WO2011084962A1 - Analogs of dehydrophenylahistins - Google Patents

Abstract

Analogs of dehydrophenylahistins are disclosed as are methods for making such compounds. Compositions and methods for treating various disease conditions including cancer and non-cancer diseases associated with vascular proliferation are also disclosed.

Description

ANALOGS OF DEHYDROPHENYLAHISTINS

Related Application

[0001] This application claims the benefit of U.S. Provisional Application No. 61/292,378, filed January 5, 2010, which is incorporated herein by reference in its entirety.

Background of the Invention

Field of the Invention

[0002] The present invention relates to compounds and methods of synthetic preparation in the fields of chemistry and medicine. More specifically, the present invention relates to compounds and procedures for making compounds useful in the treatment of cancer and the treatment of fungal infections.

Brief Description of the Related Art

[0003] It has been reported that tryprostatins A and B (which are diketopiperazines consisting of proline and isoprenylated tryptophan residues), and five other structurally-related diketopiperazines, inhibited cell cycle progression in the M phase, see Cui, C. et al. , 1996 J Antibiotics 49:527-33; Cui, C. et al. 1996 J Antibiotics 49:534-40, and that these compounds also affect the microtubule assembly, see Usui, T. et al. 1998 Biochem J 333:543-48; Kondon, M. et al. 1998 / Antibiotics 51:801-04. Furthermore, natural and synthetic compounds have been reported to inhibit mitosis, thus inhibit the eukaryotic cell cycle, by binding to the colchicine binding- site (CLC-site) on tubulin, which is a macromolecule that consists of two 50 kDa subunits (a- and β-tubulin) and is the major constituent of microtubules. See, e.g. , Iwasaki, S., 1993 Med Res Rev 13: 183-198; Hamel, E. 1996 Med Res Rev 16:207-31 ; Weisenberg, R.C. et al, 1969 Biochemistry 7:4466-79. Microtubules are thought to be involved in several essential cell functions, such as axonal transport, cell motility and determination of cell morphology. Therefore, inhibitors of microtubule function may have broad biological activity, and be applicable to medicinal and agrochemical purposes. It is also possible that colchicine (CLC)-site ligands such as CLC, steganacin, see Kupchan, S.M. et al., 1973 J Am Chem Soc 95: 1335-36, podophyllotoxin, see Sackett, D.L., 1993 Pharmacol Ther 59:163-228, and combretastatins, see Pettit, G.R. et al, 1995 J Med Chem 38:166- 67, may prove to be valuable as eukaryotic cell cycle inhibitors and, thus, may be useful as chemo therapeutic agents.

[0004] Although diketopiperazine-type metabolites have been isolated from various fungi as mycotoxins, see Horak R.M. et al, 1981 JCS Chem Comm 1265-67; Ali M. et al, 1898 Toxicology Letters 48:235-41, or as secondary metabolites, see Smedsgaard J. et al, 1996 J Microbiol Meth 25:5-17, little is known about the specific structure of the diketopiperazine-type metabolites or their derivatives and their antitumor activity, particularly in vivo. Not only have these compounds been isolated as mycotoxins, the chemical synthesis of one type of diketopiperazine-type metabolite, phenylahistin, has been described by Hayashi et al. in /. Org. Chem. (2000) 65, page 8402. In the art, one such diketopiperazine-type metabolite derivative, dehydrophenylahistin, has been prepared by enzymatic dehydrogenation of its parent phenylahistin. With the incidences of cancer on the rise, there exists a particular need for chemically producing a class of substantially purified diketopiperazine-type metabolite- derivatives having animal cell-specific proliferation-inhibiting activity and high antitumor activity and selectivity. There is therefore a particular need for an efficient method of synthetically producing substantially purified, and structurally and biologically characterized, diketopiperazine-type metabolite-derivatives.

Summary of the Invention

[0005] One embodiment disclosed herein includes a compound having the structure of Formula I and harmaceutically acceptable salts and tautomers thereof:

(I)

wherein:

Ri, R₂, R₃, and R4 are independently hydrogen, Ci_₆ alkyl, Ci_₆ alkoxy, halogen, or independently absent;

R5 is hydrogen, Ci_₆ alkyl, Ci_₆ alkoxy, halogen, absent, or a bond connecting Z₅ to the phenyl ring in formula I; Zi, Z₂, Z₃, Z₄, and Z₅ are each independently selected from the group consisting of carbon and nitrogen;

R₆ is Ci-6 alkyl;

Z is oxygen or NH;

provided that at least one of Ri, R₂, R₃, R4, and R₅ is not hydrogen or absent, or that at least one of Z_l5 Z₂, Z₃, Z₄, and Z5 is nitrogen, or that R⁶ is methyl.

[0006] Also disclosed are methods and materials for treating neoplastic tissue or preventing cancers or infection by a pathogenic fungus. These methods and materials are particularly well suited for treatment of mammalian subjects, more particularly humans, and involve administering to the subject a compound described herein. The method comprises administering to the subject a composition comprising an effective antitumor or antifungal amount of a compound described herein.

[0007] Some embodiments include the treatment of cancer using a compound described herein. Non-limiting examples of such cancer include carcinomas (e.g., those associated with skin cancer, cervical cancer, prostate cancer, pancreatic cancer, anal carcinoma, esophageal cancer, hepatocellular carcinoma, laryngeal cancer, renal cell carcinoma, stomach cancer, testicular cancer, thyroid cancer, breast cancer, lung cancer, ovarian cancer, and colon cancer), sarcomas (e.g., osteosarcoma, chondrosarcoma, fibrosarcoma, Kaposi's sarcoma, uterine sarcoma, and rhabdomyosarcoma), melanomas (e.g., those associated with skin cancer and eye cancer), teratomas, and hematological malignancies (e.g., leukemias, lymphomas, and mylelomas).

[0008] Further embodiments relate to methods for treating a condition in an animal, which methods can include administering to the animal a compound as described herein in an amount that is effective to reduce vascular proliferation or in an amount that is effective to reduce vascular density. Exemplary conditions include neoplasms, such as cancers, as well as other conditions associated with or which rely upon vascularization, including for example, immune and non-immune inflammation, rheumatoid arthritis, chronic articular rheumatism, psoriasis, diabetic retinopathy, neovascular glaucoma, retinopathy of prematurity, macular degeneration, corneal graft rejection, retrolental fibroplasia, rubeosis, capillary proliferation in atherosclerotic plaques, osteoporosis, and the like. In some embodiments, the disease is not cancer.

[0009] Other embodiments relate to methods of inducing vascular collapse in an animal. The methods can include treating said animal with a therapeutically effective amount of a compound as described herein, for example. The therapeutically effective amount of said compound can cause tubulin depolymerization in the vasculature.

[0010] Preferably the animal can be a human. Preferably the disease can be a tumor, a diabetic retinopathy, an age-related macular degeneration, and the like. In some aspects the disease is not cancer or cancer can be specifically excluded from the methods and uses.

[0011] Still further embodiments relate to pharmaceutical compositions for treating or preventing vascular proliferation comprising a pharmaceutically effective amount of a compound disclosed herein together with a pharmaceutically acceptable carrier therefor. The vascular proliferation can be a symptom of a disease, for example, cancer, age-related macular degeneration and diabetic retinopathy. .

[0012] Some embodiments relate to methods of preferentially targeting tumor vasculature over non-tumor tissue vasculature. The methods can include the step of administering to an animal, preferably a human, a compound having the structure of Formula (I) as described herein. The non-tumor tissue can be, for example, skin, muscle, brain, kidney, heart, spleen, gut, and the like. The tumor vasculature can be preferentially targeted over non-tumor tissue vasculature, for example, by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% and 90%.

[0013] Other embodiments relate to methods of preferentially targeting tumor vasculature over non-tumor tissue vasculature, which methods can include administering to an animal an agent that preferentially targets tumor vasculature over non-tumor tissue vasculature.

[0014] Further embodiments relate to uses of a compound as described herein in the preparation of a medicament for the treatment of a condition associated with increased vasculature or which relies upon vasculature. In some aspects the condition can be cancer, while in others, cancers particular types or all cancers are specifically excluded. The condition can be any other that is associated with hypervascularization, associated with vasculature or which relies upon vasculature. Examples include immune and nonimmune inflammation, rheumatoid arthritis, chronic articular rheumatism, psoriasis, diabetic retinopathy, neovascular glaucoma, retinopathy of prematurity, macular degeneration, corneal graft rejection, retrolental fibroplasia, rubeosis, capillary proliferation in atherosclerotic plaques, osteoporosis, and the like. Brief Description of the Drawings

[0015] FIGURE 1 is a graph depicting the fluorescence emission spectra of tubulin in the presence of increasing concentrations of KPU-134.

[0016] FIGURE 2 is a graph depicting the change in fluorescence emission maxima with K_<j fit.

Detailed Description of the Preferred Embodiment

[0017] Each reference cited herein, including the U.S. patents cited herein, is to be considered incorporated by reference in its entirety into this specification, to the full extent permissible by law.

[0018] The disclosure provides methods for the synthetic preparation of compounds, including novel compounds, including dehydrophenylahistin analogs, and provides methods for producing pharmaceutically acceptable cell cycle inhibitors, antitumor agents and antifungal agents in relatively high yield, wherein said compounds and/or their derivatives are among the active ingredients in these cell cycle inhibitors, antitumor agents and antifungal agents. Other objects include providing novel compounds not obtainable by currently available, non-synthetic methods. It is also an object to provide a method of treating cancer, particularly human cancer, comprising the step of administering an effective tumor-growth inhibiting amount of a member of a class of new anti-tumor compounds. This invention also provides a method for preventing or treating a pathogenic fungus in a subject which involves administering to the subject an effective anti-fungal amount of a member of a class of new anti-fungal compounds, e.g., administering a compound disclosed herein in an amount and manner which provides the intended antifungal effect. In the preferred embodiment of the compounds and methods of making and using such compounds disclosed herein, but not necessarily in all embodiments of the present invention, these objectives are met.

[0019] Some embodiments include compounds of Formula (I), described above.

[0020] Also provided are pharmaceutically acceptable salts and pro-drug esters of the compound of Formula (I) and provides methods of synthesizing such compounds by the methods disclosed herein. [0021] The term "pro-drug ester," especially when referring to a pro-drug ester of the compound of Formula (I) synthesized by the methods disclosed herein, refers to a chemical derivative of the compound that is rapidly transformed in vivo to yield the compound, for example, by hydrolysis in blood or inside tissues. The term "pro-drug ester" refers to derivatives of the compounds disclosed herein formed by the addition of any of several ester-forming groups that are hydrolyzed under physiological conditions. Examples of pro-drug ester groups include pivoyloxymethyl, acetoxymethyl, phthalidyl, indanyl and methoxymethyl, as well as other such groups known in the art, including a (5- R-2-oxo-l,3-dioxolen-4-yl)methyl group. Other examples of pro-drug ester groups can be found in, for example, T. Higuchi and V. Stella, in "Pro-drugs as Novel Delivery Systems", Vol. 14, A.C.S. Symposium Series, American Chemical Society (1975); and "Bioreversible Carriers in Drug Design: Theory and Application", edited by E. B. Roche, Pergamon Press: New York, 14-21 (1987) (providing examples of esters useful as prodrugs for compounds containing carboxyl groups).

[0022] The term "pro-drug ester," as used herein, also refers to a chemical derivative of the compound that is rapidly transformed in vivo to yield the compound, for example, by hydrolysis in blood. The term "pro-drug ester" refers to derivatives of the compounds disclosed herein formed by the addition of any of several ester-forming groups that are hydrolyzed under physiological conditions. Examples of pro-drug ester groups include pivoyloxymethyl, acetoxymethyl, phthalidyl, indanyl and methoxymethyl, as well as other such groups known in the art, including a (5-R-2-oxo-l,3-dioxolen-4- yl)methyl group. Other examples of pro-drug ester groups can be found in, for example, T. Higuchi and V. Stella, in "Pro-drugs as Novel Delivery Systems", Vol. 14, A.C.S. Symposium Series, American Chemical Society (1975); and "Bioreversible Carriers in Drug Design: Theory and Application", edited by E. B. Roche, Pergamon Press: New York, 14-21 (1987) (providing examples of esters useful as prodrugs for compounds containing carboxyl groups).

[0023] The term "pharmaceutically acceptable salt," especially when referring to a pharmaceutically acceptable salt of the compound of Formula (I), refers to any pharmaceutically acceptable salts of a compound, and preferably refers to an acid addition salt of a compound. Preferred examples of pharmaceutically acceptable salt are the alkali metal salts (sodium or potassium), the alkaline earth metal salts (calcium or magnesium), or ammonium salts derived from ammonia or from pharmaceutically acceptable organic amines, for example C1-C7 alkylamine, cyclohexylamine, triethanolamine, ethylenediamine or tris-(hydroxymethyl)-aminomethane. With respect to compounds synthesized by the method that are basic amines, the preferred examples of pharmaceutically acceptable salts are acid addition salts of pharmaceutically acceptable inorganic or organic acids, for example, hydrohalic, sulfuric, phosphoric acid or aliphatic or aromatic carboxylic or sulfonic acid, for example acetic, succinic, lactic, malic, tartaric, citric, ascorbic, nicotinic, methanesulfonic, p-toluensulfonic or naphthalenesulfonic acid.

[0024] Preferred pharmaceutical compositions disclosed herein include pharmaceutically acceptable salts and pro-drug esters of the compound of Formula (I) synthesized by the method disclosed herein. Accordingly, if the manufacture of pharmaceutical formulations involves intimate mixing of the pharmaceutical excipients and the active ingredient in its salt form, then it is preferred to use pharmaceutical excipients which are non-basic, that is, either acidic or neutral excipients.

[0025] The term "halogen atom," as used herein, means any one of the radio- stable atoms of column 7 of the Periodic Table of the Elements, i.e., fluorine, chlorine, bromine, or iodine, with fluorine and chlorine being preferred.

[0026] The term "alkyl," as used herein, means any unbranched or branched, substituted or unsubstituted, saturated hydrocarbon, with CrC₆ unbranched, saturated, unsubstituted hydrocarbons being preferred, with methyl, ethyl, iosbutyl, and tert-butyl being most preferred. Among the substituted, saturated hydrocarbons, CrC₆ mono- and di- and per-halogen substituted saturated hydrocarbons and amino-substituted hydrocarbons are preferred, with perfluromethyl, perchloromethyl, perfluoro-tert-butyl, and perchloro-tert-butyl being the most preferred. The term "substituted" has its ordinary meaning, as found in numerous contemporary patents from the related art. See, for example, U.S. Patent Nos. 6,583,143, 6,509,331; 6,506,787; 6,500,825; 5,922,683; 5,886,210; 5,874,443; and 6,350,759. Specifically, the definition of substituted is as broad as that provided in U.S. Patent No. 6,583,143, which defines the term substituted as any groups such as alkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, heterocycle and heterocyclealkyl, wherein at least one hydrogen atom is replaced with a substituent. The term "substituted" is also as broad as the definition provided in U.S. Patent No. 6,509,331, which defines the term "substituted alkyl" such that it refers to an alkyl group, preferably of from 1 to 10 carbon atoms, having from 1 to 5 substituents, and preferably 1 to 3 substituents, selected from the group consisting of alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyacylamino, cyano, halogen, hydroxyl, carboxyl, carboxylalkyl, keto, thioketo, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclic, heterocyclooxy, hydroxyamino, alkoxyamino, nitro,— SO-alkyl,— SO-substituted alkyl,— SO-aryl,— SO- heteroaryl, — S0₂-alkyl, — S0₂- substituted alkyl, — S0₂-aryl and -S0₂-heteroaryl. The other above-listed patents also provide standard definitions for the term "substituted" that are well-understood by those of skill in the art. The term "cycloalkyl" refers to any non- aromatic hydrocarbon ring, preferably having five to twelve atoms comprising the ring. The term "acyl" refers to alkyl or aryl groups derived from an oxoacid, with an acetyl group being preferred.

[0027] The term "alkenyl," as used herein, means any unbranched or branched, substituted or unsubstituted, unsaturated hydrocarbon including polyunsaturated hydrocarbons, with CrC₆ unbranched, mono-unsaturated and di- unsaturated, unsubstituted hydrocarbons being preferred, and mono-unsaturated, di- halogen substituted hydrocarbons being most preferred. In the Ri and R4 positions, of the compound of structure (I) a z-isoprenyl moiety is particularly preferred. The term "cycloalkenyl" refers to any non-aromatic hydrocarbon ring, preferably having five to twelve atoms comprising the ring.

[0028] The terms "aryl," "substituted aryl," "heteroaryl," and "substituted heteroaryl," as used herein, refer to aromatic hydrocarbon rings, preferably having five, six, or seven atoms, and most preferably having six atoms comprising the ring. "Heteroaryl" and "substituted heteroaryl," refer to aromatic hydrocarbon rings in which at least one heteroatom, e.g., oxygen, sulfur, or nitrogen atom, is in the ring along with at least one carbon atom.

[0029] The term "alkoxy" refers to any unbranched, or branched, substituted or unsubstituted, saturated or unsaturated ether, with Ci-C₆ unbranched, saturated, unsubstituted ethers being preferred, with methoxy being preferred, and also with dimethyl, diethyl, methyl-isobutyl, and methyl-tert-butyl ethers also being preferred. The term "cycloalkoxy" refers to any non-aromatic hydrocarbon ring, preferably having five to twelve atoms comprising the ring.

[0030] The terms "purified," "substantially purified," and "isolated" as used herein refer to the compound being free of other, dissimilar compounds with which the compound is normally associated in its natural state, so that the compound of the invention comprises at least 0.5%, 1%, 5%, 10%, or 20%, and most preferably at least 50% or 75% of the mass, by weight, of a given sample.

[0031] The compound of Formula (I) may be chemically synthesized or produced from reagents known and available in the art. For example, modifications of diacyldiketopiperazine (diacetyldiketopiperazine) have been described, for example, by Loughlin et ah, 2000 Bioorg Med Chem Lett 10:91 or by Brocchini et al. in WO 95/21832. The diacyldiketopiperazine (diacetyldiketopiperazine) may be prepared, for example, by diacetylation of inexpensive 2,5-piperazinedione (TCI Cat. No. G0100, 25 g) with sodium acetate and sodium anhydride. The diacetyl structure of the activated deketopiperazine can be replaced with other acyl groups, to include carbamates such as Boc (t-butoxycarbonyl), Z (benzoyloxycarbonyl).

[0032] The imidazolecarboxaldehyde may be prepared, for example, according the procedure disclosed in Hayashi et ah, 2000 J Organic Chem 65: 8402 as depicted below:

)

88% 88% 96%

99% 68% 92%

77% 48% 59%

95% [0033] Another example of an imidazolecarboxaldehyde derivative is an imidazole-4-carboxaldehyde 15 derivative which can be produced from, for example, a commercially available beta-ketoester 18 (TCI Cat, No. P1031, 25mL) by the following route

95%

[0034] The synthetic method disclosed herein may be preferably performed in the presence of cesium carbonate as a base in DMF and in a deoxygenated atmosphere. The inert atmosphere circumvents the probable oxidation of activated a-carbon atoms of the diketopiperazine ring during the treatment with cesium carbonate (see below) as reported, for example, by Watanabe et ah, 18^th International Congress of Heterocyclic Chemistry in Yokohama, Ja an (30 July 2001), Abstract, page 225.

Air-oxidation of Activated Carbonyl Compounds with Cesium Salts

[0035] Other embodiments of the synthetic method involve modifications to the compounds used in or otherwise involved in the synthesis of compounds represented by Formula (I).

Pharmaceutical Compositions

[0036] The present invention also encompasses the compounds disclosed herein, optionally and preferably produced by the methods disclosed herein, in pharmaceutical compositions comprising a pharmaceutically acceptable carrier prepared for storage and subsequent administration, which have a pharmaceutically effective amount of the products disclosed above in a pharmaceutically acceptable carrier or diluent. Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A.R. Gennaro edit. 1985). Preservatives, stabilizers, dyes and even flavoring agents may be provided in the pharmaceutical composition. For example, sodium benzoate, ascorbic acid and esters of p-hydroxybenzoic acid may be added as preservatives. In addition, antioxidants and suspending agents may be used.

[0037] The compositions may be formulated and used as tablets, capsules, or elixirs for oral administration; suppositories for rectal administration; sterile solutions, suspensions for injectable administration; patches for transdermal administration, and sub-dermal deposits and the like. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection or infusion, or as emulsions. Suitable excipients are, for example, water, saline, dextrose, mannitol, lactose, lecithin, albumin, sodium glutamate, cysteine hydrochloride, human serum albumin and the like. In addition, if desired, the injectable pharmaceutical compositions may contain minor amounts of nontoxic auxiliary substances, such as wetting agents, pH buffering agents, and the like. If desired, absorption enhancing preparations (for example, liposomes), may be utilized.

[0038] Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or other organic oils such as soybean, grapefruit or almond oils, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents that increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.

[0039] Pharmaceutical preparations for oral use may be obtained by combining the active compounds with solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate. Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses. For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses. Such formulations can be made using methods known in the art (see, for example, U.S. Patent Nos. 5,733,888 (injectable compositions); 5,726,181 (poorly water soluble compounds); 5,707,641 (therapeutically active proteins or peptides); 5,667,809 (lipophilic agents); 5,576,012 (solubilizing polymeric agents); 5,707,615 (anti-viral formulations); 5,683,676 (particulate medicaments); 5,654,286 (topical formulations); 5,688,529 (oral suspensions); 5,445,829 (extended release formulations); 5,653,987 (liquid formulations); 5,641,515 (controlled release formulations) and 5,601,845 (spheroid formulations).

[0040] Further disclosed herein are various pharmaceutical compositions well known in the pharmaceutical art for uses that include intraocular, intranasal, and intraauricular delivery. Pharmaceutical formulations include aqueous ophthalmic solutions of the active compounds in water-soluble form, such as eyedrops, or in gellan gum (Shedden et al., 2001 Clin Ther 23(3):440-50) or hydrogels (Mayer et al., 1996 Ophthalmologica 210:101-3); ophthalmic ointments; ophthalmic suspensions, such as microparticulates, drug-containing small polymeric particles that are suspended in a liquid carrier medium (Joshi, A., 1994 J Ocul Pharmacol 10:29-45), lipid-soluble formulations (Aim et al., 1989 Prog Clin Biol Res 312:447-58), and microspheres (Mordenti, 1999 Toxicol Sci 52:101-6); and ocular inserts. Such suitable pharmaceutical formulations are most often and preferably formulated to be sterile, isotonic and buffered for stability and comfort. Pharmaceutical compositions may also include drops and sprays often prepared to simulate in many respects nasal secretions to ensure maintenance of normal ciliary action. As disclosed in Remington's Pharmaceutical Sciences (Mack Publishing, 18^th Edition), and well-known to those skilled in the art, suitable formulations are most often and preferably isotonic, slightly buffered to maintain a pH of 5.5 to 6.5, and most often and preferably include antimicrobial preservatives and appropriate drug stabilizers. Pharmaceutical formulations for intraauricular delivery include suspensions and ointments for topical application in the ear. Common solvents for such aural formulations include glycerin and water.

[0041] The compound of Formula (I) can be administered by either oral or a non-oral pathways. When administered orally, it can be administered in capsule, tablet, granule, spray, syrup, or other such form. When administered non-orally, it can be administered as an aqueous suspension, an oily preparation or the like or as a drip, suppository, salve, ointment or the like, when administered via injection or infusion, subcutaneously, intreperitoneally, intravenously, intramuscularly, or the like. Similarly, it may be administered topically, rectally, or vaginally, as deemed appropriate by those of skill in the art for bringing the compound into optimal contact with a tumor, thus inhibiting the growth of the tumor. Local administration at the site of the tumor is also contemplated, either before or after tumor resection, as are controlled release formulations, depot formulations, and infusion pump delivery.

Methods of Administration

[0042] The present invention also encompasses methods for making and for administering the disclosed chemical compounds and the disclosed pharmaceutical compositions. Such disclosed methods include, among others, (a) administration though oral pathways, which administration includes administration in capsule, tablet, granule, spray, syrup, or other such forms; (b) administration through non-oral pathways, which administration includes administration as an aqueous suspension, an oily preparation or the like or as a drip, suppository, salve, ointment or the like; administration via injection or infusion, subcutaneously, intraperitoneally, intravenously, intramuscularly, intradermally, or the like; as well as (c) administration topically, (d) administration rectally, or (e) administration vaginally, as deemed appropriate by those of skill in the art for bringing the compound into contact with living tissue; and (f) administration via controlled released formulations, depot formulations, and infusion pump delivery. As further examples of such modes of administration and as further disclosure of modes of administration, disclosed herein are various methods for administration of the disclosed chemical compounds and pharmaceutical compositions including modes of administration through intraocular, intranasal, and intraauricular pathways.

[0043] The pharmaceutically effective amount of the dehydrophenylahistin or dehydrophenylahistin analog composition required as a dose will depend on the route of administration, the type of animal, including human, being treated, and the physical characteristics of the specific animal under consideration. The dose can be tailored to achieve a desired effect, but will depend on such factors as weight, diet, concurrent medication and other factors which those skilled in the medical arts will recognize.

[0044] In practicing the methods, the products or compositions can be used alone or in combination with one another, or in combination with other therapeutic or diagnostic agents. For example, as disclosed herein, the compounds disclosed herein are effective in the treatment of cancer when used in combination with other actives, specifically other chemotherapeutics, for example biologies and the specific chemo therapeutics CPT-11, Taxotene (docataxel) and paclitaxel. The compounds disclosed herein are also effective in the treatment of cancer when used in combination with other actives, including anti-vascular agents, anti-angiogenenic agents, such as Erbuitux (Imclone/bristol-Myers) and Iressa (AstraZeneca), other VEGF inhibitors and biologies, more specifically, at least one anti-VEGF antibodies, especially monoclonal antibodies to the VEGF receptor, including DC 101, a rat monoclonal antibody, which blocks the mouse VEGF receptor 2 (flk-1). Such combinations may be utilized in vivo, ordinarily in a mammal, preferably in a human, or in vitro. In employing them in vivo, the disclosed compounds, alone or in combination with other chemotherapeutics or other biologic products, may be administered to the mammal in a variety of ways, including parenterally, intravenously, via infusion or injection, subcutaneously, intramuscularly, colonically, rectally, vaginally, nasally or intraperitoneally, employing a variety of dosage forms. Such methods may also be applied to testing chemical activity in vivo.

[0045] As will be readily apparent to one skilled in the art, the useful in vivo dosage to be administered and the particular mode of administration will vary depending upon the age, weight and mammalian species treated, the particular compounds employed, and the specific use for which these compounds are employed. The determination of effective dosage levels, that is the dosage levels necessary to achieve the desired result, can be accomplished by one skilled in the art using routine pharmacological methods. Typically, human clinical applications of products are commenced at lower dosage levels, with dosage level being increased until the desired effect is achieved. Alternatively, acceptable in vitro studies can be used to establish useful doses and routes of administration of the compositions identified by the present methods using established pharmacological methods.

[0046] In non-human animal studies, applications of potential products are commenced at higher dosage levels, with dosage being decreased until the desired effect is no longer achieved or adverse side effects disappear. The dosage may range broadly, depending upon the desired affects and the therapeutic indication. Typically, dosages may be between about 10 microgram/kg and 100 mg/kg body weight, preferably between about 100 microgram/kg and 10 mg/kg body weight. Alternatively dosages may be based and calculated upon the surface area of the patient, as understood by those of skill in the art. Administration may be oral on an every third day, every other day, daily, twice daily, or thrice daily basis.

[0047] The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. See for example, Fingl et ah, in The Pharmacological Basis of Therapeutics, 1975. It should be noted that the attending physician would know how to and when to terminate, interrupt, or adjust administration due to toxicity, or to organ dysfunctions. Conversely, the attending physician would also know to adjust treatment to higher levels if the clinical response were not adequate (precluding toxicity). The magnitude of an administrated dose in the management of the disorder of interest will vary with the severity of the condition to be treated and to the route of administration. The severity of the condition may, for example, be evaluated, in part, by standard prognostic evaluation methods. Further, the dose and perhaps dose frequency, will also vary according to the age, body weight, and response of the individual patient. A program comparable to that discussed above may be used in veterinary medicine.

[0048] Depending on the specific conditions being treated, such agents may be formulated and administered systemically or locally. A variety of techniques for formulation and administration may be found in Remington's Pharmaceutical Sciences, 18th Ed., Mack Publishing Co., Easton, PA (1990). Suitable administration routes may include oral, rectal, transdermal, vaginal, transmucosal, or intestinal administration; parenteral delivery, including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, via infusion, intraperitoneal, intranasal, or intraocular injections.

[0049] For injection or infusion, the agents may be formulated in aqueous solutions, for example, in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiological saline buffer. For such transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art. Use of pharmaceutically acceptable carriers to formulate the compounds herein disclosed for the practice of the invention into dosages suitable for systemic administration is within the scope of the invention. With proper choice of carrier and suitable manufacturing practice, the compositions disclosed herein, in particular, those formulated as solutions, may be administered parenterally, such as by intravenous injection or infusion. The compounds can be formulated readily using pharmaceutically acceptable carriers well known in the art into dosages suitable for oral administration. Such carriers enable the compounds to be formulated as tablets, pills, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated.

[0050] Agents intended to be administered intracellularly may be administered using techniques well known to those of ordinary skill in the art. For example, such agents may be encapsulated into liposomes, then administered as described above. All molecules present in an aqueous solution at the time of liposome formation are incorporated into the aqueous interior. The liposomal contents are both protected from the external micro-environment and, because liposomes fuse with cell membranes, are efficiently delivered into the cell cytoplasm. Additionally, due to their hydrophobicity, small organic molecules may be directly administered intracellularly.

[0051] Determination of the effective amounts is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein. In addition to the active ingredients, these pharmaceutical compositions may contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. The preparations formulated for oral administration may be in the form of tablets, dragees, capsules, or solutions. The pharmaceutical compositions may be manufactured in a manner that is itself known, for example, by means of conventional mixing, dissolving, granulating, dragee-making, levitating, emulsifying, encapsulating, entrapping, or lyophilizing processes.

[0052] Compounds disclosed herein can be evaluated for efficacy and toxicity using known methods. For example, the toxicology of a particular compound, or of a subset of the compounds, sharing certain chemical moieties, may be established by determining in vitro toxicity towards a cell line, such as a mammalian, and preferably human, cell line. The results of such studies are often predictive of toxicity in animals, such as mammals, or more specifically, humans. Alternatively, the toxicity of particular compounds in an animal model, such as mice, rats, rabbits, or monkeys, may be determined using known methods. The efficacy of a particular compound may be established using several art recognized methods, such as in vitro methods, animal models, or human clinical trials. Art-recognized in vitro models exist for nearly every class of condition, including the conditions abated by the compounds disclosed herein, including cancer, cardiovascular disease and various fungal infections. Similarly, acceptable animal models may be used to establish efficacy of chemicals to treat such conditions. When selecting a model to determine efficacy, the skilled artisan can be guided by the state of the art to choose an appropriate model, dose, and route of administration, and regime. Of course, human clinical trials can also be used to determine the efficacy of a compound in humans.

[0053] When used as an anti-cancer agent, or a tumor-growth-inhibiting compound, the compounds disclosed herein may be administered by either oral or a non- oral pathways. When administered orally, it can be administered in capsule, tablet, granule, spray, syrup, or other such form. When administered non-orally, it can be administered as an aqueous suspension, an oily preparation or the like or as a drip, suppository, salve, ointment or the like, when administered via injection or infusion, subcutaneously, intreperitoneally, intravenously, intramuscularly, intradermally, or the like. Similarly, it may be administered topically, rectally, or vaginally, as deemed appropriate by those of skill in the art for bringing the compound into optimal contact with a tumor, thus inhibiting the growth of the tumor. Local administration at the site of the tumor or other disease condition is also contemplated, either before or after tumor resection, or as part of an art-recognized treatment of the disease condition. Controlled release formulations, depot formulations, and infusion pump delivery are similarly contemplated.

[0054] When used as an anti-cancer agent or an anti-tumor agent, may be orally or non-orally administered to a human patient in the amount of about .0007 mg/day to about 7,000 mg/day of the active ingredient, and more preferably about 0.07 mg/day to about 70 mg/day of the active ingredient at, preferably, one time per day or, less preferably, over two to about ten times per day. Alternatively and also preferably, the compound may preferably be administered in the stated amounts continuously by, for example, an intravenous drip. Thus, for a patient weighing 70 kilograms, the preferred daily dose of the active anti-tumor ingredient would be about 0.0007 mg/kg/day to about 35 mg/kg/day including 1.0 mg/kg/day and 0.5 mg/kg/day, and more preferable, from 0.007 mg/kg/day to about 0.050 mg/kg/day, including 0.035 mg/kg/day. Nonetheless, as will be understood by those of skill in the art, in certain situations it may be necessary to administer the anti-tumor compound in amounts that excess, or even far exceed, the above-stated, preferred dosage range to effectively and aggressively treat particularly advanced or lethal tumors.

[0055] When used as an antifungal agent the preferable amount of the compound disclosed herein effective in the treatment or prevention of a particular fungal pathogen will depend in part on the characteristics of the fungus and the extent of infection, and can be determined by standard clinical techniques. In vitro or in vivo assays may optionally be employed to help identify optimal dosage ranges. Effective doses may be extrapolated from dose-response curves derived from in vitro analysis or preferably from animal models. The precise dosage level should be determined by the attending physician or other health care provider and will depend upon well known factors, including route of administration, and the age, body weight, sex and general health of the individual; the nature, severity and clinical stage of the infection; the use (or not) of concomitant therapies.

[0056] The effective dose of the compound disclosed herein will typically be in the range of about 0.01 to about 50 mg/kgs, preferably about 0.1 to about 10 mg/kg of mammalian body weight per day, administered in single or multiple doses. Generally, the compound may be administered to patients in need of such treatment in a daily dose range of about 1 to about 2000 mg per patient. [0057] To formulate the dosage including the compounds disclosed herein as a tumor- growth-inhibiting compound, known surface active agents, excipients, smoothing agents, suspension agents and pharmaceutically acceptable film-forming substances and coating assistants, and the like may be used. Preferably alcohols, esters, sulfated aliphatic alcohols, and the like may be used as surface active agents; sucrose, glucose, lactose, starch, crystallized cellulose, mannitol, light anhydrous silicate, magnesium aluminate, magnesium methasilicate aluminate, synthetic aluminum silicate, calcium carbonate, sodium acid carbonate, calcium hydrogen phosphate, calcium carboxymethyl cellulose, and the like may be used as excipients; magnesium stearate, talc, hardened oil and the like may be used as smoothing agents; coconut oil, olive oil, sesame oil, peanut oil, soya may be used as suspension agents or lubricants; cellulose acetate phthalate as a derivative of a carbohydrate such as cellulose or sugar, or methyiacetate-methacrylate copolymer as a derivative of polyvinyl may be used as suspension agents; and plasticizers such as ester phthalates and the like may be used as suspension agents. In addition to the foregoing preferred ingredients, sweeteners, fragrances, colorants, preservatives and the like may be added to the administered formulation of the compound, particularly when the compound is to be administered orally.

[0058] The compositions disclosed herein in a pharmaceutical compositions may also comprise a pharmaceutically acceptable carrier. Such compositions may be prepared for storage and for subsequent administration. Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A.R. Gennaro edit. 1985). For example, such compositions may be formulated and used as tablets, capsules or solutions for oral administration; suppositories for rectal or vaginal administration; sterile solutions or suspensions for injectable administration. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection or infusion, or as emulsions. Suitable excipients include, but are not limited to, saline, dextrose, mannitol, lactose, lecithin, albumin, sodium glutamate, cysteine hydrochloride, and the like. In addition, if desired, the injectable pharmaceutical compositions may contain minor amounts of nontoxic auxiliary substances, such as wetting agents, pH buffering agents, and the like. If desired, absorption enhancing preparations (for example, liposomes), may be utilized. [0059] The pharmaceutically effective amount of the composition required as a dose will depend on the route of administration, the type of animal being treated, and the physical characteristics of the specific animal under consideration. The dose can be tailored to achieve a desired effect, but will depend on such factors as weight, diet, concurrent medication and other factors which those skilled in the medical arts will recognize.

[0060] The products or compositions, as described above, may be used alone or in combination with one another, or in combination with other therapeutic or diagnostic agents. Specifically, the compounds can be administered or used in combination with treatments such as chemotherapy, radiation, and biologic therapies. In some embodiments the compounds can be administered or used with a chemotherapeutic agent. Examples of such chemotherapeutic s include Alkaloids, alkylating agents, antibiotics, antimetabolites, enzymes, hormones, platinum compounds, immuno therapeutics (antibodies, T-cells, epitopes), BRMs, and the like. Examples include, Vincristine, Vinblastine, Vindesine, Paclitaxel (Taxol), Docetaxel, topoisomerase inhibibitors epipodophyllotoxins (Etoposide (VP- 16), Teniposide (VM-26)), Camptothecin, nitrogen mustards (cyclophosphamide), Nitrosoureas, Carmustine, lomustine, dacarbazine, hydroxymethylmelamine, thiotepa and mitocycin C, Dactinomycin (Actinomycin D), anthracycline antibiotics (Daunorubicin, Daunomycin, Cerubidine), Doxorubicin (Adriamycin), Idarubicin (Idamycin), Anthracenediones (Mitoxantrone), Bleomycin (Blenoxane), Plicamycin (Mithramycin, Antifolates (Methotrexate (Folex, Mexate)), purine antimetabolites (6-mercaptopurine (6- MP, Purinethol) and 6- thioguanine (6-TG). The two major anticancer drugs in this category are 6-mercaptopurine and 6-thioguanine, Chlorodeoxyadenosine and Pentostatin, Pentostatin (2'-deoxycoformycin), pyrimidine antagonists, fluoropyrimidines (5- fluorouracil(Adrucil), 5-fluorodeoxyuridine (FdUrd) (Floxuridine)), Cytosine Arabinoside (Cytosar, ara-C), Fludarabine, L-ASPARAGINASE, Hydroxyurea, glucocorticoids, antiestrogens, tamoxifen, nonsteroidal antiandrogens, flutamide, aromatase inhibitors Anastrozole(Arimidex), Cisplatin, 6-Mercaptopurine and Thioguanine, Methotrexate, Cytoxan, Cytarabine, L-Asparaginase, Steroids: Prednisone and Dexamethasone. Also, proteasome inhibitors such as bortezomib or marizomib can be used in combination with the instant compounds, for example. Examples of biologies can include agents such as TRAIL antibodies to TRAIL, integrins such as alpha- V-beta-3 (ανβ3) and / or other cytokine/growth factors that are involved in angiogenesis, VEGF, EGF, FGF and PDGF, immunotherapeutics, such as proteasome inhibitors, T cells, T cells vaccines, and the like. In some aspects, the compounds can be conjugated to or delivered with an antibody. Radiation therapy includes, but is not limited to, treatment with X-ray radiation and proton beam therapy. The above-described combination methods can be used to treat a variety of conditions, including cancer and neoplastic diseases, inflammation, and microbial infections.

[0061] These products or compositions can be utilized in vivo or in vitro. The useful dosages and the most useful modes of administration will vary depending upon the age, weight and animal treated, the particular compounds employed, and the specific use for which these composition or compositions are employed. The magnitude of a dose in the management or treatment for a particular disorder will vary with the severity of the condition to be treated and to the route of administration, and depending on the disease conditions and their severity, the compositions may be formulated and administered either systemically or locally. A variety of techniques for formulation and administration may be found in Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing Co., Easton, PA (1990).

[0062] To formulate the compounds of Formula (I), preferably synthetically produced according to the methods disclosed herein, known surface active agents, excipients, smoothing agents, suspension agents and pharmaceutically acceptable film- forming substances and coating assistants, and the like may be used. Preferably alcohols, esters, sulfated aliphatic alcohols, and the like may be used as surface active agents; sucrose, glucose, lactose, starch, crystallized cellulose, mannitol, light anhydrous silicate, magnesium aluminate, magnesium methasilicate aluminate, synthetic aluminum silicate, calcium carbonate, sodium acid carbonate, calcium hydrogen phosphate, calcium carboxymethyl cellulose, and the like may be used as excipients; magnesium stearate, talc, hardened oil and the like may be used as smoothing agents; coconut oil, olive oil, sesame oil, peanut oil, soya may be used as suspension agents or lubricants; cellulose acetate phthalate as a derivative of a carbohydrate such as cellulose or sugar, or methyiacetate- methacrylate copolymer as a derivative of polyvinyl may be used as suspension agents; and plasticizers such as ester phthalates and the like may be used as suspension agents. In addition to the foregoing preferred ingredients, sweeteners, fragrances, colorants, preservatives and the like may be added to the administered formulation of the compound produced by the method, particularly when the compound is to be administered orally. [0063] The compounds disclosed herein may be orally or non-orally administered to a human patient in the amount of about 0.001 mg/kg/day to about 10,000 mg/kg/day of the active ingredient, and more preferably about 0.1 mg/kg/day to about 100 mg/kg/day of the active ingredient at, preferably, once every three days on a cyclic basis, once every other day, one time per day, twice per day, or less preferably, over two to about ten times per day. Alternatively and also preferably, the compound produced by the method may preferably be administered in the stated amounts continuously by, for example, an intravenous drip. Thus, for the example of a patient weighing 70 kilograms, the preferred daily dose of the active anti-tumor ingredient would be about 0.07 mg/day to about 700 grams/day, and more preferable, 7 mg/day to about 7 grams/day. Nonetheless, as will be understood by those of skill in the art, in certain situations it may be necessary to administer the compound in amounts that excess, or even far exceed, the above-stated, preferred dosage range to effectively and aggressively treat particularly advanced or lethal tumors.

[0064] In the case of using the compounds disclosed herein as a biochemical test reagent, the compound inhibits the progression of the cell cycle when it is dissolved in an organic solvent or hydrous organic solvent and it is directly applied to any of various cultured cell systems. Usable organic solvents include, for example, methanol, methylsulfoxide, and the like. The formulation can, for example, be a powder, granular or other solid inhibitor, or a liquid inhibitor prepared using an organic solvent or a hydrous organic solvent. While a preferred concentration of the compound produced by the method of the invention for use as a cell cycle inhibitor is generally in the range of about 1 to about 100 μg/ml, the most appropriate use amount varies depending on the type of cultured cell system and the purpose of use, as will be appreciated by persons of ordinary skill in the art. Also, in certain applications it may be necessary or preferred to persons of ordinary skill in the art to use an amount outside the foregoing range.

[0065] From a pharmaceutical perspective, certain embodiments provide methods for preventing or treating fungal infections and/or a pathogenic fungus in a subject, involve administering to the subject a composition including a compound disclosed herein, for example, administering the compound in an amount and manner which provides the intended antifungal effect.

[0066] Other embodiments include the treatment or prevention of infection in a patient by a pathogenic fungus such as those listed above or referred to below. [0067] Another embodiment relates to the treatment or prevention of infection in a patient by a pathogenic fungus which is resistant to one or more other antifungal agents, especially an agent other than compound disclosed herein, including e.g. amphotericin B or analogs or derivatives thereof (including 14(s)-hydroxyamphotericin B methyl ester, the hydrazide of amphotericin B with l-amino-4-methylpiperazine, and other derivatives) or other polyene macrolide antibiotics, including, e.g., nystatin, candicidin, pimaricin and natamycin; flucytosine; griseofulvin; echinocandins or aureobasidins, including naturally occurring and semi-synthetic analogs; dihydrobenzo[a]napthacenequinones; nucleoside peptide antifungals including the polyoxins and nikkomycins; allylamines such as naftifine and other squalene epoxidase inhibitors; and azoles, imidazoles and triazoles such as, e.g., clotrimazole, miconazole, ketoconazole, econazole, butoconazole, oxiconazole, terconazole, itraconazole or fluconazole and the like. For additional conventional antifungal agents and new agents under development, see e.g. Turner and Rodriguez, 1996 Current Pharmaceutical Design, 2:209-224. Another embodiment involves the treatment or prevention of infection in a patient by a pathogenic fungus in cases in which the patient is allergic to, otherwise intolerant of, or nonresponsive to one or more other antifungal agents or in whom the use of other antifungal agents is otherwise contra-indicated. Those other antifungal agents include, among others, those antifungal agents disclosed above and elsewhere herein.

[0068] In the foregoing methods for treatment or prevention, a compound disclosed herein, is administered to the subject in an effective antifungal amount.

[0069] Other embodiments relate to the treatment or prevention of infection by a pathogenic fungus in a patient by administration of a compound disclosed herein, in conjunction with the administration of one or more other antifungal agents, including for example, any of the previously mentioned agents or types of agents (e.g. in combination with treatment with amphotericin B, preferably in a lipid or liposome formulation; an azole or triazole such as fluconazole, for example; an aureobasidin; dihydrobenzo[alnapthacenequinone; or an echinocardin) as well as with a different compound disclosed herein.

[0070] The compound disclosed herein may be administered before, after or at the same time the other antifungal agent is administered. In certain embodiments, the combination therapy will permit the use of reduced amounts of one or both antifungal components, relative to the amount used if used alone. [0071] Still other embodiments relate to administration of a compound disclosed herein to a subject for the treatment or prevention of infection by a pathogenic fungus, where the subject is immunosuppressed or immunocompromised, e.g. as the result of genetic disorder, disease such as diabetes or HIV or other infection, chemotherapy or radiation treatment for cancer or other disease, or drug- or otherwise induced immunosuppression in connection with tissue or organ transplantation or the treatment of an autoimmune disorder. Where the patient is being or will be treated with an immunosuppressive agent, e.g., in connection with a tissue or organ transplantation, a compound disclosed herein may be co-administered with the immunosuppressive agent(s) to treat or prevent a pathogenic fungal infection.

[0072] Another aspect of this invention is the treatment or prevention of infection by a pathogenic fungus in a patient infected, or suspected of being infected, with HIV, by administration of an antifungal compound disclosed herein, in conjunction with the administration of one or more anti-HIV therapeutics (including e.g. HIV protease inhibitors, reverse transcriptase inhibitors or anti-viral agents). The compound disclosed herein may be administered before, after or at the same time as administration of the anti- HIV agent(s).

[0073] Another aspect of this invention is the treatment or prevention of infection by a pathogenic fungus in a patient by administration of an antifungal compound disclosed herein, in conjunction with the administration of one or more other antibiotic compounds, especially one or more antibacterial agents, preferably in an effective amount and regiment to treat or prevent bacterial infection. Again, the compound disclosed herein may be administered before, after or at the same time as administration of the other agent(s).

[0074] Pathogenic fungal infections which may be treated or prevented by the disclosed methods include, among others, Aspergillosis, including invasive pulmonary aspergillosis; Blastomycosis, including profound or rapidly progressive infections and blastomycosis in the central nervous system; Candidiasis, including retrograde candidiasis of the urinary tract, e.g. in patients with kidney stones, urinary tract obstruction, renal transplantation or poorly controlled diabetes mellitus; Coccidioidomycosis, including chronic disease which does not respond well to other chemotherapy; Cryptococcosis; Histopolasmosis; Mucormycosis, including e.g. craniofacial mucormycosis and pulmonary mucormycosis; Paracoccidioidomycosis; and Sporotrichosis. It should be noted that administration of a composition comprising an antifungal amount of one or more compound disclosed herein may be particularly useful for treating or preventing a pathogenic fungal infection in a mammalian subject where the fungus is resistant to one or more other antifungal therapies, or where the use of one or more other antifungal therapies is contraindicated, e.g., as mentioned above.

[0075] Antifungal pharmaceutical compositions containing at least one antifungal compound disclosed herein, are also provided for use in practicing the disclosed methods. Those pharmaceutical compositions may be packaged together with an appropriate package insert containing, inter alia, directions and information relating to their antifungal use. Pharmaceutical compositions are also provided which contain one or more compound disclosed herein together with a second antifungal agent.

Methods of Treating Cancer

[0076] Certain embodiments include the treatment of cancer using a compound described herein. Non-limiting examples of such cancer include carcinomas (e.g., those associated with skin cancer, cervical cancer, prostate cancer, pancreatic cancer, anal carcinoma, esophageal cancer, hepatocellular carcinoma, laryngeal cancer, renal cell carcinoma, stomach cancer, testicular cancer, thyroid cancer, breast cancer, lung cancer, ovarian cancer, and colon cancer), sarcomas (e.g., osteosarcoma, chondrosarcoma, fibrosarcoma, Kaposi's sarcoma, uterine sarcoma, and rhabdomyosarcoma), melanomas (e.g., those associated with skin cancer and eye cancer), teratomas, and hematological malignancies (e.g., leukemias, lymphomas, and mylelomas).

[0077] In some embodiments, the cancer is characterized by a tumor that is supported by vasculature. In such embodiments, the compounds described herein may be used reduce the tumor vasculaturization and thereby treat the cancer.

Methods of Treating Fungal Infections

[0078] Certain embodiments disclosed herein relate to methods for treating or preventing a pathogenic fungal infection, including for example Aspergillosis, including invasive pulmonary aspergillosis; Blastomycosis, including profound or rapidly progressive infections and blastomycosis in the central nervous system; Candidiasis, including retrograde candidiasis of the urinary tract, e.g. in patients with kidney stones, urinary tract obstruction, renal transplantaion or poorly controlled diabetes mellitus; Coccidioidomycosis, including chronic disease which does not respond well to other chemotherapy; Cryptococcosis; Histopolasmosis; Mucormycosis, including e.g. craniofacial mucormycosis and pulmonary mucormycosis; Paracoccidioidomycosis; and Sporotrichosis. The methods may involve administering at least one antifungal compound disclosed herein, as described above, to a human subject such that the fungal infection is treated or prevented. In certain embodiments the compound disclosed herein may be administered in conjunction with administration of one or more non-compound disclosed herein antifungal agents such as amphotericin B, or an imidazole or triazole agent such as those mentioned previously.

[0079] The pathogenic fungal infection may be topical, e.g., caused by, among other organisms, species of Candida, Trichophyton, Microsporum or Epiderinophyton or mucosal, e.g., caused by Candida albicans (e.g. thrush and vaginal candidiasis). The infection may be systemic, e.g., caused by Candida albicans, Cryptococcus neoformans, Aspergillus fumigatus, Coccidiodes, Paracocciciodes, Histoplasma or Blastomyces spp. The infection may also involve eumycotic mycetoma, chromoblastomycosis, cryptococcal meningitits or phycomycosis.

[0080] Further embodiments relate to methods for treating or preventing a pathogenic fungal infection selected from the group consisting of Candida spp. including C albicans, C tropicalis, C. kefyr, C. krusei and C galbrata; Aspergillus spp. including A. fumigatus and A. flavus; Cryptococcus neoibrmans; Blastomyces spp. including Blastomyces dermatitidis; Pneumocystis carinii; Coccidioides immitis; Basidiobolus ranarum; Conidiobolus spp.; Histoplasma capsulatum; Rhizopus spp. including R. oryzae and R. microsporus; Cunninghamella spp.; Rhizoniucor spp.; Paracoccidioides brasiliensis; Pseudallescheria boydii; Rhino sporidium seeberi; and Sporothrix schenckii. Again, the method may involve administering a non-immunosuppressive antifungal compound disclosed herein to a patient in need thereof such that the fungal infection is treated or prevented without inducing an untoward immunosuppressive effect.

[0081] Further embodiments relate to methods for treating or preventing a pathogenic fungal infection which is resistant to other antifungal therapy, including pathogenic fungal infections which are resistant to one or more antifungal agents mentioned elsewhere herein such as amphotericin B, flucytosine, one of the imidazoles or triazoles (including e.g. fluconazole, ketoconazole, itraconazole and the other previously mentioned examples). The methods may involve administering to the patient one or more antifungal compound disclosed herein, in an amount and dosing regimen such that a fungal infection resistant to another antifungal therapy in the subject is treated or prevented.

[0082] Further embodiments relate to methods for treating or preventing a pathogenic fungal infection in a patient who is allergic to, intolerant of or not responsive to another antifungal therapy or in whom the use of other antifungal agents is otherwise contra-indicated, including one or more other antifungal agents mentioned elsewhere herein such as amphotericin B, flucytosine, one of the imidazoles or triazoles (including e.g. fluconazole, ketoconazole, itraconazole and the other previously mentioned examples). The methods may involve administering to such patient one or more antifungal compound disclosed herein, in an amount such that a fungal infection is treated or prevented.

Packaged Compound disclosed herein

[0083] Certain embodiments relate to packaged compound disclosed herein, preferably packaged nonimmunosuppressive antifungal compound disclosed herein, which term is intended to include at least one compound disclosed herein, as described above, packaged with instructions for administering the compound disclosed herein as an antifungal agent without causing a untoward immunosuppressive effects within a human subject. In some embodiments, the non-immunosuppressive antifungal compound disclosed herein is a member of one of the preferred subsets of compounds described above. The compound disclosed herein can be packaged alone with the instructions or can be packaged with another compound disclosed herein, raparnycin or another ingredient or additive, e.g., one or more of the ingredients of the pharmaceutical compositions. The package can contain one or more containers filled with one or more of the ingredients of the phan-naceutical compositions. Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceutical or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.

Labeled compound disclosed herein

[0084] The compounds described herein may also comprise one or more labels, including photoaffinity labels, enzymatic labels (e.g., alkaline phosphatase, horseradish peroxidase), fluorescent labels (e.g., rhodamine, fluorescein), chemiluminescent labels, bioluminescent labels, radiolabels and the like. A photoaffinity label contains a photoactivatable group that forms a covalent link with an adjacent protein upon illumination with visible or ultraviolet (UV) light, and is often used to covalently link small molecules to proteins for binding studies. On example of a photoaffinity label is a benzophenone group, which is a well known photoaffinity label that is activated upon irradiation with UV light. Other photoaffinity labels are well known in the art, and include 8-azidoflavins, azidobenzoyl and benzoylbenzoyl groups. Methods of attaching these various labels to small molecules are also well known in the art. These photoactivatable compounds are useful in determination of protein binding sites, particularly when conjugated to a detectable label such as biotin.

[0085] The following non-limiting examples are meant to describe the preferred methods using certain preferred embodiments. Variations in the details of the particular methods employed and in the precise chemical compositions obtained will undoubtedly be appreciated by those of skill in the art.

EXAMPLE 1

Synthesis of Imidazole-type Precursers (1 J-dimethyl-2-propenyl substituted)

[0086] Reagents: a) LDA, CH₃CHO; b) Tos-Cl, pyridine; c) DBU; d) NaOH; e) C2CI2O2; f) KOOCCH₂COOEt, BuLi; g) S0₂C1₂; h) H₂NCHO, H₂0; i) LiAlH₄; j) Μη(¾; k) l,4-diacetyl-piperazine-2,5-dione, Cs₂C0₃; 1) benzaldehyde, Cs₂C0₃ 3-Hydroxy-2,2-dimethyl-butyric acid methyl ester

[0087] A solution of LDA in heptane/THF/ethylbenzene (2 M, 196 ml, 0.39 mol) was added under argon to a solution of methyl isobutyrate (45 ml, 0.39 mol) in THF (270 ml) at -60° and the resultant mixture was stirred for 30 min. A solution of acetaldehyde (27 ml, 0.48 mol) in THF (45 ml), precooled to -60°, was added slowly and the resulting solution stirred for a further 30 min. Saturated ammonium chloride (50 ml) was added and the solution was allowed to warm to room temperature. The reaction mixture was extracted with ethyl acetate, and the extracts were washed with HCl (2 M), sodium bicarbonate, then brine. The organic layer was dried over magnesium sulfate, filtered, then evaporated to give a clear oil (52.6 g). Distillation 76-82°/30 mmHg gave pure 3-hydroxy-2,2-dimethyl-butyric acid methyl ester (42.3 g, 74%). (Burk et al., J. Am. Chem. Soc , 117:4423-4424 (1995)).

[0088] 1H NMR (400 MHz, CDC1₃) δ 1.15 (d, J = 6.2 Hz, 3H); 1.17 (s, 6H); 2.66 (d, / = 6.2 Hz, 1H, -OH); 3.71 (s, 3H, -OMe); 3.87 (app quintet, J = 6.4 Hz, 1H, H3).

2,2-Dimethyl-3-(toluene-4-sulfonyloxy)-butyric acid methyl ester

[0089] To a cooled (0°) solution of 3-hydroxy-2,2-dimethyl-butyric acid methyl ester (52.0 g, 0.36 mol) in pyridine (100 ml) was added gradually, p-toluene sulfonyl chloride (69.0 g, 0.36 mol). The mixture was allowed to warm to room temperature and was stirred for 60 h. The reaction was again cooled in ice and was acidified by addition of HCl (2 M). The resultant solution was extracted with ethyl acetate, the extracts were washed with HC1, then brine, dried and evaporated to give an oil which formed a white precipitate upon standing. This mixture was dissolved in the minimum amount of ethyl acetate and then light petroleum was added to afford a white precipitate which was collected and washed with more light petroleum. The filtrate was partially evaporated and a second crop of crystals was collected and added to the first to afford 2,2-dimethyl-3-(toluene-4-sulfonyloxy)-butyric acid methyl ester (81.2 g, 76%).

[0090] 1H NMR (400 NMz, CDC1₃) δ 1.12 (s, 3H); 1.13 (s, 3H); 1.24 (d, / = 6.4 Hz, 3H); 2.45 (s, 3H, -PhMe) 3.58 (s, 3H, -OMe); 4.94 (quartet, / = 6.4 Hz, 1H, H3), 7.33 (d, / = 8.0 Hz, 2H), 7.78 (d, / = 8.0 Hz, 2H).

[0091] Evaporation of the final filtrate afforded additional crude 2,2-dimethyl- 3-(toluene-4-sulfonyloxy)-butyric acid methyl ester (19.0 g, 18%).

2,2-Dimethyl-but-3-enoic acid methyl ester

[0092] A solution of 2,2-dimethyl-3-(toluene-4-sulfonyloxy)-butyric acid methyl ester (18.06 g, 0.06 mol) in DBU (15 ml) was heated at 140-160° for 3.5 h. The mixture was allowed to cool to room temperature and was then diluted with ether. The mixture was washed with HC1 (1 M), sodium bicarbonate, then brine. The ethereal layer was dried and partially evaporated to give a concentrated solution of 2,2-dimethyl-but-3- enoic acid methyl ester (10 g). (Savu and Katzenellenbogen, J. Org. Chem, 46:239-250 (1981)). Further evaporation was avoided due to the volatility of the product (bp 102°). (Tsaconas et al, Aust. J. Chem., 53:435-437 (2000)).

[0093] 1H NMR (400 NMz, CDC1₃) δ 1.31 (s, 6H); 3.68 (s, 3H); 5.06 (d, / = 17.1 Hz, 1H, -CH=CH₂); 5.11 (d, / = 10.7 Hz, 1H, -CH=CH₂); 6.03 (dd, / = 17.1, 10.7 Hz, 1H, -CH=CH₂). 2,2-Dimethyl-but-3-enoic acid

[0094] The above ethereal solution of 2,2-dimethyl-but-3-enoic acid methyl ester (10 g) was diluted with ethanol (25 ml), sodium hydroxide (4 M, 22 ml) was added and the mixture was stirred overnight. The solution was partially evaporated to remove the ethanol and the resultant mixture was added to HC1 (1M, 100 ml). The product was extracted with ethyl acetate and the extracts were dried and evaporated to give 2,2- dimethyl-but-3-enoic acid (6.01 g, 88% 2 steps). (Hayashi et al., J. Org. Chem., 65:8402- 8405 (2000).

[0095] 1H NMR (400 MHz, CDC1₃) δ 1.33 (s, 6H); 5.11 (d, J = 10.8 Hz, 1H, - CH=CH₂); 5.15 (d, J = 17.2 Hz, 1H, -CH=CH₂); 6.05 (dd, J = 17.2, 10.8 Hz, 1H, - CH=CH₂).

[0096] Monoethyl hydrogen malonate (Wierenga and Skulnick, "Aliphatic and Aromatic β-keto Esters from Monoethyl Malonate: Ethyl 2-Butyrylacetate," Organic Syntheses Collective Volume 7, 213).

[0097] Ethyl potassium malonate (25.0 g, 0.15 mol) was suspended in water (15.6 ml) and cooled in an ice bath. Concentrated HC1 (12.5 ml) was added dropwise over 30 min, then the mixture was stirred for a further 10 min. The precipitate was filtered, then washed twice with ether. The filtrate was separated and the aqueous phase was extracted with ether. The combined ethereal solutions were dried (MgS0₄) and evaporated to afford, as an oil, monoethyl hydrogen malonate (19.2 g, 99%) which was dried under vacuum overnight (or 50 1 mm for 1 h) prior to use. 4,4-Dimethyl-3-oxo-hex-5-enoic acid ethyl ester

[0098] Oxalyl chloride (3.83 ml, 43.9 mmol) was added dropwise to a cooled (0°) solution of 2,2-dimethyl-but-3-enoic acid (5.0 g, 43.9 mmol) and DMF (1 drop) in anhydrous dichloromethane (25 ml). The mixture was stirred for 1 h at 0°, then for 16 h at room temperature. Fractional distillation (121°/760 mmHg) afforded 2,2-dimethyl-but- 3-enoyl chloride (4.1 g, 71%).

[0099] Monoethyl hydrogen malonate (7.2 g, 0.05 mol) and bipyridyl (few milligrams) were dissolved in THF (90 ml) and the system was flushed with nitrogen. The solution was cooled to -70°, then BuLi (2.5 M in hexanes, 37 ml, 0.09 mol) was added. After the addition of only -10 ml of BuLi the solution turned pink and additional THF (15 ml) was required to enable magnetic stirring. The cooling bath was removed and the remaining BuLi was added, the temperature was allowed to reach -10°, upon which the solution turned colorless. The mixture was again cooled to -60° and a solution of 2,2- dimethyl-but-3-enoyl chloride (4.1 g, 0.03 mol) in THF (12 ml) was added dropwise. After addition was complete the mixture was allowed to warm to 0° and stir for 3 h, then it was added to a 1:1 mixture of ether/lM HC1 (260 ml) at 0° and stirred for a further 1.5 h. The organic layer was removed, washed with HC1 (1 M), sodium bicarbonate solution, brine then dried and evaporated to give 4,4-dimethyl-3-oxo-hex-5-enoic acid ethyl ester (5.6 g, 98%). (Hayashi et al., J. Org. Chem., 65:8402-8405 (2000). Distillation with a Kugelrohr oven (160°/1 mmHg) afforded pure material.

[0100] 1H NMR (400 MHz, CDC1₃) δ 1.26 (s, 6H); 1.27 (t, / = 6.9 Hz, 3H, - CH2CH3); 3.51 (s, 2H); 4.18 (q, / = 6.9 Hz, 2H, -CH₂CH₃); 5.20 (d, / = 17.7 Hz, 1H, - CH=CH₂); 5.21 (d, / = 9.6 Hz, 1H, -CH=CH₂); 5.89 (dd, / = 17.7, 9.6 Hz, 1H, - CH=CH₂). 2-Chloro-4,4-dimethyl-3-oxo-hex-5-enoic acid ethyl ester

[0101] Sulfuryl chloride (0.84 ml, 10.4 mmol) was added to a cooled (0°) solution of 4,4-dimethyl-3-oxo-hex-5-enoic acid ethyl ester (1.83 g, 9.93 mmol) in chloroform (7 ml). The resulting mixture was allowed to warm to room temperature and stir for 30 min, after which it was heated under reflux for 2 h. After cooling to room temperature the reaction mixture was diluted with chloroform, then was washed with sodium bicarbonate, water then brine. The organic phase was dried and evaporated to afford, as a brown oil, 2-chloro-4,4-dimethyl-3-oxo-hex-5-enoic acid ethyl ester (2.01 g, 93%). (Hayashi et al., J. Org. Chem., 65:8402-8405 (2000).

[0102] 1H NMR (400 MHz, CDC1₃) δ 1.28 (t, J = 7.0 Hz, 3H, -CH₂CH₃); 1.33 (s, 3H); 1.34 (s, 3H); 4.24 (q, / = 7.0 Hz, 2H, -CH₂CH₃); 5.19 (s, 1H; 5.28 (d, J = 16.9 Hz, 1H, -CH=CH₂y, 5.29 (d, J = 10.9 Hz, 1H, -CH=CH₂); 5.96 (dd, J = 16.9, 10.9 Hz, 1H, -CH=CH₂).

[0103] LC/MS t_R = 8.45 (219.3 [M(C1³⁷)+H]⁺ min.

[0104] This material was reacted without further purification.

5-(l,l-Dimeth l-allyl)-3H-imidazole-4-carboxylic acid ethyl ester

[0105] A suspension of 2-chloro-4,4-dimethyl-3-oxo-hex-5-enoic acid ethyl ester (19.4 g, 0.09 mol) and water (1.94 ml, 0.11 mol) in formamide (36.8 ml) was shaken briefly, then dispensed into 15 x 18 ml vials. The vials were sealed and heated at 150° for 5 h. After cooling to room temperature, the vials' contents were combined and extracted exhaustively with chloroform. The extracts were dried and evaporated to afford a concentrated formamide solution (14.7 g). This was added to a silica column (7 cm diameter, 11 cm height) packed in 1% MeOH/1% Et₃N in chloroform. Elution of the column with 2 L of this mixture followed by 2 L of 2% MeOH/1% Et₃N in chloroform afforded, in the early fractions, a compound suspected of being 5-( 1 , 1 -dimethyl-allyl)- oxazole-4-carboxylic acid ethyl ester (1.23 g. 7%).

[0106] HPLC (214nm) t_R = 8.68 (50.4%) min.

[0107] 1H NMR (400 MHz, CDC1₃) δ 1.40 (t, J = 7.2 Hz, 3H, -CH₂CH₃); 1.54 (s, 6H); 4.38 (t, / = 7.2 Hz, 2H, -CH₂CH₃); 5.03 (d, / = 17.4 Hz, 1H, -CH=CH₂); 5.02 (d, / = 10.4 Hz, 1H, -CH=CH₂); 6.26 (dd, / = 17.4, 10.4 Hz, 1H, -CH=CH₂); 7.83 (s, 1H).

[0108] LCMS t_R = 8.00 (210.1 [M+H]⁺, 361.1 [2M+H]⁺) min.

[0109] Recovered from later fractions was the desired 5-(l,l-dimethyl-allyl)- 3H-imidazole-4-carboxylic acid ethyl ester (3.13 g, 17%). (Hayashi et al., J. Org. Chem., 65:8402-8405 (2000)).

[0110] HPLC (214nm) t_R = 5.52 (96.0%) min.

[0111] 1H NMR (400 MHz, CDC1₃) δ 1.38 (t, / = 7.0 Hz, 3H); 1.57 (s, 6H); 4.35 (q, / = 7.0 Hz, 2H); 5.04-5.14 (m, 2H, -CH=CH₂); 6.28 (dd, / = 18.0, 10.4 Hz, 1H, - CH=CH₂); 7.52 (s, 1H).

[0112] LC/MS t_R = 5.30 (209.1 [M+H]⁺, 417.2 [2M+H]⁺) min.

[0113] Additional 5-(l,l-dimethyl-allyl)-3H-imidazole-4-carboxylic acid ethyl ester was also recovered from the column (3.59 g, 19%) which was of lower purity but still sufficient for further reaction.

[0114] Another byproduct isolated from a similar reaction (smaller scale) by further elution of the column with 5% MeOH/1% Et₃N in chloroform was a compound suspected of being 5-(l,l-dimethyl-allyl)-3H-imidazole-4-carboxylic acid (0.27 g, 9%).

[0115] HPLC (245nm) t_R = 5.14 (68.9%) min.

[0116] 1H NMR (400 MHz, CD₃OD) δ 1.45 (s, 6H); 4.97 (d, / = 10.6 Hz, 1H, -CH=CH₂); 5.01 (d, / = 17.7 Hz, 1H, -CH=CH₂); 6.28 (dd, / = 17.7, 10.6 Hz, 1H, - CH=CH₂); 7.68 (s, 1H).

[0117] LCMS t_R = 4.72 (181.0 [M+H]⁺, 361.1 [2M+H]⁺) min.

5-( 1, 1 -Dimethyl-allyl)-3H-imidazol-4-yl] -methanol

[0118] A solution of 5-(l,l-dimethyl-allyl)-3H-imidazole-4-carboxylic acid ethyl ester (3.13 g, 15.0 mmol) in THF (60 ml) was added dropwise to a suspension of lithium aluminium hydride (95% suspension, 1.00 g, 25.0 mmol) in THF (40 ml) and the mixture was stirred at room temperature for 4 h. Water was added until the evolution of gas ceased, the mixture was stirred for 10 min, then was filtered through a sintered funnel. The precipitate was washed with THF, then with methanol, the filtrate and washings were combined, evaporated, then freeze-dried to afford [5-(l,l-dimethyl-allyl)-3H-imidazol-4- yl]-methanol (2.56 g, 102%). Residual water was removed by azeotroping with chloroform prior to further reaction. (See Hayashi et al., J. Org. Chem., 65:8402-8405 (2000)).

[0119] HPLC (240nm) t_R = 3.94 (56.8%) min.

[0120] 1H NMR (400 MHz, CD₃OD) δ 1.43 (s, 6H); 4.57 (s, 2H); 5.01 (d, J = 10.5 Hz, 1H, -CH=CH₂); 5.03 (d, / = 17.7 Hz, 1H, -CH=CH₂); 6.10 (dd, / = 17.7, 10.5 Hz, 1H, -CH=CH₂); 7.46 (s, 1H).

[0121] LC/MS t_R = 3.77 (167.3 [M+H]⁺) min.

5-(l,l-Dimethyl-allyl)-3H-imidazole-4-carbaldehyde

[0122] Manganese dioxide (20 g, 0.23 mol) was added to a solution of [5-(l,l- dimethyl-allyl)-3H-imidazol-4-yl]-methanol (2.56 g, 0.02 mol) in acetone (300 ml) and the resulting mixture was stirred at room temperature for 5 h. The mixture was filtered through filter paper and the residue was washed with acetone. The filtrate and washings were combined and evaporated to afford 5-(l,l-dimethyl-allyl)-3H-imidazole-4- carbaldehyde (1.82 g, 51%). (Hayashi et al., J. Org. Chem., 65:8402-8405 (2000)).

[0123] HPLC (240nm) t_R = 4.08 (91.5%) min.

[0124] 1H NMR (400 MHz, CDC1₃) δ 1.56 (s, 6H); 5.16 (d, J = 10.6 Hz, 1H, - CH=CH₂); 5.19 (d, J = 17.3 Hz, 1H, CH=CH₂); 6.22 (dd, J = 17.3, 10.6 Hz, 1H, - CH=CH₂); 7.75 (s, 1H), 10.02 (s, 1H, HCO).

[0125] LC/MS t_R = 3.75 (165.2 [M+H]⁺) min. 1 -Acetyl-3-[ 5 '-( l -dimethyl-allyl)-lH midazol-4'-Z-ylmethylene]-piperazine-2,5-dion^

[0126] To a solution of 5-(l,l-dimethyl-allyl)-3H-imidazole-4-carbaldehyde (1.78 g, 0.01 mol) in DMF (35 ml) was added l,4-diacetyl-piperazine-2,5-dione (8.59 g, 0.04 mol) and the mixture was evacuated, then flushed with argon. The evacuation- flushing process was repeated a further two times, then cesium carbonate (3.53 g, 0.01 mol) was added. The evacuation-flushing process was repeated a further three times, then the resultant mixture was heated at 45° for 5 h. The reaction mixture was partially evaporated (heating under high vacuum) until a small volume remained and the resultant solution was added dropwise to ice- water (50 ml). The yellow precipitate was collected, washed with water, then freeze-dried to afford l-acetyl-3-[5'-(l,l-dimethyl-allyl)-lH- imidazol-4'-ylmethylene]-piperazine-2,5-dione (1.18 g, 36%).

[0127] HPLC (214nm) t_R = 6.01 (72.6%) min.

[0128] 1H NMR (400 MHz, CDC1₃) δ 1.53 (s, 6H); 2.64 (s, 3H); 4.47 (s, 2H); 5.19 (d, / = 17.3 Hz, 1H, -CH=CH₂); 5.23 (d, / = 10.7 Hz, 1H, -CH=CH₂); 6.06 (dd, / = 17.3, 10.7 Hz, 1H, -CH=CH₂); 7.16 (s, 1H), 7.59 (s, 1H), 9.47 (bs, 1H); 12.11 (bs, 1H) [observed -2% l,4-diacetyl-piperazine-2,5-dione contamination δ 2.59 (s, 6H); 4.60 (s, 4H).]

[0129] LC/MS t_R = 6.65 (303.3 [M+H]⁺, 605.5 [2M+H]⁺) min. (n.b. different system used).

EXAMPLE 2

Synthesis of Imidazole-type Precursers (t-butyl substituted)

Synthesis of 3-Z-Benzylidene-6-(5'^: ' -tert-butyl-lH-imidazol-4" ' -Z-ylmethylene)

2,5-dione (2)

[0130] Reagents: g) S0₂C1₂; h) H₂NCHO, H₂0; I)LiAlH₄; j) Mn0₂; k) 1,4- diacetyl-piperazine-2,5-dione, Cs₂C0₃; 1) benzaldehyde, Cs₂C0₃

2-Chloro-4,4-dimeth l-3-oxo-pentanoic acid ethyl ester

[0131] Sulfuryl chloride (14.0 ml, 0.17 mol) was added to a cooled (0°) solution of ethyl pivaloylacetate (27.17 g, 0.16 mol) in chloroform (100 ml). The resulting mixture was allowed to warm to room temperature and was stirred for 30 min, after which it was heated under reflux for 2.5 h. After cooling to room temperature, the reaction mixture was diluted with chloroform, then washed with sodium bicarbonate, water then brine.

[0132] The organic phase was dried and evaporated to afford, as a clear oil, 2- chloro-4,4-dimethyl-3-oxo-pentanoic acid ethyl ester (33.1 g, 102%). (Durant et al., "Aminoalkylimidazoles and Process for their Production." Patent No. GB1341375 (Great Britain, 1973)).

[0133] HPLC (214nm) t_R = 8.80 (92.9%) min.

[0134] 1H NMR (400 MHz, CDC1₃) δ 1.27 (s, 9H); 1.29 (t, J = 7.2 Hz, 3H); 4.27 (q, / = 7.2 Hz, 2H); 5.22 (s, 1H).

[0135] ¹³C NMR (100 MHz, CDC1₃) δ 13.8, 26.3, 45.1, 54.5, 62.9, 165.1,

203.6.

5-tert-Butyl-3H-imidazole-4-carboxylic acid ethyl ester

[0136] A solution of 2-chloro-4,4-dimethyl-3-oxo-pentanoic acid ethyl ester (25.0 g, 0.12 mol) in formamide (47.5 ml) and water (2.5 ml) was shaken, then dispensed into 15 x 8 ml vials. All vials were sealed and then heated at 150° for 3.5 h. The vials were allowed to cool to room temperature, then water (20 ml) was added and the mixture was exhaustively extracted with chloroform. The chloroform was removed to give a concentrated formamide solution (22.2 g) which was added to a flash silica column (6 cm diameter, 12 cm height) packed in 1% MeOH/1% Et₃N in chloroform. Elution of the column with 2.5 L of this mixture followed by 1 L of 2% MeOH/1% Et₃N in chloroform gave, in the early fractions, a product suspected of being 5-tert-butyl-oxazole-4-carboxylic acid ethyl ester (6.3 g, 26%).

[0137] HPLC (214nm) t_R = 8.77 min.

[0138] 1H NMR (400 MHz, CDC1₃) δ 1.41 (t, J = 7.2 Hz, 3H); 1.43 (s, 9H); 4.40 (q, / = 7.2 Hz, 2H); 7.81 (s, 1H).

[0139] ¹³C NMR (100 MHz, CDC1₃) δ 14.1, 28.8, 32.5, 61.3, 136.9, 149.9, 156.4, 158.3. [0140] ESMS m/z 198.3 [M+H]⁺, 239.3 [M+CH₄CN]⁺.

[0141] LC/MS t_R = 7.97 (198.1 [M+H]⁺) min.

[0142] Recovered from later fractions was 5-tert-butyl-3H-imidazole-4- carboxylic acid ethyl ester (6.20 g, 26%). (Durant et al., "Aminoalkylimidazoles and Process for their Production." Patent No. GB1341375 (Great Britain, 1973)).

[0143] HPLC (214nm) t_R = 5.41 (93.7%) min.

[0144] 1H NMR (400 MHz, CDC1₃) δ 1.38 (t, J = 7.0 Hz, 3H); 1.47 (s, 9H); 4.36 (q, / = 7.2 Hz, 2H); 7.54 (s, 1H).

[0145] ¹³C NMR (100 MHz, CDC1₃) δ 13.7, 28.8, 32.0, 59.8, 124.2, 133.3, 149.2, 162.6.

[0146] ESMS m/z 197.3 [M+H]⁺, 238.3 [M+CH₄CN]⁺.

[0147] Further elution of the column with 1L of 5% MeOh/1% Et₃N gave a compound suspected of being 5-tert-butyl-3H-imidazole-4-carboxylic acid (0.50 g, 2%).

[0148] HPLC (245nm) t_R = 4.68 (83.1%) min.

[0149] 1H NMR (400 MHz, CD₃OD) δ 1.36 (s, 9H); 7.69 (s, 1H).

[0150] 1H NMR (400 MHz, CDC1₃) δ 1.37 (s, 9H); 7.74 (s, 1H).

[0151] 1H NMR (400 MHz, CD₃SO) δ 1.28 (s, 9H); 7.68 (s, 1H).

[0152] ESMS m/z 169.2 [M+H]⁺, 210.4 [M+CH₄CN]⁺.

(5-tert-Butyl-3H-imidazol-4-yl)-methanol

[0153] A solution of 5-tert-butyl-3-imidazole-4-carboxylic acid ethyl ester (3.30 g, 16.8 mmol) in THF (60 ml) was added dropwise to a suspension of lithium aluminium hydride (95% suspension, 0.89 g, 22.2 mmol) in THF (40 ml) and the mixture was stirred at room temperature for 3 h. Water was added until the evolution of gas ceased, the mixture was stirred for 10 min, then was filtered through a sintered funnel. The precipitate was washed with THF, then with methanol, the filtrate and washings were combined and evaporated. The residue was freeze-dried overnight to afford, as a white solid (5-tert-butyl-3H-imidazol-4-yl)-methanol (2.71 g, 105%). (Durant et al., "Aminoalkylimidazoles and Process for their Production." Patent No. GB1341375 (Great Britain, 1973)).

[0154] HPLC (240nm) t_R = 3.70 (67.4%) min.

[0155] 1H NMR (400 MHz, CD₃OD) δ 1.36 (s, 9H); 4.62 (s, 2H); 7.43 (s, 1H).

[0156] ¹³C NMR (100 MHz, CD₃OD) δ 31.1, 33.0, 57.9, 131.4, 133.9, 140.8.

[0157] LC/MS t_R = 3.41 (155.2 [M+H]⁺) min.

[0158] This material was used without further purification.

5-tert-Butyl-3H-imidazole-4-carbaldehyde

[0159] Manganese dioxide (30 g, 0.35 mol) was added to a heterogeneous solution of (5-tert-butyl-3H-imidazol-4-yl)-methanol (4.97 g, 0.03 mol) in acetone (700 ml) and the resulting mixture was stirred at room temperature for 4 h. The mixture was filtered through a pad of Celite and the pad was washed with acetone. The filtrate and washings were combined and evaporated. The residue was triturated with ether to afford, as a colorless solid, 5-tert-butyl-3H-imidazole-4-carbaldehyde (2.50 g, 51%).

[0160] HPLC (240nm) t_R = 3.71 (89.3%) min.

[0161] 1H NMR (400 MHz, CDC1₃) δ 1.48 (s, 9H); 7.67 (s, 1H); 10.06 (s, 1H).

[0162] LC/MS t_R = 3.38 (153.2 [M+H]⁺) min.

[0163] Evaporation of the filtrate from the trituration gave additional 5-tert- butyl-3H-imidazole-4-carbaldehyde (1.88 g, 38%).

l-Acetyl-3-(5'-tert-butyl-lH mdazol-4'-Z-ylmethylene)-piperazine-2,5-dione

[0164] To a solution of 5-tert-butyl-3H-imidazole-4-carbaldehyde (2.50 g, 164.4 mmol) in DMF (50 ml) was added l,4-diacetyl-piperazine-2,5-dione (6.50 g, 32.8 mmol) and the solution was evacuated, then flushed with argon. The evacuation-flushing process was repeated a further two times, then cesium carbonate (5.35 g, 16.4 mmol) was added. The evacuation-flushing process was repeated a further three times, then the resultant mixture was stirred at room temperature for 5 h. The reaction mixture was partially evaporated (heat and high vacuum) until a small volume remained and the resultant solution was added dropwise to water (100 ml). The yellow precipitate was collected, then freeze-dried to afford l-acetyl-3-(5'-tert-butyl-lH-imidazol-4'-Z- ylmethylene)-piperazine-2,5-dione (2.24 g, 47%).

[0165] HPLC (214nm) t_R = 5.54 (94.4%) min.

[0166] 1H NMR (400 MHz, CDC1₃) δ 1.47 (s, 9H); 2.65 (s, 3H), 4.47 (s, 2H); 7.19 (s, 1H); 7.57 (s, 1H), 9.26 (s, 1H), 12.14 (s, 1H).

[0167] ¹³C NMR (100 MHz, CDC1₃+CD₃0D) δ 27.3, 30.8, 32.1, 46.5, 110.0, 123.2, 131.4, 133.2, 141.7, 160.7, 162.8, 173.0

[0168] LC/MS t_R = 5.16 (291.2 [M+H]⁺, 581.6 [2M+H]⁺) min.

Analytical Conditions

NMR Conditions

[0169] 1H NMR (400 MHz) analysis was performed on a Varian Inova Unity 400 MHz NMR machine. Samples were run in deuterated chloroform containing 0.1% TMS (unless otherwise specified). Chemical shifts (ppm) are referenced relative to TMS (0.00 ppm) or CH₃OH at 3.30 ppm for samples run CD₃OD. Coupling constants are expressed in hertz (Hz).

Analytical HPLC Conditions

[0170] System 6 conditions:

[0171] RP-HPLC was done on a Rainin Microsorb-MV C18 (5 μπι, 100A) 50 x 4.6 mm column.

[0172] Buffer A: 0.1% aqueous TFA

[0173] Buffer B: 0.1% TFA in 90% aqueous MeCN

[0174] Gradient: 0 - 100% Buffer B over 11 min

[0175] Flow rate: 1.5 mL/min

LCMS Conditions

[0176] LCMS were run on a Perkin-Elmer Sciex API- 100 instrument.

[0177] LC conditions:

[0178] Reverse Phase HPLC analysis

[0179] Column: Monitor 5 μπι CI 8 50x4.6 mm [0180] Solvent A: 0.1 % TFA in water

[0181] Solvent B: 0.085% TFA in 90% aqueous MeCN

[0182] Gradient: 0-100% B over 11.0 min

[0183] Flow rate: 1.5 mL/min

[0184] Wavelength: 214 nm

[0185] MS conditions:

[0186] Ion Source: Ionspray

[0187] Detection: Ion counting

[0188] Flow rate to the mass spectrometer: 300 μΙ7ητίη after split from column (1.5 mL/min).

ESMS Conditions

[0189] ESMS was done on a Perkin Elmer/Sciex-API ΙΠ LC/MS/MS using an electro spray inlet.

[0190] Solvent: 0.1% AcOH in 60% aqueous MeCN

[0191] Flow rate: 25 μΐνηιίη

[0192] Ionspray: 5000 V

[0193] Orifice plate: 55 V

[0194] Acquisition time: 2.30 min

[0195] Scan range: 100-1000 amu/z

[0196] Scan step size: 0.2 amu/z

Preparative RP-HPLC Purification Conditions

[0197] Reverse phase HPLC purification was carried out using Nebula with the Waters XterraMS column (19x50 mm, 5 μπι, C18) using the following conditions:

[0198] Solvent A: 0.1 % aqueous TFA

[0199] Solvent B: 0.1% TFA in 90% aqueous MeCN

[0200] Gradient: 5-95% B over 4 min

[0201] Flow rate: 20 mL/min

[0202] Wavelength: 214 nm

[0203] Abbreviations are as follows: br s: broad singlet; BuLi : n-butyl lithium; d: doublet; DBU: l,8-diazabicyclo[5.4.0]undec-7-ene; ESMS: electrospray mass spectrometry; HCl: hydrochloric acid; HPLC: high performance liquid chromatography; LCMS: liquid chromatography mass spectrometry; LD: lithium diisopropylamide; +: molecular ion; m: multiplet; MeCN: acetonitrile; M: mass spectrometry; MW: molecular weight; NMR: nuclear magnetic resonance; q: quartet; s: singlet; : triplet; t_R: retention time; TFA: trifluoroacetic acid; THF: tetrahydrofuran

EXAMPLE 3

Synthesis of Oxazole-type Precursers

CN COO

99% 2 67 % 57 %

5 6

Scheme 1. General synthetic scheme of oxazole-type tBu-dehydroPLH derivatives.

Ethyl 5-(tert-butyl)oxazole-4-carboxylate (2)

[0204] According to the report by Suzuki et al. (JOC, 38, 3571-3575 (1973)), to a stirring solution of ethyl isocyanoacetate 1 (25 g, 221 mmol, caution: bad smell, it should be treated in a draft chamber, Wako Pure Chemical, Osaka, Japan, Cat. No. 055-06672, Rf 0.70 [CHCl₃:MeOH = 10:0.5]) in anhydrous THF (200 mL, Kanto Chemical, Tokyo, Japan, Cat. No. 40993-05) was added DBU (34.3 mL, 243 mmol, Nacalai Tesque, Kyoto, Japan, Cat. No. 11117-05) and pivalic anhydride (49.3 mL, 243 mmol, Wako Pure Chemical, Osaka, Japan, Cat. No. 168-19661) at 4 °C. After 10 min, the ice-bath was removed and the mixture was stirred overnight at room temperature.

start ng mate a reaction mixture

[0205] Then, the solvent of the obtained dark brown reaction mixture was removed by evaporation in vacuo. AcOEt (200 mL) was added to the obtained residue, then this mixture was washed with 10% Na₂C0₃ (x 3), 10% citric acid (x 3) and saturated NaCl (x 3), and dried over anhydrous Na₂S0₄, and the solvent was evaporated in vacuo. The residual oil was dissolved in CHC1₃ (20 mL) and applied to silica-gel column chromatography (6 x 30 cm, Merck 107734 silica gel 60, 70-230 mesh, prepared with Hexane: AcOEt = 20:1) and eluted with Hexane: AcOEt (20:1 to 4:1, the desired 2 is eluted at 8:1), to give an pale yellow oil of 2 (66.4 g, 99%). Rf 0.68 (CHCl₃:MeOH = 10:0.5), 1H NMR (300MHz CDC1₃) δ 7.70 (s, 1H), 4.39 (q, J = 7.2 Hz, 2H), 1.46 (s, 9H), 1.41 (t, J = 7.2 Hz, 3H); HRMS(EI): m/z 197.1050 (M⁺) (Calcd for Ci₀Hi₅NO₃: 197.1052).

5-(tert-butyl)oxazole-4-carboxaldehyde (4)

[0206] To a solution of Ethyl 5-(tert-butyl)oxazole-4-carboxylate 2 (20 g, 101 mmol) in anhydrous THF (250 mL) was added L1AIH₄ (3.84 g, 101 mmol, Kanto Chemical, Tokyo, Japan, Cat. No. 24115-35) portionwise under argon atmosphere at - 60°C, and the bath temperature was gradually increased up to -40°C with stirring for 2 h. (In case that the temperature was more than -40°C, the reduction of the oxazole ring (oxazolidine formation) predominantly proceeded). After the reaction mixture was quenched with saturated NH₄C1 aq (50 ml) at -60°C (Please avoid rapid elevation of temperature. Some impurity increased.), AcOEt (250 ml) was added and stirred for a few minutes, and the resulting precipitate was removed by celite filtration (Celite-535, Nacalai Tesque, Kyoto, Japan, Cat. No. 07509-05). The filtrate was washed with water (x 2), 10% citric acid (x 3, a byproduct, oxazolidine, can be removed by this separation) and saturated NaCl (x 3), dried over anhydrous Na₂S0₄, and concentrated in vacuo to give an oil of corresponding oxazole alcohol 3 (10.6 g, 67%). Rf 0.50 (CHCl₃:MeOH = 10:0.5), 1H NMR (300MHz CDC1₃) δ 7.71 (s, 1H), 4.66 (s, 2H), 1.36 (s, 9H); HRMS(EI): m/z 155.0852 (M⁺) (Calcd for C₈Hi₃N0₂: 155.0854). This oil was used to the next oxidation without further purification.

isonitrile 1

starting

[0207] To a solution of oxazole alcohol 3 (9.9 g, 64 mmol) in acetone (200 mL, Aldrich, Cat. No. 179124) was added Mn0₂ (27.7 g, 319 mmol, 5 equiv., Wako Pure Chemical, Osaka, Japan, Cat. No. 138-09675), and the mixture was stirred at room temperature overnight. After celite filtration to remove Mn0₂, the solvent was removed by evaporation, and the residual pale brown oil was dissolved in CHC1₃ and applied to silica-gel column chromatography (prepared with CHC1₃), then eluted with CHCl₃:MeOH (100:1 to 50:1), to give an pale yellow oil of aldehyde 4 (5.54 g, 57% (38% in two steps)). Rf 0.65 (CHCl₃:MeOH = 10:0.5), 1H NMR (300MHz CDC1₃) δ 10.10 (s, 1H), 7.77 (s, 1H), 1.47 (s, 9H); HRMS(EI): m/z 153.0794 (M⁺) (Calcd for C₈HnN0₂: 153.0790). l-Acetyl-3-{(Z)-l-[5-(tgrt-butyl)-4- xazolyl1methylidene}1-2.5-piperazinedione (6)

[0208] Using a 100 mL round bottom flask which is connected to the vacuum pump through three-way cock, to a solution of 5-(tert-butyl)oxazole-4-carboxaldehyde 4 (1.0 g, 7.1 mmol) in anhydrous DMF (10 mL, Kanto, Chemical, Tokyo, Japan, Cat. No. 11339-05) was added N,N'-diacetyl-2,5-piperazinedione 5 (2.1 g, 10.6 mmol), then the solution was repeatedly evacuated in a short time to remove oxygen and flushed with Ar. Then, Cs₂C0₃ (3.9 g, 12.0 mmol, Aldrich, Cat. No. 202126-25) was added to this solution and the evacuation-flushing process was repeated again. The resultant mixture was stirred for 6 h at room temperature. After the solvent was removed by evaporation, the residue was purified by column chromatography (2 x 30 cm) on silica using CHC1₃ as an eluant to give 1.15 g (56 %) of a pale yellow solid 6. mp 145-147 °C; 1H NMR (300MHz CDC1₃) δ 11.22 (br s, 1H), 7.83 (s, 1H), 7.09 (s, 1H), 4.48 (s, 2H), 2.65 (s, 3H), 1.45 (s, 9H) ; HRMS (EI) m/z 291.1217 (M⁺) (calcd for Ci₄Hi₇N₃0₄: 291.1219).

[0209] During the reaction and purification, the apparatus or flask was coved with aluminum foil to avoid isomerization as much as possible.

EXAMPLE 4

Synthesis of KPU-246

[0210] KPU-246 may be synthesized by reacting the aldehyde

with the precursor from Example 3 in anhydrous DMF in the presence of Cs₂C0₃. The solvent can then be evaporated and the residue dissolved in a mixture of EtOAc and water, then, washed with 5% citric acid (x 1), 5% NaHC0₃ (x 1) and saturated NaCl (x 3), dried over Na₂S0₄ and concentrated in vacuo. The resulting residue can then be dissolved in CHC1₃ and applied to on silica-gel column chromatography, eluted with CHCl₃:MeOH = 80:1 to 20:1, to give the final product. EXAMPLE 5

Synthesis of KPU-113, KPU-114, and KPU-115

[0211] KPU-113, KPU-114, and KPU-115 can be synthesized by reacting the

EXAMPLE 6

Synthesis of Benzophenone Analogs

[0212] Benzophenone analogs of phenylahistins according to Formula I may be synthesized by the following route:

where R¹, R², R³, R⁴, R⁵, R⁶, Zi, Z₂, Z₃, Z₄, Z₅, and Z are as defined above, EDC is 1- ethyl-3-(3-dimethylaminopropyl)carbodiimide, and PDC is pyridinum dichromate. Several such compounds were synthesized and are listed in Table 1 in the following example.

EXAMPLE 7

Biological Characteristics of Benzophenone Analogs

[0213] The biological characteristics of synthesized analogs were evaluated in HT29 human colon cells.

[0214] HT-29 (ATCC HTB-38) a human colorectal adenocarcinoma can be maintained in McCoy's complete medium (McCoy's 5A medium with L-glutamine and 25mM HEPES supplemented with 10% FBS, ImM Na pyruvate, IX NEAA, 2mM L- glutamine, and Pen/Strep at lOOIU/ml and 100μg/ml, respectively). Cell lines can be cultured at 37 °C, 5% C0₂ in a 95% humidified incubator.

[0215] For tumor cytotoxicity assays HT-29 cells can be seeded at 5,000 cells/well in 90 μΐ complete media into a Corning 3904 black-walled, clear-bottom tissue culture plate and the plate incubated overnight to allow cells to establish and enter log phase growth. 20 mM stock solutions of dehydrophenylahistin analogs can be prepared in 100% DMSO and stored at -20 °C. 10X concentrated serial dilutions of the compounds can be prepared in appropriate culture medium for final concentrations ranging from 2.0 x 10^"5 M to 2.0 x 10^"10 M. Ten μΐ volumes of the 10X serial dilutions can be added to the test wells in triplicate and the plates returned to the incubator for 48 hours. The final concentration of DMSO can be 0.25% in all samples.

[0216] Following 48 hours of drug exposure 10 μΐ of 0.2 mg/ml resazurin (obtained from Sigma-Aldrich Chemical Co.) in Mg²⁺, Ca²⁺ free PBS can be added to each well and the plates returned to the incubator for 3-4 hours. The plates can then be removed and resazurin fluorescence measured using 530 nm excitation and 590 nm emission filters in a Fusion fluorimeter (Packard Instruments). Resazurin dye without cells can be used to determine the background, which is then subtracted from the data for all experimental wells. The data can be analyzed using Prism software (GraphPad Software). The data can be normalized to the average of the cells treated with media only (100% cell growth) and IC₅₀ values determined. [0217] The cytotoxic effects of various compounds were examined in HT-29 human colon cells. These compounds were synthesized using the methods described above

[0218] IC₅₀ values are summarized in Table 1 below.

Table 1. Activity of phenylahistin or dehydrophenylahistin

and of dehydrophenylahistin derivatives

EXAMPLE 8

Prodrugs

[0219] The E-form of the compounds may be used as a prodrug of dehydroPLH or of one or more of its analogs, including those analogs described herein. One of the undesired properties of anti-tubulin drugs involves its low selectivity between tumor and intact tissues, although these drugs belong to one of the molecular target therapies. This causes undesired side effects. However, if the compounds functions selectively only in tumor tissues, negative side effects of anti- microtubule drugs can be reduced. Since the dehydroPLH (Z-form) can be produced from its E-isomer by visible light irradiation, the E-form is administered and photo irradiation is performed only at the tumor site, then only the tumor is damaged by photo-produced Z-form and the adverse effect to the intact tissues is reduced.

[0220] The E-form can be protected chemically by the addition of a bulky but biodegradable acyl group, which is introduced into the diketopiperazine ring as a prodrug. This acyl group can be cleaved by the protease in the body. Therefore, the acylated-E- compound is maintained before administration, then after administration it is changed to the real E-form, which can migrate to the bioactive Z-form by the local photo irradiation.

EXAMPLE 9

Pharmaceutical Formulations

1) Formulations Administered Intravenously, by Drip, Injection, Infusion or The Like

[0221] Vials containing 5 g of powdered glucose are each added aseptically with 10 mg of a compound synthesized by the method and sealed. After being charged with nitrogen, helium or other inert gas, the vials are stored in a cool, dark place. Before use, the contents are dissolved in ethanol and added to 100 ml of a 0.85% physiological salt water solution. The resultant solution is administered as a method of inhibiting the growth of a cancerous tumor in a human diagnosed as having such a tumor at between approximately 10 ml/day to approximately 1000 ml/day, intravenously, by drip, or via a subcutaneous or intraperitoneal injection, as deemed appropriate by those of ordinary skill in the art.

2) Formulation to be Administered Orally Or The Like

[0222] A mixture obtained by thoroughly blending 1 g of a compound synthesized by the method, 98 g of lactose and 1 g of hydroxypropyl cellulose is formed into granules by any conventional method. The granules are thoroughly dried and sifted to obtain a granule preparation suitable for packaging in bottles or by heat sealing. The resultant granule preparations are orally administered at between approximately lOOml/day to approximately 1000 ml/day, depending on the symptoms, as deemed appropriate by those of ordinary skill in the art of treating cancerous tumors in humans. 3) Formulation to be Administered Topically

[0223] Administration to an individual of an effective amount of the compound can also be accomplished topically by administering the compound(s) directly to the affected area of the skin of the individual. For this purpose, the compound administered or applied is in the form of a composition including a pharmacologically acceptable topical carrier, such as a gel, an ointment, a lotion, or a cream, which includes, without limitation, such carriers as water, glycerol, alcohol, propylene glycol, fatty alcohols, triglycerides, fatty acid esters, or mineral oils. Other topical carriers include liquid petroleum, isopropyl palmitate, polyethylene glycol, ethanol (95%), polyoxyethylene monolaurate (5%) in water, or sodium lauryl sulfate (5%) in water. Other materials such as anti-oxidants, hurnectants, viscosity stabilizers, and similar agents may be added as necessary. Percutaneous penetration enhancers such as Azone may also be included. In addition, in certain instances, it is expected that the compound may be disposed within devices placed upon, in, or under the skin. Such devices include patches, implants, and injections which release the compound into the skin, by either passive or active release mechanisms.

EXAMPLE 10

Assays For Activity Against Pathogenic Fungi

[0224] Comparative activity of a compound disclosed herein against a pathogenic fungus, relative to known antifungal compounds recited above, for use in determining the compound disclosed herein 's AF/IS value is measured directly against the fungal organism, e.g. by microtiter plate adaptation of the NCCLS broth macrodilution method described in Diagn Micro and Infect Diseases 21:129-133 (1995). Antifungal activity can also be determined in whole-animal models of fungal infection. For instance, one may employ the steroid-treated mouse model of pulmonary mucormycosis (Goldaill, L.Z. & Sugar, A.M. 1994 J Antimicrob Chemother 33:369-372). By way of illustration, in such studies, a number of animals are given no compound disclosed herein, various doses of compound disclosed herein (and/or combinations with one or more other antifungal agents), or a positive control (e.g. Amphotericin B), respectively, beginning before, at the time of, or subsequent to infection with the fungus. Animals may be treated once every 24 hours with the selected dose of compound disclosed herein, positive control, or vehicle only. Treatment is continued for a predetermined number of days, e.g. up to ten days. Animals are observed for some time after the treatment period, e.g. for a total of three weeks, with mortality being assessed daily. Models can involve systemic, pulmonary, vaginal and other models of infection with or without other treatments (e.g. treatment with steroids) designed to mimic a human subject susceptible to infection.

[0225] To further illustrate, one method for determining the in vivo therapeutic efficacies (ED₅₀, e.g. expressed in mg compound disclosed herein /kg subject), is a rodent model system. For example, a mouse is infected with the fungal pathogen such as by intravenous infection with approximately 10 times the 50% lethal dose of the pathogen (10⁶ C. albicans cells /mouse). Immediately after the fungal infection, dehydrophenylahistin compounds are given to the mouse at a predetermined dosed volume. The ED₅₀ is calculated by the method of Van der Waerden (Arch Exp Pathol Pharmakol 195:389-412, 1940) from the survival rate recorded on 20th day postinfection. Generally, untreated control animals die 7 to 1 3 days post-infection.

[0226] In another illustrative embodiment, C. albicans Wisconsin (C43) and C. tropicalis (CI 12), grown on Sabouraud dextrose agar (SDA) slants for 48 h at 28°C, are suspended in saline and adjusted to 46% transmission at 550 nm on a spectrophotometer. The inoculum is further adjusted by hemacytometer and confirmed by plate counts to be approximately 1 or 5 x 10 CFU/ml. CF-1 mice are infected by injection 1 or 5 x 10⁶ CFU into the tail vein. Antifungal agents are administered intravenously or subcutaneously in ethanokwater (10:90), 4 h post infection and once daily thereafter for 3 or 4 more days. Survival is monitored daily. The ED₅₀ can be defined as that dose which allows for 50% survival of mice.

EXAMPLE 11

Evaluating vascular targeting activity

[0227] Tumors and neoplastic conditions can be treated using the compounds disclosed herein. The occlusion of the blood supply in tumors with vascular targeting agents (VTAs) induces regression of the tumors. The compounds disclosed herein, for example, can be used as VTAs. Many VTAs exhibit their vascular effects by interacting at the colchicine-binding site on microtubules. This interaction induces a characteristic, rapid collapse and occlusion of established vasculature in the tumor and therefore compromises the integrity of existing vessels leading to necrosis. [0228] Vascular collapse can occur, for example, within 30-60 minutes of exposure to the VTA and involves changing the shape of the immature and proliferating, but not the quiescent and mature, endothelial cells in the central portion of the tumor. This differential effect on vascular cells provides a rationale for the selective effects on the tumor due to the higher percentage of proliferating immature endothelial cells in the tumor blood vessels versus normal blood vessels. VTAs can be classified into three overlapping spectra of activity: (1) potent vascular and cytotoxic effects, (2) potent vascular with weak cytotoxic effects, and (3) potent cytotoxic with weak vascular effects.

Combination Therapy with Microtubule Targeting Agents

[0229] The findings that VTAs selectively damage the vasculature in the central part of the tumor versus the periphery, which recovers functionality, support using these agents in combination with chemo therapeutics (e.g., Taxol, Vinblastine and Cisplatin), radiation and angiogenesis inhibitors directed against VEGF and EGF. The new VTAs will supplement rather than supplant these therapies and should provide for greater antitumor activities.

[0230] While not being bound by any particular theory, it is believed that neovascularization in tumors result in spatial and temporal heterogeneity yielding a decline in average blood flow with increasing tumor growth. This heterogeneity is believed to cause regions of hypoxia, acidosis, and general nutrient depletion in some regions of the tumor. These oxygen-deficient or hypoxic cells can demonstrate therapeutic resistance to radiation treatment. VTAs, such as those disclosed herein, may result in extensive tumor necrosis in the central part of tumors with surviving cells found only at the tumor periphery. The viable rim of tumor cells presumably survives because they derive nutritional support from nearby normal tissue blood vessels that are typically non-responsive to VTAs. Because the rim tumor cells are likely to be well oxygenated, they will be sensitive to radiation treatment. Accordingly, combining the VTAs disclosed herein with radiation therapy provide complementary treatment of tumors. The VTA therapy reduces or eliminates the poorly oxygenated and hence radioresistant tumor cell subpopulations while the radiation therapy destroys cells not affected by VTAs.

[0231] Accordingly, in various embodiments, a tumor is treated by combination therapy of a VTA disclosed herein and radiation. The VTA may be administered by any suitable method, including the methods disclosed herein. The radiation treatment may be any suitable radiation treatment such as those currently used to treat tumors, including but not limited to X-ray radiation and proton beam therapy. Tumors that may be treated by this combination approach included cancerous tumors of any type or origin including but not limited to carcinomas (e.g., those associated with skin cancer, cervical cancer, anal carcinoma, esophageal cancer, hepatocellular carcinoma, laryngeal cancer, renal cell carcinoma, stomach cancer, testicular cancer, and thyroid cancer), sarcomas (e.g., osteosarcoma, chondrosarcoma, fibrosarcoma, Kaposi's sarcoma, and rhabdomyosarcoma), melanomas (e.g., those associated with skin cancer and eye cancer), teratomas, and myelomas. In some embodiments, combination therapy is used on advanced large tumors that are more resistant to radiation mono-therapy.

Treatment of other conditions

[0232] In addition to cancer, other diseases may be treated using the VTAs disclosed herein. Conditions include other neoplasms, retinopathies, and any other condition or disease that relies upon blood supply, preferably blood supply from new vasculature in order to remain viable and/or proliferate.

[0233] Many conditions are associated with excessive or inappropriate vasculature. Examples of conditions associated with excessive vasculature include inflammatory disorders such as immune and non-immune inflammation, rheumatoid arthritis, chronic articular rheumatism and psoriasis; disorders associated with inappropriate or inopportune invasion of vessels such as diabetic retinopathy, neovascular glaucoma, retinopathy of prematurity, macular degeneration, corneal graft rejection, retrolental fibroplasia, rubeosis, capillary proliferation in atherosclerotic plaques and osteoporosis; and cancer associated disorders, including for example, solid tumors, tumor metastases, blood born tumors such as leukemias, angiofibromas, Kaposi sarcoma, benign tumors such as hemangiomas, acoustic neuromas, neurofibromas, trachomas, and pyogenic granulomas, as well as other cancers which require vascularization to support tumor growth. Additional examples of vasculature-dependent diseases include, for example, Osier- Webber Syndrome; myocardial angiogenesis; plaque neovascularization; telangiectasia; hemophiliac joints and wound granulation. Furthermore, excessive vasculature is also associated with clinical problems as part of biological and mechanical implants (tissue/organ implants, stents, etc.). The instant compounds and compositions can be used to target vasculature, in preferably to preferentially target disease vasculature over non disease tissue vasculature, and thus the compounds and compositions can be used in the treatment of such conditions. Other diseases in which vascularization plays a role, and to which the instant compounds and compositions can be used, are known by those of skill in the art.

[0234] Examples of retinopathies include age-related macular degeneration (ARMD), diabetic retinopathy, and the like. Pathological angiogenesis is a major contributing factor to a number of retinopathies that collectively are major cause of blindness in the developed world. Kahn and Hiller Am J Ophthalmol (1974) 78, 58-67, which is incorporated herein by reference in its entirety. For example, retinal and disk neovascularization occurs in 30-50% of patients with diabetic retinopathy for more than 20 years. Yanko et al Retina (2003) 23, 518-522, which is incorporated herein by reference in its entirety. Furthermore, subretinal neovascularization is a serious complication in -10% of patients with macular degeneration. Ferris et al Arch Ophthalmol (1984), 102, 1640-1642, which is incorporated herein by reference in its entirety.

[0235] Vascular targeting agents such as Combretastatin A-4 (CA-4) have been shown to cause the disruption of neovessels in non-neoplastic tissue. Griggs et al Br J Cancer (2001) 84, 832-835, which is incorporated herein by reference in its entirety. Additionally, CA-4P was shown to inhibit the retinal neovascularization that occurs during proliferative retinopathy. Griggs et al Am J Path (2002) 160, 1097-1103, which is incorporated herein by reference in its entirety. Finally, CA-4P Phosphate was demonstrated to suppress the development of VEGF induced retinal neovascularization and inhibit the development and/ or cause partial regression of choroidal neovascularization. Nambu et al Invest Ophthalmology & Visual Sci (2003) 44, 3650- 3655, which is incorporated herein by reference in its entirety. The compounds disclosed herein can be used to treat retinopathies. For example, the methodologies of Griggs (2001 and 2002) and Nambu are used to treat retinopathies. Furthermore, the compounds and compositions disclosed herein can be used to treat such retinopathies by applying the compounds and/or compositions to the target area in an effective amount for reducing vascular density and/or vascular proliferation. EXAMPLE 12

In Vitro Action on Microtubules

Purification of Microtubule Protein and Tubulin

[0236] Microtubule protein (MTP) was prepared as previously described (Farrell KW and Wilson L. (1987) Tubulin-colchicine complexes differentially poison opposite microtubule ends. Biochemistry 23(16):3741-8, which is incorporated herein by reference in its entirety). MTP preparations consisting of 70% tubulin and 30% microtubule-associated proteins (MAPs) were isolated from bovine brain by three cycles of warm polymerization and cold depolymerization in PEM100 (100 mM 1-4 piperazinediethansulfonic acid (Pipes), 1 mM MgS0₄, 1 mM EGTA, pH 6.8) and 1 mM GTP. MTP was drop-frozen in liquid nitrogen and stored at -70°C until use. Tubulin was purified from microtubule protein by phosphocellulose chromatography (PC-tubulin) and stored in PEM50 (50 mM Pipes, 1 mM MgS0₄, 1 mM EGTA, pH 6.8). Protein concentration was determined by a Bradford assay (Sigma Chemicals, St. Louis, MO) using bovine serum albumin as the standard (Bradford, 1976).

Test Agents

[0237] Stock solutions of tert-butyl dehydrophenylahistin and KPU-134 are prepared in DMSO. Colchicine (Sigma Chemicals, St. Louis, MO) (CLC) is prepared in water. All agents are shielded from ambient light with amber Eppendorf tubes. Serial dilutions are made in DMSO and/or PEM50 to the desired concentrations.

Fluorescence Spectroscopy

[0238] Fluorescence measurements are performed using a Perkin-Elmer LS50B spectrofluorimeter. PC-tubulin (0.2 mg/ml) can be incubated in PEM50, 2 mM GTP, 3% DMSO, with 0 - 30 μΜ tert-butyl dehydrophenylahistin. The interaction of drug with tubulin is reported by 4,4'-dianilino- Ι,Γ-binaphthyl- 5,5'-disulfonic acid, dipotassium salt (bis-ANS; Molecular Probes, Eugene, OR) fluorescence.

[0239] The bis-ANS fluorophore probes the hydrophobic surface of proteins and a change in intensity of the bis-ANS fluorescence signal is a result of a change in the solvent accessible surface area of a protein. If there is some conformational change that changes the tubulin-bis-ANS interaction upon ligand binding, then bis-ANS can be used to report binding. [0240] PC-tubulin was incubated with 0 - 20 μΜ KPU-134. Bis-ANS (25 μΜ) was then added and relative fluorescence intensities of samples were measured. KPU-134 quenched bis-ANS fluorescence in a concentration-dependent manner (Figure 1 - fluorescence emission spectra of tubulin in the presence of increasing KPU-134).

[0241] The K<j can be determined by fitting experimental data using the equation F = ((-F_max x L)/(Kd + L)) + Fo where F is the fluorescence intensity of bis-ANS- tubulin in the presence of total ligand concentration L, F_max is the bis-ANS fluorescence intensity of fully liganded tubulin, and Fo is bis-ANS fluorescence in the absence of drug. Fmax can be determined by plotting 1/(F₀-F) versus 1/L and extrapolating to 1/L = 0. The fraction of binding sites B occupied by drug can be determined using the following relationship: B = (F₀-F)/(F₀-F_max). The concentration of free ligand can be determined with Lfree = L - B[C] in which [C] is the molar concentration of ligand-binding sites, assuming a single binding site per tubulin dimer.

[0242] For KPU-134 and tubulin as measured by non-linear regression analysis of bis-ANS fluorescence intensity at the emission maximum, K<j = 0.214 μΜ (Figure 2 - change in fluorescence emission maxima with ¾ fit). This is compared to ¾ values of 3.2 μΜ for colchicine and 1.46 μΜ for t-butyl dehydrophenylahistin.

EXAMPLE 13

Combination Treatment with Radiation

Testing of Combination Efficacy

[0243] The efficacy of combination therapy using a compound disclosed herein and radiation against any particular tumor may be tested by the following method. A tumor xenograft is intitated by intramscular injection of tumor cells into hind limbs of nude mice. The tumors are allowed to grow to a desired size. One set of mice are administered a compound disclosed herein and irradiated with radiation therapy. Another set is not administered a compound disclosed herein but is irradiated with radiation therapy. A third set is untreated. Irradiation is conducted using a 6-MV Clinac 600c linear accelerator operating at a dose rate of 400 cGy/min. Total radiation dose is varied among the mice. Clonogenic cell survival is assessed for all tumors using an in vivo to in vitro clonogenic cell curvival assay. 24 h after treatment, the mice are killed and their tumors excised and dissociated into single cell suspensions using a combination of mechanical and enzymatic dissociation procedures. Cells are plated with complete media (Eagle's minimum essesntial medium supplemented with 10% fetal calf serum). Cell survival is determined using a double agar layer assay. 2 mL layers comprising 0.5% agar in complete media are prepared. After solidification, tumor cells are added in a 2 mL volume of 0.33% agar in complete media. After 2 weeks incubation, colonies of more than 50 cells are counted. Tumor surviving fractions are determined by multiplying the calculated fraction of surviving cells by the ratio of cells recovered in treated vs. untreated tumors. Comparison of the tumor surviving fractions of combination therapy treatment with radiation mono-therapy at various radiation doses illustrates whether the combination approach provides increased efficacy as compared with the mono-therapy approach.

Treatment of a Human with Cancer

[0244] A human patient suffering from a cancer characterized by a tumor is administered a compound described herein. The tumor is also irradiated with X-ray radiation. More of the tumor is necrosed than had radiation therapy been used alone.

[0245] The examples described above are set forth solely to assist in the understanding of the invention. Thus, those skilled in the art will appreciate that the disclosed methods and compounds encompass and may otherwise provide further derivatives of dehydrophenylahistins.

[0246] One skilled in the art would readily appreciate that the present invention is well adapted to obtain, for example, the ends and advantages mentioned, as well as others inherent. The methods and procedures described herein are presently representative of preferred embodiments and are exemplary and are not intended as limitations on the scope of the invention. Changes therein and other uses will occur to those skilled in the art which are encompassed within the spirit of the invention.

[0247] It will be readily apparent to one skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention.

[0248] As noted above, all patents and publications mentioned in the specification are indicative of the levels of those skilled in the art to which the invention pertains. All patents and publications are hereby incorporated by reference herein to the extent allowable by law, such that each individual patent and publication may be treated as specifically and individually indicated to be incorporated by reference.

[0249] The invention illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions indicates the exclusion of equivalents of the features shown and described or portions thereof. It is recognized that various modifications are possible within the scope of the invention. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be falling within the scope of the invention.

Claims

WHAT IS CLAIMED IS:

1. A compound having the structure of Formula I and pharmaceutically acceptable salts and tautomers thereof:

(I)

wherein:

Ri, R₂, R₃, and R4 are independently hydrogen, Ci_₆ alkyl, Ci_₆ alkoxy, halogen, or independently absent;

R5 is hydrogen, Ci_₆ alkyl, Ci_₆ alkoxy, halogen, absent, or a bond connecting Z₅ to the phenyl ring in formula I;

Zi, Z₂, Z₃, Z₄, and Z5 are each independently selected from the group consisting of carbon and nitrogen;

R₆ is Ci_6 alkyl;

Z is oxygen or NH;

provided that at least one of R_l5 R₂, R3, R4, and R5 is not hydrogen or absent, or that at least one of Zi, Z₂, Z₃, Z₄, and Z₅ is nitrogen, or that R⁶ is methyl.

2. The compound of claim 1, wherein R₆ is tert-butyl.

3. The compound of any one of the preceding claims, wherein Zi, Z₂, Z₃, Z₄, are each carbon.

4. The compound of any one of the preceding claims, wherein R⁵ is hydrogen or absent.

1, selected from the group consisting of:

-66-

-67-

-68-

and pharmaceutically acceptable salts and tautomers thereof.

6. A compound, selected from the group consisting of:

and pharmaceutically acceptable salts and tautomers thereof.

7. A method for treating a neoplastic disease, comprising administering to a patient in need a compound according to any one of the preceding claims.

8. The method of claim 7, wherein the neoplastic disease is cancer.

9. The method of claim 8, wherein the cancer comprises a tumor.

10. The method of Claim 8, wherein the cancer is a human colorectal adenocarcinoma.

11. The method of Claim 8, wherein the cancer is a prostate adenocarcinoma.

12. The method of Claim 8, wherein the cancer is a leukemia.

13. The method of Claim 8, wherein the cancer is a lung cancer.

14. The method of Claim 8, wherein the cancer is a breast cancer.

15. The method of Claim 8, wherein the cancer is an ovarian cancer.

16. The method of Claim 8, wherein the cancer is a melanoma.

17. The method of Claim 8, wherein the cancer is a uterine sarcoma.

18. The method of Claim 8, wherein the cancer is a prostate cancer.

19. The method of Claim 8, wherein the cancer is a pancreatic cancer.

20. The method of Claim 8, wherein the cancer is taxol resistant.

21. The method of Claim 8, wherein the cancer is a mitoxantrone resistant.