 1960, pp131-138] was dissolved in a mixture of water (150 ml and aqueous ammonia solution (9 ml) and heated to 90 0 C. Aliquots (2-3 ml) of a suspension of Raney Nickel were added to the reaction mixture until TLC and LC/MS analysis showed the reaction to be complete. The reaction mixture was allowed to cool to ambient temperature and filtered through a pad of celite.
• Phosphorous oxychloride was added to 7H-Pyrrolo[2,3-d]pyrimidine-4-ol (1.15 g, 8.5 mmol) and the reaction was heated under N 2 atmosphere to 100 0 C for 2.5 hours. The initial suspension becomes homogeneous dark suspension which was then allowed to cool to room temperature. Excess phosphorous oxychloride was removed in vacuo and the residue was cooled in ice bath and crushed ice was added with stirring. The mixture was diluted with water (20 ml) and extracted with ethyl acetate (2 x 30 ml). The combined organic extracts were washed with sat NaCI (aq) solution, then dried over anhydrous Na 2 SO 4 . Mixture was filtered and filtrate solvents removed in vacuo to afford a white solid (0.811 g; (62%).
• the title compound was prepared from 4-Chloro-7H-Pyrrolo[2,3-d]pyrimidine (0.805 g; 5.24 mmol) using the method of example 1 step 1.
• Product was purified by flash chromatography on silica gel (25g) eluting with 2-25% gradient of ethyl acetate in hexane. This afforded the title compound as colorless oil, 1.31g (87%).
• This compound had activity 'B' in the fluorescence polarization assay described below.
• step 1 The product from step 1 was de-protected using the method of example 1 step 5.
• the final product was purified by HPLC (performed at pH 4) to afford the title compound as an off-white solid, 7 mg, 10%.
• This compound had activity 1 B' in the fluorescence polarization assay described below.
• This compound had activity 'B' in the fluorescence polarization assay described below.
• This compound had activity 'B' in the fluorescence polarization assay described below.
• EtMgBr (0.04 ml, 0.11 mmol, 3M solution in Et 2 O) was added to a solution of 4-[(2- methyl-4-fluoro-phenyl]-2-methylsulfanyl-7-(2-trimethylsilanyl-ethoxymethyl)-7H- pyrrolo[2,3-d]pyrimidine-5-carbonitrile (example 12 step 1) (50 mg, 0.11 mmol) at O 0 C. After 20 min. the reaction was partitioned between EtOAc (2 x 15 ml) and water (15 ml). The organics were then passed through a hydrophobic frit and evaporated in vacuo to give the crude protected product.
• This compound had activity 'A' in the fluorescence polarization assay described below.
• This compound had activity 'A' in the fluorescence polarization assay described below.
• 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 85 0 C (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 title compound was prepared using the method out lined in example 1 step 5.
• This compound had activity 'A' in the fluorescence polarization assay described below.
• This compound had activity 'A' in the fluorescence polarization assay described below.
• This compound had activity 'A' in the fluorescence polarization assay described below.
• This compound had activity 'A' in the fluorescence polarization assay described below.
• This compound had activity 'A' in the fluorescence polarization assay described below.
• This compound had activity 'B' in the fluorescence polarization assay described below.
• This compound had activity 'B' in the fluorescence polarization assay described below.
• This compound had activity 'A 1 in the fluorescence polarization assay described below.
• the title compound was made by way of method of example 22 step 3 (diazotization and quench with aqueous Iodine / sodium iodide solution).
• Step 4 4-(2-Chloro-4-cyano-5-methoxy-phenyl)-2-methylsulfanyl-7-(2-trimethylsilanyl- ethoxymethyl)-7H-pyrrolo[2,3-d]pyrimidine-5-carbonitrile
• the title compound was prepared by way of the method of example 26 step 1 (boronic acid formation and subsequent cross coupling).
• Step 5 4-(2-Chloro-4-cyano-5-methoxy-phenyl)-2-methanesulfonyl-7-(2-trimethylsilanyl- ethoxymethyl)-7H-pyrrolo[2,3-d]pyrimidine-5-carbonitrile
• the title compound was prepared by way of the method of example 22 step 1 (oxidation with mcpba).
• This compound had activity 'A' in the fluorescence polarization assay described below.
• This compound had activity 'A' in the fluorescence polarization assay described below.
• This compound had activity 'C in the fluorescence polarization assay described below.
• This compound had activity 'A' in the fluorescence polarization assay described below.
• This compound had activity 'C in the fluorescence polarization assay described below.
• the title compound was prepared by the route outlined in scheme 2 and by the way of the methods of examples 2 and 22, using 1-Bromo-2-chloro-4-methoxy-5- methoxymethoxy-benzene and 4-chloro-2-methylsulfanyl-7-(2-trimethylsilanyl- ethoxymethyl)-7H-pyrrolo[2,3-d]pyrimidine-5-carbonitrile in the appropriate step (cross coupling).
• the title compound was prepared by the route outlined in scheme 4 and by the way of the methods of example 12 (step 1), using 4-(2-Chloro-4-methoxy-5-methoxymethoxy- phenyl)-2-methylsulfanyl-7-(2-trimethylsilanyl-ethoxymethyl)-7H-pyrrolo[2,3-d]pyrimidine- 5-carbonitrile.
• the title compound was prepared by the methods of example 1 step 5. using 4-[2- Chloro-5-(2-diethylamino-ethoxy)-4-methoxy-phenyl]-2-isopropylsulfanyl-7-(2- trimethylsilanyl-ethoxymethyl)-7H-pyrrolo[2,3d]pyrimidine-5-carbonitrile and TBAF / ethylenediamine in THF.
• This compound had activity 'B' in the fluorescence polarization assay described below.
• This compound had activity 'B' in the fluorescence polarization assay described below.
• the title compound was prepared by the routes outlined in scheme 2 and 4 and by the way of the methods of example 12 (step 2), using Ethyl thioglycolate, sodium hydride and 4-(2-Chloro-4-methoxy-5-methoxymethoxy-phenyl)-2-methanesulfonyl-7-(2- trimethylsilanyl-ethoxymethyl)-7H-pyrrolo[2,3-d]pyrimidine-5-carbonitrile (example 45, step 6).
• the resultant crude product was purified by flash chromatography on SiO 2 eluting with Hexane then 30% EtOAc/Hexane to afford the title compound as an oil, 240mg, 81%.
• the title compound was prepared by the way of the methods of example 45 step 7, using [4-(2-Chloro-4-methoxy-5-methoxymethoxy-phenyl)-5-cyano-7-(2-trimethylsilanyl- ethoxymethyl)-7H-pyrrolo[2,3-d]pyrimidin-2-ylsulfanyl]-acetic acid ethyl ester and pyridine p-toluenesulfonate in the appropriate step (MOM deprotection). After a full aqueous work up the title compound was isolated as a cream-coloured foam and used without further purification, 172mg, 81%.
• the reaction mixture was stirred 18hrs at RT and then partitioned between EtOAc and water.
• the organic layer was separated and the aqueous extracted with a further portion of EtOAc and these combined organic layers were washed successively with saturated sodium bicarbonate solution and saturated brine solution.
• the organics were dried (Na 2 SO 4 ) and evaporated in vacuo.
• the resultant crude product was purified by flash chromatography on SiO 2 eluting with 30% EtOAc/Hexane - 50% EtOAc/Hexane (gradient) to afford the title compound as an oil, 120mg, 60%.
• the title compound was prepared by way of the method of example 1 step 5, using [4- ⁇ 2- chloro-5-[2-(3,3-difluoro-pyrrolidin-1-yl)-ethoxy]-4-methoxy-phenyl ⁇ -5-cyano-7-(2- trimethylsilanyl-ethoxymethyl ⁇ H-pyrrolop.S-dlpyrimidin ⁇ -ylsulfanyrj-acetic acid ethyl ester, tetrabutyl ammonium fluoride solution 1M and 1 ,2-diaminoethane in THF.
• the resultant crude product was purified by flash chromatography on SiO 2 eluting first with
• This compound had activity 'A' in the fluorescence polarization assay described below.
• the title compound was prepared by the way of the methods of example 1 step 5, using 4-(1,3-dihydro-isoindole-2-carbonyl)-2-methylsulfanyl-7-(2-trimethylsilanyl- ethoxymethyl)-7H-pyrrolo[2,3-d]pyrimidine-5-carbonitrile tetrabutyl ammonium fluoride solution 1M and 1,2-diaminoethane in THF.
• 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.
• 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.
• Chemicals are of the highest purity commercially available and all aqueous solutions are made up in AR water.
• BSA bovine serum albumen
• E. coli expressed human full-length HSP90 protein purified >95% (see, e.g., Panaretou et al., 1998) and stored in 50 ⁇ L aliquots at -80°C .
• the Z' factor is calculated from zero controls and positive wells. It typically gives a value of 0.7 - 0.9.
• Control wells are at either side of the 96 well plates, where 40 ⁇ l of medium is added.
• Gl 50 Growth inhibition in BT474 cells as described.
• Hsp90-related protein TRAP1 is a mitochondrial protein with distinct functional properties, J. Biol. Chem. 5, 3305-3312.
• Hsp90 inhibitors Nature 425. 407-410. Kasibhatla SR, Hong K, Zhang L, Biamonte MA, Boehm MF, Shi J, Fan J. PCT
• HDAC6 regulates Hsp90 acetylation and chaperone-dependent activation of the glucocorticoid receptor, MoI. Cell 18, 601-607.
• Hsp90 drives a molecular 'clamp' via transient dimerization of the N-terminal domains

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