HK1091831B

HK1091831B - Isoxazole compounds as inhibitors of heat shock proteins

Info

Publication number: HK1091831B
Application number: HK06112447.8A
Authority: HK
Inventors: M‧J‧德赖斯代尔; B‧W‧迪莫克; H‧芬奇; P‧韦伯; E‧麦克唐纳德; K‧E‧詹姆斯; 张桂明; T‧P‧马修斯
Original assignee: 癌症研究技术有限公司; 癌症研究所; 弗奈利斯（R&D）有限公司
Priority date: 2003-02-11
Filing date: 2004-02-09
Publication date: 2010-11-19

Description

Isoxazole compounds

Technical Field

The present invention relates to substituted isoxazoles having HSP900 inhibitory activity, to the use of these compounds in medicine, to diseases which respond to inhibition of HSP900 activity, such as cancer, and to pharmaceutical compositions comprising these compounds.

Background

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

Heat Shock Proteins (HSP)

Exposing cells to a number of environmental stresses, including heat shock, alcohol, heavy metals and oxidative stress, results in the accumulation of many chaperones by the cells, the latter of which are commonly referred to as Heat Shock Proteins (HSPs). Induction of HSPs protects cells from initial stress damage, enhances defense, and allows the stress-tolerant state to be maintained. However, it is clear that certain HSPs may also play a major chaperone role in normal, unstressed conditions through proper folding, degradation, localization and function of a growing list of important cellular proteins.

There are many multigene families of HSPs, each with variations in cellular expression, function, and localization. They can be classified according to molecular weight, for example, HSP70, HSP90 and HSP 27.

Some diseases in humans are the result of protein misfolding (reviewed in Tytell et al 2001; Smith et al 1998). The development of therapies that disrupt the cellular chaperone mechanism may therefore prove beneficial. Under some conditions, (e.g., Alzheimer's disease, prion disease, and Huntington's disease), misfolded proteins can cause aggregation of proteins that lead to neurodegenerative diseases. Misfolded proteins can also result in loss of function of the wild-type protein, resulting in deregulated molecular and physiological function in the cell.

HSPs are also implicated in cancer. For example, evidence of differences in HSP expression that may be correlated with the stage of tumor progression (Martin et al, 2000; Conroy et al, 1996; Kawanishi et al, 1999; Jameel et al, 1992; Honng et al, 2000; Lebeau et al, 1991). Since HSP90 is involved in a variety of key tumorigenic pathways, certain natural products with anticancer activity were found to target these chaperones. Inhibition of HSP function has been proposed as an attractive view that can be useful for the treatment of cancer. The first chaperone inhibitor is now in clinical trials.

HSP90

HSP90 constitutes about 1-2% of the total cellular protein and is usually present in the cell as a dimer with one of many other proteins (see, e.g., Pratt, 1997). It is essential for the survival of cells, and it shows a dual chaperoning function (Young et al, 2001). It plays a key role in cellular stress by interacting with many proteins whose native conformation is altered by various environmental stresses, such as heat shock, to ensure adequate protein folding and to prevent non-specific aggregation (Smith et al, 1998). In addition, recent results indicate that HSP90 may also play a role in buffering the effects of mutations by correcting improper folding of the mutant protein (Rutherford and Lindquist, 1998). However, HSP90 also plays an important regulatory role. Under normal physiological conditions, along with its endoplasmic reticulum homolog GRP94, HSP90 plays a housekeeping role in the cell, maintaining conformational stability and maturation of several key client proteins (client proteins). These can be subdivided into three groups: (a) steroid hormone receptors, (b) serine/threonine or tyrosine kinases (e.g., ERBB2, RAF-1, CDK4 and LCK) and (c) apparently unrelated collections of proteins, e.g., mutant p53 and the catalytic subunit of telomerase hTERT. All of these proteins play key regulatory roles in many physiological and biochemical processes of the cell. New HSP90 client proteins are continually being identified.

The highly conserved HSP90 family in humans consists of four genes, namely the cytoplasmic HSP90 alpha and HSP90 beta isoforms (Hickey et al, 1989), GRP94 in the endoplasmic reticulum (Argon et al, 1999) and HSP75/TRAP1 in the mitochondrial matrix (Felts et al, 2000). It is believed that all family members have similar modes of action, but bind to different client proteins depending on their intracellular localization. For example, ERBB2 is known to be a specific client protein of GRP94(Argon et al, 1999) and the receptors for tumor necrosis factor type 1 (TNFR1) and RB, both of which are shown to be clients of TRAP1 (Song et al, 1995; Chen et al, 1996).

HSP90 is involved in a complex series of interactions with many client proteins and regulatory proteins (Smith, 2001). Although precise molecular details remain to be elucidated, biochemical and X-ray crystallography studies conducted over the past few years (Prodromou et al, 1997; Stebbins et al, 1997) have provided an increasingly detailed understanding of the chaperone function of HSP 90.

After early debate on this problem, it is now clear that HSP90 is an ATP-dependent chaperone (Prodromou et al, 1997) and that dimerization of the nucleotide binding region is essential for ATP hydrolysis, which in turn is essential for chaperone function (Prodromou et al, 2000 a). Binding of ATP leads to the formation of a circular dimer structure in which the N-terminal regions are in closer contact with each other, resulting in a conformational transition known as the "clip mechanism" (Prodromou and Pearl, 2000 b).

Known HSP90 inhibitors

The earliest discovered class of HSP90 inhibitors were the benzoquinone ansamycins, which included the compounds herbimycin a and geldanamycin. They have been shown to reverse the malignant phenotype of fibroblasts transformed with the v-Src oncogene (Uehara et al, 1985), followed by strong antitumor activity in animal models in vivo (Schulte et al, 1998) and in vitro (Supko et al, 1995).

Immunoprecipitation and affinity matrix studies have shown that the major mechanism of geldanamycin action involves binding to HSP90 (Whitesell et al, 1994; Schulte and Neckers, 1998). In addition, X-ray crystallography studies have shown that it competes for the ATP-binding site and inhibits the intrinsic ATPase activity of HSP90 (Prodromou et al, 1997; Panaretou et al, 1998). This in turn prevents the formation of mature multimeric complexes capable of chaperoning the client protein HSP 90. As a result, the client protein is targeted for degradation via the ubiquitin proteasome pathway. 17-allylamino, 17-demethoxygeldanamycin (17AAG) retains the inhibitory properties of HSP90, leading to client protein depletion and anti-tumor activity in cultured cells and xenograft models (Schulte et al, 1998; Kelland et al, 1999), but with significantly less hepatotoxicity than geldanamycin (Page et al, 1997). 17AAG is now being evaluated in phase I clinical trials.

Radicicol is a macrocyclic antibiotic that has been shown to reverse the malignant phenotype of v-Src and v-Ha-Ras transformed fibroblasts (Kwon et al, 1992; Zhao et al, 1995). It has been demonstrated that many signal proteins are degraded by inhibition of HSP90 (Schulte et al, 1998). X-ray crystallographic data confirmed that radicicol also binds to the N-terminal region of HSP90 and inhibits intrinsic ATPase activity (Roe et al, 1998). Radicicol lacks anti-tumor activity in vivo due to the unstable chemical nature of the compound.

Coumarin antibiotics are known to bind to bacterial DNA gyrase at ATP binding sites similar to HSP 90. It has been shown that coumarin, novobiocin, and HSP90 bind at the carboxylic acid end, i.e., at a site different from the site occupied by the benzoquinone ansamycin and radicicol bound at the N-terminus (Marcu et al, 2000 b). However, this still leads to inhibition of HSP function and degradation of many HSP90 chaperone signaling proteins (Marcu et al, 2000 a). After neomycin, geldanamycin was unable to bind HSP 90; this suggests that some interaction between the N-terminal and C-terminal regions must exist and is consistent with the notion that both sites are important for the chaperone properties of HSP 90.

Purine-based HSP90 inhibitors, PU3, have been shown to cause degradation of signaling molecules, including ERBB2, and to cause cell cycle arrest and differentiation of breast cancer cells (Chiosis et al, 2001).

HSP90 as therapeutic target

The chaperone HSP90 is now being assessed as a new target for anticancer drug development due to its involvement in the regulation of many of the vital signaling pathways driving the tumor phenotype, and the discovery that certain biologically active natural products exert their effects through HSP90 activity (Neckers et al, 1999).

The major mechanisms of action of geldanamycin, 17AAG and radicicol include binding to HSP90at ATP binding sites located in the N-terminal region of the protein, resulting in inhibition of the ATPase activity intrinsic to HSP90 (see, e.g., Prodromou et al, 1997; Stebbins et al, 1997; Panaretou et al, 1998).

Inhibition of HSP90 ATPase activity prevents recruitment of co-chaperones and promotes the formation of a class of HSP90 heterocomplexes from which these client proteins are targeted for degradation via the ubiquitin proteasome pathway (see, e.g., Neckers et al, 1999; Kelland et al, 1999).

Treatment with HSP90 inhibitors results in the selective degradation of important proteins involved in cell proliferation, cell cycle regulation and apoptosis and processes important in cancer.

Inhibition of HSP90 function has been shown to result in selective degradation of important signaling proteins involved in cell proliferation, cell cycle regulation, apoptosis, and processes important in cancer and often deregulated (see, e.g., Hostein et al, 2001). The very attractive rationale for the clinical use of drugs against this target is that by simultaneously depleting proteins associated with the transformed phenotype, one could potentially achieve strong antitumor effects and therapeutic advantages against cancer cells over normal cells. These downstream events of HSP90 inhibition are thought to be responsible for the antitumor activity of HSP90 inhibitors in cultured cells and animal models (see, e.g., Schulte et al, 1998; Kelland et al, 1999).

Disclosure of Invention

The present invention relates to the use of a class of substituted isoxazole compounds as HSP90 inhibitors, for example for inhibiting cancer cell proliferation. The invention also includes the novel isoxazole compounds themselves and pharmaceutical compositions containing them.

The invention provides the use of a compound of formula (A) or (B), or a salt, N-oxide, hydrate or solvate thereof, or a prodrug thereof, in the preparation of a composition capable of inhibiting HSP90 activity.

In the formula

R₁Is a group of the general formula (IA):

-Ar¹-(Alk¹)_p-(Z)_r-(Alk²)_s-Q (IA)

in the formula (I) in any compatible combination

Ar¹Is an optionally substituted aryl or heteroaryl group,

Alk¹and Alk²Is optionally substituted divalent C₁-C₆Alkylene or C₂-C₆An alkenylene group which is a radical of an alkylene group,

p, r and s are independently 0 or1,

z is-O-, -S-, -C-O-and- (C-S) -and-SO₂-，-C(＝O)O-，-C(＝O)NR^A-，-C(＝S)NR^A-，-SO₂NR^A-，-NR^AC(＝O)-，-NR^ASO₂-or-NR^A-, in which R^AIs hydrogen or C₁-C₆Alkyl, and

q is hydrogen or an optionally substituted carbocyclic or heterocyclic group;

R₂is (i) as for R₁A group of the general formula (IA) as defined in (A);

(ii) a carboxamide group; or

(iii) A non-aromatic carbocyclic or heterocyclic ring in which the carbon atoms of the ring are of the formula- (Alk)¹)_p-(Z)_r-(Alk²)_sThe radical-Q is optionally substituted and/or the nitrogen atom of the ring is substituted by a radical of the formula- (Alk)¹)_p-(Z)_r-(Alk²)_sThe radical-Q is optionally substituted, where Q, Alk¹，Alk²Z, p, r and s are as defined for the group (IA); and

R₃is hydrogen, optionally substituted cycloalkyl, cycloalkenyl, C₁-C₆Alkyl radical, C₁-C₆Alkenyl, or C₁-C₆An alkynyl group, or a carboxyl, carboxamide or carboxylate group.

In general, the class of compounds of formula (I) defined above is believed to be novel and the invention includes all novel members of that class and their salts, hydrates and solvates, as well as prodrugs thereof.

As used herein:

the term "carboxy" refers to a group of the formula-COOH;

the term "carboxylate group" refers to a group of the formula-COOR, wherein R is an actual or abstract organic radical derived from a hydroxy compound ROH; and

the term "carboxamide group" refers to a compound of the formula-CONR_aR_bA group of formula (II) wherein-NR_aR_bIs actual or abstract HNR from ammonia or amines_aR_bDerived primary or secondary (including cyclic) amine groups.

The term "(C) as used herein_a-C_b) Alkyl ", wherein a and b are integers, refers to straight or branched chain alkyl groups having a to b carbon atoms. Thus for example when a is 1 and b is 6, the term includes methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl and n-hexyl.

The term "divalent (C) as used herein_a-C_b) Alkylene ", wherein a and b are integers, refers to a saturated hydrocarbon chain having a to b carbon atoms and two unsaturations.

The term "(C) as used herein_a-C_b) Alkenyl ", wherein a and b are integers, refers to straight or branched chain alkenyl groups having a to b carbon atoms and containing at least one double bond in the E or Z configuration. Including, for example, vinyl and allyl.

The term "divalent (C) as used herein_a-C_b) Alkenylene ", wherein a and b are integers, refers to a hydrocarbon chain having a to b carbon atoms, at least one double bond and two unsaturation (unsaturation) valencies.

The term "(C) as used herein_a-C_b) Alkynyl ", wherein a and b are integers, refers to straight or branched chain alkynyl groups having a to b carbon atoms, at least one triple bond. Including, for example, ethynyl, prop-2-ynyl.

The term "divalent (C) as used herein_a-C_b) Alkynylene ", wherein a and b are integers, refers to a straight or branched chain alkynyl group having a to b carbon atoms, at least one triple bond and two unsaturations.

The term "cycloalkyl" as used herein refers to a saturated carbocyclic group containing 3 to 8 carbon atoms and includes, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl.

The term "cycloalkenyl" as used herein refers to carbocyclic radicals containing from 3 to 8 carbon atoms and at least one double bond, including, for example, cyclopentenyl, cyclohexenyl, cycloheptenyl and cyclooctenyl.

The term "aryl" as used herein refers to a mono-, di-or tri-cyclic carbocyclic aromatic group. Examples of such radicals are phenyl, biphenyl and naphthyl.

The term "carbocycle" as used herein refers to a cyclic group wherein the ring atoms are all carbon and includes monocyclic aryl, cycloalkyl and cycloalkenyl groups.

The term "heteroaryl" as used herein refers to a mono-, bi-or tri-cyclic aromatic group containing one or more heteroatoms selected from S, N and O. Examples of such groups are thienyl, benzothienyl, furyl, benzofuryl, pyrrolyl, imidazolyl, benzimidazolyl, thiazolyl, benzothiazolyl, isothiazolyl, benzisothiazolyl, pyrazolyl, oxazolyl, benzoxazolyl, isoxazolyl, benzisoxazolyl, isothiazolyl, triazolyl, benzotriazolyl, thiadiazolyl, oxadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, indolyl and indazolyl.

The non-limiting term "heterocycle" as used herein includes "heteroaromatic ring" as defined above and particularly denotes a group comprising a mono-, bi-or tri-cyclic non-aromatic group containing one or more heteroatoms selected from S, N or O, a monocyclic non-aromatic group containing one or more of said heteroatoms covalently linked to another such group or to a monocyclic carbocyclic group, examples of such organic groups being pyrrolyl, furyl, thienyl, piperidinyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, thiadiazolyl, pyrazolyl, pyridinyl, pyrrolidinyl, pyrimidinyl, morpholinyl, piperazinyl, indolyl, morpholinyl, benzofuranyl, pyranyl, isoxazolyl, benzimidazolyl, methylenedioxyphenyl, ethylenedioxyphenyl, maleimido, succinimidyl.

Unless otherwise indicated herein, the term "substituted" as used in any part herein denotes substitution with up to four compatible substituents, each of which may independently be, for example, (C)₁-C₆) Alkyl, (C)₁-C₆) Alkoxy, hydroxy (C)₁-C₆) Alkyl, mercapto (C)₁-C₆) Alkyl, (C)₁-C₆) Alkylthio, halogen (including fluorine, bromine and chlorine), trifluoromethyl, trifluoromethoxy, nitro, nitrile (-CN), oxo, phenyl, -COOH, -COOR^A、-COR^A、-SO₂R^A、-CONH₂、-SO₂NH₂、-CONHR^A、-SO₂NHR^A、-CONR^AR^B、-SO₂NR^AR^B、-NH₂、-NHR^A、-NR^AR^B、-OCONH₂、-OCONHR^A、-OCONR^AR^B、-NHCOR^A、-NHCOOR^A、-NR^BCOOR^A、-NHSO₂OR^A、

-NR^BSO₂OH、-NR^BSO₂OR^A、-NHCONH₂、-NR^ACONH₂、-NHCONHR^B、-NR^ACONHR^B、-NHCONR^AR^Bor-NR^ACONR^AR^BWherein R is^AAnd R^BIndependently is (C)₁-C₆) An alkyl group. The "optional substituent" may be any one of the above-mentioned substituents. In the above substituents, (C)₁-C₆) Alkyl, halo, trifluoromethyl, trifluoromethoxy, trifluoromethanesulfonyl and phenyl are those groups that are generally considered lipophilic. Other listed alkyl-containing substituents may be lipophilic, depending on the particular alkyl group present.

The term "salt" as used herein includes base addition, acid addition and quaternary salts. The acidic compounds of the present invention may be reacted with a base, for example, an alkali metal hydroxide, such as sodium and potassium hydroxides; alkaline earth metal hydroxides such as hydroxides of calcium, barium and magnesium; organic bases such as N-methyl-D-glucamine, choline, tris (hydroxymethyl) amino-methane, L-arginine, L-lysine, N-ethylpiperidine, dibenzylamine and the like form salts, including pharmaceutically or veterinarily acceptable salts. Those basic compounds (I) may form salts with inorganic acids, for example hydrohalic acids, such as hydrochloric or hydrobromic acid, sulphuric acid, nitric acid or phosphoric acid, and the like, and with organic acids, for example acetic acid, tartaric acid, succinic acid, fumaric acid, maleic acid, malic acid, salicylic acid, citric acid, methanesulphonic acid, p-toluenesulphonic acid, benzoic acid, benzenesulphonic acid, glutamic acid, lactic acid and mandelic acid, and the like, including pharmaceutically or veterinarily acceptable salts.

The term "lipophilic" as used herein with respect to a substituent means that it has a positive substituent hydrophobicity constant (π). (a positive pi value indicates that the substituent is more lipophilic than hydrogen, while a negative pi value indicates that the substituent is less lipophilic, i.e., more hydrophilic than hydrogen).

Some compounds of the invention contain one or more actual or potential chiral centers due to the presence of asymmetric carbon atoms. The presence of several asymmetric carbon atoms gives rise to a number of diastereomers with R or S stereochemistry at each chiral center. The present invention includes all such diastereomers and mixtures thereof.

One aspect of the present invention includes compounds of the above general formula (A) or (B), and salts, N-oxides, hydrates or solvates thereof and prodrugs thereof, with the exception of the following three (X), (Y) and (Z) which are commercially available

Compound (a):

with the proviso that those are excluded, the present invention specifically includes the substituents R therein₁，R₂And R₃Is as follows under the heading "radical R₁"," radical R₂The "and" radical R₃"and on the other hand includes the use of these compounds in the treatment of diseases responsive to inhibition of HSP90 activity.

Radical R ₁

In general, it is generally preferred that R is present₁Organic radical Ar in the radical¹Is optionally substituted phenyl, preferably with one optional substituent being the hydroxyl group in the 2-position relative to the point at which the phenyl ring is attached to the isoxazole ring. In other words, the group R₁Preferably having the formula (IB)

Wherein Alk¹，Alk²P, R, s, Z and Q are as described above for R₁As defined, R represents one or more optional substituents. At such a junctionIn the structure, it is more preferable that the ring carbon atom adjacent to the hydroxyl group is unsubstituted. In the following for R₁In the further discussion of (a), this preference holds true except for any other possibilities mentioned.

In the simplest configuration of the invention, p, R and s can be 0 and Q can be hydrogen, so that R is₁Is an optionally substituted aryl or heteroaryl group. In this case, R₁May be, for example, optionally substituted phenyl, preferably 2-hydroxyphenyl which may be further substituted by one or more of hydroxy, methyl, ethyl, methoxy, ethoxy, chloro or bromo. Generally preferred is where R is₁Are compounds of 2, 4-dihydroxyphenyl which are substituted in the 5-position by a small lipophilic group, for example having the same or a smaller molecular volume than tert-butyl, such as methyl, ethyl, isopropyl, isobutyl, tert-butyl, chlorine or bromine, especially ethyl, isopropyl or chlorine. In the 5-substituted 2, 4-dihydroxyphenyl compounds of the present invention, the hydroxyl group may be protected by a group which can be cleaved in vivo to release the hydroxyl group. Known such groups of the prodrug type which can be cleaved to hydroxyl include alkylcarbonyloxy groups, such as methylcarbonyloxy, and alkylaminocarbonyloxy groups (such as dialkylamino-or isopropylamino-carbonyloxy).

In other simple structures to which the invention relates, p, r and s may again be 0, Q may be an optionally substituted carbocyclic or heterocyclic ring, such as a benzene or pyridine ring. In this case, Q is an optionally substituted Ar¹Direct substituents of the ring.

In more complex structures to which the invention relates, one or more of p, r and s may be 1 and Q may be hydrogen or an optionally substituted carbocyclic or heterocyclic ring. For example, p and/or s may be 1 and r may be 0, such that Q is linked to Ar through an alkylene or alkenylene group¹Above, e.g. optionally substituted C₁-C₃An alkylene group. In other cases, p, r and s may be 1, in which case Q is attached to Ar through an alkylene or alkenylene group interrupted by a Z group containing a heteroatom¹The above. At itIn which case p and s may be 0 and r may be 1, in which case Q is attached to Ar through a group containing a heteroatom Z¹The above.

R of the above type₁Specific examples of groups are present in the compounds of the examples herein.

Radical R ₂

When R is₂Are of type (i), i.e. groups of formula (IA), examples include phenyl, 2-, 3-, or 4-pyridyl, 2-or 3-furyl, 2-or 3-thienyl, or thiazolyl, wherein the optional substituents include any of those groups listed in the "substituted" definitions above, such as methoxy, ethoxy, methylenedioxy, ethylenedioxy, fluoro, chloro, bromo and trifluoromethyl. For example R₂May be at 4-bit by C₁-C₆Alkoxy, for example methoxy or ethoxy, or phenyl substituted by fluorine, chlorine, bromine, piperazinyl, N-methylpiperazinyl or piperidinyl.

Presently preferred R₂Substituents include those having the following partial structure:

substituted amino-NR in the formula¹⁰R¹¹Are solubilizing groups. Many such solubilizing groups are known in medicinal chemistry. Examples include (2-or 3-) morpholinyl, piperidinyl, piperazinyl, pyrrolidinyl, ethylamino, isopropylamino, diethylamino, cyclohexylamino, cyclopentylamino, methoxyethylamino, piperidin-4-yl, N-acetylpiperazinyl, methylsulfonylamino, thiomorpholinyl dioxide, 4-hydroxyethylpiperidinyl and 4-hydroxypiperidinyl.

Our co-pending international patent application PCT/GB2003/005275 discloses classes that inhibit HSP90Similar to the pyrazole compounds of isoxazoles to which the present invention relates, isoxazoles are believed to bind in a similar manner to the HSP90 target. Those pyrazole compounds correspond to the prior isoxazole R₂Has a carboxamide group in the position (a). Thus, when R of the present isoxazoles is₂When they are carboxamide groups of the above-mentioned type (ii), examples include those of the pyrazole compounds present in PCT/GB2003/005275, for example of the formula-CONR^B(Alk)_nR^AOf the formula (II) in

Alk is a divalent alkylene, alkenylene or alkynylene radical, e.g. -CH₂-，-CH₂CH₂-，-CH₂CH₂CH₂-，-CH₂CH ═ CH-, or-CH₂CCCH₂And Alk group may be optionally substituted.

n is a number of 0 or1,

R^Bis hydrogen, or C₁-C₆Alkyl or C₂-C₆Alkenyl, for example methyl, ethyl, n-or i-propyl, or allyl.

R^AIs hydroxy or optionally substituted carbocyclyl, such as hydroxy and/or chloro substituted phenyl and 3, 4 methylenedioxyphenyl; or a heterocyclic group such as pyridyl, furyl, thienyl, N-piperazinyl or N-morpholinyl, all of which may be substituted.

Or R^AAnd R^BTogether with the nitrogen atom to which they are attached, form an N-heterocyclic ring which may optionally contain one or more additional heteroatoms selected from O, S and N, which may optionally be substituted on one or more ring carbon or nitrogen atoms. Examples of such N-heterocycles include morpholino, piperidinyl, piperazinyl and N-phenylpiperazinyl.

Radical R ₃

R₃Can be, for example, hydrogen, methyl, ethyl, n-or i-propyl, trifluoromethyl, hydroxyethyl, methylsulfonylAminomethyl or the above-mentioned pair R₂Carboxamide group-CONR in question^B(Alk)_nR^A. Carboxamide groups are presently preferred, especially ethylaminocarbonyl or isopropylaminocarbonyl.

A particular subgroup of compounds to which the invention relates includes regioisomers of formula (ID) and formula B, as well as salts, hydrates and solvates thereof, and prodrugs thereof:

wherein each R independently represents an optional substituent, R³Represents a carboxamide group.

A preferred subgroup of compounds to which the present invention relates includes regioisomers of formula (IE) and formula (B), and salts, hydrates and solvates thereof, and prodrugs thereof:

in the formula R₃Represents a carboxamide group (e.g. ethylaminocarbonyl CH)₃CH₂NHC (═ O) -or isopropylaminocarbonyl (CH)₃)₂CHNHC(＝O)-)；R₉represents-CH₂NR¹⁰R¹¹or-NR¹⁰R¹¹Wherein the substituted amino group is-NR¹⁰R¹¹Is a solubilizing group, (e.g., (2-or 3-) morpholinyl, piperidinyl, piperazinyl, pyrrolidinyl, ethylamino, isopropylamino, diethylamino, cyclohexylamino, cyclopentylamino, methoxyethylamino, piperidin-4-yl, N-acetylpiperazinyl, N-methylpiperazinyl, methanesulfonylamino, thiomorpholinyl dioxide, 4-hydroxyethylpiperidinyl, or 4-hydroxypiperidinyl); r₈Represents an optional substituent, especially a small parentAn aliphatic group (e.g., ethyl, isopropyl, bromo, or chloro).

Specific compounds to which the invention relates include those of the examples, especially the following compounds, and salts, N-oxides, hydrates and solvates thereof, and prodrugs thereof:

5- (2, 4-dihydroxy-5-isopropyl-phenyl) -4- (4-morpholin-4-ylmethyl-phenyl) -isoxazole-3-carboxylic acid ethylamide

5- (2, 4-dihydroxy-5-isopropyl-phenyl) -4- (4-piperidin-1-ylmethyl-phenyl) -isoxazole-3-carboxylic acid ethylamide

4- (4-diethylaminomethyl-phenyl) -5- (2, 4-dihydroxy-5-isopropyl-phenyl) -isoxazole-3-carboxylic acid ethylamide

5- (2, 4-dihydroxy-5-isopropyl-phenyl) -4- [4- (4-methyl-piperazin-1-ylmethyl) -phenyl ] -isoxazole-3-carboxylic acid ethylamide

5- (2, 4-dihydroxy-5-isopropyl-phenyl) -4- (4-ethylaminomethyl-phenyl) -isoxazole-3-carboxylic acid ethylamide

5- (2, 4-dihydroxy-5-isopropyl-phenyl) -4- [4- (isopropylamino-methyl) -phenyl ] -isoxazole-3-carboxylic acid ethylamide

4- (4-cyclohexylaminomethyl-phenyl) -5- (2, 4-dihydroxy-5-isopropyl-phenyl) -isoxazole-3-carboxylic acid ethylamide

4- [4- (tert-butylamino-methyl) -phenyl ] -5- (2, 4-dihydroxy-5-isopropyl-phenyl) -isoxazole-3-carboxylic acid ethylamide

5- (2, 4-dihydroxy-5-isopropyl-phenyl) -4- {4- [ (2-methoxy-ethylamino) -methyl ] -phenyl } -isoxazole-3-carboxylic acid ethylamide

5- (2, 4-dihydroxy-5-isopropyl-phenyl) -4- (4-morpholin-4-ylmethyl-phenyl) -isoxazole-3-carboxylic acid isopropylamide

5- (2, 4-dihydroxy-5-isopropyl-phenyl) -4- [4- (4-methyl-piperazin-1-ylmethyl) -phenyl ] -isoxazole-3-carboxylic acid isopropylamide

5- (5-tert-butyl-2, 4-dihydroxy-phenyl) -4- [4- (4-methyl-piperazin-1-ylmethyl) -phenyl ] -isoxazole-3-carboxylic acid ethylamide

5- (5-tert-butyl-2, 4-dihydroxy-phenyl) -4- (4-piperidin-1-ylmethyl-phenyl) -isoxazole-3-carboxylic acid ethylamide

5- (2, 4-dihydroxy-5-isobutyl-phenyl) -4- (4-morpholin-4-ylmethyl-phenyl) -isoxazole-3-carboxylic acid ethylamide

5- (2, 4-dihydroxy-5-isobutyl-phenyl) -4- (4-piperidin-1-ylmethyl-phenyl) -isoxazole-3-carboxylic acid ethylamide

5- (5-tert-butyl-2, 4-dihydroxy-phenyl) -4- (4-morpholin-4-ylmethyl-phenyl) -isoxazole-3-carboxylic acid ethylamide

5- (5-tert-butyl-2, 4-dihydroxy-phenyl) -4- (4-diethylaminomethyl-phenyl) -isoxazole-3-carboxylic acid ethylamide

3- (5-chloro-2, 4-dihydroxy-phenyl) -4- (4-morpholin-4-ylmethyl-phenyl) -isoxazole-5-carboxylic acid ethylamide

4- (4-diethylaminomethyl-phenyl) -5- (4, 6-dihydroxy-2' -methyl-biphenyl-3-yl) -isoxazole-3-carboxylic acid ethylamide

4- (4-diethylaminomethyl-phenyl) -5- (4' -fluoro-4, 6-dihydroxy-biphenyl-3-yl) -isoxazole-3-carboxylic acid ethylamide

4- (4-diethylaminomethyl-phenyl) -5- (4, 6-dihydroxy-biphenyl-3-yl) -isoxazole-3-carboxylic acid ethylamide

5- (2' -fluoro-4, 6-dihydroxy-biphenyl-3-yl) -4- (4-pyrrolidin-1-ylmethyl-phenyl) -isoxazole-3-carboxylic acid ethylamide

5- (4, 6-dihydroxy-biphenyl-3-yl) -4- (4-morpholin-4-ylmethyl-phenyl) -isoxazole-3-carboxylic acid ethylamide

5- (2, 4-dihydroxy-5-phenethyl-phenyl) -4- (4-morpholin-4-ylmethyl-phenyl) -isoxazole-3-carboxylic acid ethylamide

5- (5-chloro-2, 4-dihydroxy-phenyl) -4- (4-piperidin-1-ylmethyl-phenyl) -isoxazole-3-carboxylic acid isopropylamide

4- (4-diethylaminomethyl-phenyl) -5- (5-ethyl-2, 4-dihydroxy-phenyl) -isoxazole-3-carboxylic acid ethylamide

5- (5-ethyl-2, 4-dihydroxy-phenyl) -4- [4- (4-methyl-piperazin-1-ylmethyl) -phenyl ] -isoxazole-3-carboxylic acid ethylamide

5- (5-ethyl-2, 4-dihydroxy-phenyl) -4- (4-morpholin-4-ylmethyl-phenyl) -isoxazole-3-carboxylic acid ethylamide

5- (5-chloro-2, 4-dihydroxy-phenyl) -4- (4-diethylaminomethyl-phenyl) -isoxazole-3-carboxylic acid ethylamide

5- (5-chloro-2, 4-dihydroxy-phenyl) -4- [4- (4-methyl-piperazin-1-ylmethyl) -phenyl ] -isoxazole-3-carboxylic acid ethylamide

5- (5-chloro-2, 4-dihydroxy-phenyl) -4- (4-morpholin-4-ylmethyl-phenyl) -isoxazole-3-carboxylic acid ethylamide

The compounds to which the present invention relates can be prepared by literature methods, such as those in the preparation examples herein, and otherwise analogously.

For example, some compounds of formula (IA) can be prepared by reacting hydroxylamine with a compound of formula (III):

wherein ring A corresponds to R of compound (IA)₁Group, R₂And R₃Are as defined for formula (I). The compounds prepared in this way can then be chemically modified to introduce the desired substituents, giving rise to other compounds of the general formula (A),for example wherein R₁Is a phenyl ring which optionally already carries substituents, the bromine substituents introduced being capable of introducing further substituents, in general via sp2 coupling, at the bromine position.

In an additional route to some compounds of formula (A), isoxazole rings are formed by the reaction of compound (IV) and hydroxylamine

R 'in the formula'₁And R'₃Is a substituent type R as defined above₁And R₃Followed by introduction of further substituents R₂(e.g. by bromination or iodination of a ring carbon atom in (V) and sp2 coupling, and/or R on the resulting isoxazole¹ ₁，R¹ ₃And R₂Substituent is modified) to yield isoxazole (V).

In addition, some isoxazole regioisomers (B) can be prepared from isoxazoles (a) by reaction with trimethyloxonium boron trifluoride, and the compounds prepared in this way can then be further chemically modified to introduce the desired substituents, yielding further compounds of the general formula (IA).

It will be appreciated that in the above synthesis it may be desirable to protect any reactive group, for example a hydroxyl group, and thereafter deprotect it. Further synthetic details are described in the examples herein.

The compounds of the invention are HSP90 inhibitors and are therefore useful in the treatment of diseases that respond to inhibition of HSP90 activity. Such as cancer, viral diseases such as Hepatitis C (HCV) (Waxman, 2002), immunosuppression such as in transplantation (Bijlmakers, 2000 and Yorgin, 2000), anti-inflammatory diseases (Bucci, 2000) such as rheumatoid arthritis, asthma, multiple sclerosis, type I diabetes, lupus, psoriasis, inflammatory bowel disease, cystic fibrosis (Fuller, 2000), and angiogenesis-related diseases (Hur, 2002 and Kurebayashi, 2001): diabetic retinopathy, hemangiomas, psoriasis, endometriosis and tumor angiogenesis. The Hsp90 inhibitors of the invention may also protect normal cells from chemotherapy-induced toxicity and are useful in diseases where the underlying cause is the inability to undergo apoptosis. These Hsp90 inhibitors may also be useful in diseases that induce cellular stress or heat shock protein effects, for example, to avoid hypoxia-ischemic injury of tissues due to elevation of Hsp70 in the heart (Hutter, 1996 and Trost, 1998) and brain (Plumier, 1997 and Rajder, 2000). Hsp90 inhibitors may also be useful in diseases where the major causative factor is protein misfolding or aggregation, such as scrapie/CJD, Huntingdon's disease, and Alzheimer's disease (Sittler, 2001; Trazelt, 1995 and Winklhofer, 2001).

Accordingly, the present invention also provides:

(i) a method of treatment of a disease or condition responsive to inhibition of HSP90 activity in a mammal, particularly a human being, which method of treatment comprises administering to the mammal an amount of a compound of formula (a) or (B), or a salt, hydrate, solvate thereof, as defined above, effective to inhibit said HSP90 activity; and

(ii) a compound of formula (a) or (B) as defined above, or a salt, hydrate, solvate thereof, for use as a human or veterinary medicament, particularly for the treatment of a disease or condition responsive to inhibition of HSP90 activity;

(iii) a pharmaceutical composition comprising a compound of formula (A) or (B) as defined and illustrated above, and a pharmaceutically acceptable carrier. In particular, the invention includes solutions or suspensions of such compounds in a sterilized, physiologically acceptable carrier, such as saline.

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 and diet, time of administration, route of administration, rate of excretion, drug combination and the pathogenic mechanism involved, and the severity of the particular disease undergoing therapy. In general, suitable dosages for oral formulations are generally in the range of 0.1-3000mg, administered once, twice or three times daily, or given by infusion or other routes as equivalent daily dosages. However, as is customary in the art, the optimum dose level and frequency of administration will be determined by clinical trials.

The compounds of the invention may be prepared for administration by any route consistent with pharmacokinetic properties. Compositions for oral administration may be in the form of tablets, capsules, powders, granules, lozenges, liquids or gels, such as oral, topical or sterile injectable pharmaceutical solutions or suspensions. Tablets or capsules for oral administration may be in unit dosage form and may include conventional excipients such as binding agents, for example syrup, gum arabic, gelatin, sorbitol, tragacanth, or polyvinylpyrrolidone; fillers such as lactose, sucrose, corn starch, calcium phosphate, sorbitol or glycine; tablet lubricants, such as magnesium stearate, talc, polyethylene glycol or silicon dioxide; a disintegrant such as potato starch, or an acceptable wetting agent such as sodium lauryl sulfate. The tablets may be coated according to methods 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 vehicle before use. These liquid preparations may contain conventional additives, for example suspending agents, such as sorbitol, syrup, methyl cellulose, glucose syrup, gelatin, hydrogenated edible fats; emulsifiers, such as lecithin, sorbitan monooleate or gum arabic; non-aqueous vehicles (which may include edible oils), such as almond oil, fractionated coconut oil, oily fats such as glycerin, propylene glycol or ethanol; preservatives, for example methyl or propyl p-hydroxybenzoate or sorbic acid, may, if desired, also contain conventional flavouring or colouring agents.

For topical application to the skin, the medicament may be formulated in a cream, lotion or ointment. Cream or ointment formulations for use in medicine are conventional formulations known in the art. For example as described in standard textbooks of pharmacy, for example the british pharmacopoeia.

The active ingredient may also be administered by injection in a sterile vehicle. Depending on the vehicle or concentration used, the drug may be suspended or dissolved in the vehicle. Preferably, adjuvants, such as local anesthetics, preservatives and buffering agents, can be dissolved in the vehicle.

The compounds of the invention are also useful in vitro assays that rely on inhibition of HSP90 activity, for example in screening for alternative classes of HSP90 inhibitors in which test compounds compete with or replace the compounds of the invention. Thus, in another aspect, the invention includes a method of inhibiting HSP90 activity comprising contacting in vitro an HSP90 enzyme with a compound of formula (a) or (B) as defined and described above.

The following examples illustrate the preparation and activity of specific compounds of the invention.

Detailed Description

Examples 1 to 4

Scheme 1: preparation of bromine intermediates and subsequent arylation

Example 1

4- [4- (4-methoxy-phenyl) -3-methyl-isoxazol-5-yl ] -benzene-1, 3-diol

Step 1

1- (2, 4-dihydroxy-phenyl) -2- (4-methoxy-phenyl) -ethanone

Resorcinol (4.4g, 40mmol) and 4-methoxyphenylacetic acid (6.6g, 40mmol) were heated in boron trifluoride ether complex (25ml, 0.2mol) under nitrogen at 90 ℃ for 90 minutes to give a pale red solution. The solution was allowed to cool and poured into aqueous sodium acetate (200ml, 10%) and the mixture stirred to give a pale yellow precipitate. The solid was filtered off and washed with water (200 ml). The solid was dissolved in ethyl acetate (250ml) and washed with water (200 ml). The solution was dried over anhydrous magnesium sulfate and concentrated to give a yellow semi-solid. Trituration with ether (100ml) gave 1- (2, 4-dihydroxy-phenyl) -2- (4-methoxy-phenyl) -ethanone as a pale orange solid, which was dried in vacuo (2.2g),

LC Retention time 2.39 min [ M + H]⁺259.2 (run time 3.75 minutes)

N.M.R(DMSO-d₆)7.95(d J 8.9Hz ArH)7.2(d J 8.7Hz 2ArH)6.9(d J 8.7Hz2ArH)6.4(d J 9.9ArH)6.25(s ArH)4.2(s 2CH₂)3.75(s 3OCH₃)

Step 2

7-hydroxy-3- (4-methoxy-phenyl) -2-methyl-chromen-4-one

Acetic anhydride (3ml, 30mmol) was added to a suspension of potassium carbonate (4.0g, 29mmol) and 1- (2, 4-dihydroxy-phenyl) -2- (4-methoxy-phenyl) -ethanone (1.95g, 7.5mmol) in DMF (10ml) and the resulting suspension was heated at 115 ℃ for-90 min. The mixture was allowed to cool and poured into water (200ml) to give an off-white precipitate. The solid was filtered off and washed with water (100ml) and diethyl ether (2X 40ml) to give 7-hydroxy-3- (4-methoxy-phenyl) -2-methyl-chromen-4-one as an off-white powder, which was dried in vacuo (1.65g),

LC Retention time 2.26 min [ M + H]⁺283.2 (run time 3.75 minutes)

N.M.R(DMSO-d₆)7.8(d J 8.7Hz ArH)7.2(d J 8.8Hz 2ArH)7.0(d J 8.8Hz2ArH)6.9(d J 8.7ArH)6.8(s ArH)3.8(s 3OCH₃)2.2(s 3CH₃)

Step 3

4- [4- (4-methoxy-phenyl) -3-methyl-isoxazol-5-yl ] -benzene-1, 3-diol

Hydroxylamine hydrochloride (0.35g, 5mmol) was added to a suspension of 7-hydroxy-3- (4-methoxy-phenyl) -2-methyl-chromen-4-one (0.14g, 0.5mmol) in pyridine (3ml) and the mixture was heated under reflux for 4 hours. The solution was allowed to cool and poured into water (50ml) and extracted with diethyl ether (50 ml). The extract was washed with water (3X 50ml) and saturated aqueous sodium chloride (30 ml). The solution was dried over anhydrous magnesium sulfate and concentrated to give a light brown gum.

The crude product was purified by column chromatography, eluting on silica gel with ethyl acetate/n-hexane (1: 2) to give a colourless gum. Trituration with n-hexane gave 4- [4- (4-methoxy-phenyl) -3-methyl-isoxazol-5-yl ] -benzene-1, 3-diol as a white powder, which was dried in vacuo (0.087g),

LC Retention time 2.20 min [ M + H]⁺298.2 (run time 3.75 minutes)

N.M.R(DMSO-d₆)7.1(d J 8.8Hz 2ArH)6.85(d J 8.6Hz ArH)6.8(d J 8.8Hz2ArH)6.25(s ArH)6.15(d J 8.6Hz ArH)3.65(s 3OCH₃)2.15(s 3CH₃)

Example 2

4-bromo-6- [4- (4-methoxy-phenyl) -3-methyl-isoxazol-5-yl ] -benzene-1, 3-diol

Benzyltrimethylammonium tribromide (3.95g, 10mmol) was added portionwise to an ice-cooled suspension of 4- [4- (4-methoxy-phenyl) -3-methyl-isoxazol-5-yl ] -benzene-1, 3-diol (example 1) (2.95g, 10mmol) in dichloromethane (50ml) and the mixture was stirred at room temperature for-60 min. Ethyl acetate (300ml) was added to the solution, and the mixture was washed with water (3X 200ml) and a saturated aqueous solution (50ml) of sodium chloride. The solution was dried over anhydrous magnesium sulfate and concentrated to give a light brown solid. The crude product was purified by column chromatography eluting with ethyl acetate/n-hexane (1: 2) on silica gel to give 4-bromo-6- [4- (4-methoxy-phenyl) -3-methyl-isoxazol-5-yl ] -benzene-1, 3-diol as a white solid which was dried in vacuo (3.42g),

LC Retention time 2.38 min [ M + H]⁺378.2 (run time 3.75 minutes)

N.M.R (acetone-d 6)7.35(s ArH)7.2(d J8.8.8 Hz 2ArH)6.9(d J8.8.8 Hz 2ArH)6.65(s ArH)3.8(s 3 OCH)₃)2.25(s 3CH₃)

Example 3

5- [4- (4-methoxy-phenyl) -3-methyl-isoxazol-5-yl ] -biphenyl-2, 4-diol

Step 1

5- (2, 4-bis-benzyloxy-5-bromo-phenyl) -4- (4-methoxy-phenyl) -3-methyl-isoxazole

Benzyl bromide (0.36ml, 3mmol) was added to a suspension of 4-bromo-6- [4- (4-methoxy-phenyl) -3-methyl-isoxazol-5-yl ] -benzene-1, 3-diol (example 2) (0.55g, 1.5mmol) and cesium carbonate (0.85g, 2.6mmol) in DMF (5ml) and the mixture was stirred at room temperature for 18 h. Water (100ml) was added and the mixture was extracted with diethyl ether (2 × 30 ml). The combined extracts were washed with water (4X75ml) and saturated aqueous sodium chloride (50 ml). The solution was dried over anhydrous magnesium sulphate and concentrated to give a pale brown gum which was triturated with n-hexane to give 5- (2, 4-bis-benzyloxy-5-bromo-phenyl) -4- (4-methoxy-phenyl) -3-methyl-isoxazole as an off-white solid which was dried in vacuo (0.5 g).

LC Retention time 3.08 min [ M + H]⁺558.4 (run time 3.75 minutes)

N.M. R (deuterated chloroform) 7.55(s ArH)7.35-7.25(m 5ArH)7.2(m 3ArH)6.95(d J8.8Hz 2ArH)6.85(m 2ArH)6.7(d J8.8.8 Hz 2ArH)6.35(s ArH)4.95(s2CH 2₂)4.6(s2CH₂)3.75(s 3OCH₃)2.25(s 3CH₃)

Step 2

5- (4, 6-bis-benzyloxy-biphenyl-3-yl) -4- (4-methoxy-phenyl) -3-methyl-isoxazole

Potassium phosphate (0.1g, 0.5mmol) was added to 5- (2, 4-bis-benzyloxy-5-bromo-phenyl) -4- (4-methoxy-phenyl) -3-methyl-isoxazole (0.14g, 0.25mmol) and phenylboronic acid (0.095g, 0.75mmol) in1, 4-dioxane (4ml) under nitrogen. Four (triphenylphosphine) palladium (0) (catalytic amount) were added and the suspension was heated at 80 ℃ for 18 h. The suspension was cooled and ethyl acetate (25ml) was added. The mixture was washed with water (3X 25ml) and saturated aqueous sodium chloride (25 ml). The solution was dried over anhydrous magnesium sulfate and concentrated to give a light brown gum, triturated with n-hexane to give 5- (4, 6-bis-benzyloxy-biphenyl-3-yl) -4- (4-methoxy-phenyl) -3-methyl-isoxazole as an off-white solid, and dried in vacuo.

LC Retention time 3.08 min [ M + H]⁺554.4 (run time 3.75 minutes)

N.M. R (deuterated chloroform) 7.4(m 2ArH)7.35(s ArH)7.3-7.1(m 11ArH)6.95(d J8.8Hz 2ArH)6.9(m 2ArH)6.7(d J8.8.8 Hz 2ArH)6.45(s ArH)4.9(s 2 CH)₂)4.7(s2CH₂)3.75(s 3OCH₃)2.25(s 3CH₃)

Step 3

7-hydroxy-3- (4-methoxy-phenyl) -2-methyl-6-phenyl-chromen-4-one

Ammonium formate (3.2g, 50mmol) was added to a solution of 5- (4, 6-bis-benzyloxy-biphenyl-3-yl) -4- (4-methoxy-phenyl) -3-methyl-isoxazole (1.4g, 2.5mmol) in methanol (20 ml)/ethyl acetate (10ml) under a nitrogen atmosphere. Palladium on carbon (10%) (catalytic amount) was added and the suspension was heated at 60 ℃ for-18 hours. The suspension was cooled and ethyl acetate (150ml) was added. The suspension was filtered. The filtrate was washed with water (3X 100ml) and saturated aqueous sodium chloride (50 ml). The solution was dried over anhydrous magnesium sulfate and concentrated to give a light brown gum which was triturated with methanol to give 7-hydroxy-3- (4-methoxy-phenyl) -2-methyl-6-phenyl-chromen-4-one as an off-white solid which was dried in vacuo.

LC Retention time 2.58 min [ M + H ] +359.2 (run time 3.75 min)

N.M.R(DMSO-d₆)7.9(s ArH)7.5-7.3(m 5ArH)7.25(d J 8.8Hz 2ArH)7.1(sArH)7.05(d J 8.8Hz 2ArH)3.85(s 3OCH₃)2.2(s 3CH₃)

Step 4

5- [4- (4-methoxy-phenyl) -3-methyl-isoxazol-5-yl ] -biphenyl-2, 4-diol

Hydroxylamine hydrochloride (75mg, 1.08mmol) was added to a suspension of 7-hydroxy-3- (4-methoxy-phenyl) -2-methyl-6-phenyl-chromen-4-one (105mg, 0.29mmol) in pyridine (2ml) and the mixture was heated under reflux for 6 hours to give a pale yellow solution. The solution was allowed to cool and water (20ml) was added and the mixture extracted with diethyl ether (2X 10 ml). The combined extracts were washed with water (2X 20ml) and saturated aqueous sodium chloride (10 ml). The solution was dried over anhydrous magnesium sulfate and concentrated. The crude product was purified by column chromatography (silica gel) eluting with ethyl acetate/n-hexane (1: 1) to give the title compound as an off-white powder (80mg)

LC Retention time 2.56 min [ M + H]⁺374.3 (run time 3.75 minutes)

N.M.R (acetone-d)₆)7.5-7.3(m 5ArH)7.2(d J 8.8Hz 2ArH)7.0(d J 8.8Hz2ArH)6.9(d J 8.6Hz ArH)6.35(s ArH)6.1(d J 8.7Hz ArH)3.85(s 3OCH₃)2.25(s 3CH₃)

Example 4

4-chloro-6- [4- (4-methoxy-phenyl) -3-methyl-isoxazol-5-yl ] -benzene-1, 3-diol

Hydroxylamine hydrochloride (0.7g, 10mmol) was added to a suspension of 6-chloro-7-hydroxy-3- (4-methoxy-phenyl) -2-methyl-benzopyran-4-one (prepared similarly to the second step of example 1) (0.32g, 1.0mmol) in pyridine (4ml), and the mixture was heated under reflux for 6 hours to give a pale yellow solution. The solution was allowed to cool and water (20ml) was added and the mixture extracted with diethyl ether (2X 10 ml). The combined extracts were washed with water (2X 20ml) and saturated aqueous sodium chloride (10 ml). The solution was dried over anhydrous magnesium sulfate and concentrated. The crude product was purified by column chromatography (silica gel) eluting with ethyl acetate/n-hexane (1: 1) to give 4-chloro-6- [4- (4-methoxy-phenyl) -3-methyl-isoxazol-5-yl ] -benzene-1, 3-diol (0.103g) as an off-white powder

LC Retention time 2.37 min [ M + H]⁺332.2 (run time 3.75 minutes)

N.M.R (acetone-d)₆)7.2(d J 8.8Hz 2ArH)7.15(s ArH)6.9(d J 8.8Hz 2ArH)6.6(sArH)3.85(s 3OCH₃)2.25(s 3CH₃)

The compounds of examples 1-4 had IC50 values for HSP90 in range a when tested using the Malachite Green ATPase assay described below (Malachite Green ATPase assay). In the table below, the last column, with the exception of example 12b, is based on the same results given for the compound in question, the activity quoted for example 12b being determined by the fluorescence polarization assay described below.

Examples 5-16 were prepared using the reactions described in examples 1-4. Other details of preparation examples 6 and 7 are similar to those of examples 86 and 87.

Commercially available from interbiscreen

^ξCommercially available from Enamine

Prepared from a protected bromoresorcinol intermediate and copper (I) cyanide in dimethylformamide at 150 ℃

Fluorescence polarization assay: 'A' < 10 uM; 'B' > 10uM

Example 14

4- [4- (4-methoxy-phenyl) -3-methyl-isoxazol-5-yl ] -6-phenethyl-benzene-1, 3-diol

Prepared by the coupling reaction of styrylboronic acid with the bromoisoxazole compound of the first step of example 2, followed by reduction and treatment with hydroxylamine, as described above, is similar to example 3.

LC Retention time 2.56 min [ M + H]⁺402 (run time 3.75 minutes)

Example 15

4- [4- (4-methoxy-phenyl) -3-methyl-isoxazol-5-yl ] -2, 6-bis- (4-methyl-piperazin-1-ylmethyl-benzene-1, 3-diol

Scheme 2: mannich reaction

N-methylpiperazine (0.125ml, 1.1mmol) was added to a suspension of 4- [4- (4-methoxy-phenyl) -3-methyl-isoxazol-5-yl ] -benzene-1, 3-diol (0.15g, 0.5mmol) and paraformaldehyde (0.040g) in1, 4-dioxane (4ml) and the mixture was heated under reflux for 18 hours to give a light brown yellow solution. The solution was cooled and ethyl acetate (25ml) was added. The mixture was washed with water (3X 25ml) and saturated aqueous sodium chloride (25 ml). The solution was dried over anhydrous magnesium sulfate and concentrated to give a light brown gum. Trituration with n-hexane afforded 4- [4- (4-methoxy-phenyl) -3-methyl-isoxazol-5-yl ] -2, 6-bis- (4-methyl-piperazin-1-ylmethyl) -benzene-1, 3-diol as a light brown powder (0.121 g).

LC Retention time 1.61 min [ M + H]⁺522.6 (run time 3.75 minutes)

N.M.R (acetone-d)₆)7.2(d J 8.8Hz 2ArH)6.95(s ArH)6.8(d J 8.8Hz 2ArH)3.85(s 3OCH₃)3.75(s 2CH₂)3.65(s 2CH₂)2.9-2,0(br s 16CH₂)2.3(s 3CH₃)2.25(s 3CH₃)2.2(s 3CH₃)

Example 16

2, 4-dihydroxy-5- [4- (4-methoxy-phenyl) -3-methyl-isoxazol-5-yl ] -benzoic acid methyl ester

Scheme 3: formation of esters

Step 1

N-butyllithium (100 μ l) was added to a solution of 5- (2, 4-bis-benzyloxy-5-bromo-phenyl) -4- (4-methoxy-phenyl) -3-methyl-isoxazole (154mg, 0.28mmol) in tetrahydrofuran (2.5ml) under a nitrogen atmosphere at-78 ℃. The solution was stirred at-70 ℃ for 30 minutes to give an orange solution. The ions were quenched with methyl chloroformate (100 μ l, 3 equivalents) and allowed to warm to room temperature for 30 minutes. The solution was quenched with saturated aqueous ammonium chloride (5 ml). The mixture was extracted with ethyl acetate (3X 5 ml). The combined extracts were washed with water (2X 5ml) and saturated aqueous sodium chloride (5 ml). The solution was dried over anhydrous magnesium sulfate and concentrated. The crude product was purified by column chromatography (silica gel) eluting with ethyl acetate in n-hexane (20% to 60% ethyl acetate gradient eluent) to give 2, 4-bis-benzyloxy-5- [4- (4-methoxy-phenyl) -3-methyl-isoxazol-5-yl ] -benzoic acid methyl ester (72 mg).

LC Retention time 4.95 min [ M + H]⁺536.4 (run time 7.5 minutes)

N.M.R(DMSO-d₆)7.8(s ArH)7.55(d J 7.1Hz 2ArH)7.4(t J 6.2Hz 2ArH)7.35(d J 6.1Hz ArH)7.3(m 3ArH)7.1(m 4ArH)7.0(s ArH)6.9(d 8.8Hz 2ArH)5.3(s 2CH₂)5.1(s 2CH₂)3.78(s OCH₃)3.76(s OCH₃)2.28(s CH₃)

Step 2

Ammonium formate (172mg, 20 equivalents) was added to a solution of 2, 4-bis-benzyloxy-5- [4- (4-methoxy-phenyl) -3-methyl-isoxazol-5-yl ] -benzoic acid methyl ester (72mg, 0.13mmol) in methanol (2 ml)/ethyl acetate (1ml) under a nitrogen atmosphere. 10% palladium on carbon (catalytic amount) catalyst was added and the suspension was heated at 60 ℃ overnight. The solution was cooled and ethyl acetate (5ml) was added. The solution was washed with water (2X 5ml) and saturated aqueous sodium chloride (5 ml). The solution was dried over anhydrous magnesium sulphate and concentrated and the crude product purified by column chromatography (silica gel) eluting with ethyl acetate in n-hexane (25% to 45% ethyl acetate gradient eluent) to give 2, 4-dihydroxy-5- [4- (4-methoxy-phenyl) -3-methyl-isoxazol-5-yl ] -benzoic acid methyl ester (7.0 mg).

LC Retention time 2.49 min [ M + H]⁺356.3 (run time 3.75 minutes)

N.M.R(CDCl₃)δ＝10.85(s ArOH)7.52(s ArOH)7.12(d J 8Hz 2ArH)6.98(s ArH)6.91(d J8Hz 2ArH)6.45(s ArH)3.78(s 3OCH₃)3.71(s 3OCH₃)2.21(s 3CH₃).

The compounds of examples 14-16 had HSP90IC50 in the ranges 'A', 'B' and 'B', respectively, when tested by the Malachite Green ATPase assay (Malachite Green ATPase assay) described below.

Similarly, the preparations of examples 17 to 20 were quenched with N-formylpiperidine, phenylthioisocyanate, 2-methoxyphenyl isocyanate and benzaldehyde, respectively. The final deprotection reaction was carried out with boron trichloride as described in example 23 (last reaction of scheme 5). Example 21 is a by-product of the first step of example 16. The cited activities were obtained from the Malachite Green ATPase assay (Malachite Green ATPase assay) described below.

Example 22

4-benzyl-6- [4- (4-methoxy-phenyl) -3-methyl-isoxazol-5-yl ] -benzene-1, 3-diol

Scheme 4: synthesis of benzylresorcinol

2-benzoyl-5-ethoxycarbonyloxy-phenyl carbonate ethyl ester

Triethylamine (10ml, 72.2mmol) was added to a solution of 2, 4-dihydroxybenzophenone (1) (5.4g, 23.3mmol) in THF (50ml) and the solution was cooled to 0 deg.C. Ethyl chloroformate (6.9ml, 72.2mmol) was slowly added and the suspension was stirred at 0 ℃ for-30 minutes and at room temperature for-3 hours. Water (150ml) was added to the reaction solution, and the mixture was extracted with diethyl ether (150 ml). The extract was washed with water (2X 150ml) and saturated aqueous sodium chloride (100 ml). The solution was dried over anhydrous magnesium sulfate and concentrated to give 4-benzyl-benzene-1, 3-diol as a pale green gum which solidified on standing (8.2 g).

LC Retention time 2.73 min [ M + H]⁺359.2 (run time 3.75 minutes)

Delta (deuterated chloroform) 7.7(m 2ArH)7.5(m 2ArH)7.35(m 2ArH)7.15(m 2ArH)4.25(qj7.1hz 2 CH)₂)4.05(q J 7.1Hz 2CH₂)1.35(t J 7.1Hz 3CH₃)1.15(t J 7.1Hz 3CH₃)

4-benzyl-benzene-1, 3-diol

A solution of sodium borohydride (1.85g, 49mmol) in water (30ml) was added to an ice-cold solution of 2-benzoyl-5-ethoxycarbonyloxy-phenyl carbonate ethyl ester (3.6g, 10mmol) in THF (30 ml). The mixture was stirred at 0 ℃ for-60 minutes and at room temperature for-60 hours to give a pale red suspension. Water (150ml) was added to the reaction solution, and the mixture was extracted with diethyl ether (150 ml). The extract was washed with water (2X 100ml) and saturated aqueous sodium chloride (50 ml). The solution was dried over anhydrous magnesium sulfate and concentrated to give a pale yellow gum. The gum was dissolved in aqueous sodium hydroxide (20ml, 10%) and the solution heated to reflux for-60 minutes. The solution was allowed to cool and acidified with hydrochloric acid (5ml, 37%). The mixture was extracted with diethyl ether (50 ml). The extract was washed with water (3X 40ml) and saturated aqueous sodium chloride (30 ml). The solution was dried over anhydrous magnesium sulfate and concentrated to give 4-benzyl-benzene-1, 3-diol as a dark red gum (2.1 g).

LC Retention time 2.28 min [ M + H]⁺No ion (run time 3.75 min)

Delta (deuterated chloroform) 7.2(m 3ArH)7.1(m 2ArH)6.85(d J8.1.1 Hz ArH)6.3(d J8.1.1 HzArH)6.2(s ArH)3.85(s 2 CH)₂)

In the synthesis of scheme 1, which gives example 23, 4-benzyl-benzene-1, 3-diol was used as starting material.

Example 23

3- {2, 4-dihydroxy-5- [4- (4-methoxy-phenyl) -3-methyl-isoxazol-5-yl ] -phenyl } -acrylic acid

Scheme 5: heck reaction and deprotection of boron trichloride

Step 1

3- {2, 4-bis-benzyloxy-5- [4- (4-methoxy-phenyl) -3-methyl-isoxazol-5-yl ] -phenyl } -acrylic acid tert-butyl ester

Diisopropylethylamine (1ml, 5.7mmol) was added to a suspension of 5- (2, 4-bis-benzyloxy-5-bromo-phenyl) -4- (4-methoxy-phenyl) -3-methyl-isoxazole (0.56g, 1.0mmol) in tert-butyl acrylate (1ml, 6.8mmol) and 1-butanol (8ml) under a nitrogen atmosphere. Bis (tri-o-tolylphosphine) palladium (II) dichloride (catalytic amount) is added and the suspension is heated at 140 ℃ for 18 hours to give a yellow/green solution. The solution was allowed to cool and concentrated to give a yellow/green gum. The crude product was purified by column chromatography (silica gel) to give 3- {2, 4-bis-benzyloxy-5- [4- (4-methoxy-phenyl) -3-methyl-isoxazol-5-yl ] -phenyl } -acrylic acid tert-butyl ester as a yellow/green gum after elution with ethyl acetate/n-hexane solution (1: 9) (315 mg). The starting material (170mg) was recovered.

LC Retention time 3.23 min [ M + H]⁺604.6 (run time 3.75 minutes)

N.M. R (deuterated chloroform) 7.85(d J16.1.1 Hz CH)7.6(s ArH)7.4-7.25(m 8ArH)7.05(dJ 8.8Hz 2ArH)6.9(m 2ArH)6.8(d J8.8.8 Hz 2ArH)6.5(s ArH)6.35(d J16.1.1 Hz CH)5.05(s 2 CH)₂)4.75(s 2CH₂)3.75(s 3OCH₃)2.25(s 3CH₃)1.5(s 9CCH₃)

Step 2

A solution of boron trichloride (2ml of a 1.0M solution in dichloromethane) was slowly added to a solution of 3- {2, 4-bis-benzyloxy-5- [4- (4-methoxy-phenyl) -3-methyl-isoxazol-5-yl ] -phenyl } -acrylic acid tert-butyl ester (50mg, 0.09mmol) in dichloromethane (1ml) at-78 ℃ (dry ice/acetone) under nitrogen. The resulting solution was stirred at-78 ℃ for-1 hour and at room temperature for-90 minutes. The solution was cooled to-78 ℃ and water (2ml) was added and the mixture stirred at room temperature for-30 minutes. Ethyl acetate (30ml) was added to the solution, which was washed with water (2X 5ml) and saturated aqueous sodium chloride (10 ml). The solution was dried over anhydrous magnesium sulfate and concentrated to give a pale yellow gum. Trituration with n-hexane gave a yellow solid, the solid was filtered off, washed with n-hexane and dried under vacuum to give 3- {2, 4-dihydroxy-5- [4- (4-methoxy-phenyl) -3-methyl-isoxazol-5-yl ] -phenyl } -acrylic acid as a yellow powder.

LC Retention time 2.08 min [ M + H]⁺368.3 (run time 3.75 minutes)

N.M.R (acetone-d)₆)7.85(d J 16.1Hz CH)7.5(s ArH)7.25(d J 8.8Hz 2ArH)6.95(d J 8.8Hz 2ArH)6.6(s ArH)6.35(d J 16.1Hz CH)3.8(s 3OCH₃)2.25(s3CH₃)

Similarly, 4- [4- (4-methoxy-phenyl) -3-methyl-isoxazol-5-yl ] -6-styryl-benzene-1, 3-diol (example 24) was prepared by deprotecting 5- (2, 4-bis-benzyloxy-5-styryl-phenyl) -4- (4-methoxy-phenyl) -3-methyl-isoxazole (styrylboronic acid and bromoisoxazole intermediate prepared by coupling example 3) with boron trichloride.

LC Retention time 2.08 min[M+H]⁺368.3 (run time 3.75 minutes)

The compounds of examples 22-24 each had a range when tested by the Malachite Green ATPase assay (Malachite Green ATPase assay) described below; HSP90IC50 within 'a', 'B', and 'C'.

Scheme 6: synthesis of 5-carbonamido isoxazoles

Example 25

5- (5-chloro-2, 4-dihydroxyphenyl) -4- (4-methoxy-phenyl) -isoxazole-3-carboxylic acid ethylamide

Step 1

1- (5-chloro-2, 4-dihydroxy-phenyl) -ethanones

Acetic acid (17.5ml) was added dropwise to a suspension of 4-chlororesorcinol (42.5g, 0.293mmol) in boron trifluoride etherate (200ml) under nitrogen. The reaction mixture was heated at 90 ℃ for 3.5 hours and then cooled to room temperature. After about 1 hour of cooling, a solid formed. The mixture was poured into 700ml of a 10% mass/volume aqueous solution of sodium acetate. The mixture was stirred vigorously for 2.5 hours. The light brown solid formed was filtered off and washed with water and air dried overnight to give 1- (5-chloro-2, 4-dihydroxy-phenyl) -ethanone (31.6g, 58%). LCMS: [ M-H ]]⁺185.

Step 2

1- (2, 4-bis-benzyloxy-5-chloro-phenyl) -ethanone

Benzyl bromide (30mL) was added to a mixture of 1- (5-chloro-2, 4-dihydroxy-phenyl) -ethanone (20g, 0.107 mol) and potassium carbonate (37g, 2.5 eq) in acetonitrile (350 mL). The mixture was heated to reflux for 6 hours, then cooled and stirred overnight. The mixture was filtered and the solid was washed with dichloromethane (3 × 100 mL). The combined organic extracts were evaporated in vacuo to leave a pale yellow solid which was triturated with n-hexane (350 mL)/ethyl acetate (15mL) and filtered to give 1- (2, 4-bis-benzyloxy-5-chloro-phenyl) -ethanone as an off-white solid (35.4g, 90%). 1H NMR (400MHz) was consistent with the structure.

Step 3

4- (2, 4-bis-benzyloxy-5-chlorophenyl) -2-hydroxy-4-oxo-but-2-enoic acid ethyl ester

Metallic sodium (1.35g, 0.058mol) was added in small pieces to stirred anhydrous ethanol under nitrogen over 20 minutes. The reaction mixture was then stirred for an additional 10 minutes until all the sodium had reacted to give a homogeneous solution. 1- (2, 4-bis-benzyloxy-5-chloro-phenyl) -ethanone (10.0g, 0.027mol) was added in portions over 2-3 minutes and the resulting suspension was stirred for 5 minutes before ethyl oxalate (6ml, 0.043mol) which provided a thick, yellow precipitate was added. The reaction mixture was heated to reflux (to give a homogeneous brown solution) for 4h, then allowed to cool to room temperature and acetic acid (6ml) was added. The solid formed was triturated, filtered, washed with ethanol and dried to give a yellow solid (12.0 g)，95％)。¹H NMR(400MHz，CDCl₃)δ1.2(t，3H)，4.19(q，2H)，5.05(s，2H)，5.10(s，2H)，6.50(s，1H)，7.22-7.41(m，10H)，7.97(s，1H)。

Step 4

5- (2, 4-bis-benzyloxy-5-chlorophenyl) -isoxazole-3-carboxylic acid ethyl ester

Hydroxylamine hydrochloride (0.89 g; 12.8mmol) was added to a suspension of ethyl 4- (2, 4-bis-benzyloxy-5-chlorophenyl) -2-hydroxy-4-oxo-but-2-enoate (5.00 g; 10.7mmol) in absolute ethanol (100 ml). The reaction mixture was heated to reflux for 4 hours and then cooled to room temperature (during which time the mixture was still heterogeneous but turned to a lighter yellow in color). The mixture was filtered and the filtered solid washed with water (2X 20ml) and ethanol (2X 20ml) and dried under vacuum at 45 ℃. 4.49g (91%) of 5- (2, 4-bis-benzyloxy-5-chlorophenyl) -isoxazole-3-carboxylic acid ethyl ester are obtained as a fluffy yellow solid. LCMS: [ M + H ]]⁺466，464(³⁷Cl；³⁵Cl).).¹HNMR(400MHz，CDCl₃)δ1.42(t，3H)，4.42(q，2H)，5.13(s，2H)，5.14(s，2H)，6.62(s，1H)，7.01(s，1H)，7.35-7.43(m，10H)，8.00(s，1H).

Step 5

5- (2, 4-bis-benzyloxy-5-chlorophenyl) -isoxazole-3-carboxylic acid ethylamide

A solution of ethylamine in methanol (2.0M; 40 mL; 80mmol) was added to a stirred solution of ethyl 5- (2, 4-bis-benzyloxy-5-chlorophenyl) -isoxazole-3-carboxylate (4.40 g; 9.51mmol) in absolute ethanol (50mL)) In suspension. The reaction mixture was heated to 80 ℃ (oil bath temperature) for 5 hours. The reaction mixture was allowed to cool to room temperature and left overnight. The product was formed as a colourless solid and the reaction mixture was further cooled in an ice-water bath, filtered and washed with cold ethanol (2X 20 ml). The colorless product was dried under vacuum to give 3.42g (78%) 5- (2, 4-bis-benzyloxy-5-chlorophenyl) -isoxazole-3-carboxylic acid ethylamide. LCMS: [ M + H ]]⁺465，463(³⁷Cl；³⁵Cl).¹HNMR(400MHz，CDCl₃)δ1.25(t，3H)，3.48(m，2H)，5.10(s，2H)，5.2(s，2H)，6.59(s，1H)，6.83(brt，1H)，7.08(s，1H)，7.30-7.41(m，10H)，7.97(s，1H)。

Step 6

5- (2, 4-bis-benzyloxy-5-chlorophenyl) -4-bromo-isoxazole-3-carboxylic acid ethylamide

A solution of bromine in acetic acid (0.6M; 7.2 mL; 4.32mmol) was added to a stirred suspension of 5- (2, 4-bis-benzyloxy-5-chlorophenyl) -4-bromo-isoxazole-3-carboxylic acid ethylamide (2.00 g; 4.32mmol) and sodium acetate (0.708 g; 8.64mmol) in acetic acid (30mL) at room temperature. The mixture was heated to 80 ℃ and became homogeneous within 5-10 minutes to give a dark red solution. After heating for 2.5 hours, the solution was yellow in color. TLC analysis indicated the presence of starting material and product. Over the next 2 hours, 2.0ml (1.2mmol) of bromine in acetic acid was added. The reaction mixture was allowed to cool to room temperature and acetic acid was removed under vacuum to give a solid residue which was partitioned between diethyl ether (200ml) and water (200 ml). The two phases were separated and the organic phase was washed with water (3X 100ml), saturated aqueous sodium bicarbonate (2X 100ml) and saturated aqueous sodium chloride (1X 200 ml). The organic phase was dried over sodium sulfate, filtered and the filtrate solvent was removed in vacuo to give a yellow oil. Purifying the oil by flash chromatography on silica gel, eluting with 1-20% ethyl acetate in n-hexane. The product was obtained as a colorless solid, 1.2g (52%). LCMS: [ M + H ]]⁺543，541(⁸¹Br；⁷⁹Br).¹H NMR(400MHz，CDCl₃)δ1.26(t，1H)，3.50(m，2H)，5.01(s，2H)，5.12(s，2H)，6.62(s，1H)，6.74(br t，1H)，2.28-7.41(m，10H)，7.53(s，1H)。

Step 7

5- (2, 4-bis-benzyloxy-5-chlorophenyl) -4- (4-methoxy-phenyl) -isoxazole-3-carboxylic acid ethylamide

To a mixture of 4-methoxyphenylboronic acid (0.178g, 1.17mmol) and 5- (2, 4-bis-benzyloxy-5-chlorophenyl) -4-bromo-isoxazole-3-carboxylic acid ethylamide (0.507g, 0.94mmol) was added sodium bicarbonate (237mg, 2.82mmol) followed by DMF (5mL) and water (1.0 mL). The mixture was evacuated and degassed and flushed with nitrogen (three times), then the next mixture was bubbled with nitrogen for 5 minutes. Dichlorobis (triphenylphosphine) palladium (II) (66mg, 0.094mmol) was added and the reaction mixture was heated at 90 ℃ for 2h under nitrogen (the reaction mixture turned dark brown in color). An additional 10mg of dichlorobis (triphenylphosphine) palladium (II) were added and the reaction mixture was heated at 90 ℃ for 15 hours and then allowed to cool to room temperature. Most of the solvent was removed under vacuum and the residue partitioned between ethyl acetate (50mL) and water (50 mL). The mixture was filtered through a celite pad to remove residual palladium, then the two phases were separated, and the organic layer was washed with water (2 × 30mL), saturated aqueous sodium chloride (50mL), and then dried over sodium sulfate. The mixture was filtered and the filtrate solvent was removed in vacuo to give a yellow oil (598 mg). The crude reaction product was purified by first adsorption on silica gel followed by flash chromatography on silica gel (20g IST) with a solvent gradient of 1-20% ethyl acetate in n-hexane. This gave 5- (2, 4-bis-benzyloxy-5-chlorophenyl) -4- (4-methoxy-phenyl) -isoxazole-3-carboxylic acid ethylamide as a colorless solid (0.223g, 40%). LCMS: [ M + H ]]⁺571，569(³⁷Cl；³⁵Cl).¹H NMR(400MHz，CDCl₃)δ1.21(t，3H)，3.44(m，2H)，3.79(s，3H)，4.73(s，2H)，6.45(s，1H)，6.65(t，1H)，6.80(d，2H)，7.14 to 7.44(m，8H)，6.95(m 2H).

Step 8

To an ice-cooled solution of 5- (2, 4-bis-benzyloxy-5-chlorophenyl) -4- (4-methoxy-phenyl) -isoxazole-3-carboxylic acid ethylamide (0.213mg, 0.374mmol) in dichloromethane (5mL) was added a 1.0M solution of boron trichloride in dichloromethane (1.12 mL; 1.12mmol) under a nitrogen atmosphere. The reaction mixture was stirred at 0 ℃ for 15 minutes and then at room temperature for 35 minutes. The reaction mixture was again cooled to 0 ℃ and quenched by addition of saturated aqueous sodium bicarbonate (5 mL). After stirring for 5 min, the dichloromethane was removed under vacuum and the residue was partitioned between ethyl acetate (30mL) and water (30 mL). The two phases were separated and the organic layer was washed with water (30mL), saturated aqueous sodium chloride (30mL) and then dried over sodium sulfate. The mixture was filtered and the filtrate solvent removed in vacuo to give a foamy, colorless solid which was purified by first adsorption onto silica gel and then flash chromatography on silica gel (10g IST) eluting with 50% ethyl acetate in n-hexane. This gave 5- (5-chloro-2, 4-dihydroxyphenyl) -4- (4-methoxy-phenyl) -isoxazole-3-carboxylic acid ethylamide as a colourless solid (0.097 g; 67%). LCMS: [ M + H ]]⁺391，389(³⁷Cl；³⁵Cl).¹H NMR(400MHz，d₆-DMSO)1.08(t，3H)，3.22(m，2H)，3.73(s，3H)，6.59(s 1H)，6.87(d，1H)，7.13-7.17(m，3H)，8.88(br t，1H)，10.09(s，1H)，10.62(s，1H)。

Example 25 has activity 'a' in the fluorescence polarization assay described below.

Similarly, example 26 was prepared by coupling of Boc-protected 4-piperazinyl phenylboronate as above. This boronic ester was prepared from 1- (4-bromophenyl) piperazine via boc protection followed by coupling with bis (tetramethylpinacolato) diboron under Pd catalysis to form the boronic ester example 27 was prepared analogously example 27a was prepared from 5- (2, 4-bis-benzyloxy-5-chlorophenyl) -4-bromo-isoxazole-3-carboxylic acid ethylamide by deprotection:

fluorescence polarization assay

Scheme 7:preparation of 5- (2, 4-bis-benzyloxy-5-chloro-phenyl) -4-iodo-3-methyl-isoxazole intermediates

Example 28

4-chloro-6- [ 3-methyl-4- (3-morpholin-4-ylmethyl-phenyl) -isoxazol-5-yl ] -benzene-1, 3-diol

Step 1

1- (2, 4-bis-benzyloxy-5-chloro-phenyl) -3-hydroxy-but-2-en-1-one:

to a solution of ketone (15g) in EtOAc (200mL) was added sodium platelet (3.0 g). The suspension was stirred at room temperature for 15 minutes and then heated to reflux overnight. The reaction was quenched with acetic acid and the yellow precipitate was filtered off. The precipitate was triturated in n-hexane to give bright yellow crystals. NMR showed this to be the desired product-mainly enol-trace ketone.

Step 2

5- (2, 4-bis-benzyloxy-5-chloro-phenyl) -3-methyl-isoxazole:

the diketone (4.0g) was suspended in 80% aqueous EtOH. Hydroxylamine hydrochloride (3.4g) and sodium acetate (4.0g) were added and the pH was adjusted to 8/9 with 2M NaOH. The solution was refluxed for 24 hours (due to R)_fValues very similar were difficult to monitor by TLC). After this time, the solution was acidified to pH 5 with 1M HCl and poured into water. The white precipitate was filtered off and washed with water and triturated with n-hexane to give a white solid. Note: the compound may also be washed with ether to remove trace impurities if necessary, but is generally not required. NMR showed this to be the desired product.

Step 3

5- (2, 4-bis-benzyloxy-5-chloro-phenyl) -4-iodo-3-methyl-isoxazole:

isoxazole (2g) was placed in a mixture of acetic acid (24ml) and water (30 ml). Iodine monochloride (2g excess) was added and the solution was heated at 80 ℃ for 2-3 hours. After cooling to room temperature, 10% Na was added₂SO₃Aqueous (sodium sulfite) solution (50 ml). A viscous orange solid/oil was separated from the mixture and washed with water, then dissolved in acetone and filtered. Acetone was removed under vacuum to give a viscous orange oil which solidified to an orange solid overnight. NMR and LCMS indicated this to be the desired product.

Step 4

3- [5- (2, 4-bis-benzyloxy-5-chloro-phenyl) -3-methyl-isoxazol-4-yl ] -benzaldehyde

After addition of 1M sodium bicarbonate solution (1.1ml, 3.0 equiv.) and Pd (Ph)₃P)₂Cl₂(21mg, 0.08 equiv.) 5- (2, 4-bis-benzyloxy-5-chloro-phenyl) -4-iodo-3-methyl-isoxazole (200mg, 0.38mmol) and 3-formylphenylboronic acid (85mg, 1 equiv.) were dissolved in DMF (12ml) with stirring. The reaction mixture was transferred to three microwave tubes, which were sealed, and the mixture therein degassed before being irradiated in a CEM microwave apparatus with an initial power of 200W to 150 ℃ for 15 minutes. On cooling, the reaction mixtures were combined and partitioned between ethyl acetate (10ml) and water (10 ml). The aqueous layer was separated and extracted again with ethyl acetate (10 ml). The combined organic layers were washed with water (2X 20ml), saturated brine (20ml), dried over sodium sulfate before concentration in vacuo, and purified by flash chromatography on silica gel eluting with 25% ethyl acetate in n-hexane.

LCMS t_R＝9.06，MS m/z 510.4[M+H]⁺

Step 5

4- {3- [5- (2, 4-bis-benzyloxy-5-chloro-phenyl) -3-methyl-isoxazol-4-yl ] -benzyl } -morpholine

Reacting 3- [5- (2, 4-bis-benzyloxy-5-chloro-phenyl) -3-methyl-isoxazol-4-yl]Benzaldehyde (25mg, 0.05mmol) and morpholine (0.3ml) were mixed with DCE (0.5ml) in a microwave tube. Sodium triacetoxyborohydride (15mg, 1.4 equivalents) was added, the tube sealed and a nitrogen atmosphere introduced. After 1 hour, more sodium triacetoxyborohydride (15mg) was added and the reaction stirred overnight. TLC analysis indicated that the reaction was not complete, so one drop of acetic acid was added, the reaction was stirred again overnight, after which the reaction was performed with 1M NaHCO₃The solution (7ml) was quenched and extracted into EtOAc (5 ml). The solution was over MgSO₄Drying and removal of the solvent under vacuum gave 13mg of crude product as an off-white powder which was used in the deprotection step.

Step 6

Deprotection of 4- {3- [5- (2, 4-bis-benzyloxy-5-chloro-phenyl) -3-methyl-isoxazol-4-yl ] -benzyl } -morpholine following the procedure shown previously and purification of the crude product by preparative TLC eluting with 10% ethanol in dichloromethane afforded 0.6mg (7% yield) of the product as a white powder.

LCMS t_R＝5.46，MS m/z 399.3[M-H]^-

Example 28 had an active ` A ` in a fluorescence polarization assay as described below.

Example 29

1- {3- [5- (2, 4-bis-benzyloxy-5-chloro-phenyl) -3-methyl-isoxazol-4-yl ] -benzyl } -piperidine-4-carboxamide

Prepared using a procedure analogous to that for 4-chloro-6- [ 3-methyl-4- (3-morpholin-4-ylmethyl-phenyl) -isoxazol-5-yl ] -benzene-1, 3-diol except that morpholine is replaced with iso-3-piperidinecarboxylic acid amide and sodium triacetoxyborohydride (3 equivalents) and acetic acid (1 drop) are initially added. The reaction was complete after 18 hours. The crude product obtained after work-up was used for the deprotection step.

1- {3- [5- (5-chloro-2, 4-dihydroxy-phenyl) -3-methyl-isoxazol-4-yl ] -benzyl } -piperidine-4-carboxamide

Deprotection of 1- {3- [5- (2, 4-bis-benzyloxy-5-chloro-phenyl) -3-methyl-isoxazol-4-yl ] -benzyl } -piperidine-4-carboxamide as indicated previously and purification of the crude product by preparative TLC eluting with 10% ethanol in dichloromethane afforded 0.7mg (3% yield) of the product as a white powder.

LCMS t_R＝5.36，MS m/z 442.3[M+H]⁺

Example 29 had an active ` A ` in the fluorescence assay as described below.

In a similar manner, example 30 was prepared:

fluorescence polarization assay

Example 31

Scheme 8

Step 1

5- (2, 4-bis-benzyloxy-5-chloro-phenyl) -4-iodo-isoxazole-3-carboxylic acid ethylamide

The mixture became homogeneous at heating reflux (oil bath 100 ℃ C.)Previously, 5- (2, 4-bis-benzyloxy-5-chloro-phenyl) -isoxazole-3-carboxylic acid ethylamide (0.90g, 1.94mmol), N-iodosuccinimide (0.44g, 1 eq) and cerium (IV) ammonium nitrate (0.53g, 0.5 eq) were suspended in acetonitrile (55 ml). After 18 hours, the solution was cooled and the solvent was removed in vacuo to give a thick orange oil. The oil was partitioned between DCM (25ml) and water (10ml), the organic layer was retained and washed with Na₂SO₄The extract was washed with saturated brine (2X 25ml) before drying. After removal of DCM in vacuo, 0.88g (77% yield) of the product is obtained as an orange/tan powder.

LCMS t_R＝8.75，MS m/z 589.1[M+H]⁺

Step 2

1- (3-bromo-phenyl) -4-methyl-piperazine

1, 3-dibromobenzene (0.90ml, 7.49mmol), N-methylpiperazine (0.28ml, 2.50mmol) and dry toluene (7ml) were added by syringe to a dry, argon-filled flask. In BINAP (47mg) and Pd₂dba₃(23mg) the solution was mixed well before addition, the flask was again filled with argon and DBU (0.93g, 2.5 eq.) was added via syringe. The reaction mixture was warmed to 60 ℃ before starting the reaction with a single addition of freshly ground sodium tert-butoxide. The reaction was stirred at 60 ℃ overnight and TLC analysis appeared to show some piperazine was still present, so the reaction was heated to 100 ℃ and stirred for an additional 24 hours before it was partitioned between EtOAc (20ml) and water (20 ml). The aqueous layer was re-extracted with EtOAc and the combined organic layers were washed with 1.6M HCl solution (2X 10 mL). The acidic solution containing the product was basified with a similar volume of 1M NaOH solution to the acid solution and then pH 8.5 was brought by careful addition of solid sodium bicarbonate before extraction back to EtOAc (2 × 15 ml). The extract was washed with saturated brine and MgSO₄Drying and evaporation to dryness gave 0.50g (78% yield) of pure product as a yellow oil,

LCMS t_R＝4.55，MS m/z 255.4/257.3[M+H]⁺

Step 3

1-methyl-4- [3- (4, 4, 5, 5-tetramethyl- [1, 3, 2] dioxaborolan-2-yl) -phenyl ] -piperazine

PdCl to sealed microwave tube filled with argon₂To a solution of DCM (10mg, 0.012mmol) in dry toluene (4ml) was added 1- (3-bromo-phenyl) -4-methyl-piperazine (100mg, 0.39mmol), Et3N (0.11ml, 2 equivalents), and 4, 4, 5, 5-tetramethyl-1, 3, 2-dioxaborolan (0.09ml, 1.5 equivalents). The microwave tubes were evacuated and back-filled with argon before being irradiated in a CEM microwave reactor with an initial power of 200W for 1 hour at 100 ℃. The reaction mixture was partitioned between more toluene (6ml) and water (10ml), the organic layer was separated and washed with water (1X 10ml), MgSO₄Drying and then evaporation under vacuum left a purple/brown residue which, without purification, could be used in suzuki coupling.

LCMS t_R＝0.97，MS m/z 303.5[M+H]⁺

Step 4

5- (2, 4-bis-benzyloxy-5-chloro-phenyl) -4- [3- (4-methyl-piperazin-1-yl) -phenyl ] -isoxazole-3-carboxylic acid ethylamide

Coupling 5- (2, 4-bis-benzyloxy-5-chloro-phenyl) -4-bromo-isoxazole-3-carboxylic acid ethylamide (38mg, 0.07mmol) and 1-methyl-4- [3- (4, 4, 5, 5-tetramethyl- [1, 3, 2] dioxaborolan-2-yl) -phenyl ] -piperazine (31mg, 2 equiv) using the suzuki method described above gives 37mg (83% yield) of the crude product as a brown oil for the deprotection step.

Step 5

5- (5-chloro-2, 4-dihydroxy-phenyl) -4- [3- (4-methyl-piperazin-1-yl) -phenyl ] -isoxazole-3-carboxylic acid ethylamide

Reacting 5- (2, 4-bis-benzyloxy-5-chloro-phenyl) -4- [3- (4-methyl-piperazin-1-yl) -phenyl]-isoxazole-3-carboxylic acid ethylamide deprotected as shown previously. The precipitate formed during the reaction was separated and partitioned between EtOAc and water. The aqueous phase was retained and basified with solid sodium bicarbonate and the product extracted with EtOAc (2 × 10 ml). The combined organic layers were washed with saturated brine (10ml) and MgSO₄Dried and evaporated in vacuo to yield 5.2mg (20% yield) of product as a tan powder.

LCMS t_R＝5.58，MS m/z 457.3[M+H]⁺

δ_H(d⁴-MeOH)，7.17(1H，m，Ar-H)，7.09(1H，s，Ar-H)，6.94(1H，m，Ar-H)，6.80(1H，m，Ar-H)，6.49(1H，s，Ar-H)，3.13(4H，t，NCH₂CH₂N-CH₃)，2.69(2H，q，CONHCH₂CH₃)，2.61(4H，t，NCH₂CH₂N-CH₃)，2.37(3H，s，NCH₂CH₂N-CH₃)，1.19(3H，t，CONHCH₂CH₃).

Example 31 has an activity 'a' in the fluorescence polarization assay as follows:

examples 32-38 in the table were prepared similarly, but with the following changes:

1. for example 36, the dioxaborolane intermediate was prepared as follows:

scheme(s)

Step 1

1- (4-bromo-phenyl) -4-methyl-piperazine

1- (4-bromo-phenyl) -piperazine (1g, 4.1mmol) and potassium carbonate (1.8g, 3 equivalents) in DMF (15ml) were treated with methyl iodide (250. mu.l, 1.1 equivalents) and the solution was stirred at room temperature overnight. The reaction was quenched with deionized water (10ml) and extracted with ethyl acetate. The organic phase was washed with sodium bicarbonate to remove any dimethylated impurities, dried, and the solvent was removed to give 1- (4-bromo-phenyl) -4-methyl-piperazine in 73% yield.

LC retention time 2.21 min [ M + H ] +256 (run time 3.75 min).

Step 2

1-methyl-4- [4- (4, 4, 5, 5-tetramethyl- [1, 3, 2] dioxaborolan-2-yl) -phenyl ] -piperazine

1- (4-bromo-phenyl) -4-methyl-piperazine (750mg, 3mmol) and bis (pinacolato) diborane (1.1g, 1.5 equivalents) and potassium acetate (900mg, 3 equivalents) in DMSO (15 ml). The suspension was degassed before adding PdCl2(dppf) (catalytic amount) and stirred at 80 ℃. After 3 hours, bis (pinacolato) diborane (1 eq) was added and stirred for an additional 2 hours. The suspension was partitioned between ethyl acetate and water. Purification by column chromatography eluting with a gradient of 0-8% methanol in dichloromethane afforded 1-methyl-4- [4- (4, 4, 5, 5-tetramethyl- [1, 3, 2] dioxaborolan-2-yl) -phenyl ] -piperazine in 62% yield.

LC Retention time 1.83 min [ M + H ] +303 (run time 3.75 min)

2. For examples 37 and 38, the boronic acid intermediate was used instead of dioxaborolane, the former being prepared as follows:

4- [ (2-Methylsulfonyl) -ethylaminomethyl ] -phenylboronic acid (intermediate of example 37)

4-Aminomethylphenylboronic acid hydrochloride (560mg, 3mmol) in ethanol (5ml) was treated with methylvinylsulfone (260. mu.l, 1 equiv.) and triethylamine (1.2ml, 3 equiv.). The solution was stirred at 100 ℃ for 2 hours. Ethanol was removed in vacuo and partitioned between water and butanol to give 4- [ (2-methanesulfonyl) -ethylaminomethyl ] -phenylboronic acid in 94% yield.

LC retention time 0.39 min [ M + H ] +258 (run time 3.75 min).

4- [ N-methyl-S, S-dioxo-thiomorpholinyl ] -phenylboronic acid (intermediate of example 38)

4-Aminomethylphenylboronic acid hydrochloride (456mg, 2.4mmol) in ethanol (8ml) was treated with vinylsulfone (244. mu.l, 1 equiv.) and triethylamine (2 equiv.). The solution was stirred at 100 ℃ for 3 hours. Ethanol was removed under vacuum and the product was obtained in 88% yield in water and butanol.

LC retention time 1.65 min [ M + H ] +270 (run time 8 min).

Example 39

Scheme 9：

Step 1

5- (2, 4-bis-benzyloxy-5-chloro-phenyl) -4- (3-chloro-phenyl) -isoxazole-3-carboxylic acid ethylamide

5- (2, 4-bis-benzyloxy-5-chloro-phenyl) -4-bromo-isoxazole-3-carboxylic acid ethylamide (60mg, 0.11mmol) and 3-chlorobenzeneboronic acid (23mg, 1.3 equiv) were coupled together using the suzuki method described previously to give 35mg (55% yield) of the crude product as a brown powder which was used in the next step.

Step 2

5- (2, 4-bis-benzyloxy-5-chloro-phenyl) -4- (3-chloro-phenyl) -isoxazol-3-ylmethyl ] -ethyl-amine

To a solution of 5- (2, 4-bis-benzyloxy-5-chloro-phenyl) -4- (3-chloro-phenyl) -isoxazole-3-carboxylic acid ethylamide (36mg, 0.06mmol) in anhydrous THF under argon was added 1M borane-THF complex (1ml) and the solution refluxed overnight. After cooling, the solution was poured intoSPE Flash SCX-25 g cartridge, elution Rapid with methanol (2X 20 ml). The desired product was then recovered eluting with a mixture of 10% aqueous ammonia in methanol (2X 10ml) to yield 23mg (65% yield) of a pale yellow powder after evaporation in vacuo.

LCMS(LCT)t_R＝8.18，MS m/z 558.8[M+H]⁺

Example 39 has an activity 'a' in fluorescence polarization assay as follows:

example 40 was prepared similarly:

fluorescence polarization assay

Examples

Scheme 10:

step 1

5- (2, 4-bis-benzyloxy-5-chloro-phenyl) -4- (4-formyl-phenyl) -isoxazole-3-carboxylic acid ethylamide

5- (2, 4-bis-benzyloxy-5-chloro-phenyl) -4-iodo-isoxazole-3-carboxylic acid ethylamide (prepared as in example 31) (2g, 3.4mmol), 4-formylboronic acid (0.612g, 4.08mmol), NaHCO₃(10.2ml, 1M aqueous solution, 10.2mmol), PdCl₂(PPh₃)₂(119mg, 0.17mmol) and DMF (50ml) were combined together. Then the mixture was heated at 80 ℃ for 1 hour by adding N₂The mixture was degassed by bubbling for 5 minutes. The mixture was evaporated under vacuum and partitioned between EtOAc (3 × 50ml) and water (50 ml). Combined and dried (Na)₂SO₄) The organic layer was evaporated in vacuo to give a crude oil. It was dissolved in EtOAc and passed through SiO₂Stopper, washed with EtOAc. The filtrate was evaporated in vacuo and the resulting oil was taken up in Et₂O trituration afforded 5- (2, 4-bis-benzyloxy-5-chloro-phenyl) -4- (4-formyl-phenyl) -isoxazole-3-carboxylic acid ethylamide as a pale solid (1.577g, 82%), LC/MS: RT 2.908 min, 567.3 (MH)⁺)。

Step 2

5- (2, 4-bis-benzyloxy-5-chloro-phenyl) -4- (4-morpholin-4-ylmethyl-phenyl) -isoxazole-3-carboxylic acid ethylamide

Acetic acid (0.37ml, 6.44mmol) was added dropwise to 5- (2, 4-bis-benzyloxy-)5-chloro-phenyl) -4- (4-formyl-phenyl) -isoxazole-3-carboxylic acid ethylamide (730mg, 1.29mmol), morpholine (0.225ml, 2.58mmol), 3A powdered molecular sieve (730mg) and MeOH (21 ml). In N₂Stirring was continued overnight. The mixture was then evaporated under vacuum and the crude product obtained was in CH₂Cl₂(3X 40ml) and saturated NaHCO3 solution (40 ml). Combined and dried (Na)₂SO₄) Evaporation of the excess organic layer under vacuum gave crude 5- (2, 4-bis-benzyloxy-5-chloro-phenyl) -4- (4-morpholin-4-ylmethyl-phenyl) -isoxazole-3-carboxylic acid ethylamide as a yellow solid (810mg), LC/MS: RT 2.365 min, 638.4 (MH)⁺)。

Step 3

At 0 deg.C, BCl₃(1M CH₂Cl₂Solution, 3.87ml, 3.87mmol) was added dropwise to crude 5- (2, 4-bis-benzyloxy-5-chloro-phenyl) -4- (4-morpholin-4-ylmethyl-phenyl) -isoxazole-3-carboxylic acid ethylamide (810mg, 1.29mmol) of CH₂Cl₂(30ml) in solution. The reaction was then allowed to reach room temperature. Then slowly add saturated NaHCO₃Aqueous solution (40ml) and the resulting mixture was concentrated in vacuo. It was partitioned between EtOAc (3 × 50ml) and water (50 ml). Combined and dried (Na)₂SO₄) The organic layer was evaporated under vacuum. By flash chromatography with CH₂Cl₂-10％MeOH/1％NH₃/CH₂Cl₂Elution yielded 5- (5-chloro-2, 4-dihydroxy-phenyl) -4- (4-morpholin-4-ylmethyl-phenyl) -isoxazole-3-carboxylic acid ethylamide as a yellow foam (380mg, 64% over 2 steps). LC/MS: RT 1.751 min, 458.2 (MH)⁺)。

Example 41 has an activity 'a' in fluorescence polarization assay as follows:

in the following table, examples 42-64 were prepared in a similar manner to example 41 using the appropriate aldehyde or ketone.

Fluorescence polarization assay

Prepared from ethyl resorcinol starting material

Additional compounds 41a-s were prepared in a manner analogous to example 41:

fluorescence polarization assay

Prepared from ethyl resorcinol starting material

Prepared by reducing an aldehyde intermediate

Prepared by alkylation of intermediate phenol

From naphthyl aldehyde

Example 65

Reduction scheme:

step 1

1- (2, 4-bis-benzyloxy-phenyl) -ethanones

35g of 2, 4-dihydroxyacetophenone (0.230mol, 1 eq.) were dissolved in 500ml of acetonitrile. 79.5g of potassium carbonate (0.575mol, 2.5 eq) and 86.6g of benzyl bromide (0.506mol, 2.2 eq) are added. The mixture was refluxed for 64 hours, cooled and the acetonitrile was removed under reduced pressure. The residue was partitioned between water and ethyl acetate. The residue is mainly mono-benzylated resorcinol.

The crude product (43g) was then dissolved in 250ml of DMF. Potassium carbonate (29g, 0.210mol, 1.2 equiv.) and 25ml benzyl bromide (0.210mol, 1.2 equiv.) were added and the mixture was stirred overnight. The solvent was removed under reduced pressure and the residue was partitioned between ethyl acetate and water. After removal of the solvent, the residue was triturated with n-hexane to remove excess benzyl bromide.

LC-MS[M+H]+＝333

Yield: 51.2g (67%)

Step 2

1- (2, 4-bis-benzyloxy-5-bromo-phenyl) -ethanone

51.2g1- (2, 4-bis-benzyloxy-phenyl) -ethanone (0.154mol, 1 eq) was dissolved in 250ml DMF. 27.42g of N-bromosuccinimide (0.154mol, 1 eq.) in 100ml of DMF were added dropwise. The mixture was stirred at room temperature overnight. The reaction mixture is poured onto 700ml of water and the precipitate is filtered off. The filter cake was washed with water and the colourless solid was recrystallised from 370ml of acetonitrile.

LC-MS [ M + H ] + ═ 411 and 413

Yield: 58.15g (92%)

Step 3

4- (2, 4-bis-benzyloxy-5-bromo-phenyl) -2, 4-dioxo-butyric acid ethyl ester

9.75g of sodium (0.424mol, 3 eq.) are dissolved in 500ml of absolute ethanol (1.5 h). 58g of 1- (2, 4-bis-benzyloxy-5-bromo-phenyl) -ethanone (0.141mol, 1 eq) and 30.98g of diethyl oxalate (0.212mol, 1.5 eq) were added and the mixture refluxed for 2 hours. After cooling, the mixture was poured onto 220ml of 2N aqueous HCl and the product was extracted into 700ml of dichloromethane. The solvent was removed under reduced pressure and the yellow residue was triturated with 150ml of diethyl ether.

Yield: 69.24g (96%)

1H NMR(400MHz，CDCl3)δ1.27(t，3H)，4.27(q，2H)，5.13(d，2H)，6.54(s，1H)，7.37(m，10H)，8.17(s，1H)。

Step 4

5- (2, 4-bis-benzyloxy-5-bromo-phenyl) -isoxazole-3-carboxylic acid ethyl ester

69.3g 4- (2, 4-bis-benzyloxy-5-bromo-phenyl) -2, 4-dioxo-butyric acid ethyl ester (0.135mol, 1 eq) were dissolved in 750ml ethanol. Hydroxylamine hydrochloride (14.11 g) (0.203mol, 1.5 equivalents) was added. The mixture was refluxed for 2.5 hours and cooled. Then it was poured onto 1000ml of water and the precipitate was filtered off. The filter cake is washed with 500ml of water, followed by 75ml of diethyl ether and dried.

Yield: 67.62g (99%)

1H NMR(400MHz，CDCl3)δ1.39(t，3H)，4.41(q，2H)，5.11(d，2H)，5.15(d，2H)，6.58(s，1H)，6.99(s，1H)，7.35(m，10H)，8.16(s，1H)。

Step 5

5- (2, 4-bis-benzyloxy-5-bromo-phenyl) -isoxazole-3-carboxylic acid ethylamide

Ethyl 5- (2, 4-bis-benzyloxy-5-bromo-phenyl) -isoxazole-3-carboxylate was suspended in ethanol and ethylamine (2M in methanol, 3 eq.) and the resulting yellow suspension was heated to reflux (80 ℃) under nitrogen at which temperature all reagents went into solution. The solution was heated for 14 hours and then cooled to room temperature. A white solid formed and the solid was filtered off and washed with more ethanol before drying in vacuo.

LC-MS Retention time 2.868 min [ M + H ] + ═ 507&509 (run time 3.75 min)

Step 6

5- (2, 4-bis-benzyloxy-5-styrylphenyl) -isoxazole-3-carboxylic acid ethylamide

To a mixture of trans-2-phenylvinylboronic acid (0.472g, 3.2mmol) and 5- (2, 4-bis-benzyloxy-5-bromophenyl) -isoxazole-3-carboxylic acid ethylamide (1.079g, 2.13mmol) was added sodium potassium bicarbonate (536mg, 6.39mmol) followed by DMF (25mL) and water (5 mL). The mixture was degassed by evacuation and flushed with nitrogen (three times), followed by bubbling nitrogen through the mixture for 5 minutes.

Dichlorobis (triphenylphosphine) palladium (II) (149mg, 0.21mmol) was added and the reaction mixture was heated under nitrogen at 80 ℃ for several hours (after 10 minutes the reaction mixture became dark brown in color). The reaction mixture was allowed to cool to room temperature and most of the solvent was removed under vacuum. The resulting residue was partitioned between ethyl acetate (100mL) and water (100mL) and the mixture was filtered through a pad of celite to remove the palladium residue. The two phases were separated and the organic phase was washed with water (2X 50mL) and saturated aqueous sodium chloride (100mL) and dried over sodium sulfate. The mixture was filtered and the filtrate solvent was removed under vacuum to give a brown solid (800 mg). The celite cake was washed with dichloromethane and then dried over sodium sulfate. The mixture was filtered and the filtrate solvent was removed under vacuum to give a brown solid (541 mg). The combined batches were triturated and purified with an ethyl acetate-n-hexane mixture. This gave 5- (2, 4-bis-benzyloxy-5-styrylphenyl) -isoxazole-3-carboxylic acid ethylamide as a light brown solid (808mg, 71%). LCMS: [ M + H ] +531.1H NMR (400MHz, CDCl3) delta 1.12(t, 3H), 3.37(M, 2H), 4.95(s, 2H), 5.07(s, 2H), 6.46(s, 1H), 6.70(brt, 1H), 7.11(s, 1H), 7.17(d, 1H), 7.23(d, 1H), 7.32-7.44(M, 15H), 8.09(s, 1H).

Step 7

5- (2, 4-bis-benzyloxy-5-phenethylphenyl) -isoxazole-3-carboxylic acid ethylamide

Palladium on carbon catalyst (10%; 50mg) was added to a degassed solution of 5- (2, 4-bis-benzyloxy-5-styrylphenyl) -isoxazole-3-carboxylic acid ethylamide (690mg, 1.30mmol) in1, 4-dioxane (50mL) under a nitrogen atmosphere. The reaction mixture was hydrogenated for a total of 4.75 hours, and palladium on carbon catalyst (50mg) was added at 0.75 hours and 2.5 hours. The reaction mixture was filtered through a pad of celite, and the pad was washed with 1, 4-dioxane (20mL) and dichloromethane (20 mL). The combined filtrate solvents were removed under reduced pressure to give a cream-colored solid, which was purified by flash chromatography on silica gel (20g, IST) eluting with 10-50% ethyl acetate in n-hexane. This gave 5- (2, 4-bis-benzyloxy-5-phenethylphenyl) -isoxazole-3-carboxylic acid ethylamide as a pale yellow solid (609mg, 88%). LCMS: [ M + H ]]+533.1H NMR(400MHz，CDCl3)1.26(t，3H)，2.86-2.96(m，4H)，3.49(m，2H)，5.03(s，2H)，5.18(s，2H)，6.56(s，1H)，6.81(t，1H)，7.07(s，1H)，7.15-7.20(m，3H)，7.23-7.28(m，2H)，7.31-7.42(m，10H)，7.73(s，1H)。

Step 8

5- (2, 4-bis-benzyloxy-5-phenethylphenyl) -4-bromo-isoxazole-3-carboxylic acid ethylamide

N-bromosuccinimide (207mg, 1.16mmol) was added to a suspension of 5- (2, 4-bis-benzyloxy-5-phenethylphenyl) -isoxazole-3-carboxylic acid ethylamide (564mg, 1.06mmol) in acetonitrile (20 mL). Ammonium cerium nitrate (209mg, 0.53mmol) was added and the mixture heated to reflux (to give a homogeneous orange solution) and stirred for 30 min. The reaction mixture was cooled to room temperature and the acetonitrile was removed in vacuo. The residue was partitioned between ethyl acetate (50mL) and water (50mL) and the two phases were separated. The organic phase was washed with saturated aqueous sodium chloride (50mL) and dried over sodium sulfate. The mixture was filtered and the filtrate solvent was removed in vacuo to give a yellow oil which was purified by flash chromatography on silica gel (20g, IST) eluting with 10-30% ethyl acetate in n-hexane. 5- (2, 4-bis-benzyloxy-5-phenethylphenyl) -4-bromo-isoxazole-3-carboxylic acid ethylamide was obtained as a yellow oil (326mg, 53%). LCMS: [ M + H ] +613, 611.

Step 9

5- (2, 4-bis-benzyloxy-5-phenethyl-phenyl) -4- (4-morpholin-4-ylmethyl-phenyl) -isoxazole-3-carboxylic acid ethylamide

To a mixture of 4-morpholin-4-ylmethyl-phenylppinacol borane (0.215g, 0.71mmol) and 5- (2, 4-bis-benzyloxy-5-phenethylphenyl) -4-bromo-isoxazole-3-carboxylic acid ethylamide (0.347g, 0.57mmol) was added sodium bicarbonate (142mg, 1.69mmol) followed by DMF (10mL) and water (2.0 mL). The mixture was degassed by evacuation and flushed with nitrogen (three times), followed by bubbling nitrogen into the mixture for 5 minutes. Dichlorobis (triphenylphosphine) palladium (II) (40mg, 0.057mmol) was added and the reaction mixture was heated at 80 ℃ for 5h under nitrogen (the reaction mixture turned dark brown in color). An additional 20mg (0.029mmol) of dichlorobis (triphenylphosphine) palladium (II) were added and the reaction mixture was heated at 80 ℃ for 15 hours and then allowed to cool to room temperature. Most of the solvent was removed under vacuum and the residue was partitioned between ethyl acetate (50mL) and water (50 mL). The mixture was filtered through a pad of celite to remove the palladium residue, and then the two phases were separated. The organic phase was washed with water (2X 50mL) and saturated aqueous sodium chloride (50mL) and then dried over sodium sulfate. The mixture was filtered and the filtrate solvent was removed in vacuo to give a brown oil and the crude reaction product was purified by flash chromatography on silica gel (20g, IST) using a gradient of 30-70% ethyl acetate in n-hexane to elute the solvent. 5- (2, 4-bis-benzyloxy-5-phenethyl-phenyl) -4- (4-morpholin-4-ylmethyl-phenyl) -isoxazole-3-carboxylic acid ethylamide was obtained as a yellow oil (0.110g, 27%). LCMS: [ M + H ] +708.

Step 10

5- (2, 4-dihydroxy-5-phenethyl-phenyl) -4- (4-morpholin-4-ylmethyl-phenyl) -isoxazole-3-carboxylic acid ethylamide hydrochloride

To an ice-bath cooled solution of 5- (2, 4-bis-benzyloxy-5-phenethyl-phenyl) -4- (4-morpholin-4-ylmethyl-phenyl) -isoxazole-3-carboxylic acid ethylamide (0.109g, 0.15mmol) in dichloromethane (4mL) was added a 1.0M solution of boron trichloride in dichloromethane (0.45 mL; 0.45mmol) under nitrogen. The reaction mixture was stirred at 0 ℃ for 20 minutes and then at room temperature for 3.5 hours. The reaction mixture was again cooled to 0 ℃ and quenched by addition of saturated aqueous sodium bicarbonate (5 mL). After stirring for 5 min, the dichloromethane was removed under vacuum and the residue was partitioned between ethyl acetate (20mL) and water (20 mL). The two phases were separated and the organic phase was washed with water (20mL) and saturated aqueous sodium chloride (20mL) and then dried over sodium sulfate. The mixture was filtered and the filtrate solvent was removed in vacuo to give a light brown oil which was adsorbed onto silica gel and purified by flash chromatography on silica gel (10g, IST) eluting with 0-5% methanol in ethyl acetate. This gave a colourless oil which was triturated with 1.0M HCl in ether (5mL) to give 5- (2, 4-dihydroxy-5-phenethyl-phenyl) -4- (4-morpholin-4-ylmethyl-phenyl) -isoxazole-3-carboxylic acid ethylamide hydrochloride (0.019 g; 24%). LCMS: [ M + H ] +528.1H NMR (400MHz, d6-DMSO) delta 1.08(t, 3H), 2.60(M, 4H), 2.90-3.30(M, 6H), 3.67(M, 2H), 3.87(M, 2H), 4.30(s, 2H), 6.46(s, 1H), 6.84(s, 1H), 7.05-7.49(M, 5H), 7.40-7.68(M, 4H), 8.90(brs, 1H), 9.67(s, 1H), 9.89(s, 1H), 10.75(brs, 1H).

Example 65 has an activity 'a' in fluorescence polarization assay as follows:

the examples in the following table were prepared using a method similar to that of example 64 and were active in fluorescence polarization assays, as described below.

Additional examples 75a-v in the table below were also prepared by a method similar to example 65.

Example 76

Reaction scheme

Step 1

3- (2, 4-bis-benzyloxy-5-bromo-phenyl) -4- (4-methoxy-phenyl) -5-methyl-isoxazole

Trimethyloxonium boron trifluoride (Aldrich; 70mg, 0.47mmol) was added to a stirred solution of 5- (2, 4-bis-benzyloxy-5-bromo-phenyl) -4- (4-methoxy-phenyl) -3-methyl-isoxazole (example 3, step 1) (120mg, 0.22mmol) in dichloromethane (3ml) and stirring was continued for 3 h. The resulting mixture was concentrated in vacuo to leave a white semisolid which was mixed with hydroxylamine hydrochloride (70mg, 1.0mmol), potassium carbonate (120mg, 0.87mmol) and methanol (2mL) and heated at reflux for 18 h. The reaction mixture was partitioned between water (20mL) and ethyl acetate (2 × 10mL), and the combined organic phases were dried over anhydrous magnesium sulfate and evaporated in vacuo to leave a colorless oil. The crude product was purified by column chromatography (silica gel (10g)), eluting with n-hexane, followed by ether/n-hexane (1: 1) to give 3- (2, 4-bis-benzyloxy-5-bromo-phenyl) -4- (4-methoxy-phenyl) -5-methyl-isoxazole (44mg, 37%) as a white solid.

LC Retention time 5.55 min [ M + H]⁺556.0 and 558.0 (run time 8.00 minutes)

N.m. r (deuterated chloroform) 7.64(s ArH)7.356.76(m 14ArH)6.34(sArH)4.90(s2 CH)₂)4.60(s 2CH₂)3.79(s 3CH₃)2.46(s 3CH₃)

Step 2

4-bromo-6- [4- (4-methoxy-phenyl) -5-methyl-isoxazol-3-yl ] -benzene-1, 3-diol

Boron trichloride solution (1M dichloromethane solution, 1ml, 1mmol) was added to a solution of 3- (2, 4-bis-benzyloxy-5-bromo-phenyl) -4- (4-methoxy-phenyl) -5-methyl-isoxazole (38mg, 0.068mmol) in dichloromethane (1ml) and stirring was continued for 1 hour. The reaction mixture was partitioned between water (20ml) and dichloromethane (2X 20ml), the combined organic phases were dried over anhydrous magnesium sulphate and evaporated under vacuum to leave a brown oil. The crude product was purified by column chromatography (silica gel (10g)), eluting with n-hexane followed by n-hexane/ether (3: 1 then 1: 1) to give 4-bromo-6- [4- (4-methoxy-phenyl) -5-methyl-isoxazol-3-yl ] -benzene-1, 3-diol (11mg, 43%) as a colourless oil.

LC Retention time 2.52 min [ M + H]⁺376.1 and 378.1 (run time 3.75 minutes)

N.M.R(DMSO-d₆)10.40(s OH)9.69(s OH)7.22(ArH)7.10-6.89(m 4ArH)6.5(s ArH)3.7(s OCH₃)2.46(s CH₃)

The compound was active 'a' in Hsp90 fluorescence polarization assay.

Example 76A

The following compounds are commercially available (interbiscreen) and are active 'B' in fluorescence polarization assays:

the following compounds were prepared according to example 76:

example 77

Preparation of 5- (5-tert-butyl-2, 4-dihydroxy-phenyl) -4- (4-morpholin-4-ylmethyl-phenyl) -isoxazole-3-carboxylic acid ethylamide

The reaction scheme is as follows:

step 1

1- (5-tert-butyl-2, 4-dihydroxy-phenyl) -ethanones

Sulfuric acid (4ml, 75mmol) was added to a suspension of 2, 4-dihydroxyacetophenone (22.8g, 150mmol) in a mixture of 2-methyl-2-propanol (35g, 470mmol) and trifluoroacetic acid (80ml) under a nitrogen atmosphere. And heating the obtained suspension for 3-3 hours at the oil bath temperature of 75 ℃ to obtain a light red solution. The resulting solution was allowed to cool and poured into ice/water (350ml) to give a light pink precipitate. The solid was filtered off and washed with water (600ml) and n-hexane (200ml) to give a pale pink powder. Vacuum drying (40 ℃) gave 1- (5-tert-butyl-2, 4-dihydroxy-phenyl) -ethanone (28.2g, 92%) as a pale orange powder.

LC Retention time 2.74 min [ M + H]⁺209.1 (run time 3.75 minutes)

N.M. R (deuterated chloroform) 7.35(s ArH)6.05(s ArH)7.35(m 2ArH)2.35(s 3 CH)₃)1.15(s 9CH₃)

Step 2

1- (2, 4-bis-benzyloxy-5-tert-butyl-phenyl) -ethanone

Benzyl bromide (10ml, 84mmol) was added to a solution of acetophenone (13.5g, 65mmol) in DMF (50ml), potassium carbonate (20g, 145mmol) was added and the suspension was stirred at room temperature for-4 hours. The resulting suspension was poured into water (200ml) to give a pale orange precipitate. The solid was filtered off and washed with water. The solid was dissolved in dichloromethane (150ml) and the solution was washed with water (2X 100ml) and saturated aqueous sodium chloride (100 ml). The solution was dried over anhydrous sodium sulfate and concentrated to give a pale red oil.

The oil was dissolved in 2-methyl-2-propanol (100ml) and potassium tert-butoxide (7.5g, 67mmol) was added to give a pale yellow precipitate, benzyl bromide (8ml, 67mmol) was added and the mixture heated under reflux for-1 h. The resulting suspension was allowed to cool and poured into water (250ml) to give a pale orange precipitate. The solid was filtered off and washed with water. The solid was dissolved in ethyl acetate (150ml) and washed with water (2X 200ml) and saturated aqueous sodium chloride (100 ml). The solution was dried over anhydrous sodium sulfate, concentrated to an orange semi-solid and triturated with methanol to give a pale pink solid. The solid was filtered off and dried under vacuum (40 ℃) to give 1- (2, 4-bis-benzyloxy-5-tert-butyl-phenyl) -ethanone as a pale pink powder (9.1g, 36%).

LC Retention time 3.03 min [ M + H]⁺389.3 (run time 3.75 minutes)

N.M. R (deuterated chloroform) 7.65(s ArH)7.25-7.15(m 10ArH)6.35(s ArH)4.95(s2 CH)₂)4.9(s 2CH₂)2.4(s 3CH₃)1.2(s 9CH₃)

Step 3

4- (2, 4-bis-benzyloxy-5-tert-butyl-phenyl) -2-hydroxy-4-oxo-but-2-enoic acid ethyl ester

Sodium ethoxide (2.8g, 41mmol) was added to a suspension of 1- (2, 4-bis-benzyloxy-5-tert-butyl-phenyl) -ethanone (7.8g, 20mmol) in ethanol (40 ml). Diethyl oxalate (4ml, 29.5mmol) was added and the resulting suspension was heated under reflux for-2 hours to give a pale red solution. The solution was allowed to cool and poured into water (200ml), the mixture was acidified with hydrochloric acid (50ml, 1M) and extracted with dichloromethane (150 ml). The extract was washed with water (2X 200ml) and saturated aqueous sodium chloride (100 ml). The solution was dried over anhydrous sodium sulfate and concentrated to a yellow gum. Trituration with n-hexane afforded a yellow solid. The solid was filtered off, washed with n-hexane and dried under vacuum (40 ℃) to give 4- (2, 4-bis-benzyloxy-5-tert-butyl-phenyl) -2-hydroxy-4-oxo-but-2-enoic acid ethyl ester (9.1g, 93%) as a yellow powder.

N.M. R (deuterated chloroform) 8.0(s ArH)7.5-7.35(m 11ArH)6.6(s ArH)5.2(s 2 CH)₂)5.15(s 2CH₂)4.3(q J 7.1Hz 2CH₂)1.4(s 9CH₃)1.25(t J 7.1Hz 3CH₃)

Step 4

5- (2, 4-bis-benzyloxy-5-tert-butyl-phenyl) -isoxazole-3-carboxylic acid ethyl ester

Hydroxylamine hydrochloride (3.6g, 52mmol) was added to a solution of ethyl 4- (2, 4-bis-benzyloxy-5-tert-butyl-phenyl) -2-hydroxy-4-oxo-but-2-enoate (9.0g, 18.5mmol) in ethanol (75ml) and the suspension was heated under reflux for 4 h. The resulting solution was allowed to cool and poured into water (200ml) to give an off-white precipitate. The precipitate was filtered off and dissolved in dichloromethane (150 ml). The solution was washed with water (150ml) and saturated aqueous sodium chloride (50 ml). The solution was dried over anhydrous sodium sulfate and concentrated to give an off-white solid. The solid was washed with n-hexane and dried under vacuum (40 ℃) to give 5- (2, 4-bis-benzyloxy-5-tert-butyl-phenyl) -isoxazole-3-carboxylic acid ethyl ester (8.0g, 89%) as a light brown powder.

LC Retention time 3.13 min [ M + H]⁺486.5 (run time 3.75 minutes)

N.M. R (deuterated chloroform) 7.85(s ArH)7.4-7.25(m 10ArH)6.9(s ArH)6.5(s ArH)5.1(s 2 CH)₂)5.0(s 2CH₂)4.35(q J 7.1Hz 2CH₂)1.4(s 9CH₃)1.35(t J 7.1Hz 3CH₃)

Step 5

5- (2, 4-bis-benzyloxy-5-tert-butyl-phenyl) -isoxazole-3-carboxylic acid ethylamide

Ethyl 5- (2, 4-bis-benzyloxy-5-tert-butyl-phenyl) -isoxazole-3-carboxylate (10.0g, 20.6mmol) was added to a solution of ethylamine in methanol (60ml, 2.0M) and the suspension heated for 2 hours at an oil bath temperature of 75 ℃. The resulting solution was allowed to cool and concentrated to give a light brown oil, dichloromethane (150ml) was added and the solution was washed with water (100ml) and saturated aqueous sodium chloride (75 ml). The solution was dried over anhydrous sodium sulfate and concentrated to give a brown oil which solidified on standing (9.9g, quantitative).

LC Retention time 3.02 min [ M + H]⁺485.3 (run time 3.75 minutes)

N.M. R (deuterated chloroform) 7.8(s ArH)7.4-7.2(m 10ArH)7.0(s ArH)6.75(br t J5.4 HzNH)6.5(s ArH)5.1(s 2 CH)₂)5.0(s 2CH₂)3.4(dq J 5.4Hz，7.1Hz 2CH₂)1.35(s 9CH₃)1.15(t J 7.1Hz 3CH₃)

Step 6

5- (2, 4-bis-benzyloxy-5-tert-butyl-phenyl) -4-iodo-isoxazole-3-carboxylic acid ethylamide

N-iodosuccinimide (9.0g, 40mmol) was added to a suspension of 5- (2, 4-bis-benzyloxy-5-tert-butyl-phenyl) -isoxazole-3-carboxylic acid ethylamide (9.9g, 20.4mmol) in acetonitrile (60 mL). Cerium ammonium nitrate (0.25g, 0.46mmol) was added and the suspension stirred for-18 hours. The resulting suspension was concentrated and the residue was dissolved in dichloromethane (125ml), and the resulting solution was washed with an aqueous sodium metabisulfite solution (2X 100ml, 5%), water (100ml) and a saturated aqueous sodium chloride solution (100 ml). The solution was dried over anhydrous sodium sulfate and concentrated to give a pale red gum. Trituration with ethanol (25ml) gave an off-white solid which was filtered off and washed with ethanol. Drying in vacuo (40 ℃) gave 5- (2, 4-bis-benzyloxy-5-tert-butyl-phenyl) -4-iodo-isoxazole-3-carboxylic acid ethylamide as an off-white powder (7.75g, 62%).

LC Retention time 3.07 min [ M + H]⁺611.2 (run time 3.75 minutes)

N.M. R (deuterated chloroform) 7.45-7.25(m 11ArH)6.8(br t J5.4 Hz NH)6.6(s ArH)5.05(s 4 CH)₂)3.5(dq J 5.4Hz，7.1Hz 2CH₂)1.35(s 9CH₃)1.2(t J 7.1Hz 3CH₃)

Step 7

5- (2, 4-bis-benzyloxy-5-tert-butyl-phenyl) -4- (4-formyl-phenyl) -isoxazole-3-carboxylic acid ethylamide

Aqueous potassium phosphate (25ml, 1.2M) was added to a solution of 5- (2, 4-bis-benzyloxy-5-tert-butyl-phenyl) -4-iodo-isoxazole-3-carboxylic acid ethylamide (6.1g, 10mmol) and 4-formylphenylboronic acid (2.35g, 15.7mmol) in1, 4-dioxane (75ml) under a nitrogen atmosphere. Dichloro-bis (tri-o-tolylphosphine) palladium (II) (catalytic amount) was added and the mixture heated for-1 hour with an oil bath temperature of 100 ℃. The mixture was cooled, and the aqueous layer was separated and extracted with ethyl acetate (100 ml). The combined organic layers were concentrated to give a light brown gum.

The crude product was purified by column chromatography (silica gel (600ml)) eluting with ethyl acetate/hexanes (1: 3) to give 5- (2, 4-bis-benzyloxy-5-tert-butyl-phenyl) -4- (4-formyl-phenyl) -isoxazole-3-carboxylic acid ethylamide as a pale yellow foam (5.18g, 88%).

LC Retention time 3.01 min [ M + H]⁺589.4 (run time 3.75 minutes)

N.M.R (deuterated chloroform) 9.75 (sCHO) 7.5(d J6.9.9 Hz 2ArH)7.2(d J6.9.9 Hz 2ArH)7.15-7.0(m 8ArH)6.8(m 2ArH)6.65(br t J5.4 Hz NH)6.2 (sArH) 4.8(s 2 CH)₂)4.5(s 2CH₂)3.2(dq J 5.4Hz，7.1Hz 2CH₂)1.1(s 9CH₃)1.05(t J 7.Hz 3CH₃)

Step 8

5- (2, 4-bis-benzyloxy-5-tert-butyl-phenyl) -4- (4-morpholin-4-ylmethyl-phenyl) -isoxazole-3-carboxylic acid ethylamide

Sodium cyanoborohydride (65mg, 1.03mmol) was added to a solution of 5- (2, 4-bis-benzyloxy-5-tert-butyl-phenyl) -4- (4-formyl-phenyl) -isoxazole-3-carboxylic acid ethylamide (125mg, 0.21mmol), morpholine (50 μ l, 0.57mmol) and acetic acid (catalytic amount) in methanol (4ml) and the solution was stirred for-72 hours. Dichloromethane (50ml) was added and the solution was washed with water (2X 50ml) and saturated aqueous sodium chloride (50 ml). The solution was dried over anhydrous sodium sulfate and concentrated to give a colorless gum.

The crude product was purified by column chromatography (silica gel (20g)) eluting with ethyl acetate/hexanes (1: 1) to give 5- (2, 4-bis-benzyloxy-5-tert-butyl-phenyl) -4- (4-morpholin-4-ylmethyl-phenyl) -isoxazole-3-carboxylic acid ethylamide as a colourless oil (35mg, 25%).

LC Retention time 2.56 min [ M + H]⁺660.8 (run time 3.75 minutes)

N.M. R (deuterated chloroform) 7.35-7.05(m 15ArH)6.7(br t J5.4 Hz NH)6.4(s ArH)4.9(s 2 CH)₂)4.75(s 2CH₂)3.6(t J 4.5Hz 4CH₂)3.(s 2CH₂)3.35(dq J 5.4Hz，7.1Hz2CH₂)2.35(br s 4CH₂)1.15(t J 7.1Hz 3CH₃)1.1(s 9CH₃)

Step 9

A solution of boron trichloride (1ml of a 1.0M solution in dichloromethane) was added to a solution of 5- (2, 4-bis-benzyloxy-5-tert-butyl-phenyl) -4- (4-morpholin-4-ylmethyl-phenyl) -isoxazole-3-carboxylic acid ethylamide (35mg, 0.05mmol) in dichloromethane (1ml) at-20 ℃ (ice/methanol) under a nitrogen atmosphere. The resulting solution was stirred at 0 deg.C (ice/water) for 90 minutes. Methanol (2ml) was added and the solution was concentrated to a brown gum.

The crude product was purified by preparative HPLC to give 5- (5-tert-butyl-2, 4-dihydroxy-phenyl) -4- (4-morpholin-4-ylmethyl-phenyl) -isoxazole-3-carboxylic acid ethylamide (formate) as a white powder (21mg, 75%).

LC Retention time 1.97 min [ M + H]⁺480.5 (run time 3.75 minutes)

N.M.R(DMSO-d₆)8.8(t J 5.6Hz NH)7.25(d J 7.2Hz 2ArH)7.15(d J 7.2Hz2ArH)6.7(s ArH)6.45(s ArH)3.45(br s 4CH₂)3.2(dq J 5.6Hz，7.2Hz 2CH₂)2.3(br s 4CH₂)1.1(s 9CH₃)1.05(t J 7.2Hz 3CH₃)

This compound has an activity 'a' in Hsp90 fluorescence polarization assay.

Examples 77a-f were prepared in a similar manner to the compound of example 77.

Example 78

Preparation of 5- (2, 4-dihydroxy-5-isopropyl-phenyl) -4- (4-morpholin-4-ylmethyl-phenyl) -isoxazole-3-carboxylic acid ethylamide

Reaction scheme

Step 1

1- (2, 4-bis-benzyloxy-phenyl) -ethanones

Potassium carbonate (2.5 equiv.) was added to a solution of 2 ', 4' -dihydroxyacetophenone (1 equiv.) in acetonitrile (400mL) and the suspension was stirred at room temperature. Benzyl bromide (2.5 equivalents) was added dropwise over 10 minutes and the mixture was heated to refluxFor 18 hours. The mixture was cooled and evaporated under vacuum to give a slurry. The slurry was partitioned between water and ethyl acetate and the two layers were separated. The aqueous layer was further extracted with dichloromethane and the organic extracts were combined and dried (MgSO)₄) Evaporated under vacuum. The product was triturated with n-hexane, filtered and washed with cold n-hexane and dried under vacuum (45 ℃) to give 1- (2, 4-bis-benzyloxy-phenyl) -ethanone as a white powder.

LC Retention time 2.704 min [ M + H]⁺333.3

Step 2

2, 4-bis-benzyloxy-1-isopropenyl-benzene

Methyltriphenylphosphonium bromide (1.1 eq) was suspended in THF under nitrogen and cooled to 0 ℃. A1.6M n-butyllithium solution in n-hexane (1.1 equiv.) was added dropwise and stirred for 30 minutes. 1- (2, 4-bis-benzyloxy-phenyl) -ethanone (1 eq) was dissolved in anhydrous (an.) THF and added dropwise to the suspension. After the addition was complete, the ice bath was removed and the reaction mixture was stirred at room temperature under nitrogen overnight. Methanol was added to the reaction mixture and the resulting solution was evaporated under vacuum. N-hexane was added to the resulting oil and heated at reflux for 30 minutes, followed by filtration through celite. The liquid was evaporated in vacuo to give an oil which was purified by column chromatography eluting with 30% ethyl acetate in n-hexane. To give 2, 4-bis-benzyloxy-1-isopropenyl-benzene. Rf retention time 0.722, 3: 1 n-hexane: ethyl acetate.

Step 3

4-isopropyl-benzene-1, 3-diol

2, 4-bis-benzyloxy-1-isopropenyl-benzene was dissolved in ethanol to prepare a solution, which was added to 10% palladium on carbon pre-wetted with water. Hydrogen was introduced into the flask and the mixture was shaken for 16 hours. The catalyst was filtered from the reaction mixture by an appropriate method and the liquid was concentrated in vacuo to give 4-isopropyl-benzene-1, 3-diol as a white crystalline solid.

LC Retention time 2.088 min [ M + H]⁺153.1

Step 4

1- (2, 4-dihydroxy-5-isopropyl-phenyl) -ethanones

4-isopropyl-benzene-1, 3-diol (1 eq) was dissolved in BF₃.OEt₂(6 equiv.) and acetic acid (2 equiv.) is added. The solution was heated at 90 ℃ for 16 hours and then allowed to cool to room temperature. The solution was added dropwise to 10% NaOAc (aq) and allowed to stand for 4 hours before extraction into EtOAc. The organic phases were combined and washed with saturated NaHCO₃(aqueous solution) washing with MgSO 2₄Dried, filtered and concentrated under vacuum. The residual oil was purified by column chromatography eluting with dichloromethane to give 1- (2, 4-dihydroxy-5-isopropyl-phenyl) -ethanone as a white solid.

LC Retention time 2.633 min [ M + H]⁺195.1

Step 5

1- (2, 4-bis-benzyloxy-5-isopropyl-phenyl) -ethanone

1- (2, 4-dihydroxy-5-isopropyl-phenyl) -ethanone (1 eq) was dissolved in DMF and potassium carbonate (2.2 eq) was added followed by benzyl bromide (2.2 eq). The suspension was heated to 150 ℃ for 16 hours under nitrogen and stirring. The solution was cooled to room temperature and the mixture was poured into 1M HCl (aq) and then extracted into ethyl acetate. The organic phases were combined and washed again with 1MHCl (aq) and then five times with saturated brine. The organic phase was dried over MgSO4, filtered and concentrated in vacuo to give a solid which was triturated with ether: n-hexane (1: 1) and purified to give 1- (2, 4-bis-benzyloxy-5-isopropyl-phenyl) -ethanone.

LC Retention time 3.575 min [ M + H]⁺375.2

Step 6

4- (2, 4-bis-benzyloxy-5-isopropyl-phenyl) -2-hydroxy-4-oxo-but-2-enoic acid ethyl ester

Sodium (2.8 equiv.) was added to ethanol under nitrogen at room temperature and stirred for 25 minutes to yield sodium ethoxide. 1- (2, 4-bis-benzyloxy-5-isopropyl-phenyl) -ethanone (1 eq) was dissolved in another portion of ethanol and added to a solution of sodium ethoxide. Diethyl oxalate (1.64 eq) was added and the reaction mixture was heated to reflux for 4 hours. The mixture was allowed to cool to room temperature and sufficient 1M aqueous HCl was added to acidify the reaction mixture, which was then concentrated in vacuo. The resulting gum was partitioned between dichloromethane and brine, and the organic phase was MgSO₄Drying, filtration and evaporation in vacuo gave 4- (2, 4-bis-benzyloxy-5-isopropyl-phenyl) -2-hydroxy-4-oxo-but-2-enoic acid ethyl ester as a yellow gum.

LC Retention time 3.057 min [ M + H]⁺475

Step 7

5- (2, 4-bis-benzyloxy-5-isopropyl-phenyl) -isoxazole-3-carboxylic acid ethyl ester

4- (2, 4-bis-benzyloxy-5-isopropyl-phenyl) -2-hydroxy-4-oxo-but-2-enoic acid ethyl ester (1 eq) was dissolved in ethanol with stirring. Hydroxylamine hydrochloride (1.2 equivalents) was added and the solution heated to reflux under nitrogen for 4 hours. The reaction mixture was cooled to room temperature and concentrated under vacuum. The residue was partitioned between saturated brine and dichloromethane. The organic phase is MgSO₄Drying, filtration and concentration in vacuo afforded 5- (2, 4-bis-benzyloxy-5-isopropyl-phenyl) -isoxazole-3-carboxylic acid ethyl ester as a solid.

LC Retention time 3.059 min [ M + H]⁺472

Step 8

5- (2, 4-bis-benzyloxy-5-isopropyl-phenyl) -isoxazole-3-carboxylic acid ethylamide

Ethyl 5- (2, 4-bis-benzyloxy-5-isopropyl-phenyl) -isoxazole-3-carboxylate was dissolved in an excess of 2M ethylamine in methanol and heated at 120 ℃ for 600 seconds in a Smith synthesizer (Smith synthesizer) microwave. The solution was concentrated in vacuo to give a solid, which was triturated with n-hexane and purified to give 5- (2, 4-bis-benzyloxy-5-isopropyl-phenyl) -isoxazole-3-carboxylic acid ethylamide.

LC Retention time 2.979 min [ M + H]⁺471.3

Step 9

5- (2, 4-bis-benzyloxy-5-isopropyl-phenyl) -4-iodo-isoxazole-3-carboxylic acid ethylamide

5- (2, 4-bis-benzyloxy-5-isopropyl-phenyl) -isoxazole-3-carboxylic acid ethylamide (1 eq) was dissolved in anhydrous acetonitrile, N-iodosuccinimide (2.0 eq) was added, followed by cerium ammonium nitrate (0.05 eq) and the solution was stirred at room temperature overnight. The reaction mixture was concentrated in vacuo and the resulting gum partitioned between ethyl acetate and saturated brine. The organic phase is MgSO₄Dried, filtered and concentrated under vacuum. The residue was purified by column chromatography eluting with 9: 1 n-hexane: ethyl acetate to give 5- (2, 4-bis-benzyloxy-5-isopropyl-phenyl) -4-iodo-isoxazole-3-carboxylic acid ethylamide as an oil.

LC Retention time 2.975 min [ M + H]⁺597.2

Step 10

5- (2, 4-bis-benzyloxy-5-isopropyl-phenyl) -4-iodo-isoxazole-3-carboxylic acid ethylamide (1 eq) was dissolved in anhydrous DMF and 1MNa was added₂CO₃(aq) followed by the addition of 4-formylphenylboronic acid (2 eq) and then a catalytic amount of PdCl2(PPh3) 2. The solution was sparged with nitrogen at room temperature for 10 minutes, after which the temperature was raised to 80 ℃ for 15 minutes under nitrogen atmosphere. The reaction mixture was cooled to room temperature, and diluted with ethyl acetate. The solution was washed with saturated brine and then MgSO₄Dried, filtered, and concentrated under vacuum to give an oilA compound (I) is provided. Purification by column chromatography eluting with 10% EtOAc in hexanes afforded 5- (2, 4-bis-benzyloxy-5-isopropyl-phenyl) -4-iodo-isoxazole-3-carboxylic acid ethylamide as a white solid.

LC Retention time 2.981 min [ M + H]⁺575.3

Step 11

5- (2, 4-bis-benzyloxy-5-isopropyl-phenyl) -4- (4-morpholin-4-ylmethyl-phenyl) -isoxazole-3-carboxylic acid ethylamide

5- (2, 4-bis-benzyloxy-5-isopropyl-phenyl) -4-iodo-isoxazole-3-carboxylic acid ethylamide (1 eq) was dissolved in methanol and added to powdered 3A molecular sieves. Morpholine (2 equivalents) was added followed by sodium cyanoborohydride (2 equivalents). Acetic acid (5 equivalents) was added dropwise and the suspension was stirred under nitrogen at room temperature for 16 hours. The reaction mixture was diluted with dichloromethane and saturated NaHCO₃(aqueous solution) washing. The organic phase is MgSO₄Dried, filtered and concentrated under vacuum. The resulting gum was purified by flash chromatography eluting with 1% MeOH in DCM to give 5- (2, 4-bis-oxy-5-isopropyl-phenyl) -4- (4-morpholin-4-ylmethyl-phenyl) -isoxazole-3-carboxylic acid ethylamide as a colorless oil.

LC Retention time 4.42 min [ M + H]⁺646.2 method B

Step 12

Under nitrogen atmosphere, 5- (2, 4-bis-oxy-5-isopropyl-phenyl) -4- (4-morpholin-4-ylmethyl-phenyl) -isoxazole-3-carboxylic acid ethylamide (1 eq) was dissolved in anhydrous DCM and cooled to 0 ℃. Dropwise addition of 1M BCl₃The solution was stirred under these conditions for 30 minutes. Methanol (2ml) was added and the reaction mixture was concentrated in vacuo. The sample was purified by preparative LC/MS to give 5- (2, 4-dihydroxy-5-isopropyl-phenyl) -4- (4-morpholin-4-ylmethyl-phenyl) -isoxazole-3-carboxylic acid ethylamide as a white solid.

LC Retention time 1.991 min [ M + H]⁺466.3

This compound has an activity 'a' in Hsp90 fluorescence polarization assay.

In a similar manner to the preparation of the compound of example 78, examples 78a-u were prepared.

Example 78v

Phosphoric acid 4-chloro-5- (diethoxy-phosphoryloxy) -2- [ 3-ethylcarbamoyl-4- (4-methoxy-phenyl) -isoxazol-5-yl ] -phenyl ester diethyl ester

To a vial of 5- (5-chloro-2, 4-dihydroxy-phenyl) -4- (4-methoxy-phenyl) -isoxazole-3-carboxylic acid ethylamide (11mg, 2.1 × 10)^-2mmol) and MgO (25mg), 10 drops of diethyl chlorophosphate were added. The resulting mixture was heated at 70 ℃ with stirring for 1 hour and the progress of the reaction was monitored by TLC. After cooling, MeOH (1ml) and DCM (1ml) were added. After filtration, the solvent was evaporated to give a yellow oil. The diphosphate was isolated by preparative TLC in 4mg yield. R_f＝0.35；¹H NMR δ 7.95(1H, s, wide); 7.74(1H, s); 7.55(1H, s); 7.32(2H, d, J ═ 9.0 Hz); 6.90(2H, d, J ═ 9.0 Hz); 4.30(8H, q); 3.80(3H, s)) (ii) a 3.40(2H, q); 1.35(12H, t) and 1.25(3H, t). LCMS: (M +1)⁺661.1(RT ═ 7.60 minutes)

Example 79

Reaction scheme

Step 1

1- (2, 4-dihydroxy-phenyl) -2-methyl-propan-1-one

Resorcinol (1 equivalent) was dissolved in BF₃.OEt₂(6 equiv.) and isobutyric acid (1 equiv.) was added. The solution was heated at 90 ℃ for 1.5 hours and then cooled to room temperature. This solution was added dropwise to 10% NaOAc (aq) and allowed to stand for 4 hours before extraction into EtOAc. The organic phases were combined and washed with saturated NaHCO₃Washed (aq), then dried over magnesium sulfate, filtered and concentrated in vacuo to give 1- (2, 4-dihydroxy-phenyl) -2-methyl-propan-1-one as a red oil, which was used without further purification.

LC Retention time 2.279 min [ M + H]⁺181.1

Step 2

4-isobutyl-benzene-1, 3-diol

Ethyl chloroformate (3 equivalents) was slowly added to a cooled (0 ℃ C.) solution of 1- (2, 4-dihydroxy-phenyl) -2-methyl-propan-1-one (1 equivalent) and triethylamine (3 equivalents) in THF. The mixture was warmed to room temperature, stirred for 3 hours before filtration and the solid washed with cold THF. The combined filtrates were cooled to 0 ℃ and a solution of sodium borohydride (4 equivalents) in water (same volume of water as the THF filtrate) was slowly added. The mixture was warmed to room temperature, stirred for 3 hours and diluted with water. The mixture was extracted twice with diethyl ether, the combined extracts were concentrated to dryness and resuspended in 10% aqueous sodium hydroxide (4 eq). After refluxing for 90 minutes, the mixture was cooled, acidified with 5m aqueous cl solution and extracted twice with ether. The organic extracts were dried over magnesium sulfate, filtered and concentrated to give 4-isobutyl-benzene-1, 3-diol as a cloudy oil, which was used without purification.

NMR was consistent with the structure.

Example 3:

1- (2, 4-dihydroxy-5-isobutyl-phenyl) -ethanones

4-isobutyl-benzene-1, 3-diol (1 eq) was dissolved in BF₃.OEt₂(6 equiv.) and acetic acid (2 equiv.) is added. The solution was heated at 90 ℃ for 16 hours and then allowed to cool to room temperature. The solution was added dropwise to 10% NaOAc (aq) and allowed to stand for 4 hours before being extracted twice with ether. The organic phases were combined and washed with saturated NaHCO₃(aqueous solution) washing with MgSO 2₄Drying, filtering and concentrating under vacuum to obtain 1-, (2, 4-dihydroxy-5-isobutyl-phenyl) -ethanone, which can be used without purification.

NMR was consistent with the structure.

Step 4

1- (2, 4-bis-benzyloxy-5-isobutyl-phenyl) -ethanone

1- (2, 4-dihydroxy-5-isobutyl-phenyl) -ethanone (1 eq) was dissolved in DMF and potassium carbonate (4.4 eq) was added followed by benzyl bromide (4.4 eq). The suspension was heated to 150 ℃ for 16 hours under nitrogen and stirring. The solution was cooled to room temperature, filtered and concentrated to dryness, and the solid was purified by column chromatography (silica gel, n-hexane: ethyl acetate 4: 1) and then recrystallized from ethyl acetate: n-hexane to give 1- (2, 4-bis-benzyloxy-5-isobutyl-phenyl) -ethanone as colorless crystals.

LC Retention time 3.030 min [ M + H]⁺389.3

Step 5

4- (2, 4-bis-benzyloxy-5-isobutyl-phenyl) -2, 4-dioxo-butyric acid ethyl ester

Sodium (3 equivalents) was added to ethanol under nitrogen at room temperature and stirred until completely dissolved. 1- (2, 4-bis-benzyloxy-5-isobutyl-phenyl) -ethanone (1 eq) was added followed by diethyl oxalate (1.5 eq) and the reaction mixture was heated under reflux for 4 h. The mixture was cooled to room temperature and acidified with 2M HCl (aq) to give 4- (2, 4-bis-benzyloxy-5-isobutyl-phenyl) -2, 4-dioxo-butyric acid ethyl ester as a yellow precipitate, which was filtered off.

LC Retention time 3.254 min [ M + H]⁺489.3

Step 6

5- (2, 4-bis-benzyloxy-5-isobutyl-phenyl) -isoxazole-3-carboxylic acid ethyl ester

4- (2, 4-bis-benzyloxy-5-isobutyl-phenyl) -2, 4-dioxo-butyric acid ethyl ester (1 eq) was dissolved in ethanol with stirring. Hydroxylamine hydrochloride (1.2 eq) was added and the solution heated under reflux for 2 hours. The reaction mixture was cooled to room temperature to obtain a precipitate. Filtration gave the precipitate as a white solid, 5- (2, 4-bis-benzyloxy-5-isobutyl-phenyl) -isoxazole-3-carboxylic acid ethyl ester.

LC Retention time 3.261 min [ M + H]⁺486.3

Step 7

5- (2, 4-bis-benzyloxy-5-isobutyl-phenyl) -isoxazole-3-carboxylic acid ethylamide

Ethyl 5- (2, 4-bis-benzyloxy-5-isobutyl-phenyl) -isoxazole-3-carboxylate was dissolved in 2M solution of ethylamine in methanol (10 eq) and heated at 120 ℃ for 600 seconds under microwaves in a Smith synthesizer (Smith synthesizer). The solution was concentrated in vacuo to afford 5- (2, 4-bis-benzyloxy-5-isobutyl-phenyl) -isoxazole-3-carboxylic acid ethylamide as a white solid, which was used without additional purification.

LC Retention time 3.112 min [ M + H]⁺485.3

Step 8

5- (2, 4-bis-benzyloxy-5-isobutyl-phenyl) -4-iodo-isoxazole-3-carboxylic acid ethylamide

5- (2, 4-bis-benzyloxy-5-isobutyl-phenyl) -isoxazole-3-carboxylic acid ethylamide (1 eq) and N-iodosuccinimide (2.0 eq) were dissolved in acetonitrile, cerium ammonium nitrate (0.1 eq) was added and the solution was stirred at room temperature overnight. The reaction mixture was concentrated in vacuo and the resulting gum partitioned between ethyl acetate and saturated brine. The organic phase is MgSO₄Dried, filtered and concentrated under vacuum. The residue was purified by column chromatography eluting with 4: 1 n-hexane: ethyl acetate to give 5- (2, 4-bis-benzyloxy-5-isobutyl-phenyl) -4-iodo-isoxazole-3-carboxylic acid ethylamide as an oil.

LC Retention time 3.089 min [ M + H]⁺611.2

Step 9

5- (2, 4-bis-benzyloxy-5-isobutyl-phenyl) -4- (4-formyl-phenyl) -isoxazole-3-carboxylic acid ethylamide

5- (2, 4-bis-benzyloxy-5-isobutyl-phenyl) -4-iodo-isoxazole-3-carboxylic acid ethylamide (1 eq) was dissolved in DMF and 1MNa was added₂CO₃(aq) (3 equiv.) followed by addition of 4-formylphenylboronic acid (2 equiv.), and catalytic amount of PdCl₂(PPh₃)₂. The solution was bubbled with nitrogen at room temperature for 10 minutes, after which the temperature was raised to 80 ℃ for 2 hours under nitrogen atmosphere. The reaction mixture was cooled to room temperature, and diluted with ethyl acetate. For solutionsWashed with saturated brine and then MgSO₄Dried, filtered, and concentrated under vacuum to give an oil. Column chromatography purification eluting with 10% EtOAc in hexanes afforded 5- (2, 4-bis-benzyloxy-5-isobutyl-phenyl) -4- (4-formyl-phenyl) -isoxazole-3-carboxylic acid ethylamide as a white solid.

LC Retention time 5.57 min [ M + H]⁺589.1 method B

Step 10

5- (2, 4-bis-benzyloxy-5-isobutyl-phenyl) -4- (4-morpholin-4-ylmethyl-phenyl) -isoxazole-3-carboxylic acid ethylamide

5- (2, 4-bis-benzyloxy-5-isobutyl-phenyl) -4- (4-formyl-phenyl) -isoxazole-3-carboxylic acid ethylamide (1 eq) was dissolved in methanol and powdered 3A molecular sieve was added. Morpholine (2 eq) was added followed by acetic acid (5 eq) and after stirring for 30 minutes, sodium cyanoborohydride (2 eq) was added in portions. The suspension was stirred under nitrogen at room temperature for 16 hours. The reaction mixture was filtered through celite and concentrated to dryness. Column chromatography purification eluting with 5% MeOH in DCM gave 5- (2, 4-bis-benzyloxy-5-isobutyl-phenyl) -4- (4-morpholin-4-ylmethyl-phenyl) -isoxazole-3-carboxylic acid ethylamide as a colorless oil.

LC Retention time 4.53 min [ M + H]⁺660.2 method B

Step 11

Under a nitrogen atmosphere, 5- (2, 4-bis-benzyloxy-5-isobutyl-phenyl) -4- (4-morpholin-4-ylmethyl-phenyl) -isoxazole-3-carboxylic acid ethylamide (1 eq) was dissolved in anhydrous DCM and cooled to 0 ℃. A solution of 1MBCl3 in dichloromethane (9 equivalents) was added dropwise and the solution stirred for 30 minutes. Methanol (2ml) was added and the reaction mixture was concentrated in vacuo. The sample was purified by preparative LC/MS to give 5- (2, 4-dihydroxy-5-isobutyl-phenyl) -4- (4-morpholin-4-ylmethyl-phenyl) -isoxazole-3-carboxylic acid ethylamide as a white solid.

LC Retention time 1.902 min [ M + H]⁺480.3

This compound has an activity 'a' in Hsp90 fluorescence polarization assay.

Example 80 was prepared in a similar manner to the preparation of the compound of example 79. The sample was purified by preparative LC/MS to give the compound as a white solid.

Example 81

N- [5- (5-chloro-2, 4-dihydroxy-phenyl) -4- (4-fluoro-phenyl) -isoxazol-3-ylmethyl ] -methanesulfonamide

Example 82

N- [5- (5-chloro-2, 4-dihydroxy-phenyl) -4- (4-fluoro-phenyl) -isoxazol-3-ylmethyl ] -acetamide

5- (2, 4-bis-benzyloxy-5-chloro-phenyl) -4- (4-fluoro-phenyl) -isoxazole-3-carboxamide

5- (2, 4-bis-benzyloxy-5-chloro-phenyl) -4-iodo-isoxazole-3-carboxamide (0.45g, 0.80mmol) and 4-fluorophenylboronic acid (0.17g, 1.5 equivalents) were cross-coupled using the standard conditions described above. The crude product, an orange solid (0.40g), was used in the next step without purification.

LCMS(LCQ)t_R＝8.70，MS m/z 529.1[M+H]⁺

C- [5- (2, 4-bis-benzyloxy-5-chloro-phenyl) -4- (4-fluoro-phenyl) -isoxazol-3-yl ] -methylamine

To a solution of 5- (2, 4-bis-benzyloxy-5-chloro-phenyl) -4- (4-fluoro-phenyl) -isoxazole-3-carboxamide (0.40g, 0.76mmol) in anhydrous THF (20ml) under argon was added 1M borane-THF complex (1ml) and the solution was refluxed overnight. After cooling the reaction was quenched with methanol (10ml) and the product usedSPE FlashSCX-25 g purified to give 0.30g powder (77% yield).

LCMS(LCQ)t_R＝7.54，MS m/z 515.2[M+H]⁺

C- [5- (2, 4-bis-benzyloxy-5-chloro-phenyl) -4- (4-fluoro-phenyl) -isoxazol-3-yl ] -methylamine (100mg, 0.19mmol) was dissolved in DCM (3ml) before addition of methanesulfonyl chloride (17 μ l, 1.1 equivalents) and triethylamine (30 μ l, 1.1 equivalents). The solution was stirred at room temperature overnight. Then evaporated to dryness under vacuum to leave the crude benzyl-protected product as a blue residue (90 mg). Deprotection with boron trichloride using standard procedure described above and purification by preparative TLC (10% ethanol in DCM) and soxhlet extraction (soxhlet extraction) of silica gel with ether gave the pure compound as a nearly colourless solid (8mg, 10% yield).

LCMS(LCQ)tR＝6.65，MS m/z 411.2[M-H]-

δH(d4-MeOH)，7.19(2H，m，Ar-H)，7.04(1H，s，Ar-H)，7.03(2H，m，Ar-H)，6.34(1H，s，Ar-H)，4.27(2H，s，CH2NH)，2.81(3H，s，SO2CH3).

N- [5- (5-chloro-2, 4-dihydroxy-benzyl) -4- (4-fluoro-phenyl) -isoxazol-3-ylmethyl ] -acetamide

To a solution of C- [5- (2, 4-bis-benzyloxy-5-chloro-phenyl) -4- (4-fluoro-phenyl) -isoxazol-3-yl ] -methylamine (100mg, 0.19mmol) in DCM was added acetic anhydride (130 μ l, 7.0 equivalents) and triethylamine (81 μ l, 3.0 equivalents). The solution was stirred at room temperature until the amine was consumed. Removal of the solvent under vacuum left the crude benzyl-protected product as a pale yellow oil. Deprotection with boron trichloride using standard procedure described above and purification by preparative TLC and soxhlet extraction (soxhlet extraction) of silica gel with diethyl ether gave the pure compound as a colourless solid (10mg, 14% yield).

LCMS(LCQ)t_R＝6.57，MS m/z 377.1[M+H]⁺

δ_H(d⁴-MeOH)，7.17(2H，m，Ar-H)，7.01(1H，s，Ar-H)，6.98(2H，m，Ar-H)，6.32(1H，s，Ar-H)，4.37(2H，s，CH₂NH)，1.77(3H，s，COCH₃).

Examples 83, 84 and 85

5- (5-ethyl-4-hydroxy-2-methoxy-phenyl) -4- (4-morpholin-4-ylmethyl-phenyl) -isoxazole-3-carboxylic acid acetyl (83); 5- (5-ethyl-2-hydroxy-4-methoxy-phenyl) -4- (4-morpholin-4-ylmethyl-phenyl) -isoxazole-3-carboxylic acid ethylamide (84); 5- (5-Ethyl-2, 4-dimethoxy-phenyl) -4- (4-morpholin-4-ylmethyl-phenyl) -isoxazole-3-carboxylic acid ethylamide (85)

To an argon-filled flask containing 5- (5-ethyl-2, 4-dihydroxy-phenyl) -4- (4-morpholin-4-ylmethyl-phenyl) -isoxazole-3-carboxylic acid ethylamide (25mg, 0.055mmol) and n.n- (diisopropyl) aminomethylpolystyrene [ PS-DIEA ] (35mg, 3.83mmol/g, 2.4 equivalents) was added anhydrous DCM (2.3ml) and anhydrous methanol (0.25 ml). While stirring gently, a 2M (trimethylsilyl) diazomethane solution in n-hexane (28. mu.l, 1.0 equiv) was added and the solution was stirred at room temperature overnight. The solution was bubbled with argon for 10 minutes, the resin was filtered off and the volatiles were removed under vacuum. The crude residue was purified by semi-preparative HPLC to give 5- (5-ethyl-4-hydroxy-2-methoxy-phenyl) -4- (4-morpholin-4-ylmethyl-phenyl) -isoxazole-3-carboxylic acid ethylamide (83) (5.52mg, 21%), 5- (5-ethyl-2-hydroxy-4-methoxy-phenyl) -4- (4-morpholin-4-ylmethyl-phenyl) -isoxazole-3-carboxylic acid ethylamide (84) (1.14mg, 4%), 5- (5-ethyl-2, 4-dimethoxy-phenyl) -4- (4-morpholin-4-ylmethyl-phenyl) -isoxazole-3-carboxylic acid ethylamide (5 85) (1.46mg, 5%), and unmethylated starting material.

83：LCMS(LCT)t_R＝4.95，MS m/z 466.4[M+H]⁺

84：LCMS(LCT)t_R＝5.14，MS m/z 466.4[M+H]⁺

(85)：LCMS(LCT)t_R＝5.45，MS m/z 480.4[M+H]⁺

NMR data were assigned.

Example 86

Ethyl 5- (5-chloro-2-hydroxy-phenyl) -4- (4-morpholin-4-ylmethyl-phenyl) -isoxazole-3-carboxamide

Step 1

2-Benzoyloxy-5-chloro-benzoic acid methyl ester

5-chloro-2-hydroxy-benzoic acid methyl ester (2.5g, 13.4mmol), K₂CO₃A mixture of (3.7g, 26.8mmol) and bromobenzyl (2.98g, 17.4mmol) in acetone (30ml) was refluxed for 12 hours. After cooling, the acetone was evaporated. EtOAc (100ml) was added and filtered. The organic layer was then washed with 1M HCl (1X 80ml) and saturated brine (2X 80ml) and Na₂SO₄And (5) drying. Filtration and evaporation of the solvent gave a yellow semisolid (3.2 g).¹H NMR(d₆-acetone) δ 7.73(1H, d); 7.60-7.30(1H +5H, m); 7.28(1H, d); 5.30(2H, s) and 3.90(3H, s).

Step 2

1- (2-benzyloxy-5-chloro-phenyl) -2- (triphenyl-lambda)⁵Phospholene (phospholidene)) -ethanone

To a stirred suspension of triphenylphosphonium bromide (2.14g, 6.0mmol) in dry THF (30ml) at room temperature was added a 1.6M solution of n-butyllithium in n-hexane (5.25ml, 8.39 mmol). Suspending orange in waterThe suspension was stirred for 3 hours. Then a solution of methyl 2-benzoyloxy-5-chloro-benzoate (0.83g, 3.0mmol) in THF (8ml) was added slowly. The resulting mixture was stirred at 60 ℃ for 2 hours, cooled and filtered. DCM (100ml) was added to the filtrate, and the combined organic layers were washed with saturated brine (2X 80 ml). After filtration and evaporation of the solvent, a yellow oil (2.0g) was obtained. Then purified by chromatography eluting with EtOAc: n-hexane/1: 1 to give 0.97g of a solid. R_f＝0.43.¹H NMR(d₆-acetone) δ 7.80-7.52(20H, m); 7.40-7.20(1H +1H +1H, m); 5.25(2H, s); 4.72(1H, s, trans-H) and 4.62(1H, s, cis-H). (M +1)⁺521.2(RT 5.94 min.)

Step 3

4- (2-benzyloxy-5-chloro-phenyl) -2, 4-dioxo-3- (triphenyl-lambda)⁵-phospholene (phospholidene)) -ethyl butyrate

To 1- (2-benzyloxy-5-chloro-phenyl) -2- (triphenyl-lambda) at room temperature⁵Phospholene (phospholidene)) -ethanone (0.49g, 0.94mmol) and NEt₃A solution of (96mg, 0.94mmol) and DMAP (12mg, 0.09mmol) in dry toluene (20ml) was added a solution of ethyl chlorooxoacetate (0.38g, 2.78mmol) in toluene (5 ml). The mixture was stirred for 2 hours and poured into water (50 ml). The organic phase was separated and the aqueous layer was extracted with EtOAc (2X 40 mL). The combined organic layers were then washed with saturated NaHCO₃The solution (2X 40ml), saturated citric acid (1X 40ml) and saturated brine (1X 40ml) were washed and dried. The crude oil (0.36g) was purified by chromatography eluting with EtOAc. R_f＝0.88.¹HNMR(d₆-acetone) δ 7.75-7.40(15H, m); 7.30(1H, dd); 7.15(1H, d); 7.05(1H, d); 5.10(2H, s); 3.60(2H, q) and 1.10(3H, s). LCMS: (M +1)⁺621.2(RT 6.49 min.)

Step 4

3- (2-benzoyloxy-5-chloro-benzoyl) -3-bromo-3H-aziridine-2-carboxylic acid ethyl ester

To 4- (2-benzyloxy-5-chloro-phenyl) -2, 4-dioxo-3- (triphenyl-. lamda.) -at room temperature⁵A solution of-phosphidenyl (phosphanylidene)) -ethyl butyrate (0.143g, 0.23mmol) in DCM (8ml) was added TMSN₃(40mg, 0.35mmol) and NBS (62mg, 0.35mmol) in DCM (6 ml). The resulting solution was stirred for 2 hours. After evaporation of the solvent, the crude product was purified by preparative TLC. Yellow solid (38mg) was obtained. R_f0.73 (EtOAc: n-hexane 1: 2).¹H NMR(d₆-acetone) δ 7.80(1H, d); 7.60(1H, dd); 7.40(5H, m); 7.30(1H, d); 5.20(2H, s); 4.10(2H, q) and 1.00(3H, t). LCMS: (M +1)⁺438.0(RT 7.32 min.)

Step 5

5- (2-benzoyloxy-5-chloro-phenyl) -4-bromo-isoxazole-3-carboxylic acid ethyl ester

Ethyl 3- (2-benzoyloxy-5-chloro-benzoyl) -3-bromo-3H-azacyclopropene-2-carboxylate (55mg, 0.12mmol) in dry toluene was heated under reflux for 2 hours. After evaporation of the solvent, a crude solid (34mg) was obtained and purified by preparative TLC (EtOAc: n-hexane/1: 2). R_f0.73 (fluorescence).¹HNMR(d₆-acetone) δ 7.60(1H, d); 7.50(1H, dd); 7.40(1H, d); 7.30(5H, m); 5.25(2H, s); 4.42(2H, q) and 1.40(3H, t). LCMS: (M +1)⁺438.0(RT 7.09 minutes)

Step 6

Ethyl 5- (2-benzyloxy-5-chloro-phenyl) -4-bromo-isoxazole-3-carboxamide

To ethyl 5- (2-benzoyloxy-5-chloro-phenyl) -4-bromo-isoxazole-3-carboxylate (30mg, 6.8 × 10)^- ²mmol) in EtOH (1ml) was added ethylamine (70% in water, 1 ml). In solution inThe reaction mixture was heated in a microwave reactor (200W) at 100 ℃ for 1 hour. After this time, the solvent was evaporated and the compound was purified by preparative TLC to give a solid (20 mg). R_f0.39 (EtOAc: n-hexane/1: 4).¹H NMR(d₆-acetone) δ ═ 8.10(1H, s, broad peak); 7.50(1H, d); 7.45-7.35(1H +1H, m); 7.25(5H, m); 5.20(2H, s); 3.40(2H, q) and 1.20(3H, t). LCMS: (M +1)⁺437.1(RT 6.57 min.)

Step 7

Ethyl 5- (2-benzyloxy-5-chloro-phenyl) -4- (4-morpholin-4-ylmethyl-phenyl) -isoxazole-3-carboxamide

Ethyl 5- (2-benzyloxy-5-chloro-phenyl) -4-bromo-isoxazole-3-carboxamide (30mg, 5.6 × 10)^-2mmol)，Pd(Ph₃P)₄(4mg，3.5×10^-2mmol), 4[4- (4, 4, 5, 5-tetramethyl- [1, 3, 2]]Dioxaborolan-2-yl) -benzyl]Morpholine (63mg, 0.2mmol) and 1M NaHCO₃A mixture of the solution (0.2ml) in DME (1ml) was stirred under argon at 80 ℃ for 16 h. After cooling, the solution was diluted with water (8ml) and usedEtOAc (2X 20mL) extraction. The combined organic layers were washed with saturated brine (1X 20ml) and dried. After filtration and evaporation of the solvent, the crude product was purified by preparative TLC to yield 30mg of a solid. R_f＝0.44(EtOAc).¹H NMR(d₆-acetone) δ ═ 8.25(1H, s, broad peak); 7.60(1H, d); 7.55(1H, dd); 7.45(1H, d); 7.30-6.90(9H, m); 5.00(2H, s); 3.55(4H, m); 3.45(2H +2H, s + q); 2.30(4H, m) and 1.20(3H, t). LCMS: (M +1)⁺532.2(RT 4.39 min.)

Step 8

To ethyl 5- (2-benzyloxy-5-chloro-phenyl) -4- (4-morpholin-4-ylmethyl-phenyl) -isoxazole-3-carboxamide (25mg, 4.7 × 10) at 0 deg.c^-2mmol) of the solution in DCM (5ml) was added 1M BCl₃Solution of DCM (0.15 ml). The resulting cloudy yellow solution was then stirred at 0 ℃ for 15 minutes and at room temperature for 3-4 hours until the solution became clear. After this time, the solution was quenched with MeOH (1 ml). Then saturated NaHCO was added₃(1ml) and extracted with EtOAc (2X 2ml) and dried. After filtration and evaporation of the solvent, the crude oil was purified by preparative TLC (EtOAc: MeOH/50: 1) to give 12mg of a solid.¹H NMR(d₄-MeOD) δ 7.60(2H, d); 7.50-7.30(1H +1H +1H, m); 7.00(2H, d); 3.70(4H, m); 3.60(2H, s); 3.50(2H, q); 2.60(4H, m) and 1.25(3H, t). LCMS: (M +1)⁺442.2(RT 3.54 min.)

The 4-hydroxy isomer was prepared in analogy to the 2-hydroxy counterpart as follows:

example 87

Ethyl 5- (3-chloro-4-hydroxy-benzyl) -4- (4-morpholin-4-ylmethyl-phenyl) -isoxazole-3-carboxamide

Step 1

4-Benzoyloxy-3-chloro-benzoic acid methyl ester

Methyl 3-chloro-4-hydroxy-benzoate (1.0g, 5.36mmol) gave a crude solid (1.57 g).¹H NMR(d₆-acetone) δ ═ 8.00(1H, d); 7.95(1H, dd); 7.60-7.40(5H, m); 7.35(1H, d); 5.40(2H, s) and 3.90(3H, s).

Step 2

1- (4-benzyloxy-3-chloro-phenyl) -2- (triphenyl-lambda)⁵-phosphideoalkyl) -ethanones

Methyl 4-benzoyloxy-3-chloro-benzoate (1.5g, 5.40mmol) gave a crude solid (2.5 g). R_f0.31 (EtOAc: n-hexane/1: 1).¹H NMR(d₆-acetone) δ ═ 8.05(1H, d); 7.90(1H, dd); 7.85-7.35(20H, m); 7.20(1H, d); 5.30(2H, s); 4.60(1H, s, trans-H) and 4.50(1H, s, cis-H). LCMS: (M +1)⁺521.2(RT 5.29 min.)

Step 3

4- (4-benzyloxy-3-chloro-phenyl) -2, 4-dioxo-3- (triphenyl-lambda)⁵-phosphidinyl) -butyric acid ethyl ester

1- (4-benzyloxy-3-chloro-phenyl) -2- (triphenyl-lambda)⁵Phosphonylidene) -ethanone (1.84g, 3.53mmol) gave a crude solid (1.43g).¹H NMR(d₆-acetone) δ -8.00-7.35 (22H, m); 7.20(1H, d); 5.35(2H, s); 3.55(2H, q) and 1.14(3H, s). LCMS: (M +1)⁺621.2(RT 7.29 min.)

Step 4

3- (4-benzoyloxy-3-chloro-benzoyl) -3-bromo-3H-aziridine-2-carboxylic acid ethyl ester

The crude product was purified by column purification and preparative TLC, 4- (4-benzyloxy-3-chloro-phenyl) -2, 4-dioxo-3- (triphenyl-. lamda.) -⁵Phospholene-butyric acid ethyl ester (0.74g, 1.19mmol) produced a solid (0.168g) R_f0.24 (EtOAc: n-hexane/1: 6).¹H NMR(d₆-acetone) δ ═ 8.00(1H, d); 7.90(1H, dd); 7.50(1H, d); 7.40(5H, m); 5.40(2H, s); 4.05(2H, q) and 0.95(3H, t). LCMS: (M +1)⁺438.1(RT 7.27 min.)

Step 5

5- (4-benzoyloxy-3-chloro-phenyl) -4-bromo-isoxazole-3-carboxylic acid ethyl ester

The crude product was subjected to preparative TLC and recrystallisation (EtOH) followed by ethyl 3- (4-benzoyloxy-3-chloro-benzoyl) -3-bromo-3H-aziridine-2-carboxylate (68mg, 0.1)6mmol) gave a solid (20 mg). R_f0.26 (fluorescent) (EtOAc: n-hexane/1: 4).¹H NMR(d₆-acetone) δ ═ 8.00(1H, d); 7.90(1H, dd); 7.50(1H, d); 7.40(5H, m); 5.35(2H, s); 4.45(2H, q) and 1.40(3H, t). LCMS: (M +1)⁺438.0(RT 7.39 min.)

Step 6

Ethyl 5- (4-benzyloxy-3-chloro-phenyl) -4-bromo-isoxazole-3-carboxamide

5- (4-benzoyloxy-3-chloro-phenyl) -4-bromo-isoxazole-3-carboxylic acid ethyl ester (10mg, 2.3X 10)^-2mmol) gave a solid (8 mg). R_f0.53 (EtOAc: n-hexane/1: 2).¹H NMR(d₆-acetone) δ ═ 8.15(1H, s, broad peak); 8.00(1H, d); 7.90(1H, dd); 7.50(1H, d); 7.40(5H, m); 5.32(2H, s); 3.42(2H, q) and 1.20(3H, t).

Step 7

Ethyl 5- (4-benzyloxy-3-chloro-phenyl) -4- (4-morpholin-4-ylmethyl-phenyl) -isoxazole-3-carboxamide

Ethyl 5- (4-benzyloxy-3-chloro-phenyl) -4-bromo-isoxazole-3-carboxamide (10mg, 2.3 × 10)^-2mmol) gave a crude solid (10mg) which was used in the next step without further purification.

Step 8

Ethyl 5- (3-chloro-4-hydroxy-phenyl) -4- (4-morpholin-4-ylmethyl-phenyl) -isoxazole-3-carboxamide

After purification of the crude product twice by preparative TLC (EtOAc: MeOH/50: 1), ethyl 5- (4-benzyloxy-3-chloro-phenyl) -4- (4-morpholin-4-ylmethyl-phenyl) -isoxazole-3-carboxamide (8mg, 1.5X 10)^-2mmol) gave a crude solid (2 mg).¹H NMR(d₄-MeOD) δ 7.70(2H, d); 7.60(1H, d); 7.45(1H +1H, m); 7.00(2H, d); 3.80(4H, m); 3.75(2H, s); 3.50(2H, q); 2.82(4H, m) and 1.25(3H, t). LCMS: (M +1)⁺442.2(RT 4.47 min.)

Example 88

3- [4- (4-bromo-phenyl) -isoxazol-5-yl ] -5-chloro-2, 6-dihydroxy-benzaldehyde

Step 1

3- (4-bromo-phenyl) -6-chloro-7-hydroxy-4-oxo-4H-benzopyran-8-carbaldehyde

3- (4-bromo-phenyl) -6-chloro-7-hydroxy-chromen-4-one (0.35g, 1mmol) and hexamethylenetetramine (0.14g, 1mmol) were dissolved in glacial acetic acid (20ml) and heated at 100 ℃ overnight. Warm 6m hcl (10ml) was added and the mixture was heated for an additional 1 hour before the mixture was poured into water. The precipitate formed was filtered off, washed and dried to give the pure desired product as a light brown solid.

LCMS(LCQ)t_R＝8.27，MS m/z 377.3/379.2[M-H]^-

Step 2

3- [4- (4-bromo-phenyl) -isoxazol-5-yl ] -5-chloro-2, 6-dihydroxy-benzaldehyde

To a solution of 3- (4-bromo-phenyl) -6-chloro-7-hydroxy-4-oxo-4H-benzopyran-8-carbaldehyde (53.5mg, 0.14mmol) in EtOH (6ml) was added hydroxylamine hydrochloride (100mg, 1.4 mmol). The resulting mixture was heated to reflux for 16 hours. EtOH was evaporated and EtOAc (20mL) was added. The organic layer was washed with saturated NaHCO₃And (5) washing and drying. When the resulting oil was triturated with ether, a solid (33mg) was obtained.¹H NMR(d₆-DMSO) δ ═ 9.83(1H, s); 8.70(1H, s); 8.21(1H, s); 7.78(2H, d) and 7.68(2H, s). LCMS: (M +1)⁺394.1(RT 8.60 min.)

Example 89

5- (5-ethyl-2-hydroxy-4-methoxy-phenyl) -4- (4-fluoro-phenyl) -isoxazole-3-carboxylic acid hydroxyamide

Step 1

1- (5-ethyl-2, 4-dihydroxy-phenyl) -2- (4-fluoro-phenyl) -ethanone

Ethyl resorcinol (5.37g, 39mmol) and 4-fluorophenylacetic acid (6.00g, 39mmol) were dissolved in BF etherate₃(40 ml). The solution was heated at 80 ℃ for 4 hours. When cooled, is smallWater (100ml) was added cautiously and the solution was extracted with EtOAc (2X 80 ml). The organic layer was washed with saturated NaHCO₃Care was taken (2X 100ml) and washed with saturated brine (2X 100ml) and Na₂SO₄And (5) drying. After purification with decolorizing charcoal, a dark green slurry (10.5g) was obtained. R_f0.4 (EtOAc: n-hexane/1: 3). the compound was used in the next step without purification.¹H NMR(d₆-acetone) δ 7.80(1H, s); 7.35(2H, m); 7.00(1H, m); 6.35(1H, s); 4.35(2H, s); 2.55(2H, q) and 1.10(3H, t).

Step 2

4- (5-Ethyl-2, 4-dihydroxy-phenyl) -3- (4-fluoro-phenyl) -2, 4-dioxo-butyric acid ethyl ester

To a solution of 1- (5-ethyl-2, 4-dihydroxy-phenyl) -2- (4-fluoro-phenyl) -ethanone (10.3g, 37.6mmol) in dry pyridine (100ml) was added ethyl chlorooxoacetate (15.4g, 112.8mmol) at 0 ℃. The solution was stirred at 0 ℃ for 4 hours and at room temperature for 16 hours. The aqueous layer was neutralized with 1M HCl and extracted with DCM (2X 100 ml). The combined DCM layers were then washed with 2M HCl (2X 80ml), saturated NaHCO₃(1X 100ml), washed with saturated brine (1X 100ml) and washed with Na₂SO₄And (5) drying. After filtration and evaporation of the solvent, a dark brown oil (11.4g) was obtained. R_f0.22 (EtOAc: n-hexane/1: 2). LCMS shows that it is the desired compound [ (M-1) in a ratio of about 6: 1^-＝373.1，RT＝7.27]And cyclized benzopyran carboxylic acid ester [ (M-1)^-＝355.4，RT＝7.83]A mixture of (a). A small sample was purified by preparative TLC for spectroscopic analysis.¹H NMR(d₆-acetone) δ 7.75(1H, s); 7.30(2H, m); 7.00(1H, m); 6.45(1H, s); 4.65(1H, s); 4.25(2H, q); 2.55(2H, q) and 1.10(6H, t)

Step 3

6-Ethyl-3- (4-fluoro-phenyl) -7-hydroxy-4-oxo-4H-benzopyran-2-carboxylic acid ethyl ester

4- (5-Ethyl-2, 4-dihydroxy-phenyl) -3- (4-fluoro-phenyl) -2, 4-dioxo-butyric acid ethyl ester at 100 DEG C

(3.22g, 8.6mmol) was refluxed in a mixture of 0.8M HCl and MeOH (20ml/20ml) for 3 hours. After this time, MeOH was evaporated and the aqueous layer was extracted with EtOAc (2 × 60 ml). The combined organic layers were washed with saturated NaHCO₃(1X 80ml), saturated brine (2X 80ml) and water (1X 80ml) and washed with Na₂SO₄And (5) drying. After purification with decolorizing charcoal and evaporation of the solvent, a brown viscous solid was obtained. The solid was extracted with hot ether to give a dark yellow solid (0.26g)_f0.43 (EtOAc: n-hexane/1: 2), LCMS: (M +1)⁺＝357.3(RT＝7.83).¹H NMR(d₆-acetone) δ ═ 9.75(1H, s); 7.80(1H, s); 7.25(2H, m); 7.10(1H, m); 6.90(1H, s); 4.05(2H, q); 2.70(2H, q); 1.20(3H, t) and 0.95(3H, t).

Step 4

6-Ethyl-3- (4-fluoro-phenyl) -7-methoxy-4-oxo-4H-benzopyran-2-carboxylic acid ethyl ester

Methyl iodide (0.10ml, 12 equiv.) was added to a solution of ethyl 6-ethyl-3- (4-fluoro-phenyl) -7-hydroxy-4-oxo-4H-benzopyran-2-carboxylate (50mg, 0.14mmol) and potassium carbonate (58mg, 3.0 equiv.) in acetone and the mixture was refluxed overnight. The volatiles were then evaporated in vacuo and the residue partitioned between water (15ml) and EtOAc (15 ml). Saturated saline solution for organic layerWashing with MgSO 2₄Dried and evaporated to dryness under vacuum to give the product as white crystals (45mg, 87% yield)

δ_H(CDCl₃)，7.96(1H，s，Ar-H)，7.27(2H，m，Ar-H)，7.12(2H，m，Ar-H)，6.92(1H，s，Ar-H)，4.16(2H，q，CO₂CH₂CH₃)，3.95(3H，s，OCH3)，2.71(3H，q，CH₂CH₃)，1.24(3H，t，CO₂CH₂CH₃)，1.04(3H，t，CH₂CH₃)

Step 5

To a solution of ethyl 6-ethyl-3- (4-fluoro-phenyl) -7-methoxy-4-oxo-4H-benzopyran-2-carboxylate (25mg, 0.068mmol) in ethanol (2.5ml) was added hydroxylamine (50% aqueous solution, 1ml) and the solution was stirred for 48 hours. Volatiles were evaporated under reduced pressure and the residue was purified by preparative TLC (10% MeOH in DCM) to give the desired product as a light brown solid (3mg, 12% yield).

LCMS(LCT)t_R＝6.54，MS m/z 373.17[M+H]⁺

δ_H(d₆Acetone), 10.73(1H, broad s), 8.59(1H, broad s), 7.39(2H, m, Ar-H), 7.07(2H, m, Ar-H), 7.00(1H, s, Ar-H), 6.55(1H, s, Ar-H), 3.82(3H, s, OCH)₃)，2.48(2H，q，CH₂CH₃) 1.30(1H, broad s), 1.01(3H, t, CH)₂CH₃)。

Example 90

5- (5-ethyl-2, 4-dihydroxy-phenyl) -4- (4-fluoro-phenyl) -isoxazole-3-carboxylic acid hydroxyamide

To a solution of ethyl 6-ethyl-3- (4-fluoro-phenyl) -7-hydroxy-4-oxo-4H-benzopyran-2-carboxylate (25mg, 0.070mmol) in ethanol (2.5ml) was added hydroxylamine (50% aqueous solution, 1ml) and the solution was stirred for 48 hours. Volatiles were evaporated under reduced pressure and the residue was purified by preparative TLC (15% MeOH in DCM) to give the desired product as a brown solid (2mg, 82% yield).

LCMS(LCT)t_R＝5.63，MS m/z 359.13[M+H]⁺

δ_H(d₆-acetone), 10.72(1H, broad s, CONH), 8.69(1H, broad s, Ar-OH), 8.59(1H, broad s, Ar-OH), 7.39(2H, m, Ar-H), 7.06(2H, m, Ar-H), 6.99(1H, s, Ar-H), 6.52(1H, s, Ar-H), 2.49(2H, q, CH₂CH₃) 1.31(1H, broad s), 1.08(3H, t, CH)₂CH₃).

Biological results

The intrinsic atpase activity of HSP90 can be determined using yeast HSP90 as a model system. The assay was used to determine HSP90 inhibitory activity of some of the compounds of the examples herein, based on the use of malachite green for the determination of inorganic phosphate.

Malachite Green ATP enzyme assay

Material

The chemical reagents were of the highest purity available and all aqueous solutions were made up in AR water. Due to the need to reduce contamination by inorganic phosphates, precautions need to be taken with respect to the solutions and instruments used in the assay. The glassware and pH meter were rinsed with twice distilled or deionized water prior to use. However, if possible, plastic vessels should be used. Gloves are required for all steps.

(1) Greiner 384-well (Greiner 781101) or Costar 384-well flat bottom polystyrene multiwell plates (VWR).

(2) Assay buffer (a)100mM Tris-HCl, pH7.4, (b)150mM KCl, (c)6mM MgCl₂Storage at room temperature.

(3) 0.0812% (w/v) malachite green (M9636, Sigma Aldrich Ltd., Poole, UK). Storing at room temperature.

(4) 2.32% (w/v) polyvinyl alcohol USP (P1097, Sigma Aldrich Ltd, Poole, UK) in boiling water (see Note 1), allowed to cool and stored at room temperature.

(5) 5.72% (w/v) ammonium molybdate in 6M hydrochloric acid, stored at room temperature.

(6) 34% (w/v) sodium citrate. Storing at room temperature.

(7)100mM ATP, disodium salt, special quality (47699, Sigma Aldrich.) storage at-20 ℃.

(8) The yeast HSP90 protein expressed in E.coli is > 95% pure (see, e.g., Panaretou et al, 1998) and is stored in 50uL aliquots at-80 ℃.

Method

1. Test compounds were diluted to 500 μ M in AR water (DMSO concentration would be 2.5%). Transfer 2.5. mu.l of these compounds directly from daughter plate to assay plate gave a final assay concentration of 100. mu.M. To obtain 12 IC50 data points, 1: 2 serial dilutions were made to generate assay concentrations ranging from 100. mu.M to 97.6nM (2.5% DMSO), each concentration being transferred to 2.5. mu.l of assay plate. Assay plate column one contains no compound as a negative control. Another row of wells without compound was also used as background.

2. ATP was prepared by diluting 100mM stock solution to 925 μ M with assay buffer and adding 5 μ l of diluted ATP each to each well, including controls (final assay concentration of 370 μ M).

3. Add 5. mu.l buffer to the background row.

4. The enzyme preparation was diluted to 1.05. mu.M with assay buffer, 5. mu.l each to each compound well and negative control column.

5. The bottom of the well was collected, the plate covered with a sealed lid and incubated overnight at 37 ℃.

6. The first thing in the morning was to prepare malachite green reagent. Add 2 parts malachite green solution, 1 part polyvinyl alcohol solution, 1 part ammonium molybdate solution and 2 parts AR grade water.

7. The mixture was inverted and left to stand for about 1 hour until the color changed from brown to golden yellow.

8. Mu.l of malachite green reagent was added to each well and left for 5 minutes to develop color.

9. Add 5. mu.l of sodium citrate reagent to each well (see Note 2)

10. The lid was replaced with a new seal and the plate was shaken on a plate shaker for at least 15 minutes.

11. Absorbance at 620nM is measured using an appropriate plate reader (e.g., Victor, Perkinelmer Life Sciences, Milton Keynes, UK). Under these conditions, the control absorbance is 0.9 to 1.4, the background is 0.2-0.35, and the signal to noise ratio is-12 the Z' factor calculated using the data obtained under these conditions is between 0.6 and 0.9.

Note

(1) The dissolution of polyvinyl alcohol in boiling water is difficult and requires stirring for 2 to 3 hours.

(2) The time interval between addition of malachite green reagent and sodium citrate is as short as possible to reduce non-enzymatic hydrolysis of ATP. Once sodium citrate was added, the color was stable for up to 4 hours at room temperature.

(3) Compounds can be added to the assay plate using a Biomek FX Robot (Beckman Coulter). A multi-point 384 feeder (Thermo Labsystems, basigstoke, UK) can be conveniently used to add reagents to the plates.

(4) Assay conditions were optimized for time, protein and substrate concentrations to achieve minimum protein concentration while maintaining a poor signal to noise ratio.

(5) The signal-to-noise ratio (S/N) is calculated using the following formula:

(S-B)/√ S (SD of S)²+ (SD of B)²

(6) To determine the specific activity of HSP90, a series of concentrations of inorganic phosphate (0-10 μ M) were prepared and the absorbance at 620nm was determined as described. Specific activity was calculated from the calibration curve obtained.

The compounds tested in the above assay were assigned to one of two activity ranges, i.e. a ═ 50 μ M; b > 50 μ M, those assignments are published above.

Growth inhibition assays are also used to assess candidate HSP90 inhibitors

Cytotoxicity was estimated by sulforhodamine b (sulforhodamine b) (srb) assay: 50% inhibition System of concentration (IC) ₅₀ ) And (4) calculating.

Day one

1) The cell number was determined by a hemocytometer.

2) Using an 8-channel pipette, 160. mu.l of the cell suspension (3600 cells/well or 2X 10⁴Cells/ml) were added to each well of a 96-well microtiter plate.

3) At 37 ℃ in CO₂Incubate overnight in the incubator.

The next day

4) Stock solutions of the drugs were prepared and each drug was serially diluted with medium to give the final concentration in the wells.

5) Using a pipette, 40 μ l of drug (5x final concentration) was added to the quadruplicate wells.

6) Control wells can be on either side of a 96-well plate, with 40 μ l of added media.

7) Culturing in CO₂The incubator was kept for 4 days (48 hours).

Day six

8) The medium was decanted into a water tank and the plate was slowly immersed in 10% ice-cooled trichloroacetic acid (TCA). Left on ice for 30 minutes

9) The plate was washed three times with tap water by immersing the plate in a tap water bath and pouring off the tap water.

10) Drying in an incubator

11) To each well was added 100 μ l of 0.4% SRB in 1% acetic acid (except for the last row of 96-well plates (right), which is a 0% control, i.e. no drug, no color developer. The first row is 100% control group with no drug, but with color developer). And standing for 15 minutes.

12) The unbound SRB developer was washed off four times with 1% acetic acid.

13) The plates were dried in an incubator.

14) The SRB was dissolved using 100. mu.l of 10mM tris base and the plate was placed on a plate shaker for 5 minutes.

15) The absorbance at 540nm was measured using a plate reader. The average absorbance was calculated for each of the four wells. Expressed as a percentage of control, untreated wells.

16) Plotting the% absorbance values against log drug concentration, determining IC₅₀。

For example, for the SRB growth arrest assay, the compound of example 2 gave an IC50 in the 'a' range (< 50 uM).

Some compounds of the examples were also evaluated using fluorescence polarization assays:

fluorescence polarization assay

Fluorescence polarization { also known as fluorescence anisotropy } measures the rotation of a fluorescent substance in solution, where the larger the molecule, the greater the fluorescence emission polarization. When a fluorophore is excited with polarized light, the emitted light is also polarized. The size of the molecule is proportional to the polarization of the fluorescent emission.

Fluorescent labeled probe-RBT 0045864-FAM-

Binds to HSP90{ full-length human, full-length yeast or N-terminal domain HSP90} and measures anisotropy { probe: rotation of protein complexes }

The assay compound was added to the assay plate, allowed to equilibrate, and the anisotropy was measured again. Any change in anisotropy is due to competitive binding of the compound to HSP90, thus releasing the probe.

Material

The chemicals were of the highest purities available and all aqueous solutions were made with AR grade water.

1) Costar 96-well Black assay plate #3915

2) Assay buffer (a)100mM Tris pH 7.4; (b)20mM KCl; (c)6mM MgCl₂Storage at room temperature.

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

4) 20mM probe in 100% DMSO stock concentration. Storing at room temperature in dark. Working concentration was 200nM diluted with AR grade water and stored at 4 ℃. The final assay concentration was 80 nM.

5) Coli (E.coli) expressed full-length human HSP90 protein, > 95% pure (see, e.g., Panaretou et al 1998) and stored in 50. mu.L aliquots at-80 ℃.

Scheme(s)

1) Add 100. mu.l of 1 Xbuffer to wells 11A and 12A (═ FP BLNK)

2) Preparation of assay mixture-all reagents were placed on ice and the bucket was capped. Because the probe is sensitive to light.

i.Final Concⁿ

1 × Hsp90FP buffer 10ml 1 ×

·BSA 10mg/ml(NEB) 5.0μl 5μg/ml

Probe 200. mu.M 4.0. mu.l 80nM

Human full-length Hsp906.25. mu.l 200nM

3) Each 100. mu.l aliquot of the assay mixture was added to all other wells.

4) The plates were sealed and allowed to equilibrate in the dark for 20 minutes at room temperature.

Compound dilution plate-1X 3 dilution series

1) - { # VWR 007/008/257} in a clean 96-well v-bottom plate 10. mu.l of 100% DMSO was added to wells B1 to H11.

2) Add 17.5. mu.l of 100% DMSO to wells A1 to A11

3) Mu.l of compound was added to A1. This gave 2.5mM {50X } stock compound-assuming 20mM compound.

4) This operation was repeated for holes a2 through a 10. Controls are in columns 11 to 12.

5) Transfer 5 μ l from line A to line B, and not to column 12. And (4) fully mixing.

6) Transfer 5. mu.l from row B to row C. And (4) fully mixing.

7) This operation is repeated for row G.

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

9) This produced a1 × 3 dilution series from 50 μ M to 0.07 μ M.

10) Prepare 20. mu.l of 100. mu.M standard compound in well B12.

11) After the first incubation, the assay plate was fused^TMalpha-FP plate reader (Packard BioScience, Pangbourne, Berkshire, UK).

12) After the first reading, 2. mu.l of the diluted compound was added to each well of columns 1-10. Column 11{ provides a standard curve } only B11-H11 plus compound. Mu.l of 100mM standard compound { is a positive control } is added to well B12-H12

13) The Z' factor was calculated from the 0 control and positive wells. It usually gives a value of 0.7-0.9.

The compounds tested in the above assay were assigned to one of two activity ranges, i.e. a ═ 10 μ M; b ═ 10 μ M, those assignments reported above. For example, the compound of example 2 gave an IC50 in the range 'a'.

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Claims

1. A compound of the formula (A) or (B) or a salt thereof,

in the formula R₁Is 2, 4-dihydroxyphenyl which is substituted in the 5-position by methyl, ethyl, isopropyl, isobutyl, tert-butyl, chlorine or bromine; r₂Is (i) at the 4-position by methoxy, ethoxy, fluoro, chloro, bromo, piperazinyl, N-methylpiperazinyl or piperazinePyridyl-substituted phenyl, or (ii) a group of the formula:

in the formula-NR¹⁰R¹¹Is morpholinyl, piperidinyl, piperazinyl, pyrrolidinyl, ethylamino, isopropylamino, diethylamino, cyclohexylamino, cyclopentylamino, methoxyethylamino, N-acetylpiperazinyl, methanesulfonylamino, thiomorpholinyl dioxide, 4-hydroxyethylpiperidinyl or 4-hydroxypiperidinyl; and R₃Is a group of formula-CONHR wherein R is hydrogen, methyl, ethyl or isopropyl.

2. The compound of claim 1, wherein the compound is one of the formula (a).

3. A compound of claim 2, wherein R is₃Is ethylaminocarbonyl or isopropylaminocarbonyl.

4. A compound according to claim 1, characterized in that said compound is 5- (2, 4-dihydroxy-5-isopropyl-phenyl) -4- (4-morpholin-4-ylmethyl-phenyl) -isoxazole-3-carboxylic acid ethylamide or a salt thereof.

5. A pharmaceutical composition comprising a compound of claim 4, and a pharmaceutically acceptable carrier.

6. A pharmaceutical composition according to claim 5, wherein the composition is in the form of a solution or suspension of the compound in a sterile, physiologically acceptable carrier.

7. The pharmaceutical composition of claim 5, wherein said composition is in the form of a solution or suspension of said compound in sterilized saline.

8. Use of a compound according to claim 4 for the manufacture of a medicament for the treatment of: viral diseases, rheumatoid arthritis, asthma, multiple sclerosis, type I diabetes, lupus, psoriasis, inflammatory bowel disease; cystic fibrosis, diabetic retinopathy, hemangiomas, endometriosis, chemotherapy-induced toxicity; hypoxia-ischemic injury of tissues and scrapie/CJD, huntington's chorea or alzheimer's disease due to elevation of Hsp70 in the heart and brain.

9. Use of a compound of claim 4 in the manufacture of a medicament for the treatment of cancer.