THIAZOLIDIN-4-ONES HAVING ANTI-HEPATITIS B ACTIVITY

FIELD OF THE INVENTION

The present invention relates to compounds, compositions and method of treatment for viral infections and the diseases associated therewith, particularly those infections and associated diseases caused by viruses in the hepadnavirus family, particularly the hepatitis B virus (HBV).

BACKGROUND OF THE INVENTION

Despite various treatment options available for patients infected with HBV, sustained treatment success as evidenced by decrease of HBV DNA in serum, and/or anti-HBe and/or HBs seroconversion is frequently limited to a relatively small patient population for various reasons.

For example, interferon alpha has been widely used for the treatment of chronic HBV infection for a number of years. However, interferon is effective only in certain subpopulations of chronic hepatitis B patients and is poorly tolerated. In other cases, lamivudine (3'-thia-2',3'-dideoxycytidine), a particularly strong inhibitor of HBV replication, is used to treat HBV infection. However, resistance against this nucleoside analog is increasingly common, and has limited its efficacy in a high proportion of patients. The most recently approved treatment for HBV is adefovir dipivoxil (9-(2-((-bis((pivaloyloxy)methoxy)phosphinyl)methoxy)ethyl)a denine), and while this nucleoside analog is active against the lamivudine-resistant viruses, the sustained viral response rate is below 20%, and nephrotoxicity typically limits the maximum tolerated dose and/or treatment duration.

More recent developments in HBV research have resulted in various clinical trials for selected compounds with promising antiviral activity. For example, various nucleoside analogs have been reported to exhibit significant anti-HBV activity (e.g., 2'-fluoro-5-methyl-beta-L-arabinofuranosyluracil (Bukwang) and 2'-deoxy-5-fluoro- 3'-thiacytidine (Gilead); 2'-deoxy-L-thymidine and 2'-deoxy-L-cytidine (both Idenix)).

Similarly, carbocyclic nucleoside analogs (2-amino-l,9-dihydro-9-((lS,3R,4S)-4- hydroxy-3 -(hydroxymethyty-Z-methylenecyclopentyl-όH-purin-β-one monohydrate ; Bristol-Myers Squibb)), as well as acyclic nucleoside analogs with liver targeting properties (Remofovir; Ribapharm), were reported as having anti-HBV activity in clinical trials.

However, while most of the recently discovered drugs with anti-HBV activity exhibited promising in vitro antiviral activity, low response rates and the emergence of resistance limit the efficacy of these clinical candidates. Therefore, although various compositions and methods for HBV treatment are known in the art, there is still a need to provide new and/or improved compositions and methods for treatment of HBV infections in human patients.

SUMMARY OF THE INVENTION

In accordance with one aspect, the present invention provides a compound and composition for preventing and treating HBV infections in mammals and for preventing and treating diseases associated with HBV infection. The compounds of the invention have the following structure:

in which R ¹ represents a substituent selected from alkyl and branched alkyl of 1-8 carbons optionally substituted with phenyl and substituted phenyl, heterocyclic and substituted heterocyclic moieties and fused aryl and heteroaryl which can contain

functionalities such as halogen, alkyl, alkoxy, cyano, nitro, CF ₃ , and NR ⁷ R ⁸ , wherein R ⁷ and R ⁸ can be the same or different and are H, C]-C ₈ alkyl and branched alkyl which can be optionally substituted with OR ⁷ and NR ⁷ R ⁸ . R ¹ can also be phenyl and substituted phenyl, heterocyclic and substituted heterocyclic moieties and fused aryl and heteroaryl which can contain functionalities such as halogen, alkyl, alkoxy, cyano, nitro, CF ₃ , and NR ⁷ R ⁸ wherein R ⁷ and R ⁸ can be the same or different and are H, C ₁ - C ₈ alkyl and branched alkyl which can be optionally substituted with OR ⁷ and NR ⁷ R ⁸ .

At least one of R ² and R ³ is H, and the other may be H or a Ci-C ₈ linear or branched alkyl which can be optionally substituted with OR ⁷ and NR ⁷ R ⁸ .

R ⁴ and R ⁵ can be aryl such as phenyl and substituted phenyl, heterocyclic and substituted heterocyclic which can contain functionalities such as halogen, alkyl, alkoxy, cyano, nitro, CF ₃ , and NR ⁷ R ⁸ wherein R ⁷ and R ⁸ can be the same or different and are H, C ₁ -C ₈ alkyl and branched alkyl which can be optionally substituted with OR ⁷ and NR ⁷ R ⁸ ; R ⁶ can be H, halogen, Ci-C ₈ alkyl and branched alkyl, R ⁴ and R ⁵ can optionally be Ci-C ₈ alkyl and branched alkyl, R ⁴ and R ⁵ can also be alkyl and branched alkyl of 1-8 carbons optionally substituted with phenyl and substituted phenyl, heterocyclic and substituted heterocyclic moieties and fused aryl and heteroaryl which can contain functionalities such as halogen, alkyl, alkoxy, cyano, nitro, CF ₃ , and NR ⁷ R ⁸ wherein R ⁷ and R ⁸ can be the same or different and are H, Ci- C ₈ alkyl and branched alkyl which can be optionally substituted with OR ⁷ and NR ⁷ R ⁸ .

Additionally, R ⁴ can be halogen, CF ₃ , alkyl and branched alkyl, cycloalkyl, alkenyl and alkynyl and substituted alkyl, branched alkyl, cycloalkyl, alkenyl and alkynyl. These may also be substituted with O, N, or S atoms and these may contain alkyl and branched alkyl, cycloalkyl, alkenyl and alkynyl and substituted alkyl, branched alkyl, cycloalkyl, alkenyl and alkynyl, aryl and substituted aryl, heteroaryl and substituted heteroaryl, heterocyclic and substituted heterocyclic.

W is selected from O, N and substituted N, and S; X can be S, O, or N, provided that when X = N then X' is C double-bonded to X; X' can be N, C double- bonded to X, or CR ⁹ , where R ⁹ can be lower alkyl, lower cycloalkyl, aryl, substituted

aryl, CF ₃ , heteroaryl CF ₃ CF ₂ , CF ₃ CH ₂ , or CH ₃ CF ₂ ; and Y and Z are the same or different and can be CR or N.

In accordance with a second aspect, the present invention provides a compound and composition for preventing and treating HBV infections in mammals and for preventing and treating diseases associated with HBV infection. These compounds of the invention have the following structure:

in which R represents a substituent selected from linear or branched alkyl of 1-8 carbons optionally substituted with phenyl, substituted phenyl, heterocyclic, substituted heterocyclic, fused aryl and fused heteroaryl moieties which can contain functionalities such as halogen, alkyl, alkoxy, cyano, nitro, CF ₃ , and NR ⁷ R ⁸ , wherein R ⁷ and R ⁸ can be the same or different, and are H, Ci-C ₈ linear or branched alkyl which can be optionally substituted with OR ⁷ and NR ⁷ R ⁸ ; R ¹ can also be phenyl, substituted phenyl, heterocyclic, substituted heterocyclic, fused aryl or fused heteroaryl, which can contain functionalities such as halogen, alkyl, alkoxy, cyano, nitro, CF ₃ , and NR ⁷ R ⁸ wherein R ⁷ and R ⁸ can be the same or different and are H or Ci-C ₈ linear or branched alkyl which can be optionally substituted with OR ⁷ and NR ⁷ R ⁸ .

At least one of R ² and R ³ is H, and the other can be H, or Ci-C ₈ linear or branched alkyl which can be optionally substituted with OR ⁷ and NR ⁷ R ⁸ .

R ⁴ and R ⁵ can be aryl such as phenyl and substituted phenyl, heterocyclic and substituted heterocyclic which can contain functionalities such as halogen, alkyl, alkoxy, cyano, nitro, CF ₃ , and NR R wherein R and R can be the same or different and are H, Ci-C ₈ alkyl and branched alkyl which can be optionally substituted with OR ⁷ and NR ⁷ R ⁸ ; R ⁶ can be H, halogen, Ci-C ₈ alkyl and branched alkyl, R ⁴ and R ⁵ can optionally be Ci-C ₈ alkyl and branched alkyl, R ⁴ and R ⁵ can also be alkyl and branched alkyl of 1-8 carbons optionally substituted with phenyl and substituted phenyl, heterocyclic and substituted heterocyclic moieties and fused aryl and heteroaryl which can contain functionalities such as halogen, alkyl, alkoxy, cyano, nitro, CF ₃ , and NR ⁷ R ⁸ wherein R ⁷ and R ⁸ can be the same or different and are H, C ₁ - C ₈ alkyl and branched alkyl which can be optionally substituted with OR ⁷ and NR ⁷ R ⁸ .

X is S, O, or N, provided that when X is N X' is C and is double bonded to X.

X' is N, C double-bonded to X, or CR ⁹ , where R ⁹ is H, lower alkyl, lower cycloalkyl, aryl, substituted aryl, CF ₃ , heteroaryl CF ₃ CF ₂ , CF ₃ CH ₂ , or CH ₃ CF ₂ ;

In preferred embodiments, X is S, X' is CH, and R ⁴ and R ⁵ are aryl or heteroaryl.

In more preferred embodiments, X is S, X' is CH, R ² and R ⁶ are H, and R ⁴ and R ⁵ are aryl or heteroaryl.

In still more preferred embodiments, X is S, X' is CH, R ² and R ⁶ are H; and R ⁴ and R ⁵ are independently selected from the group consisting of phenyl, pyridyl, pyrazinyl, and pyrimidinyl and are unsubstituted or independently substituted with one or more halogen or methyl groups.

In even more preferred embodiments, X is S, X' is CH, R ² and R ⁶ are H; R ⁴ and R ⁵ are aryl or heteroaryl; and R ¹ is chosen from the group consisting of phenyl, benzyl, 2-phenethyl, tetrahydro-2-furfuryl, 5-methyl-2-furfuryl, 2-thienylethyl, 3,3- dimethylbutyl, 3-isopropyloxypropyl, beta-hydroxy-2-phenethyl, isobutyl, 3- methoxypropyl, piperonyl, and 2,2-dimethyl-3-hydroxypropyl.

In the most preferred embodiments, W X is S, X' is CH, R ² and R ⁶ are H; R ⁴ and R ⁵ are independently selected from the group consisting of phenyl, pyridyl,

pyrazinyl, and pyrimidinyl and are unsubstituted or independently substituted with one or more halogen or methyl groups; and R ¹ is chosen from the group consisting of phenyl, benzyl, 2-phenethyl, tetrahydro-2-furfuryl, 5-methyl-2-furfuryl, 2-thienylethyl, 3,3-dimethylbutyl, 3-isopropyloxypropyl, beta-hydroxy-2-phenethyl, isobutyl, 3- methoxypropyl, piperonyl, and 2,2-dimethyl-3-hydroxypropyl.

In many of the embodiments set forth above, one or more carbon atoms may present an asymmetric center. The invention encompasses isolated enantiomers and diastereomers as well as racemic and non-racemic mixtures thereof, which may readily be prepared, for example by the use of appropriately chiral starting materials.

It is particularly preferred that the compounds of the invention are included in a pharmaceutical composition that is formulated with one or more non-toxic pharmaceutically acceptable carriers. The pharmaceutical compositions may be specially formulated for oral administration in solid or liquid form, for parenteral injection, or for rectal administration.

The pharmaceutical compositions of this invention can be administered to humans and other animals orally, rectally, parenterally, intracistemally, intravaginally, intraperitoneally, topically (as by powders, ointments, or drops), bucally, or as an oral or nasal spray. The term "parenteral" administration as used herein refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intra-articular injection and infusion.

Pharmaceutical compositions for parenteral injection preferably comprise pharmaceutically acceptable sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions as well as sterile powders for reconstitution into sterile injectable solutions or dispersions just prior to use. Examples of suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils (such as olive oil), and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

Contemplated compositions may also contain adjuvants such as preservative, wetting agents, emulsifying agents, and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents such as sugars, sodium chloride, and the like, Prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.

In some cases, in order to prolong the effect of the drug, it is desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material with poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle.

Injectable depot forms are made by forming unitary or microparticulate matrices of the drug in biodegradable polymers such as polylactide-polyglycolide. Depending upon the ratio of drug to polymer and the nature of the particular polymer employed, the rate of drag release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissues.

The injectable formulations can be sterilized, for example, by filtration • through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium just prior to use.

Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active compound is mixed with at least one inert, pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and/or a) fillers or extenders such as starches,

lactose, sucrose, glucose, mannitol, and silicic acid, b) binders such as, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and acacia, c) humectants such as glycerol, d) disintegrating agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, e) solution retarding agents such as paraffin, f) absorption accelerators such as quaternary ammonium compounds, g) wetting agents such as, cetyl alcohol and glycerol monostearate, h) absorbents such as kaolin and bentonite clay, and i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets and pills, the dosage form may also comprise buffering agents.

Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like. The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They may optionally contain opacifying agents and may also be of a composition such that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions which can be used include polymeric substances and waxes. The active compounds may also be in micro-encapsulated form, if appropriate, with one or more of the above-mentioned excipients.

Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups and elixirs. In addition to the active compounds, the liquid dosage forms may contain inert diluents commonly used in the art such as, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethyl formamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. Besides inert diluents, the oral compositions may also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

Compositions for rectal or vaginal administration are preferably suppositories which can be prepared by mixing the compounds of this invention with suitable non- irritating excipients or carriers such as cocoa butter, polyethylene glycol or a suppository wax which are solid at room temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active compound.

Compounds of the present invention can also be administered in the form of liposomes. As is known in the art, liposomes are generally derived from phospholipids or other lipid substances. Liposomes are formed by mono- or multi¬ lamellar hydrated liquid crystals that are dispersed in an aqueous medium. Any non¬ toxic, physiologically acceptable and metabolizable lipid capable of forming liposomes may be used. The present compositions in liposome form may contain, in addition to a compound of the present invention, stabilizers, preservatives, excipients, and the like. The preferred lipids are the phospholipids and the phosphatidyl cholines (lecithins), both natural and synthetic. Methods to form liposomes are known in the art. See, for example, Prescott, Ed., Methods in Cell Biology, Volume XIV, Academic Press, New York, N. Y. (1976), p. 33 et seq.

The compounds of the present invention may be used in the form of pharmaceutically acceptable salts derived from inorganic or organic acids. By "pharmaceutically acceptable salt" is meant those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like and are commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable salts are well-known in the art. For example, S. M. Berge, et al. describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66: 1 et seq. The salts may be prepared in situ during the final isolation and purification of the compounds of the invention or separately by reacting a free base function with a suitable acid. Representative acid addition salts include, but are not limited to acetate, adipate, alginate, citrate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, camphorate, camphorsulfonate, digluconate, glycerophosphate, hemisulfate, heptanoate, hexanoate, fumarate, hydrochloride, hydrobromide, hydroiodide, 2- hydroxyethansulfonate (isethionate), lactate, maleate, methanesulfonate, nicotinate, 2- naphthalenesulfonate, oxalate, pamoate, pectinate, persulfate, 3-phenylpropionate,

picrate, pivalate, propionate, succinate, tartrate, thiocyanate, phosphate, glutamate, bicarbonate, p-toluenesulfonate and undecanoate. Also, the basic nitrogen-containing groups may be quaternized with such agents as lower alkyl halides such as methyl, ethyl, propyl, and butyl chlorides, bromides and iodides; dialkyl sulfates like dimethyl, diethyl, dibutyl and diamyl sulfates; long chain halides such as decyl, lauryl, myristyl and stearyl chlorides, bromides and iodides; arylalkyl halides like benzyl and phenethyl bromides and others. Water or oil-soluble or dispersible products are thereby obtained. Examples of acids which may be employed to form pharmaceutically acceptable acid addition salts include such inorganic acids as hydrochloric acid, hydrobromic acid, sulphuric acid and phosphoric acid and such organic acids as oxalic acid, maleic acid, succinic acid and citric acid.

Basic addition salts can be prepared in situ during the final isolation and purification of compounds of this invention by reacting a carboxylic acid-containing moiety with a suitable base such as the hydroxide, carbonate or bicarbonate of a pharmaceutically acceptable metal cation or with ammonia or an organic primary, secondary or tertiary amine. Pharmaceutically acceptable salts include, but are not limited to, cations based on alkali metals or alkaline earth metals such as lithium, sodium, potassium, calcium, magnesium and aluminum salts and the like and nontoxic quaternary ammonia and amine cations including ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, diethylamine, ethylamine and the like. Other representative organic amines useful for the formation of base addition salts include ethylenediamine, ethanolamine, diethanolamine, piperidine, piperazine and the like. Preferred salts of the compounds of the invention include phosphate, TRIS, and acetate.

Actual dosage levels of active ingredients in the pharmaceutical compositions of this invention may be varied so as to obtain an amount of the active compound(s) that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration. The selected dosage level will depend upon the activity of the particular compound, the route of administration, the severity of the condition being treated, and the condition and prior medical history of the patient being treated. However, it is within the skill of the art to start doses of the compound

at levels lower than required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. Generally, dosage levels of about 1 to about 500, more preferably of about 5 to about 50 mg of an active compound per kilogram of body weight per day are administered orally to a mammalian patient. If desired, the effective daily dose may be divided into multiple doses for purposes of administration, e.g., two to four separate doses per day.

The invention also provides methods for the treatment of HBV viral infection in a mammal by administration of the compounds of the invention, pharmaceutical compositions comprising one or more compounds of the invention, a method of inhibiting replication of HBV comprising contacting a compound of the invention with the replicating HBV, and the use of compounds of the invention for the preparation of a pharmaceutical composition for treatment of HBV.

The invention also provides a method of treating a patient suffering from a hepatitis B viral infection, comprising administering to the patient a therapeutically effective amount of a compound of the invention.

DETAILED DESCRIPTION OF THE INVENTION

HB V Screening Assay

HepG2 cells are transduced using a baculovirus to deliver the HBV genome essentially as previously described (Delaney, W.E., and Isom, H.C. Hepatitis B virus replication in human HepG2 cells mediated by hepatitis B virus recombinant baculovirus. Hepatology 1998; 28: 1134-1146.). Transduced cells are cultured in supplemented EMEM media with 10% fetal bovine serum in a 5% CO ₂ incubator at 37°C for three days in the presence of test compounds. The cells are lysed in a buffer containing 0.5% NP-40 and 500 microgram/ml proteinase K. A solid-phase hybridization is performed to capture the viral DNA and to label the target DNA with Digoxigenin-labeled DNA probes. The captured viral DNA is detected by ELISA using horseradish peroxidase-conjugated anti-digoxigenin antibodies. The EC ₅₀ values are determined using ExcelFit software from the inhibition values of a titration curve for each compound.

For CC ₅₀ determinations, the test compounds are co-cultured with non- transduced HepG2 for three days under the conditions described above. The Promega CellTiter 96 AQ _ueous One Solution Cell Proliferation Assay is used to measure cell proliferation/viability. The CC50 values are determined using ExcelFit™ software from the inhibition values of the titration curve for each compound.

Test Results for Selected Compounds

Table 1 below lists selected compounds with their structures and corresponding antiviral activity (CC50 values in μM; A, EC50 < 1.0 μM; B, 1.0 μM < EC50 < 10 μM; C, EC50 > 10 μM). Antiviral activity was determined using assay systems as described above. All compounds were tested for a CC50 value. ND means not determined. Further examples are provided in Table 2.

Table 1

13

14

15

16

17

Synthetic Procedures

2-[2-(l,3-Diphenyl-lH-pyrazol-4-yl)-4-oxo-thiazolidin-3-y l]-3-(4-hydroxy- phenyl)-propionic acid methyl ester

A mixture of l,3-diphenyl~lH-pyrazole-4-carbaldehyde (0.5g, 2mmol) and L- tyrosine methyl ester hydrochloride (0.46g, 2mmol) in 40 ml of toluene was added diisopropylethylamine (0.4ml, 2.4mmol) with stirring at room temperature. This mixture was stirred for 30 min and then mercaptoacetic acid (0.55ml, 8mmol) was added. The mixture was refluxed with condenser equipped with molecular sieves for 10 hours. The solvent was removed in vacuo and the resulting oily residue was dissolved in chloroform and washed with saturated sodium bicarbonate twice and water twice. The chloroform solution was dried over anhydrous sodium sulfate and evaporated to dryness. The residue was purified by column (chloroform) to give a yellow oily product.

2-[2-(l,3-Diphenyl-lH-pyrazoI-4-yl)-4-oxo-thiazolidin-3-yl]- 3-(4-hydroxy- phenyl)-propionic acid

2- [2-( 1 ,3 -Diphenyl- 1 H-pyrazol-4-yl)-4-oxo-thiazolidin-3 -yl] -3 -(4-hydroxy- phenyl)-propionic acid methyl ester (0.3g) was dissolved in 0.5 ml of dioxane and 0.3 ml of saturated solution of LiOH was added. This mixture was stirred for 3 hours at room temperature. The reaction mixture was acidified with 10% hydrochloric acid until acidic pH and diluted with 0.5 ml of ethyl acetate. The two layers were separated and the aqueous layer was extracted with ethyl acetate three times. The combined organic layer was dried over anhydrous sodium sulfate and evaporated to dryness in vacuo. The crude product was purified by preparative HPLC to give the desired product as white solid.

3-Benzyl-2-(l,3-diphenyl-lH-pyrazol-4-yl)-thiazolidin-4-o ne - typical procedures

A mixture of l,3-diphenyl-lH-pyrazole-4-carbaldehyde (50mg, 0.2mmol) and benzylamine (24mg, 0.22mmol) in 3 ml of toluene was heated to 100 ⁰ C for 4-5 hours, then mercaptoacetic acid (37mg, 0.4mmol) was added. The mixture was heated at 100 ⁰ C for 24 hours then cooled to room temperature. The solvent was removed in vacuo and the residue was purified by column (hexane/ethyl acetate, 4: 1) to give pure product as white solid.

3-benzyl-2-(l,3-diphenyl-lH-pyrazol-4-yl)-5-methyl-thiazo lidin-4-one

Synthesized by the above procedure but using thiolactic acid.

3-Benzyl~2-(l,3-diphenyl-lH-pyrazol-4-yl)-5,5-dimethyl-th iazolidin-4~one

Synthesized by the above procedure but using 2-mercaptoisobutyric acid.

3-Benzyl-2-(l,3-diphenyl-lH-pyrazol-4-yl)-oxazolidin-4-on e

A mixture of l^-diphenyl-lH-pyrazole^-carbaldehyde (0.5g, 2mmol) and benzylamine (0.24g, 2.2mmol) in 40 ml of THF was stirred for 5 hours and then mercaptoacetic acid (0.28ml, 4mmol) was added. After 10 min, DCC (0.6g) was added to the above mixture at room temperature at room temperature. The reaction mixture was stirred for additional 48 hours at room temperature. The precipitates were filtered and washed with THF. The filtration was evaporated in vacuo and the residue

was purified by column (chloroform) to give the desired product as white solid, which was re-crystallized from methanol to yield colorless crystals.

3-Benzyl-2-(l,3-diphenyl-lH-pyrazol-4-yl)-[l,3]thiazinan- 4-one

A mixture of l^-diphenyl-lH-pyrazole^-carbaldehyde (0.5g, 2mmol), N- benzyl-2-hydroxy-acetamide (0.36g, 2.2mmol), and p-toluenesulfonic acid (5mg) in 40 ml of toluene was refluxed with condenser equipped with molecular sieves for 24 hours. The solvent was removed in vacuo and the resulting residue was recrystallized from methanol to give off-white crystals.

l,3-Diphenyl-lH-pyrazole-4-carboxylic acid

To a suspension of l,3-diphenyl-lH-pyrazole-4-carbaldehyde (1.2g, 4.8mmol) in 5ml of 50% aqueous pyridine at stirring and cooling with tap water was added by small portion of potassium permanganate (0.76g, 4.8mmols) within 1 hour. The reaction temperature was maintained between 22-24°C during the addition. Keep stirring for 1 hour, and then the precipitate was filtered off, washed with 5% of sodium hydroxide. The filtrate was added 10% hydrochloric acid (40ml). The

product was filtered off, dried in vacuo to givel .3g of crude product as a dark brown solid, which was used for next step without further purification.

1 ,3-Diphenyl-lH-pyr azoIe-4-carbonyI chloride

To a stirred suspension of l,3-diphenyl-lH-pyrazole-4-carboxylic acid (200mg, 0.75mmol) and N,N-dimethylformamide (1 drop) in anhydrous benzene was added thionyl chloride (0.77ml). The resulting mixture was refluxed for 2 hours. After cooling the mixture was evaporated in vacuo. The crude residue was used in the next step without further purification.

l,3-Diphenyl-lH-pyrazole-4-carbonylic acid-(3-methoxy-propyl)-amide

A solution of the crude l,3-diphenyl-lH-pyrazole-4-carbonyl chloride (0.75mmols) in anhydrous benzene (3ml) was slowly added to a solution of 3- methoxypropylamine (0.18ml, l.δmmol) in anhydrous benzene (7ml). The mixture was stirred for 2 hours at room temperature, then was diluted with ethyl acetate and extracted with 10% hydrochloric acid solution, 10% sodium bicarbonate, and brine. The organic layer was dried over anhydrous sodium sulfate and evaporated to dryness in vacuo to afford 165mg (65%) of the desired amide. MS: 335

l,3-Diphenyl-lH-pyrazole-4-carboxylic acid (2-azido-acetyl)-(3-methoxy-propyl)- amide

To a stirred solution of l,3-diphenyl-lH-pyrazole-4-carbonylic acid-(3- methoxy-propyl)-amide (0.49mmols) in benzene (3ml) was added 2-chlororacetyl chloride (0.043ml) at room temperature under nitrogen. The mixture was heated to reflux for 2 hours, evaporated in vacuo. Dimethylsulfoxide (3ml) was added to the residue to give a solution, sodium azide (98mg) was added in one portion. After stirring for lOmin at room temperature, the mixture was poured onto water and extracted with ether (3x15ml). The combined ether layers were dried over anhydrous sodium sulfate and evaporated in vacuo. The crude residue was used in the next step without further purification.

2-(l,3-Diphenyl-lH-pyrazole-4-yl)-3-(3~methoxypropyI)-3,5-di hydro-imidazo-4- one

To a solution of l,3-diphenyl-lH-pyrazole-4-carboxylic acid (2-azido-acetyl)- (3-methoxy-propyl)-amide (0.49mmols) in benzene (5ml) was added triphenylphosphine (142mg, 0.54mmol). The mixture was stirred for 3 hours at room temperature and evaporated in vacuo. The residue was chromatbgraphed on a silica gel column (chloroform-ethyl acetate, 1:1) to afford imidazolinone. MS: 375.

2-(l,3-Diphenyl-lH-pyrazol-4-yl)-3-piperonyl-3,5-dihydro- imidazol-4-one

Synthesized by the above procedure but using piperonylamine.

Semicarbazone

A mixture of 4'-fluoroacetophenone (13.8g, 0.1 mol) and semicarbazide hydrochloride (11.2g, O.lmol), and sodium acetate (27.6g, 0.2mol) in 350 ml of methanol was stirred at room temperature for 2 hours, then 350 ml of water was added and the mixture was stirred for another 2 hours. The solid was filtered and washed with 50% methanol in water to give 16.5 g of white solid (84%) after drying in vacuo.

3-(4-Fluoro-phenyl)-lH-pyrazole-4-carbaIdehyde

POCl ₃ (3.0ml, 33mmol) was slowly added to anhydrous DMF (7.65ml, 66mmol) at 0 ⁰ C (ice-bath) with stirring. After stirring for 5 min, the semicarbazone was added portionwise to the above mixture with well-stirring. The mixture was heated to 60 ⁰ C for 5 hours and poured onto 2Og of ice. It was neutralized with NaOH

(6g in 24 ml of water) and heated at 60 ⁰ C for 20 min, then cooled to room temperature and neutralized with ION HCl to pH 6. The resulting white precipitates were filtered and washed with water. After drying in vacuo at 60 ⁰ C, 2.35 g (82%) of the aldehyde as white solid was obtained.

2-[3-(4-Fluoro-phenyl)-lH-pyrazol-4-yl]-3-(3-methoxy-prop yl)-thiazolidin-4-one

A mixture of 3-(4-fluoro-phenyl)-lH-pyrazole-4-carbaldehyde (0.95g, 5mmol) and 3-methoxypropylamine (0.49g, 5.5mmol) in 25 ml of toluene was heated to 100 ⁰ C for 2 hours, then mercaptoacetic acid (0.92g, lOmmol) was added and heated at 100 ⁰ C for another 24 hours. The solvent was removed in vacuo and residue was purified by silica gel column (chloroform/methanol, 40:1) to give oily product.

2-[l-(3-Fluoro-4-methyl-phenyl)-3-(4-fluoro-phenyl)-lH-py razol-4-yl]-3-(3- methoxy-propyl)-thiazolidin-4-one

To a 25 ml round bottle flask was added in sequence: 4-fiuoro-3- methylphenylboronic acid (92mg, 0.6mmol), 2-[3-(4-Fluoro-phenyl)-lH-pyrazol-4- yl]-3-(3-methoxy-propyl)-thiazolidin-4-one (100mg,0.3mmol), copper (II) acetate (81mg, 0.45mmol), 4A molecular sieves (250mg), pyridine (49μL), and 4 ml of anhydrous dichloromethane. The reaction mixture was stirred at ambient temperature for 2 days. The resulting mixture was filtered through Celite, washed with methanol and purified by silica gel column (hexane/ethyl acetate, 2:1) to give 38.7 mg of title compound.

3-Amino-l-phenyl-lH-pyrazole-4-carbaldehyde

In a 500ml 3-neck round-bottom flask fitted with a thermometer and addition funnel was added 2.24 g of 3-amino-l-phenyl-lH-pyrazole-4-carbonitrile, prepared according to the procedure reported in Aust. J. Chem., (1991), 44, 1795-1801, and 120ml of anhydrous toluene. The flask was cooled to -78°C and to it was added 30ml of a IM solution of DIBAL in hexane dropwise through the addition funnel while maintaining the temperature below -70 ⁰ C. Stirring was continued for 30 minutes at - 78°C and then warmed to O ⁰ C. Stirring was continued an additional 2h at 0 ⁰ C and the resulting solution was poured into 75ml 4N HCl. The mixture was stirred vigorously 20 minutes and then the aqueous layer isolated. The aqueous layer was extracted with ethyl acetate, washed with water, brine, dried over Na ₂ SO ₄ , filtered, evaporated and the residue purified over silica gel column (hexane/ethyl acetate, 3:1) to afford 1.33g (58%) of 3 -amino- 1 -phenyl- lΗ-pyrazole-4-carbaldehyde as a yellow crystalline solid.

3-Iodo-l-phenyl-lH-pyrazole-4-carbaldehyde

To a suspension of 5.4g of I ₂ in 15ml OfCH ₃ CN in alOOml 2-neck round- bottom flask under argon was added dropwise through an addition funnel 1.33g of the

carbaldehyde in 15ml OfCH ₃ CN while heating the suspension at 30 ⁰ C. The reaction temperature was maintained below 40 ⁰ C by means of an internal thermometer throughout the addition. The reaction mixture was stirred for an additional 30 minutes after addition and then poured into 125ml of cold saturated Na ₂ S ₂ O ₃ solution. The resulting yellow precipitate was filtered, washed with water, and dried under vacuum to afford 2.07 g (98%) of the title compound.

3-(3,4-Difluoro-phenyl)-l-phenyl-lH-pyrazoIe-4-carbaldehy de

To a solution of lOOmg of 3 -iodo-1 -phenyl- lH-pyrazole-4-carbaldehyde in 3ml of DME and 1.67ml of IM sodium carbonate was added 20mg of (PPli ₃ ) ₄ Pd and 79mg of 3,4-difluorophenyl boronic acid. The reaction was heated to 80 ⁰ C for 2.5h, cooled and diluted with ethyl acetate. The organic layer was isolated and dried over anhydrous Na ₂ SO ₄ , filtered, evaporated and the residue purified over silica gel column (hexane/ethyl acetate, 4:1) to afford 44 mg (46%) of the title compound.

2-[3-(3,4-Difluoro-phenyl)-l-phenyl-lH-pyrazol-4-yl]-3-[2 -(4-hydroxy-phenyl)- ethyl]-thiazolidin-4-one

A solution of 44 mg of the above aldehyde and 21mg of tyramine in 1.5ml methanol was heated to 60 ⁰ C for 18h. The solvent was evaporated off and 1.5ml toluene and 20 μl mercaptoacetic acid were added. The resulting suspension was heated to 100 ⁰ C for 24h. The resulting reaction mixture was evaporated and the residue purified over silica gel column (hexane/ethyl acetate, 2:1) to afford 17mg (23%) of the title compound.

l-Phenyl-S-aminopyrazole-S-carbonitrile

In a 3 -neck round bottom flask equipped with an overhead mechanical stirrer and a thermometer was charged 13.4g of sodium acetate and 11OmL of acetic acid. A constant stream of argon was flushed through the flask via a gas inlet adapter. While stirring vigorously, 35g of phenyl hydrazine was added to the solution, followed by 4Og of ethoxymethylenemalononitrile. The solution turned dark red brown and the internal temperature rose to 75 ⁰ C. After Ih, the solution cooled back down to ambient temperature. After stirring 18h, the resulting orange pasty reaction mixture was concentrated under vacuum and then treated with 100ml saturated aqueous sodium bicarbonate. The resulting orange solid was filtered, washed with 2x100ml portions of water and dried under vacuum to constant weight to afford 47g (78%) of the title compound as an orange crystalline solid.

l-Phenyl-5-aminopyrazole-5-carboxaldehyde

In a 3 -neck round bottom flask equipped with an addition funnel and a thermometer was added 7.97g of l-phenyl-S-aminopyrazole-S-carbonitrile followed by 300ml of toluene. The suspension was cooled to -78°C and a IM solution of DIBAL in hexane was added through the addition funnel dropwise, while maintaining the internal temperature below -70 ⁰ C. After addition, the solution was stirred an additional 20 min at -78 ⁰ C, then warmed to 0 ⁰ C. The solution was then slowly poured into 225ml of 4N HCl, the resulting solution warming to 26°C, before cooling back down. The aqueous layer was then isolated and diluted with 500 ml of water. The aqueous solution was extracted with 3x150ml portions of ethyl acetate. The combined organic layer were washed with water, then saturated NaHCO ₃ , dried over anhydrous Na ₂ SO ₄ , filtered and evaporated to yield 1.8 g of off-white solid. The aqueous layer was allowed to stand for 18h, then re-extracted with 3x250ml portions of ethyl acetate. The organic phase was washed with water, saturated NaHCO ₃ , dried over anhydrous Na ₂ SO ₄ , filtered and evaporated to yield 4.4g of a yellow solid and combined 54% yield.

l-Phenyl-S-iodopyrazole-S-carboxaldehyde

In a 2-neck 100ml round bottom flask equipped with an addition funnel and a thermometer was added 17.9g of iodine, followed by 50ml OfCH ₃ CN. The resulting mixture was thoroughly flushed with Argon and to this was added 4.23ml of t-butyl nitrite. The reaction mixture was heated to 30 ⁰ C and a solution of 4.4g of l-phenyl-5- aminopyrazole-5-carboxaldehyde in 50ml OfCH ₃ CN was added through the addition funnel dropwise, maintaining the internal temperature below 38°C. After addition, the reaction mixture was heated for an additional 20 minutes, then poured into 500ml of cold saturated Na ₂ S ₂ O ₃ solution. The resulting suspension was stirred vigorously and the solid filtered and washed with water. The product was dried in a vacuum oven to afford 5.32 g (76%) of the title compound as an orange solid.

5-(3,5-Difluorophenyl)-l-phenyl-lH-pyrazole-4-carbaldehyd e

A solution of lOOmg of l-phenyl-S-iodopyrazole-S-carboxaldehyde, 66mg of 3,5-difluorophenylboronic acid, 19mg of tetrakis-triphenylphosphine palladium(O), 1.67ml of IM sodium carbonate in 5ml of dimethoxye thane was flushed with argon and heated to 80 ⁰ C. After 3h, the solution was concentrated down to a solid residue

and extracted with CH ₂ Cl ₂ . The solution was purified over silica gel, eluting with hexane/ethyl acetate (6:1), affording 60 mg (63%) of the title compound as an off white solid.

2-[5-(3,5-Difluorophenyl)-l-phenyl-lH-pyrazol-4yl]-3-(3-m ethoxypropyl)- thazolidin-4-one

To a suspension of 35mg of 5-(3,5-difluorophenyl)-l -phenyl- lH-pyrazole-4- carbaldehyde in 2ml of toluene was added 3-methoxypropylamine. The resulting suspension was heated to 100 ⁰ C. After 6h, the resulting solution was treated with 27μl mercaptoacetic acid and heating continued for an additional 18h. The solution was evaporated and the residue purified over silica gel, eluting with hexane/ethyl acetate (3:1), affording 25 mg (47%) of the title compound as an off white foam.

Structure R1 R1 R5 R5 R4 R4 Activity CC50 PURITY

benzyl phenyl 3-pyridyl >50 94

1 ,4-benzodioxan- phenyl ttiiophene >50 95 2-methyl

tetrahydro furfuryl phenyl n >50 94

"S . thiophene

benzyl phenyl phenyl >50 94

benzyl phenyl 4-chloro phenyl >50 100

3-pyridyl methyl phenyl phenyl >50 100

2-pyridyl methyl phenyl phenyl >50 99

Structure R1 R1 R5 R5 R4 R4 Activity CC50 PURITY

benzyl 4-bromo phenyl 3-pyπdyl > 50 96

benzyl 2-fluoro phenyl 3-pyπdyl > 50 92

benzyl 4-methoxy phenyl 3-pyπdyl > 50 94

benzyl 3-chloro phenyl 3-pyrιdyl > 50 98

benzyl 4-methyl phenyl 3-pyπdyl > 50 98

benzyl phenyl methylsulfonyl > 50 100 phenyl

benzyl 4-methyl phenyl 4-fluoro phenyl > 50 100

Structure R1 R1 R5 R5 R4 R4 Activity CC50 PURITY

1-methyl-3-phenyl phenyl phenyl > 50 88 propyl

2-methoxy ethyl phenyl phenyl > 50 gg rf- 2-hydroxymethyl phenyl phenyl 25-50 94 isobutyl

2-hydroxymethyl phenyl phenyl > 50 92 isobutyl

2-dimethyl-3- dimethylamino phenyl phenyl 44.50 90 propyl

β-hydroxy phenyl phenyl 31.05 98 phenethyl

β-hydroxy phenyl phenyl 44.35 95 phenethyl

Structure R1 R1 R5 R5 R4 R4 Activity CC50 PURITY

3-morpholino phenyl 4-fluoro phenyl > 50 96 propyl

4-methyl benzyl phenyl 4-fluoro phenyl > 50

piperonyl phenyl 4-fluoro phenyl > 50 100

phβnethyl phenyl 4-fluoro phenyl > 50 100

4-hydroxy phenyl 4-fluoro phenyl 24.87 96 phenethyl

2-(2-pyridyl) ethyl phenyl 4-fluoro phenyl 41.58 84

3-methoxy propyl 4-methoxy phenyl 3-pyridyl > 50 99

Structure R1 R1 R5 RS R4 R4 Activity CC50 PURITY

3-chloro-4-fluoro piperonyl 4-fluoro phenyl >50 95 phenyl

piperonyl 4-chloro phenyl 4-fluoro phenyl >17 100

β-hydroxy-3- methoxy 4-methyl phenyl 4-fluoro phenyl >17 99 phenethyl

4-hydroxy

4-methyl phenyl 4-fluoro phenyl >17 99 phenethyl

piperonyl 4-fluoro phenyl 4-fluoro phenyl >50 100

4-hydroxy

4-chloro phenyl 4-fluoro phenyl >17 100 phenethyl

4-hydroxy

4-fluoro phenyl 4-fluoro phenyl >17 99 phenethyl

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