Patent Application
20090054421
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.
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).
Exposure of cells to a number of environmental stresses, including heat shock, alcohols, heavy metals and oxidative stress, results in the cellular accumulation of a number of chaperones, commonly known as heat shock proteins (Hsps). Induction of Hsps protects the cell against the initial stress insult, enhances recovery and leads to maintenance of a stress tolerant state. It has also become clear, however, that certain Hsps may also play a major molecular chaperone role under normal, stress-free conditions by regulating the correct folding, degradation, localization and function of a growing list of important cellular proteins.
A number of multigene families of Hsps exist, with individual gene products varying in cellular expression, function and localization. They are classified according to molecular weight, e.g., Hsp70, Hsp90, and Hsp27. Several diseases in humans can be acquired as a result of protein misfolding (reviewed in Tytell et al., 2001; Smith et al., 1998). Hence the development of therapies which disrupt the molecular chaperone machinery may prove to be beneficial. In some conditions (e.g., Alzheimer's disease, prion diseases and Huntington's disease), misfolded proteins can cause protein aggregation resulting in neurodegenerative disorders. Also, misfolded proteins may result in loss of wild type protein function, leading to deregulated molecular and physiological functions in the cell.
Hsps have also been implicated in cancer. For example, there is evidence of differential expression of Hsps which may relate to the stage of tumour progression (Martin et al., 2000; Conroy et al., 1996; Kawanishi et al., 1999; Jameel et al., 1992; Hoang et al., 2000; Lebeau et al., 1991). As a result of the involvement of Hsp90 in various critical oncogenic pathways and the discovery that certain natural products with anticancer activity are targeting this molecular chaperone suggests that inhibiting the function of Hsp90 may be useful in the treatment of cancer. To this end, the first in class natural product 17AAG is currently in Phase II 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). When cells undergo various environmental cellular stresses, 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). In addition, recent results suggest that Hsp90 may also play a role in buffering against the effects of mutation, presumably by correcting the inappropriate folding of mutant proteins (Rutherford and Lindquist, 1998). However, Hsp90 also has an important regulatory role. Under normal physiological conditions, together with its endoplasmic reticulum homologue GRP94, Hsp90 plays a housekeeping role in the cell, maintaining the conformational stability and maturation of many client proteins. These can be subdivided into three groups: (a) steroid hormone receptors (e.g. estrogen receptor, progesterone receptor) (b) Ser/Thr or tyrosine kinases (e.g. Her2, Raf-1, CDK4, and Lck), and (c) a collection of apparently unrelated proteins, e.g. mutant p53 and the catalytic subunit of telomerase hTERT. It has also been shown recently that 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.
The highly conserved Hsp90 family in humans consists of four genes, namely the cytosolic Hsp90 α and Hsp90β isoforms (Hickey et al., 1989), GRP94 in the endoplasmic reticulum (Argon et al., 1999) and Hsp75/TRAP1 in the mitochondrial matrix (Felts et al., 2000). Apart from the differences in sub-cellular localisation, very little is known about the differences in function between Hsp90α/β, GRP94 and TRAP1. Initial reports suggesting that certain client proteins were chaperoned by a specific Hsp90 (e.g. Her2 by Grp94 alone) appear to have been erroneous.
Hsp90 participates in a series of complex interactions with a range of client and regulatory proteins (Smith, 2001). Although the precise molecular details remain to be elucidated, biochemical and X-ray crystallographic studies (Prodromou et al., 1997; Stebbins et al., 1997) carried out over the last few years have provided increasingly detailed insights into the chaperone function of Hsp90.
Following earlier controversy on this issue, it is now clear that Hsp90 is an ATP-dependent molecular chaperone (Prodromou et al, 1997), with 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).
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 interruption of the chaperone cycle (through blockage of the ATP turnover) causes the loss of the co-chaperone p23 from the complex and the targeting of the client proteins 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). Of interest, 17AAG has been shown to be much more active on tumour cells than its affinity for purified Hsp90 would suggest. This has lead to the suggestion that tumour cells (but not non-tumourigenic cells) contain 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 II 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). However, this still resulted in inhibition of Hsp90 function and degradation of a number of Hsp90-chaperoned signalling proteins (Marcu et al., 2000a). 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.
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 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).
Inhibition of 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.
The preclinical development of 17AAG as an anticancer agent has been well documented (Sausville et al., 2003) and is currently undergoing Phase II clinical trials. Phase I clinical trials results have been recently published (Banerji et al., 2005; Goetz et al., 2005; Ramanathan et al., 2005 and Grem et al., 2005). Of all these trials, the one conducted by Banerji et al. proved the most positive with a maximum dose of 450 mg/m2/week achieved with PD marker responses in the majority of patients and possible antitumour activity in two patients
Inhibition of 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).
Recent work has shown that the acetylation status of Hsp90 also plays a role in the control of the chaperone cycle. Inhibition of HDAC6 by either small molecule inhibitors or through siRNA gene targeting interrupts the chaperone cycle. Such treatments cause client protein degradation in a fashion analogous to small molecule ATP site inhibitors (Kovacs et al, 2005; Aoyagi & Archer, 2005).
In one broad aspect the present invention provides a compound of formula (I), or a salt, N-oxide, hydrate, or solvate thereof:
wherein
R1 is -(Alk1)p-(Z)r-(Alk2)s-Q wherein
The invention also concerns the use of such compounds in the preparation of a composition for inhibition of HSP90 activity in vitro or in vivo
In another broad aspect, the invention 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 above 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, 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.
As used herein:
As used herein, the term “(Ca-Cb)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 (Ca-Cb)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 “(Ca-Cb)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-Cb)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, and includes aromatic monocyclic or bicyclic carbocyclic radicals fused to a non aromatic carbocyclic or heterocyclic ring. Illustrative of such radicals are phenyl, biphenyl and napthyl, and radicals of the formula:
wherein 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.
As used herein the term “carbocyclic” refers to a cyclic radical whose ring atoms are all carbon, and includes 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. 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.
As used herein the unqualified term “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. 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 (C1-C6)alkyl, (C1-C6)alkoxy, methylenedioxy, ethylentdioxy, hydroxy, hydroxy(C1-C6)alkyl, mercapto, mercapto(C1-C6)alkyl, (C1-C6)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, —COORA, —CORA, —SO2RA, —CONH2, —SO2NH2, —CONHRA, —SO2NHRA, —CONRARB, —SO2NRARB, —NH2, —NHRA, —NRARB, —OCONH2, —OCONHRA, —OCONRARB, —NHCORA, —NHCOORA, —NRBCOORA, —NHSO2ORA, —NRBSO2ORA, —NHCONH2, —NRACONH2, —NHCONHRB, —NRACONHRB, —NHCONRARB or —NRACONRARB wherein RA and RB are independently a (C1-C6)alkyl group. In the case where 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. In the case where 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.
As used herein the term “salt” includes base addition, acid addition and quaternary salts. Compounds of the invention which are acidic can form salts, including pharmaceutically or veterinarily acceptable salts, with bases such as alkali metal hydroxides, e.g. sodium and potassium hydroxides; alkaline earth metal hydroxides e.g. calcium, barium and magnesium hydroxides; with organic bases e.g. N-ethyl piperidine, dibenzylamine and the like. Those compounds (I) which are basic can form salts, including pharmaceutically or veterinarily acceptable salts with inorganic acids, e.g. with hydrohalic acids such as hydrochloric or hydrobromic acids, sulphuric acid, nitric acid or phosphoric acid and the like, and with organic acids e.g. with acetic, tartaric, succinic, fumaric, maleic, malic, salicylic, citric, methanesulphonic and p-toluene sulphonic acids and the like.
For a review on suitable salts, see Handbook of Pharmaceutical Salts: Properties, Selection, and Use by Stahl and Wermuth (Wiley-VCH, Weinheim, Germany, 2002).
The term ‘solvate’ is used herein to describe a molecular complex comprising the compound of the invention and a stoichiometric amount of one or more pharmaceutically acceptable solvent molecules, for example, ethanol. The term ‘hydrate’ is employed when said solvent is water.
Compounds with which the invention is concerned which may exist in one or more stereoisomeric form, because of the presence of asymmetric atoms or rotational restrictions, can exist as a number of stereoisomers with R or S stereochemistry at each chiral centre or as atropisomeres with R or S stereochemistry at each chiral axis. The invention includes all such enantiomers and diastereoisomers and mixtures thereof. In particular, the compounds (I) with which the invention is concerned contain an oxime group, the double bond of which may have the cis configuration shown in formula (IA) or the trans configuration shown in formula (IB). Both configurations and mixtures thereof are included within the scope of, and suitable for use in, the invention:
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 (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).
Also included within the scope of the invention are metabolites of compounds of formula (I), that is, compounds formed in vivo upon administration of the drug. Some examples of metabolites include
The compounds of formula (I) defined above and salts, N-oxides hydrates, and solvates thereof are believed novel, and the invention includes all such novel compounds per se.
R2 is optionally substituted aryl or heteroaryl, 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 or indazolyl. For example, R2 may be a radical of formula:
wherein R13 and R14 are independently hydrogen or optional substitutents, especially electron donating substituents such as C1-C3alkoxy and amino or mono- or di-C1-C3alkylamino, or cyclic amino groups, and ring A has 5 or 6 ring members including the carbons of the phenyl ring to which it is fused, and has at least one heteroatom O, S or N hetero atom as a ring member. Examples of the latter type of R2 group are:
Other specific examples of R2 radicals are optionally substituted phenyl, naphthyl, 2-, 3-, and 4 pyridyl; 2- and 3-thienyl, 2-, and 3-furyl, 1-, 2- and 3-pyrrolyl, 1-, 2- and 4-imidazolyl, 1,3- and 4-pyrazolyl. Specific examples of optional substituents which may be present in an R2 radical, such as a phenyl radical, include chloro, fluoro, methoxy, ethoxy, 1-pyrrolidinyl, and 2-oxo-pyrrolidin-1-yl of formula
Preferred optional substituents in an R2 radical, such as a phenyl radical, are electron donating substituents such as C1-C3alkoxy and amino or mono- or di-C1-C3alkylamino, or cyclic amino groups.
In a simple embodiment of the compounds with which the invention is concerned, R1 may be hydrogen or methyl.
In other embodiments R1 is a radical -(Alk1)p-(Z)r-(Alk2)s-Q as defined above. Since the radical R1 is currently believed not to be involved in any significant interaction with the HSP90 target, it is especially preferred that R1 be a solubilising radical. In some such embodiments Q is a 5- or 6-membered non-aromatic heterocyclic ring, especially a solubilising ring, such as morpholino, thiomorpholino, piperidinyl, piperazinyl and 2-oxo-pyrrolidinyl. In some cases r is 0 and at least one of p and s is 1, for example p may be 1, s may be 0, and Alk1 may be —CH2— or —CH2CH2—. In other cases r is 1, p is 1, and s is 0 or 1, for example p may be 1, s may be 0, and Alk1 may be —CH2— or —CH2CH2—. When r is 1, Z may be, for example —O—, —S—, —NH—, —N(CH3)—, N(CH2CH3)—, —C(═O)NH—, —SO2NH—, —NHC(═O)—, or —NHSO2—.
Specific examples of radicals R1 other than hydrogen and methyl include:
R3 is hydrogen or an optional substituent, as defined above. Presently it is preferred that R3 be hydrogen.
When R4 is a carboxamide or sulfonamide group, examples include those of formula —CONRB(Alk)nRA or —SO2NRB(Alk)nRA wherein
Presently it is preferred that R4 be a carboxamide group, and specific examples of R4 groups include ethylamide, iso-propylamide, tert-butylamide, cyclopropylamide, 2-methoxyethylamide, 3-dimethylamino-propylamide, 2-dimethylamino-ethylamide, 2,2,2-trifluoro-ethylamide, and methoxamide.
When R4 is a carboxylic ester group, examples include those of formula —COORC wherein RC is a C1-C6 alkyl or C2-C6 alkenyl 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(C1-C6 alkyl)- or heteroaryl(C1-C6 alkyl)- group such as benzyl or pyridylmethyl; or an optionally substituted cycloalkyl group such as cyclopentyl or cyclohexyl.
Other examples of R4 groups include:
A specific subgroup of compounds with which the invention is concerned consists of those of formula (I), wherein
Specific compounds with which the invention is concerned include those of the Examples.
There are multiple synthetic strategies for the synthesis of the compounds (I) 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”, 4th Edition (Wiley), J March, “Comprehensive Organic Transformation”, 2nd Edition (Wiley), R. C. Larock, “Handbook of Heterocyclic Chemistry”, 2nd 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, a ketone of formula (II)
may be reacted with hydroxylamine or a hydroxylamine derivative H2NOR1 to form the desired compound of the invention. In general, the desired compound will be formed as a mixture of the cis and trans isomers (I) and (IA), often with a preponderance of one isomer over the other. If a pure isomer is wanted, the isomeric mixture may be separated chromatographically. Ketones (II) may be prepared by, for example, the following general reaction scheme can be employed:
Starting material are either available commercially or can be made according to literature methods. Subsequent reactions may be carried out on R2, R3 or R4 to prepare additional compounds of formula (I).
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 (Bijlmakers, 2000 and Yorgin, 2000); Anti-inflammatory diseases (Bucci, 2000) such as Rheumatoid arthritis, Asthma, MS, Type I Diabetes, Lupus, Psoriasis and Inflammatory Bowel Disease; Cystic fibrosis (Fuller, 2000); Angiogenesis-related diseases (Hur, 2002 and Kurebayashi, 2001): diabetic retinopathy, haemangiomas, psoriasis, endometriosis and tumour angiogenesis. Also an Hsp90 inhibitor of the invention may protect normal cells against chemotherapy-induced toxicity and be useful in diseases where failure to undergo apoptosis is an underlying factor. Such an Hsp90 inhibitor may also be useful in diseases where the induction of a cell stress or heat shock protein response could be beneficial, for example, protection from hypoxia-ischemic injury due to elevation of Hsp70 in the heart (Hutter, 1996 and Trost, 1998) and brain (Plumier, 1997 and Rajder, 2000). An Hsp90 inhibitor-induced increase in Hsp70 levels could also be useful in diseases where protein misfolding or aggregation is a major causal factor, for example, neurogenerative disorders such as scrapie/CJD, Huntingdon's and Alzheimer's (Sittler, 2001; Trazelt, 1995 and Winklhofer, 2001)”.
Ketones of formula (II) above, wherein R2, R3 or R4 are as defined and discussed above in relation to formula (I), are also inhibitors of HSP90 and therefore useful in the same way as the compounds (I) with which the invention is concerned. Such ketones fall within the general class of HSP90 inhibitors disclosed in our copending application PCT/GB2004/003641.
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 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.
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 administered alone, or in combination treatments with other drugs. For example, for the treatment of cancer, the compounds may be administered together with other anticancer drugs, which will normally have a mode of action different from inhibition of HSP90. Treatment of cancer often involves such multi-drug treatments.
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.
All reagents obtained from commercial sources were used without further purification. Anhydrous solvents were obtained from commercial sources and used without further drying. Flash chromatography was performed with pre-packed silica gel cartridges (Strata SI-1; 61 Å, Phenomenex, Cheshire UK or IST Flash II, 54 Å, Argonaut, Hengoed, UK). Thin layer chromatography was conducted with 5×10 cm plates coated with Merck Type 60 F254 silica gel. Ion Exchange Chromatography was performed with IST SCX-2 pre packed cartridges (Argonaut, Hengoed, UK).
The compounds of the present invention were characterized by LC/MS (“method A”) using a Hewlett Packard 1100 series LC/MSD linked to quadripole detector (ionization mode: electron spray positive; column: Phenomenex Luna 3u C18(2) 30×4.6 mm; Buffer A prepared by dissolving 1.93 g ammonium acetate in 2.5 L HPLC grade H2O and adding 2 mL formic acid. Buffer B prepared by adding 132 mL buffer A to 2.5 L of HPLC grade acetonitrile and adding 2 mL formic acid; elution gradient 95:5 to 5:95 buffer A: buffer B over 3.5 or 7.5 minutes. Flow rate=2.0 mL/min).
Some compounds of the invention were characterised by a separate LC/MS system (“method B”) using an Agilent 1100 series ion trap XCT (ionisation mode: ESI). Column Genesis 3u C18. Solvent A: 0.1% HCOOH in H2O; solvent B Acetonitrile. Elution gradient 30:70 to 5:95 solvent A: solvent B over 5 minutes. Flow rate=1.0 mL/min).
Some compounds of the invention were characterised by a separate LC/MS system (“method C”) using a Micromass LCT/Water's Alliance 2795 HPLC system with a Discovery 5 μm, C18, 50 mm×4.6 mm i.d. column from Supelco at a temperature of 22° C. using the following solvent systems: Solvent A: Methanol; Solvent B: 0.1% Formic acid in water at a flow rate of 1 mL/min. Gradient starting with 10% A/90% B from 0-0.5 mins then 10% A/90% B to 90% A/10% B from 0.5 mins to 6.5 mins and continuing at 90% A/10% B up to 10 mins. From 10-10.5 mins the gradient reverted back to 10% A/90% where the concentrations remained until 15 mins. UV detection was at 254 nm and ionisation was positive or negative ion electrospray. Molecular weight scan range is 50-1000. Samples were supplied as 1 mg/mL in DMSO or methanol with 3 μL injected on a partial loop fill.
Nuclear magnetic resonance (NMR) analysis was performed with a Brucker DPX-400 MHz NMR spectrometer or a Bruker AV400 400 MHz NMR spectrometer The spectral reference was the known chemical shift of the solvent. Proton NMR data is reported as follows: chemical shift (δ) in ppm, integration, multiplicity (s=singlet, d=doublet, t=triplet, q=quartet, p=pentet, m=multiplet, dd=doublet of doublet, br=broad), coupling constant.
Compounds of the invention can be made, by way of example, by the synthetic route shown in scheme 1. Scheme 1 is a general scheme for the synthesis of some compounds of the invention. Suitable reagents, solvents, methodology and reaction temperatures for the synthetic procedures of scheme 1 will be known to those skilled in the art of organic chemistry. Examples of synthetic procedures used to synthesize compound of type E and F are given in examples below.
Alternatively, the compounds of type F in scheme 1 can be made by reacting functionalised oximes with ketones of type D. Scheme 2 is a general scheme for this transformation. Examples of synthetic procedures used to synthesize compound of type F by this method are given in example 1.
Aldehydes R1CHO depicted in scheme 1 can be obtained from commercial suppliers or in some cases synthesised by literature methods.
To a stirred mixture of 2-amino-4,6-dichloro-5-formyl-pyrimidine (available from Bionet Research Intermediates, UK) (5.0 g 1 eq.) and potassium carbonate (9.0 g; 2.5 equiv.) in acetonitrile (160 ml) at ambient temperature was added ethyl-2-mercaptoacetate (2.86 ml; 1.0 equiv.). The resulting mixture was stirred at reflux for three hours. After cooling, the solvents were removed in vacuo and the residue partitioned between ethyl acetate and water, the phases separated and the organic phase washed with saturated aqueous sodium chloride solution. Phases were separated and the organic phase was dried over Na2SO4 then filtered and filtrate solvents removed in vacuo. The crude product was purified by column chromatography on silica gel, eluting with ethyl acetate and hexanes, to afford 2-Amino-4-chloro-thieno[2,3-d]pyrimidine-6-carboxylic acid ethyl ester as a yellow powder (60%).
LC-MS: RT=2.371 minutes, m/z=258.0 [M+H]+ (Total run time=3.5 minutes)
To a solution of 2-amino-4-chloro-thieno[2,3-d]pyrimidine-6-carboxylic acid ethyl ester (0.295 g, 1.14 mmol), 4-methyl-3,4-dihydro-2H-benzo[1,4]oxazine-7-carbaldehyde (0.203 g, 1.14 mmol) and 3-ethyl-1-methyl-3H-imidazol-1-ium bromide (72 mg, 0.38 mmol) in DMF at ambient temperature, was added Sodium Hydride (1.1 mole equiv.). The solution turned dark immediately and was stirred for 3 hours. The resulting orange suspension obtained was filtered through a sinter glass funnel. Aqueous sodium chloride solution was added to the filtrate causing precipitation to occur. The precipitate was then filtered and dried in vacuo. The resulting orange solids were purified by preparative TLC. Rf=0.32 (EtOAc:hexane/3:2). This afforded 2-Amino-4-(4-methyl-3,4-dihydro-2H-benzo[1,4]oxazine-7-carbonyl)-thieno[2,3-d]pyrimidine-6-carboxylic acid ethyl ester as an orange powder (100 mg, 20%).
1H NMR (d6-acetone) δ=7.82 (1H, s); 7.55 (1H, dd, J=8.5 and 2.0 Hz); 7.40 (1H, d, J=2.0 Hz); 6.75 (2H, s, broad); 6.72 (1H, d, J=8.5 Hz); 4.30 (2H, q, J=7.0 Hz); 4.25 (2H, m); 3.50 (2H, m); 3.08 (3H, s) and 1.30 (3H, t, J=7.0 Hz).
LC/MS: (method C)RT=6.49 minutes, m/z=399.1 (M+H)+. (Total run time=15 minutes)
2-Amino-4-(4-methyl-3,4-dihydro-2H-benzo[1,4]oxazine-7-carbonyl)-thieno[2,3-d]pyrimidine-6-carboxylic acid ethyl ester (45 mg, 0.11 mmol) was added to a solution of ethylamine in methanol (15 ml) and the reaction mixture heated and stirred at 85° C. for 4 hours. Methanol was removed in vacuo and the residue was dissolved in ethyl acetate and resulting solution then washed with saturated sodium chloride solution. The organic layer was separated and dried over MgSO4 then filtered and the filtrate solvents removed in vacuo. A yellow oil (30 mg, 67%) was obtained after preparative TLC purification (ethyl acetate/hexanes). Rf=0.46 (EtOAc:hexane/5:1).
1H NMR (d6-acetone) δ=8.00 (1H, s, broad); 7.65 (1H, s); 7.55 (1H, dd, J=8.7 and 2.0 Hz); 7.38 (1H, d, J=2.0 Hz); 6.72 (1H, d, J=8.7 Hz); 6.55 (2H, s, broad); 4.25 (2H, m); 3.50 (2H, m); 3.38 (2H, q, J=7.0 Hz) 3.05 (3H, s) and 1.25 (3H, t, J=7.0 Hz).
LC/MS (method C) RT=6.11 min, m/z=398.2 (M+H)+. (Total run time=15 minutes)
To a solution of 2-Amino-4-(4-methyl-3,4-dihydro-2H-benzo[1,4]oxazine-7-carbonyl)-thieno[2,3-d]pyrimidine-6-carboxylic acid ethylamide (10 mg, 0.025 mmol) in ethanol (3 ml) was added methoxylamine hydrochloride (42 mg, 20 equiv.) and the resulting solution irradiated in a microwave reactor at 120° C. for 30 minutes. Ethanol was removed in vacuo and the residue partitioned between ethyl acetate and saturated sodium chloride solution. The organic layer was dried over MgSO4 and evaporated to dryness. Yellow oil (5 mg, 37%) was obtained after preparative TLC purification. Resulting product consists of a mixture of cis and trans isomers in a ratio of ca. 1:1.7. Rf=0.43 (EtOAc:hexane/5:2).
(Major isomer): 1H NMR (d6-acetone) δ=7.95 (1H, s, broad); 7.44 (1H, s); 6.91 (1H, d, J=2.0 Hz); 6.82 (1H, dd, J=8.5 and 2.0 Hz); 6.63 (1H, d, J=8.5 Hz); 6.47 (2H, s, broad); 4.25 (2H, m); 3.81 (3H, s); 3.35 (2H+2H, m); 2.85 (3H, s) and 1.13 (3H, t, J=7.0 Hz).
LC/MS: RT=6.42 minutes, m/z=427.3 (M+H)+. (Total run time=15 minutes)
(Minor isomer): 1H NMR (d6-acetone) δ=7.95 (1H, s, broad); 7.93 (1H, s); 7.05 (1H, d, J=2.0 Hz); 6.97 (1H, dd, J=8.5 and 2.0 Hz); 6.65 (1H, d, J=8.5 Hz); 6.38 (2H, s, broad); 4.25 (2H, m); 4.00 (3H, s); 3.35 (2H+2H, m); 2.91 (3H, s) and 1.13 (3H, t, J=7.0 Hz).
LC/MS (method C): RT=6.63 min, m/z=427.3 (M+H)+. (Total run time=15 minutes).
This compound had activity ‘A’ in the fluorescence polarization assay described below.
To a solution of 2-Amino-4-(4-methyl-3,4-dihydro-2H-benzo[1,4]oxazine-7-carbonyl)-thieno[2,3-d]pyrimidine-6-carboxylic acid ethylamide (step 3 example 1) (10 mg, 0.025 mmol) in ethanol (5 ml) was added hydroxylamine hydrochloride (35 mg, 20 equiv.) and the solution was refluxed for 5 hours. Ethanol was removed in vacuo and the residue partitioned between ethyl acetate and brine. The organic layer was dried over MgSO4 and evaporated to dryness. A yellow oil (4 mg, 39%) was obtained after preparative TLC purification. Resulting product consisted a mixture of cis and trans-isomers in a ratio of ca. 1:1.3. Rf=0.58 (EtOAc:hexane/5:1).
(Major isomer): 1H NMR (d6-acetone) δ=10.33 (1H, s); 7.98 (1H, s, broad); 7.45 (1H, s); 6.91 (1H, d, J=2.0 Hz); 6.83 (1H, dd, J=8.5 and 2.0 Hz); 6.63 (1H, d, J=8.5 Hz); 6.44 (2H, s, broad); 4.25 (2H, m); 3.81 (3H, s); 3.39 (2H, m); 3.31 (2H, m); 2.90 (3H, s) and 1.10 (3H, t, J=7.0 Hz).
LC/MS (method C): RT=5.94 min, m/z=413.4 (M+H)+. (Total run time=15 minutes)
(Minor isomer): 1H NMR (d6-acetone) δ=10.97 (1H, s); 7.98 (1H, s, broad); 7.91 (1H, s); 7.11 (1H, d, J=2.0 Hz); 7.03 (1H, dd, J=8.5 and 2.0 Hz); 6.67 (1H, d, J=8.5 Hz); 6.35 (2H, s, broad); 4.25 (2H, m); 3.81 (3H, s); 3.39 (2H, m); 3.31 (2H, m); 2.94 (3H, s) and 1.10 (3H, t, J=7.0 Hz).
LC/MS (method C): RT=6.09 minutes, m/z 413.4 (M+H)+. (Total run time=15 minutes)
This compound had activity ‘A’ in the fluorescence polarization assay described below.
This example was prepared by way of the method of Example 2.
1H NMR (Acetone-d6, 250 MHz) (major) δ 10.51 (1H, s), 7.35 (1H, broad s), 7.07 (1H, d, J=1.1 Hz), 6.69 (2H, d, J=1.0 Hz), 6.32 (2H, broad s), 5.92 (2H, s), 3.23 (2H, dq, J=1.6, 7.3 Hz), 1.01 (3H, t, J=7.2 Hz).
LC/MS (method C) (major) RT=5.85 min, m/z=386.2 (M+H)+, (minor) RT=5.99 min, m/z=386.2 (M+H)+. (Total run time=15 minutes)
This compound had activity ‘A’ in the fluorescence polarization assay described below.
2-Amino-4-(benzo[1,3]dioxole-5-carbonyl)-thieno[2,3-d]pyrimidine-6-carboxylic acid ethyl ester (20 mg, 0.054 mmol) (prepared by way of methods for step 1 and step 2 in Example 1) and methoxylamine hydrochloride (20 equiv.) were dissolved in methanol (1 ml) and heated for 10 minutes at 110° C. in a CEM microwave reactor (300 W, constant cooling). The solvents were removed in vacuo and the residue partitioned between water and ethyl acetate. The phases were separated and the organic phase was dried over MgSO4 and evaporated to dryness in vacuo. The residue was purified by preparative TLC eluting with 4% ethanol in dichloromethane to yield the pure product as a yellow powder (2.2 mg, 20%).
(Major isomer) 1H NMR (CDCl3, 250 MHz) δ 7.53 (1H, s), 7.13 (1H, d, J=1.5 Hz), 6.73-6.63 (1H+1H, m), 5.92 (2H, s), 5.31 (2H, broad s), 4.29 (2H, q, J=7.1 Hz), 3.86 (3H, s), 1.30 (2H, t, J=7.1 Hz).
LC/MS (method C): RT=7.85 min, m/z=401.1 (M+H)+. (Total run time=15 minutes)
(Minor isomer) LC/MS (method C): RT=7.95 min, m/z=401.1 (M+H)+. (Total run time=15 minutes)
This compound had activity ‘A’ in the fluorescence polarization assay described below.
The following examples (table 1) were synthesised by way of the general synthesis shown in scheme 1 and 2 and by the methods outlined in examples 1 and 2. The column headed “Hsp90 IC50” refers to the activity (‘A or B’) in the fluorescence polarization assay described below. Retention times are for Method A, 3.5 min run time unless otherwise stated.
Sodium hydride (0.843 g, 21.1 mmol, 60% suspension in oil) was added in one portion to a stirred suspension of 2-amino-4-chloro-thieno[2,3-d]pyrimidine-6-carboxylic acid ethyl ester (4.47 g, 17.3 mmol) and piperonal (2.61 g, 17.3 mmol) and 3-ethyl-1-methyl-3H-imidazol-1-ium bromide (1.09 g, 5.77 mmol) in DMF (54 mL) at room temperature under an atmosphere of nitrogen. The reaction mixture was stirred for 3 hours then poured into a saturated brine solution (300 mL) the resultant emulsion was filtered and the solid dried under vacuum. The solid was purified by flash column chromatography (eluting with 1:2 to 1:1 hexane/ethyl acetate) to give the title compound as a yellow powder (2.57 g, 40%). This yellow powder contained ˜20% impurities by HPLC.
Hydroxylamine hydrochloride (0.973 g, 14.0 mmol) was added to a suspension of the ethyl ester (1.30 g, ˜3.50 mmol) in ethanol (50 mL) and the resultant suspension heated at reflux for 4 hours. After this time the reaction mixture was concentrated in vacuo to provide a yellow solid which was then partitioned between ethyl acetate (50 mL) and saturated aqueous sodium bicarbonate (50 mL). The organic layer (which contained a significant amount of suspended solid) was then washed with water (50 mL) then saturated brine (50 mL) and then concentrated in vacuo to give a highly insoluble yellow solid (1.0 g, 74%). [The organic layer was not treated with drying agents or filtered due to the suspended solid]. Attempts to triturate the crude yellow solid with diethyl ether did not improve the purity. The crude solid was used in the next reaction without further purification.
2-Amino-4-(benzo[1,3]dioxol-5-yl-hydroxyimino-methyl)-thieno[2,3-d]pyrimidine-6-carboxylic acid ethyl ester (1.0 g, 2.59 mmol) was suspended in a mixture of ethanol (10 mL) and 1.0 M aqueous sodium hydroxide solution (20 mL) and then heated at reflux for one hour after which time the reaction mixture had formed a clear pale yellow solution. The reaction mixture was reduced in vacuo to ˜20 mL then diluted by addition of water (20 mL) then extracted with ethyl acetate (40 mL). The aqueous phase was then acidified by addition of dilute (˜1 M) aqueous hydrochloric acid then the resultant precipitate was collected by filtration and dried under vacuum at 50° C. for 20 hours to give the title compound as a pale yellow powder (0.890 g, 96%). The product was used without further purification in the next step.
To a solution of the carboxylic acid (40.0 mg, 0.111 mmol), 1-hydroxybenzotriazole (17.0 mg, 0.189 mmol) and cyclopropylamine (5.2 μL, 0.074 mmol) in DMF (2 mL) was added polymer supported carbodiimide resin (123 mg, 0.222 mmol, 1.2 mmol/g loading). The resultant mixture was shaken for 3 hours then polymer supported carbonate resin (236 mg, 1.11 mmol, 3.14 mmol/g loading) was added and the mixture shaken for a further 2 hours. The resin was filtered and washed with dichloromethane (5 mL). The resultant filtrate was then loaded onto an SCX-2 SPE ion exchange column (5 g) cartridge. The cartridge was then washed with dichloromethane (15 mL) then methanol (15 mL). The product was then eluted off the SPE cartridge by washing with ˜7 M solution of ammonia in methanol (15 mL). This latter fraction was concentrated in vacuo to give the title compound as a yellow solid (20.0 mg, 68%) in greater than 90% purity by proton NMR.
LC/MS: RT=2.00 min, m/z=398 (M+H)+. (Total run time=3.5 minutes Method A)
This compound had activity ‘A’ in the fluorescence polarization assay described below.
The following examples (table 2) were synthesised by way of the by the methods outlined in example 20. The column headed “Hsp90 IC50” refers to the activity of the compound (‘A or B’) in the fluorescence polarization assay described below. Retention times are for Method A, 3.5 minute run time unless otherwise stated.
A solution of 2-Amino-4-benzo[1,3]dioxol-5-yl-hydroxyimino-methyl)-thieno[2,3-d]pyrimidine-6-carboxylic acid ethylamide (example 3) (50.0 mg, 130 μmol) and 2-bromo-N,N′,diethylamine hydrobromide (37.2 mg, 143 μmol) in DMF (5 mL) was treated with potassium carbonate (90.0 mg, 650 μmol) then heated at 140° C. for 150 minutes. The reaction mixture was purified using an SCX-2 SPE ion exchange cartridge. Purification of the crude product by flash column chromatography (eluting with 1:9:490 conc NH3(aq):methanol:dichloromethane) gave the product as a pale yellow solid (25.0 mg, 40%). Proton NMR indicated a 3.3:1 ratio of isomers. The major isomer was the less polar isomer (by TLC).
LC/MS: RT=2.44 and 2.57 min (E/Z regioisomers), m/z=485 (M+H)+. (Total run time=7.5 minutes Method A)
This compound had activity ‘A’ in the fluorescence polarization assay described below.
The following compounds (table 3) were made by way of the general method of scheme 1 utilizing the methods outlined in examples 2, 20 and 27. The column headed “Hsp90 IC50” refers to the activity (‘A or B’) in the fluorescence polarization assay described below. Retention times are for Method A, 3.5 min run time unless otherwise stated.
The following examples (table 4) were synthesised by way of the general synthesis shown in scheme 1 and 2 and by the methods outlined in examples 1, 2, 20 and 26. The column headed “Hsp90 IC50” refers to the activity (‘A or B’) in the fluorescence polarization assay described below. Retention times are for Method B, 5.0 min run time unless otherwise stated.
Fluorescence polarization {also known as fluorescence anisotropy} measures the rotation of a fluorescing species in solution, where the larger molecule the more polarized the fluorescence emission. When the fluorophore is excited with polarized light, the emitted light is also polarized. The molecular size is proportional to the polarization of the fluorescence emission.
The fluoroscein-labelled probe—
binds to HSP90 {full-length human, full-length yeast or N-terminal domain HSP90} and the anisotropy {rotation of the probe:protein complex} is measured.
Test compound is added to the assay plate, left to equilibrate and the anisotropy measured again. Any change in anisotropy is due to competitive binding of compound to HSP90, thereby releasing probe.
Chemicals are of the highest purity commercially available and all aqueous solutions are made up in AR water.
The compounds tested in the above assay were assigned to one of two activity ranges, namely A=<10 μM; B=>10 μM, and those assignments are reported above.
A number of publications are cited above in order to more fully describe and disclose the invention and the state of the art to which the invention pertains. Full citations for these references are provided below. Each of these references is incorporated herein by reference in its entirety into the present disclosure.
Reviews for detailed background and drug discovery:
| Number | Date | Country | Kind |
|---|---|---|---|
| 0501535.9 | Jan 2005 | GB | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/GB06/00219 | 1/23/2006 | WO | 00 | 4/14/2008 |
