WO1998008829A1 - Pharmaceutical compounds - Google Patents

Abstract

The invention provides novel cryptophycin compounds which can be useful for disrupting the microtubulin system, as anti-neoplastic agents, and for the treatment of cancer. The invention further provides a formulation for administering the novel cryptophycin compounds.

Description

PHARMACEUTICAL COMPOUNDS

This invention relates to the fields of pharmaceutical and organic chemistry and provides novel cryptophycin compounds useful as anti-microtubule agents.

Neoplastic diseases, characterized by the proliferation of cells not subject to the normal control of cell growth, are a major cause of death in humans and other mammals. Clinical experience in cancer chemotherapy has demonstrated that new and more effective drugs are desirable to treat these diseases.

The microtubule system of eucaryotic cells is a major component of the cytoskeleton and is a dynamic assembly and disassembly; this is heterodimers of tubulin are polymerized and form microtubule. Microtubules play a key role in the regulation of cell architecture, metabolism, and division. The dynamic state of microtubules is critical to their normal function. With respect to cell division, tubulin is polymerized into microtubles that form the itotic spindle. The microtubules are then depolymerized when the mitotic spindle's use has been fulfilled. Accordingly, agents which disrupt the polymerization or depolymerization of microtubules, and thereby inhibit mitosis, comprise some of the most effective cancer chemotherapeutic agents in clinical use.

Additionally, the compounds claimed herein possess fungicidal properties as well. Further, such agents having the ability to disrupt the microtubule system can be useful for research purposes.

Certain cryptophycin compounds are known in the literature; however, cryptophycin compounds having even greater solubility and stability are desired. Further, a broader library of cryptophycin compounds could provide additional treatment options for the patient suffering from cancer. Applicants have now discovered novel cryptophycin compounds which can be prepared using total synthetic methods and are therefore well suited for development as pharmaceutically useful agents. Conveniently, commercially available amino acids are utilized to complete the cryptophycin ring system.

The presently claimed invention provides novel cryptophycin compounds of Formula I

wherein

Ar is selected from the group consisting of phenyl or any simple unsubstituted, substituted aromatic, heteroaromatic group, C₁-C₁₂ alkyl, C₂-C₁₂ alkene, C₂-C₁₂ alkyne, NR⁵¹R⁵², OR⁵³, and Formula Ar'

R⁵¹ is selected from the group consisting of hydrogen and C₁-C₃ alkyl;

R⁵² is selected from the group consisting of hydrogen and C₁-C₃ alkyl; R⁵³ is selected from the group consisting of C1-C₁₂ alkyl; R⁵⁴ is selected from the group consisting of hydrogen, Ci-Cβ alkyl, simple aromatic, phenyl, COOR⁵⁷, PO₃H, SO₃H, SO₂R⁵⁸, NR⁵⁹R⁶⁰, NHOR⁶¹, NHCHR⁶¹', CN, N0₂, halogen, OR⁶², and SR⁶³; R⁵⁵ is selected from the group consisting of hydrogen, C_-C₆ alkyl, simple aromatic, phenyl, COOR⁵⁷, PO₃H, SO₃H, SO₂R⁵⁸, NR⁵⁹R⁶⁰, NHOR⁶¹, NHCHR⁶¹', CN, N0₂, halogen, OR⁶², and SR⁶³; R⁵⁶ is selected from the group consisting of hydrogen, C₁-C₆ alkyl, simple aromatic, phenyl, COOR⁵⁷, PO₃H, SO₃H, SO₂R⁵⁸, NR⁵⁹R⁶⁰, NHOR⁶¹, NHCHR⁶¹', CN, N0₂, halogen, OR⁶², and SR⁶³; R⁵⁷ is selected from the group consisting of hydrogen and C1-C12 alkyl;

R⁵⁸ is selected from the group consisting of hydrogen and C₁-C₁₂ alkyl; R⁵⁹ is selected from the group consisting of hydrogen, (Cι~ Ce ) alkyl and fluorenylmethoxycarbonyl (F OC) ;

R⁶⁰ is selected from the group consisting of hydrogen and (Ci-Ce) alkyl;

R⁶¹ is selected from the group consisting of hydrogen, OR⁶⁴, CH₂NHR⁶⁵, NHR⁶⁵ and fluorenylmethoxycarbonyl (FMOC);

R⁶¹' is selected from the group consisting of hydrogen, OR⁶⁴,

CHNHR⁶⁵, NHR⁶⁵ and fluorenylmethoxycarbonyl (FMOC);

R⁶² is selected from hydrogen and Ci-C_δ alkyl;

R⁶³ is selected from hydrogen and C₁-C₆ alkyl; R⁶⁴ is selected from the group consisting of hydrogen, (Ci-

C₆) alkyl, CH₂NR⁶⁶R⁶⁷

R⁶⁵ is selected from the group consisting of hydrogen and Cι-C6 alkyl, NH₂, and fluorenylmethoxycarbonyl (FMOC) ; R⁶⁶ is selected from the group consisting of hydrogen and i-Cδ alkyl and fluorenylmethoxycarbonyl (FMOC) ; R⁶⁷ is selected from the group consisting of hydrogen and Ci-Cβ alkyl;

R¹ and R² are each independently selected from the group consisting of halogen, OH, SH, ammo, mono (Cj-Ce) alkylammo, di (Cι-C₆) alkylamino, tri (Ci-Ce) alkylammonium, (Cι~

C₆) alkylthio, di (Cι-C₆) alkylsulfonium, sulfate, phosphate, OR³¹, and SR³¹; provided that one selected from the group consisting of R¹ and R² is selected from the group consisting of OH, SH, OR³¹ and SR³¹; or

R¹ and R² may be taken together to form an epoxide ring, an aziridine ring, an episulfide ring, a sulfate ring, a cyclopropyl ring or monoalkylphosphate ring; or R¹ and R² may be taken together with Ciβ and C₁7 to _ orm a second bond between Ciβ and C₁₇; R³ is a lower alkyl group;

R⁷ is selected from the group consisting of H, a lower alkyl group, and the side chains of all D- and L- ammo acids; R³¹ is selected from the group consisting of P, S, (C₁-C₁₂) alkyl, B, R³²' and Si;

R³² is selected from the group consisting of ammo acid, carbohydrate, amino sugar, (saccharide) _q, C (O) C -Ce ) alkylR³⁸, and

R³⁴ is (C₁-C₄) alkyl;

R³⁵ is hydrogen or (C₁-C₃) alkyl; R³⁶ is OH, halo, (C₁-C₃) alkyl, OR³⁴, N0₂, NH₂ and heteromatic;

R³⁸ is COOR³⁹,

, NH₂, and amino acid; R³⁹ is H or (Cι-C₆) alkyl;

R⁴⁰, R⁴¹, and R⁴² are each independently selected from the group consisting of hydrogen, OR⁴³, halo, NH₂, NO₂, OP0₃(R ⁶)₂, -0(Cι-C₆)alkylρhenyl, and R⁴⁵ ; R⁴³ is C₁-C6 alkyl; R⁴⁵ is selected from the group consisting of an aromatic group and a substituted aromatic group;

R⁴⁶ is selected from the group consisting of H, Na, and -

C⁽CH₃ ⁾ ₃; q is 2, 3, or 4; or a pharmaceutically acceptable salt thereof.

The present invention provides pharmaceutical formulations, a method for disrupting a microtubule system using an effective amount of a compound of Formula I, a method for inhibiting the proliferation of mammalian cells comprising administering an effective amount of a compound of Formula I, and a method for treating neoplasia in a mammal comprising administering an effective amount of a compound of Formula I. Also, provided is a method for controlling a mycotic infection comprising administering to an animal infected with or susceptible to infection with a fungi, an antifungally effective amount of a compound of Formula I. As used herein, the term "simple alkyl" shall refer to C₁-C₇ alkyl wherein the alkyl may be saturated, unsaturated, branched, or straight chain. Examples include, but are in no way limited to, methyl, ethyl, n-propyl, ISO- propyl, n-butyl, propenyl, ethenyl, sec-butyl, n-pentyl, isobutyl, tert-butyl, sec-butyl, methylated butyl groups, pentyl, tert pentyl, sec-pentyl, methylated pentyl groups and the like. The term "alkenyl" refers to an alkyl group, as defined above, having from one to three double bonds. The term "alkynyl" refers to an alkyl group, as defined above, having at least one triple bond. It is especially preferred that alkynyl has only one triple bond. The term Ci-C_n' alkyl; wherein n' is an integer from 1 to 12 means an alkyl group having from one to the indicated number of carbon atoms. The Ci-C_n- alkyl can be straight or branched chain.

As used herein, the term "D- and L- ammo acid" refers to and includes both the D- and L- forms of the following ammo acids: alanme, leucine, isoleucme, valme, serine, glutamate, glutamme, aspartate, tryptophan, lysme, argimne, tyrosme, histid e, methionme, phenylalanine, asparagme, cyste e, glyc e, proline, and threon e. Accordingly, the term "side chains of all D- and L- ammo acids" means the group attached to the carbon that is attached to both the organic acid and the ammo group. For example, but not limited to -CH< for alanme, CH_?CH (CH-,) - for leucine, and so on.

As used herein the term "ammo acid" means an organic acid containing an am no group. The term includes both naturally occurring and synthetic ammo acids, therefore, the ammo group can be, but is not required to be, attached to the carbon next to the acid. The ammo acid group is attached to the parent molecule via the acid functionality. The term shall refer to, but is in no way limited to (CH₂) ₂NH C00H, CH₂CH (NH₂) CH₂COOH, CH₃CH_?(NH )CH₂COOH, CH3SCH2CH2 (NH₂) CHCOOH and the like.

As used herein, the term "carbohydrate" refers to a class of substituents made up of carbon, hydrogen, and oxygen wherein hydrogen and oxygen are in the same proportions as in water or nearly the proportions as water. The term "carbohydrate" further refers to an aldehyde or ketone alcohol or a compound which on hydrolysis produces and aldehyde or ketone. The term "carbohydrate" is as commonly understood by the skilled artisan. For example, the term refers to, but is in no way limited to, C₁₂H₂₂O₁₁ and C₆H₁₀O₅.

As used herein, the term "amino sugar" refers to a carbohydrate group containing from one to three amino substituents at any available position on the carbohydrate molecule .

As used herein, the term "saccharide" refers to carbohydrate subunits to form disaccharides or polysaccharides . The term means for example, but in no way limited to, lactose, maltose, sucrose, fructose, starch, and the like.

As used herein, the term "substituted phenyl" shall refer to a phenyl group with from one to three non- hydrocarbon substituents which may be independently selected from the group consisting of simple alkyl, Cl, Br, F, and I.

As used herein, the term "substituted benzyl" shall refer to a benzyl group with from one to three non- hydrocarbon substitutents which may be independently selected from the group consisting of simple alkyl, Cl, Br, F, and I wherein such substituents may be attached at any available carbon atom.

As used herein "cycloalkyl" refers to a saturated C₃-C_θ cycloalkyl group wherein such group may include from zero to three substituents selected from the group consisting of C₁-C₃ alkyl, halo, and OR²² wherein R²² is selected from hydrogen and C₁-C₃ alkyl. Such substituents may be attached at any available carbon atom. It is especially preferred that cycloalkyl refers to substituted or unsubstituted cyclohexyl .

As used herein "Lower alkoxyl group" means any alkyl group of one to five carbon atoms bonded to an oxygen atom. As used herein "lower alkyl group" means an alkyl group of one to six carbons and includes linear and non- linear hydrocarbon chains, including for example, but not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, sec-butyl, methylated butyl groups, pentyl, tert pentyl, sec-pentyl, and methylated pentyl groups. As used herein "allylically substituted alkene" means any alkene having from one to seven carbon atoms which contains an allyl substitution on it. As used herein the term "unsaturated lower alkyl" means a lower alkyl group as defined supra . , except that Ci is obviously not envisioned in this instance, wherein from one to two double bonds are present in the unsaturated lower alkyl substituent. A preferred unsaturated lower alkyl is -CH₂-CH=CH₂. The term "lower alkyl-C₃-C₅ cycloalkyl" refers to Cι-C_h alkyl substituted with a C₃-C₅cycloalkyl group. A preferred lower alkyl-C₃-C₅ cycloalkyl group is -C__₂-cyclopropyl; wherein the group is attached to the cryptophycin core structure at R⁹ via the CH₂.

As used herein "epoxide ring" means a three- membered ring whose backbone consists of two carbons and an oxygen atom. As used herein, "aziridine ring" means a three-membered ring whose backbone consists of two carbon atoms and a nitrogen atom. As used herein "sulfide ring" means a three-membered ring whose backbone consists of two carbon atoms and a sulfur atom. As used herein "episulfide ring" means a three-membered ring whose backbone consists of two carbon atoms and a sulfur atom. As used herein "sulfate group" means a five membered ring consisting of a carbon- carbon-oxygen-sulfur-oxygen backbone with two additional oxygen atoms connected to the sulfur atom. As used herein "cyclopropyl ring" means a three member ring whose backbone consists of three carbon atoms. As used herein, "inonoalkylphosphate ring" means a five membered ring consisting of a carbon-carbon-oxygen-phosphorous-oxygen backbone with two additional oxygen atoms, one of which bears a lower alkyl group, connected to the phosphorous atom.

As used herein, "simple unsubstituted aromatic group" refers to common aromatic rings having 4n+2 electrons in a monocyclic conjugated system, for example, but not limited to: phenyl, furyl, pyrrolyl, thienyl, pyridyl and the like, or a bicyclic conjugated system, for example but not limited to indolyl or naphthyl.

As used herein "simple substituted aromatic group" refers to a phenyl group substituted with a single group selected from the group consisting of halogen and lower alkyl group.

As used herein, "heteroaromatic group" refers to aromatic rings which contain one or more non-carbon substituent selected from the group consisting of oxygen, nitrogen, and sulfur. It is most preferred, but not limited to, an aromatic ring having from three to eight members wherein at least one member of the ring system is a heteroatom and the remaining members of the ring system are carbon. As used herein, "halogen" or "halo" refers to those members of the group on the periodic table historically known as halogens. Methods of halogenation include, but are not limited to, the addition of hydrogen halides, substitution at high temperature, photohalogenation, etc., and such methods are known to the skilled artisan.

As used herein, the term "mammal" shall refer to the Mammalia class of higher vertebrates. The term "animal" shall include, but is not limited to, mammals, reptiles, amphibians, and fish. The term "mammal" includes, but is not limited to, a human. The term "treating" as used herein includes prophylaxis of the named condition or amelioration or elimination of the condition once it has been established. The cryptophycin compounds claimed herein can be useful for veterinary health purposes as well as for the treatment of a human patient.

The processes to prepare the compounds of this invention most preferably are completed in the presence of a solvent. The skilled artisan can select appropriate solvents using standard methodologies. Suitable inert organic solvents include those known to the skilled artisan, for example, but not limited to, tetrahydrofuran (THF) and dimethylformamide (DMF) . DMF is especially preferred. Aqueous based solvents may be appropriate for some of the processes utilized herein. The pH of such aqueous solvents may be adjusted as desired to facilitate the process.

Some preferred characteristics of this invention are set forth the following tabular form wherein the features may be independently selected to provide preferred embodiments of this invention. The invention is in no way limited to the features described below:

A) R⁶ is chloro methoxy phenyl;

B) R⁷ is ethyl, propyl, isopropyl, butyl, isobutyl, pentyl, or isopentyl;

C) R⁷ is isopropyl;

D) R³ is methyl; E) Ar' is phenyl with substituent selected from the group consisting of NR⁵⁹R⁶⁰, NHOR⁶¹, and NHCHR⁶¹';

F) R¹ and R² form an epoxide ring; G) R⁷ is methyl

H) R² is a glycinate; I) R² is an acylate;

J) a compound of Formula I is used for disruption of a microtubule system; K) a compound of Formula I is used as an anti- neoplastic agent;

L) a compound of Formula I is used for the treatment of cancer in a mammal; M) a compound of Formula I is used as an antifungal agent;

N) a compound of Formula I is used as an antibacterial agent; 0) Ar is phenyl;

P) Ar is phenyl substituted with one or two from the group consisting of OH, OCH₃, halo, and methyl; Q) R² is selected from the group consisting of halogen, amino, monoalkylamino, dialkylamino, trialkylammonium, alkylthio, dialkylsulfonium, sulfate, phosphate, OR³¹, and SR³¹; ; R) a compound of Formula I is used for the treatment of a fungal infection; S) Ar' is para ethyl substituted phenyl; and T) Ar' is para methyl substituted phenyl.

The present invention provides a method of alleviating a pathological condition caused by hyperproliferating mammalian cells comprising administering to a subject an effective amount of a pharmaceutical or veterinary composition disclosed herein to inhibit proliferation of the cells. In a preferred embodiment of this invention, the method further comprises administering to the subject at least one additional therapy directed to alleviating the pathological condition. In a preferred embodiment of the present invention, the pathological condition is characterized by the formation of neoplasms. In a further preferred embodiment of the present invention, the neoplasms are selected from the group consisting of mammary, small-cell lung, non-small-cell lung, colorectal, leukemia, melanoma, pancreatic adenocarcmoma, central nervous system (CNS) , ovarian, prostate, sarcoma of soft tissue or bone, head and neck, gastric which includes pancreatic and esophageal, stomach, myeloma, bladder, renal, neuroendocrme which includes thyroid and non-Hodgkin's disease and Hodgkm's disease neoplasms.

As used herein "neoplastic" refers to a neoplasm, which is an abnormal growth, such growth occurring because of a proliferation of cells not subject to the usual limitations of growth. As used herein, "anti-neoplastic agent" is any compound, composition, admixture, co-mixture, or blend which inhibits, eliminates, retards, or reverses the neoplastic phenotype of a cell. Anti-mitotic agents may be classified into three groups on the basis of their molecular mechanism of action. The first group consists of agents, including colchicme and colcemid, which inhibit the formation of microtubules by sequestering tubulin. The second group consists of agents, including vmblastme and vmcristme, which induce the formation of paracrystallme aggregates of tubulin. Vmblastme and vmcristme are well known anticancer drugs: their action of disrupting mitotic spindle microtubules preferentially inhibits hyperproliferative cells. The third group consists of agents, including taxol, which promote the polymerization of tubulin and thus stabilizes microtubules.

The exhibition of drug resistance and multiple- drug resistance phenotype by many tumor cells and the clinically proven mode of action of anti-microtubule agents against neoplastic cells necessitates the development of anti-microtubule agents cytotoxic to non-drug resistant neoplastic cells as well as cytotoxic to neoplastic cells with a drug resistant phenotype. Chemotherapy, surgery, radiation therpy, therapy with biological response modifiers, and immunotherapy are currently used in the treatment of cancer. Each mode of therapy has specific indications which are known to those of ordinary skill in the art, and one or all may be employed in an attempt to achieve total destruction of neoplastic cells. Moreover, combination chemotherapy, chemotherapy utilizing compounds of Formula I in combination with other neoplastic agents, is also provided by the subject invention as combination therapy is generally more effective than the use of a single anti-neoplastic agent. Thus, a further aspect of the present invention provides compositions containing a therapeutically effective amount of at least one compound of Formula I, including the non-toxic addition salts thereof, which serve to provide the above recited benefits. Such compositions can also be provided together with physiologically tolerable liquid, gel, or solid carriers, diluents, adjuvants and excipients . Such carriers, adjuvants, and excipients may be found in the U.S . Pharmacopeia, Vol. XXII and National Formulary vol XVII, U.S. Pharmacopeia Convention, Inc. Rockville, MD (1989) . Additional modes of treatment are provided in AHFS Drug Information, 1993 e. by the American Hospital Formulary Service, pp. 522-660. Each of these references are well known and readily available to the skilled artisan. The present invention further provides a pharmaceutical composition used to treat neoplastic disease containing at least one compound of Formula I and at least one additional anti-neoplastic agent. Anti-neoplastic agents which may be utilized in combination with Formula I compounds include those provided in the Merck Index 11, pp 16-17, Merck & Co., Inc. (1989). The Merck Index is widely recognized and readily available to the skilled artisan. In a further embodiment of this invention, ant eoplastic agents may be antimetabolites which may include but are in no way limited to those selected from the group consisting of methotrexate, 5-fluorouracil, 6- mercaptopurme, cytosme, arabinoside, hydroxyurea, and 2- chlorodeoxyadenosine . In another embodiment of the present invention, the anti-neoplastic agents contemplated are alkylating agents which may include but are no way limited to those selected from the group consisting of cyclophosphamide, mephalan, busulfan, paraplatm, chlorambucil, and nitrogen mustard. In a further embodiment, the anti-neoplastic agents are plant alkaloids which may include but are in no way limited to those selected from the group consisting of vmcristme, vmblastme, taxol, and etoposide. In a further embodiment, the anti-neoplastic agents contemplated are antibiotics which may include, but are m no way limited to those selected from the group consisting of doxorubicm, daunorubicm, mitomyc C, and bleomycm. In a further embodiment, the anti-neoplastic agents contemplated are hormones which may include, but are in no way limited to those selected from the group consisting of calusterone, diomostavolone, propionate, epitiostanol, mepitiostane, testolactone, tamoxifen, polyestradiol phosphate, megesterol acetate, flutamide, nilutamide, and trilotane. In a further embodiment, the anti-neoplastic agents contemplated include enzymes which may include, but are in no way limited to those selected from the group consisting of L-Asparginase and aminoacridine derivatives such as, but not limited to, amsacrine. Additional anti- neoplastic agents include those provided by Skeel, Roland T., "Antineoplastic Drugs and Biologic Response Modifier: Classification, Use and Toxicity of Clinically Useful Agents" Handbook of Cancer Chemotherapy (3rd ed.), Little Brown & Co. (1991) .

These compounds and compositions can be administered to mammals for veterinary use. For example, domestic animals can be treated in much the same way as a human clinical patient. In general, the dosage required for therapeutic effect will vary according to the type of use, mode of administration, as well as the particularized requirements of the individual hosts. Typically, dosages will range from about 0.001 to 1000 mg/kg, and more usually 0.01 to 10 mg/kg of the host body weight. Or, alternatively, dosages within these ranges can be administered as a bolus or IV injection, until the desired therapeutic benefits are obtained. Indeed, drug dosage, as well as route of administration, must be selected on the basis of relative effectiveness, relative toxicity, growth characteristics of tumor and effect of Formula I compound on cell cycle, drug pharmacokinetics, age, sex, physical condition of the patient and prior treatment, which can be determined by the skilled artisan.

The compound of Formula I, with or without additional anti-neoplastic agents, may be formulated into therapeutic compositions as natural or salt forms. Pharmaceutically acceptable non-toxic salts include base addition salts which may be derived from inorganic bases such as for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like. Such salts may also be formed as acid addition salts with any free cationic groups and will generally be formed with inorganic acids such as for example, hydrochloric or phosphoric acids or organic acids such as acetic, oxalic, tartaric, mandelic, and the like. Additional excipients which further the invention are provided to the skilled artisan for example in the U.S. Pharmacopeia .

The suitability of particular carriers for inclusion in a given therapeutic composition depends on the preferred route of administration. For example, anti- neoplastic compositions may be formulated for oral administration. Such compositions are typically prepared as liquid solution or suspensions or in solid forms. Oral formulation usually include such additives as binders, fillers, carriers, preservatives, stabilizing agents, e ulsifiers, buffers, mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like. These compositions may take the form of solutions, suspensions, tablets, pills, capsules, sustained relsease formulations, or powders, and typically contain 1% to 95% of active ingedient. More preferably, the composition contains from about 2 % to about 70% active ingredient.

Compositions of the present invention may be prepared as injectables, either as liquid solutions, suspensions, or emulsions; solid forms suitable for solution in or suspension in liquid prior to injection. Such injectables may be administered subcutaneously, intravenously, intraperitoneally, intramuscularly, intrathecally, or intrapleurally. The active ingredient or ingredients are often mixed with diluents, carriers, or excipients which are physiologically tolerable and compatible with the active ingredient (s) . Suitable diluents and excipients are for example, water, saline, dextrose, glycerol, or the like and combinations thereof. In addition, if desired, the compositions may contain minor amounts of auxilary substances such as wetting or emulsifying agents, stabilizing or pH buffering agents.

The invention further provides methods for using Formula I compounds to inhibit the proliferation of mammalian cells by contacting these cells with a Formula I compound in an amount sufficient to inhibit the proliferation of the mammalian cell. A preferred embodiment is a method to inhibit the proliferation of hyperproliferative mammalian cells. For purposes of this invention "hyperproliferative mammalian cells" are mammalian cells which are not subject to the characteristic limitations of growth (programmed cell death for example) . A further preferred embodiment is when the mammalian cell is human. The invention further provides contacting the mammalian cell with at least one Formula I compound and at least one anti-neoplastic agent. The types of anti- neoplastic agents contemplated are discussed supra .

The invention further provides methods for using a compound of Formula I to inhibit the proliferation of hyperproliferative cells with drug-resistant phenotypes, including those with multiple drug-resistant phenotypes, by contacting said cell with a compound of Formula I in an amount sufficient to inhibit the proliferation of a hyperproliferative mammalian cell. A preferred embodiment is when the mammalian cell is human. The invention further provides contacting a Formula I compound and at least one additional anti-neoplastic agent, discussed supra .

The invention provides a method for alleviating pathological conditions caused by hyperproliferating mammalian cells for example, neoplasia, by administering to a subject an effective amount of a pharmaceutical composition containing Formula I compound to inhibit the proliferation of the hyperproliferating cells. As used herein "pathological condition" refers to any pathology arising from the proliferation of mammalian cells that are not subject to the normal limitations of growth. Such proliferation of cells may be due to neoplasms as discussed supra . In a further preferred embodiment the neoplastic cells are human. The present invention provides methods of alleviating such pathological conditions utilizing a compound of Formula I in combination with other therapies, as well as other anti-neoplastic agents. The effectiveness of the claimed compounds can be assessed using standard methods known to the skilled artisan. Examples of such methods are as follows:

Compounds of this invention have been found to be useful against pathogenic f ngi. For example, the usefulness for treating • Cryptococcus neoformans can be illustrated with test results against Cryptococcus neoformans employing yeast nitrogen base detrose agar medium. In carrying out the assay, a compound of this invention is solubilized in dimethyl sulfoxide supplemented with Tween 20. Twofold dilutions are made with sterile distilled water/10 percent DMSO to obtain final drug concentrations in the agar dilution assay plates ranging from 0.008 μg/ml to 16.0 μg/ml against an expanded panel of 84 Cryptococcus neoformans strains. The minimum inhibitory concentration against the panel of 84 Cryptococcus neoformans isolates is determined to illustrate the desired antifungal activity. The compounds are screened for minimum inhibitory concentrations against KB, a human nasopharyngeal carcinoma cell line, LoVo, a human colorectal adenocarcinoma cell line using The Corbett assay, see Corbett, T.H. et al . Cytotoxic Anticancer Drugs: Models and Concepts for Drug Discovery and Development, pp 35-87, Kluwer Academic Publishers: Norwell, 1992. see also, Valeriote, et al. Discovery and Development of Anticancer Agents; Kluwer Academic Publishers, Norwell, 1993 is used for the evaluation of compounds. The most active compounds are further evaluated for cytotoxicity against four different cell types, for example a murine leukemia, a murine solid tumor, a human solid tumor, and a low malignancy fibroblast using the Corbett assay. The compounds are further evaluated against a broad spectrum of murine and human tumors implanted in mice, including drug resistant tumors.

Tumor burden (T/C) (mean tumor burden in treated animals versus mean tumor burden in untreated animals) are used as a further assessment. T/C values that are less than 42% are considered to be active by National Cancer Institute Standards; T/C values less than 10% are considered to have excellent activity and potential clinical activity by National Cancer Institute standards.

Materials

Vinblastine, cytochalasin B, tetramethylrhodamine isothiocyanate (TRITC) -phalloidin, sulforhodamine B (SRB) and antibodies against β-tubulin and vimentin are commercially available from recognized commercial vendors. Basal Medium Eagle containing Earle's salts (BME) and Fetal Bovine Serum (FBS) are also commercially available. Cell Lines

The Jurkat T cell leukemia line and A-10 rat aortic smooth muscle cells are obtained from the American Type Culture Collection and are cultured in BME containing 10% FBS and 50μg/mL gentamycin sulfate. Human ovarian carcinoma cells (SKOV3) and a sub-line which has been selected fro resistance to vinblastine (SKVLBl) were a generous gift from Dr. Victor Ling of the Ontario Cancer Institute. Both cell lines are maintained in BME containing 10% FBS and 50μg/mL gentamycin sulfate. Vinblastine is added to a final concentration of lμg/mL to SKVLBl cells 24 hours after passage to maintain selection pressure for P-glycoprotein- overexpressing cells.

Cell Proliferation and Cycle Arrest Assays

Cell proliferation assays are performed as described by Skehan et al . For Jurkat cells, cultures are treated with the indicated drugs as described in Skehan and total cell numbers are determined by counting the cells in a hemacytometer . The percentage of cells in mitosis are determined by staining with 0.4% Giemsa in PBS followed by rapid washes with PBS. At least 1000 cells per treatment are scored for the presence of mitotic figures and the mitotic index is calculated as the ration of the cells with mitotic figures to the total number of cells counted.

Immunofluorescence Assays

A-10 cells are grown to near-confluency on glass coverslips in BME/10% FBS. Compounds in PBS are added to the indicated final concentrations and cells are incubated for an additional 24 hours. For the staining of microtubules and intermediate filaments, the cells are fixed with cold methanol and incubated with PBS containing 10% calf serum to block nonspecific binding sites. Cells are then incubated at 37_C for 60 mm. with either monoclonal anti-β-tubulm or with monoclonal anti-vimentm at dilutions recommended by the manufacturer. Bound primary antibodies are subsequently visualized by a 45-mmute incubation with fluoresce -con ugated rabbit antimouse IgG. The coverslips are mounted on microscope slides and the fluorescence patterns are examined and photographed using a Zeiss Photomicroscope 111 equipped with epifluorescence optics for fluorescem. For staining of microfilaments, cells are fixed with 3% paraformaldehyde, permeabilized with 0.2%

Triton X-100 and chemically reduced with sodium borohydride (lmg/ML) . PBS containing lOOnM TRITC-phalloidin is then added and the mixture is allowed to incubate for 45 mm. at 37_C. The cells are washed rapidly with PBS before the coverslips are mounted and immediately photographed as described above.

Effects of cryptophycms and vmblastme on Jurkat cell proliferation and cell cycle Dose-response curves for the effects of cryptophycin compounds and vmblastme on cell proliferation and the percentage of cells in mitosis are determined.

Effects of cytochalas B, vinblastine and cryptophycms on the cytoskeleton

Aortic smooth muscle (A-10) cells are grown on glass coverslips and treated with PBS, 2μM cytochalasm B, lOOnM v blastme or lOnM cryptophycin compounds . After 24 hours, microtubules and vimentm intermediate filaments are visualized by indirect immunofluorescence and microfilaments are stained using TRITC - phalloidin. The morphological effects of each drug is examined. Untreated cells displayed extensive microtubule networks complete with perinuclear microtubule organizing centers. Vimentm intermediate filaments were also evenly distributed throughout the cytoplasm, while bundles of microfilaments were concentrated along the major axis of the cell. Cytochalasin B caused complete depolymerization of microfilaments along with the accumulation of paracrystalline remnants. This compound did not affect the distribution of either microtubules or intermediate filaments. The cryptophycin treated microtubules and vimentin intermediates are observed for depletion of microtubules, and collapse of rimentin intermediate filaments.

Effects of cryptophycins and vinblastine on taxol-stabilized microtubules

A-10 cells are treated for 3 hours with 0 or lOμM taxol before the addition of PBS, lOOnM vinblastine or lOrύM cryptophycin compound. After 24 hours, microtubule organization is examined by immunofluorescence as described above. Compared with those in control cells, microtubules in taxol-treated cells were extensively bundled, especially in the cell polar regions. As before, vinblastine caused complete depolymerization of microtubules non-pretreated cells. However, pretreatment with taxol prevented microtubule depolymerization in response to vinblastine. Similarly, microtubules pretreated with taxol are observed with cryptophycin treatment.

Reversibility of microtubule depolymerization by vinblastine and cryptophycin

A-10 cells are treated with either lOOnM vinblastine or lOnM cryptophycins for 24 hr . , resulting in complete microtubule depolymerization. The cells are then washed and incubated in drug-free medium for periods of 1 hour or 24 hours. Microtubules repolymerized rapidly after the removal of vinblastine, showing significant levels of microtubules after 1 hour and complete morphological recovery by 24 hour. Cells are visualized for microtubule state after treatment with a cryptophycin compound of this invention at either 1 hour or 24 hours after removal of the cryptophycin compounds .

Effects of combinations of vinblastine and cryptophycins on cell proliferation

SKOV3 cells are treated with combinations of cryptophycins and vinblastine for 48 hours. The percentages of surviving cells are then determined and the IC₅₀S for each combination is calculated.

Toxicity of cryptophycins, vinblastine and taxol toward SKOV3 and SKVLBl cells

SKVLBl cells are resistant to natural product anticancer drugs because of their over expression of P- glycoprotein. The abilities of taxol, vinblastine and cryptophycin compounds to inhibit the growth of SKOV3 and SKVLBl cells are observed. Taxol caused dose-dependent inhibition of the proliferation of both cell lines with IC₅₀S for SKOV3 and SKVLBl cells of 1 and 8000nM, respectively. Vinblastine also inhibited the growth of both cell lines, with IC₅₀s of 0.35 and 4200nM for SKOV3 and SKVLBl cells, respectively. Cryptophycins compounds of this invention demonstrate activity with an IC₅₀S of from about 1 to about 1000pm for SKOV3 and SKVLBl cells.

Thus it can be demonstrated that the present invention provides novel cryptophycin compounds which are potent inhibitors of cell proliferation, acting by disruption of the microtubule network and inhibition of mitosis. These studies can illustrate that cryptophycin compounds disrupt microtubule organization and thus normal cellular functions, including those of mitosis. Classic anti-microtubule agents, such as colchicme and Vmca alkaloids, arrest cell division at mitosis. It seems appropriate to compare the effect of one of these agents on cell proliferation with the cryptophycin compounds. For this purpose, the Vmca alkaloid v blastme was selected as representative of the classic anti-microtubule agents. Accordingly, the effect of cryptophycin compounds and v blastme on the proliferation and cell cycle progression of the Jurkat T-cell leukemia cell line is compared. Since antimitotic effects are commonly mediated by disruption of microtubules in the mitotic spindles, the effects of cryptophycin compounds on cytoskeletal structures are characterized by fluorescence microscopy. Immunofluorescence staining of cells treated with either a cryptophycin compound or vmblastme demonstrate that both compounds cause the complete loss of microtubules. Similar studies with SKOV3 cells can show that the anti-microtubule effects of cryptophycin compounds are not unique to the smooth muscle cell line.

GC3 human Colon Carcinoma Screen

Selected wells of a 96 well plate were seeded with GC3 human colon carcinoma cells (1x10 cells in lOOμl assay medium/well) twenty four hours prior to test compound addition. Cell free assay medium was added to other select wells of the 96 well plate. The assay medium (RPMI-1640 was the medium used; however, any medium that will allow the cells to survive would be acceptable) was supplemented with 10% dialyzed fetal bovine serum and 25 mM HEPES buffer. The test compound was stored m an amber bottle prior to testing. Fresh dimethylsulfoxide stock solution (200μg/ml) was prepared immediately prior to preparation of test sample dilutions in phosphate-buffered saline (PBS) . A dilution of 1:20 dimethylsulfoxide solution m PBS was prepared such that the final concentration was 10 μg/ml. Serial 1:3 dilutions using PBS (.5ml previous sample of 1ml PBS) were prepared. Falcon 2054 tubes were used for the assay. A lOul sample of each dilution of test compound was added in triplicate to wells of GC3 plates. The plates were incubated for 72 hours at about 37 C. A 10 μl sample of stock 3- [4, 5-dimethyl-2-yl] -2, 5-diphenyltetrazolium bromide salt ("MTT" 5 mg/ml in PBS) was added to each well. The plates were incubated for about an hour at 37 C. The plates were centrifuged, media was decanted from the wells and lOOμl acid-isopropanol (0.04 N HC1 in isopropanol) was added to each well. The plate was read within one hour using a test wavelength of 570nm (SpectraMax reader) . Evaluation of compounds of Formula I suggest that the compounds can be useful in the treatment methods claimed herein. Further, the compounds will be useful for disrupting the microtubule system.

The preparation of the compounds of this invention can be completed using several protocols involving an activated ester followed by chromatography and acid induced deblocking where necessary.

Preparation of any ester wherein R¹ or R² is derived from a carboxylic acid includes a variety of technologies employing acid chlorides, anhydrides, and common activating reagents (eg., carbodiimides) . ^" Any solvent other than participating alcohols can be used. Any mild bases and/or catalysts (amines, carbonates) can be used to aid in esterification.

The conversion of carba ates to the corresponding salts could be effected with any strong acid, namely, mineral acids comprised of hydrogen halides, hydrogen sulfates, hydrogen phosphates, hydrogen nitrates, hydrogen perchlorates, or strong organic acids such as tri luoroacetic, p-toluenesulfonic, and methanesulfonic. The same acids could be used to produce new salts from the corresponding free base.

Compounds of this invention have been tested in the Gc3 assay and have IC₅₀ values ranging from less than one to about 700 nM; however, the most typical values are less than lOOnM.

The processes described herein are most preferably completed in the presence of a solvent. The artisan can select an appropriate solvent for the above described process. Inert organic solvents are particularly preferred; however, under certain conditions an aqueous solvent can be appropriate. For example, if R²⁷ is hydrogen and and R¹³ is BOC an aqueous base as solvent will be effective.

The product of the schemes provided herein can be further derivatized using standard methods to provide further cryptophycin compounds.

The artisan can utilize appropriate starting materials and reagents to prepare desired compounds using the guidance of the previous schemes and following examples. The ester starting material can be prepared, for example, as follows:

Step .

HEW TMG

Step 2

DIBAL

Step 3

SAE

Step 6

TBS-OTf EtaN

Step 8

DBU/ACN

Step 9

KCN

Step 10

DIBAL; HEW

R⁶ has the meaning defined supra .

The scheme for preparing the ester is further explained by the Preparation Section herein which provides one specific application of the scheme for the convenience of the skilled artisan.

The Scheme for preparing the ester is applicable to the Ar substituents claimed herein. The scheme illustration is not intended to limited the synthesis scheme only to the phenyl ring illustrated. Rather, the artisan can broadly apply this process to provide desired starting materials for the compounds claimed herein.

The necessary reaction time is related to the starting materials and operating temperature. The optimum reaction time for a given process is, as always, a compromise which is determined by considering the competing goals of throughput, which is favored by short reaction times, and maximum yield, which is favored by long reaction times .

(7)

ro-CPBA

RT

To further illustrate the invention the following examples are provided. The scope of the invention is m no way to be construed as limited to or by the following examples .

Preparation 1

Step 1. Methyl 5-Phenylpent-2 (E) -enoate. A solution of trimethyl phosphonoacetate (376 g, 417 mL, 2.07 mol) in THF (750 L) was stirred at 0 °C in a 3L 3-neck round bottom flask equipped with a mechanical stirrer and 2 inlet. To the chilled solution, neat tetramethyl guanidme (239 g, 260 L, 2.07 mol) was added dropwise via an addition funnel. The chilled clear pale yellow solution was stirred for 25 minutes at 0 °C. A solution of hydroc namaldehyde (90%, 253 g, 248 mL, 1.9 mol) in THF (125 mL) was added dropwise to the reaction solution slowly. Upon completion of addition, the reaction was stirred for 10 h rising to room temperature. GC indicated a 95:5 ratio of product to starting material. 500ml of water was added to the reaction vessel and the reaction stirred overnight separating into two layers. The organic layer was isolated and the aqueous layer was extracted with t-BuOMe. The organic layers were combined and dried over MgS04, then concentrated in vacuo to yield an orange oil. The crude product was distilled at 129 °C/0.3mm Hg yielding 360.5g, 91.7. yield, of a clear slightly yellow oil.

EIMS m/z 190(13; M+), 159(410, 158(39), 131(90), 130(62), 117(22), 104(12), 95(57), 91(100), 77(21), 65(59); HREIMS /z 190.0998 (C12H14O2 D -0.4 mnu) ; UV l ax (e) 210 (8400), 260 (230) n ; IR nmax 3027, 2949, 1723, 1658, 1454, 1319, 1203, 978, 700 cm^-1; ^λE NMR d (CDCI₃) 7.15-7.3 (Ph-H5;bm), 7.00 (3-H;dt, 15.6/6.6), 5.84 (2-H;dt, 15.6/1.2), 3.70 (OMe;s), 2.76 (5-H2;t, 7.2), 2.51 (4-H2; bdt, 6.6/7.2); ¹³C NMR d (CDC1₃) 166.9 (1), 148.3(3), 140.6 (Ph-1 ' ) , 128.4/128.2 (Ph2'/3'/5^,6' ) , 126.1 (Ph 4*), 121.4 (2). 51.3 (OMe) , 34.2/33.8 (4/5) .

Step 2. 5-phenyl-pent-2-en-l-ol. To a 12L 4-neck round bottom flask equipped with a thermocouple, mechanical stirrer and N2 inlet, a solution of enoate ester (310.5 g, 1.5 mol) in THF (1.5 L) was charged and chilled to -71 °C via a i-PrOH/C02 bath. To the reaction vessel, was added dropwise DIBAL (2.5 L, 1.5 M in toluene, 3.75 mol) at a rate to maintain the reaction temperature < -50 °C. Upon complete addition, the reaction was stirred overnight with the reaction temperature < -50 °C. TLC (3:1 Hexanes : EtOAc, Siθ2) indicated absence of starting material after 16 h.

The reaction temperature was allowed to raise to -15°C. The reaction was quenched slowly withlN HCl (150 mL) . At this point the reaction setup into a gelatinous solid. A spatula was employed to breakup the the semi-solid and IN HCl (200 mL) was added making the mixture more fluid. Concentrated HCl (625 mL) was charged to form a two phase system. The layers were separated and the product extracted with t- BuOMe . The organic layer was dried over MgS04 and concentrated in vacuo to yield a clear pale yellow oil, 247.8g. The crude product was distilled at 145 °C/0.25mm Hg yielding 209.7g, 86.2%.

EIMS m/z 162 (1:M+) 144 (16), 129 (7), 117 (9) 108 (6), 92 (17), 91 (100), 75 (5), 65 (12), HREIMS m/z 162, 1049 (C11H14O, D -0.4 mmu); UV lmax (e) 206 (9900), 260 (360); IR nmax 3356, 2924, 1603, 1496, 1454, 970, 746, 700 cm^-1; ¹E

NMR d 7.15-7.3 (Ph-H5;m) , 5.70 (3-H;dt, 15.6/6.0), 5.61 (2- H;dt, 15.6/4.8), 4.02 (1-H2;d 4.8), 2.68 (5-H2; t, 7.2),

2.40 (0H;bs), 2.36(4-H2; dt, 6.0/7.2); ¹³C NMR dl41.6 (Ph 1'), 131.8(3), 129.5 (2), 128.3/128.2 (Ph 2 ' /3 ' /5 ' /6 ' ) , 125.7 (Ph _')_/ 63.3 (1), 35.4/33.8 (4/5).

Step 3. (2S,3S) -2 , 3-Epoxy-5-phenyl-l-pentanol . To a 1L 3 neck round bottom flask equipped with a mechanical stirrer, thermocouple and nitrogen inlet was added CH₂CI₂ (350 mL) , dried 4 A molecular sieves (30 g) and L- (+) -diethyl tartrate (7.62 g, 0.037 mol) . The resulting mixture was cooled to - 20 °C and treated with Ti (O-i-Pr) 4 (9.2 mL, 0.031 mol), followed by the addition of t-butylhydroperoxide (4.0 M in CH₂CI₂ 182 mL, 0.78 mol) at a rate to maintain the temperature ² -20 °C. Upon complete addition, the reaction mixture was stirred for another 30 mm, and then treated with a solution of the allylic alcohol (50 g, 0.31 mol) in CH₂CI₂ (30 mL) at a rate to maintain the temperature ² -20 °C. The reaction was stirred at the same temperature for 5 h, then filtered into a solution of ferrous sulfate heptahydrate (132 g) and tartaπc acid (40 g) m water (400 mL) at 0 °C. The mixture was stirred for 20 mm, then transferred to a separatory funnel and extracted with t- BuOMe (2x200 mL) . The combined organic phase was stirred with 30% NaOH solution containing NaCl, for 1 h at 0 °C. The layers were again separated, and the aqueous phase extracted with t-BuOMe. The combined organic phase was washed with brine, dried over MgS0₄ and concentrated to yield 52.8 g as an amber oil.

Step 4. {2R, 3R) -2-hydroxy-3-methyl-5-phenylpentan-l-ol .

To a 5L 3 neck round bottom flask equipped with a mechanical stirrer, thermocouple and nitrogen inlet was added hexanes (1L) and cooled to 0 °C. A 2.0M solution of Me₃Al in hexanes (800 mL, 1.6 mol) was added, followed by a solution of the epoxide (120 g, 0.677 mol) hexanes (250 mL) /CH₂C1₂ (50 L) maintaining the temperature below 20 °C. Upon complete addition, the cloudy reaction mixture was stirred at 5 °C for 35 in, whereupon a solution of 10% HCl (300 mL) was added dropwise, followed by the addition of coned HCl (350 mL) . The layers were separated, and the organic phase was washed with brine and dried over MgS0₄. After removal of the volatiles in vacuo, 122.1 gram of an oil was obtained.

Step 5. (2R, 31?) -2-hydro-y-3-methyl-5-phenylpent-l-yl

Tosylate. To a 2L 3 neck round bottom flask equipped with a mechanical stirrer and nitrogen inlet was added the diol (58 g, 0.30 mol), dibutyltin oxide (1.5 g, 0.006 mol, 2 mol%), toluenesulfonyl chloride (57.5 g, 0.30 mol), CH₂C1₂ (580 mL) and triethylamine (42.0 mL, 0.30 mol). The resulting mixture was stirred at room temperature for 2 h (although the reaction was complete within 1 h) , filtered, washed with water and dried over MgSθ . Concentration of the volatiles in vacuo afforded 104.1 gram of a slightly amber oil.

Step 6. (21?, 3K)-2-[ ( ert-Butyldimethylsilyl) oxy] -S-methyl- B-phenylpent-l-yl Tosylate. A solution of the tosylate (100 g, 0.29 mol) and triethylamine (81.0 mL, 0.58 mol) in CH₂CI₂ (1200 mL) was treated with neat TBS-OTf (99 mL, 0.43 mol) dropwise with continued stirring for another 20 min. The reaction was washed twice with brine, dried over MgSθ₄ and concentrated to dryness. The oil was dissolved in a minimal amount of hexanes and filtered over a silica pad, eluting with hexanes :EtOAc (9:1) to yield a slightly amber oil, 134 g. Step 7. (2R- 3R, 5RS) -2- [ ( ert-Butyldimethylsilyl) oxy] -3- methyl-5-bromo-5-phenylpent-l-yl Tosylate. To a 5L 3 neck round bottom flask equipped with a mechanical stirrer, reflux condenser and nitrogen inlet was added CCI₄ (1680 mL) , TBS Ts (140 g, 0.30 mol), NBS (65g, 0.365 mol) and AIBN (16.5 g, 0.10 mol) . The mixture was degassed by evacuation under full vacuum with stirring, and backfilling with nitrogen (3x) . The reaction mixture was then heated to reflux, whereupon the color became dark brown. After 15 min at vigorous reflux, the reaction mixture became light yellow, and chromatographic analysis indicated the reaction was complete. After cooling to room temperature, the reaction was filtered and the filtrate concentrated to dryness. The residue was redissolved in hexanes and filtered again, and concentrated to dryness to afford 170.3 gram as an amber oil.

Step 8. (2R, 31?) -2- [ ( ert-Butyldimethylsilyl) oxy] -3-methyl- 5-phenγlpent- (£) -en-l-yl Tosylate. To a 2L 3 neck round bottom flask equipped with a mechanical stirrer, reflux condenser and nitrogen ^' inlet was added a solution of the bromide (100 g, 0.186 mol) in acetonitrile (700 mL) . DBU (83.6 mL, 0.557 mol) was added and the resulting dark brown solution was stirred at reflux for 15 min. After cooling to room temperature, the solvent was removed in vacuo, and the residue digested in CH₂CI₂ (200 mL) and filtered through a silica pad. The volatiles were again evaporated, and the residue dissolved in EtOAc and washed with water, brine and dried over MgSθ and concentrated to dryness. Preparative mplc (Prep 500) chromatography afforded the desired unsaturated compound (50.3 g, 60% yield over 4 steps). Step 9. (3S, 41.) -3- [ (tert-Butyldimethylsilyl) oxy] - -methyl- 6-phenylhex-5 (2_) -en-1-nitrile. The tosylate (50 g, 0.11 mol) was dissolved in DMSO (1 L) and treated with KCN (14.2 g, 0.22 mol) and water (25 mL) , and the resulting mixture was stirred at 60 °C under nitrogen for 18 h. After cooling to room temperature, the reaction mixture was partitioned between EtOAc (1 L) and water (1 L) . The aqueous phase was extracted with EtOAc (500 mL) , and the combined organic phase was washed with brine and dried over Na₂Sθ . Flash chromatography over silica with CH₂CI₂ afforded the desired nitrile in 92% yield.

Step 10. Methyl (5S, 61?) -5- [ (tert-Butyldimethylsilyl) oxy] - 6-methyl-θ-phenylocta-2 (E) ,7 (E) -dienoate. The nitrile (14.67 g, 46.5 mmol) was dissolved in toluene (200 mL) and cooled to -78 °C under nitrogen. A 1.5M solution of DIBAL in toluene (37.2 mL, 55.8 mmol) was added dropwise with vigorous stirring. Upon complete addition, the cooling bath was removed and the reaction was stirred at room temperature for 1 h. The reaction mixture was carefully poured into IN HCl and the mixture stirred at room temperature for 30 min. The layers were separated, and the organic phase was washed with a saturated aqueous solution of sodium potassium tartrate (2x) , brine and dried over Na₂S0₄. The volatiles were removed in vacuo, and the crude pale yellow oil was used directly in the subsequent condensation. The crude aldehyde from above was dissolved in THF (90 mL) and treated with trimethyl phosphonoacetate (9.03 L, 55.8 mmol) and tetramethylguanidine (7.0 mL, 55.8 mmol) at room temperature under nitrogen. The reaction mixture was stirred for 16 h, then partitioned between EtOAc (200 mL) and water (100 L) . The aqueous phase was back extracted with EtOAc (100 mL) , and the combined organic phase was washed with water, brine and dried over a₂Sθ₄. The volatiles were removed in vacuo, and the crude yellow oil (17.0 g) was chromatographed over silica gel with CH₂CI₂ : cyclohexane (1 : 1 to 2 : 1) to afford 13.67 grams of the desired ester, 78.5%.

Example 1

Synthesis of Allyl (2S) -2- [2' {tert-Butoxycarbonyl) amino-1' - (S) -methyl ] -4-methylpentanoate : (2a)

°— X DMAP.DCC 0«_ X_ .II

(2.)

(1)

846 mg (5.02 mmol, 0.95 eq) of allyl (2S) -2-hydroxy-4- methylpentanoate (1), lg (5.29 mmol) N-(tert- Butoxycarbonyl) -1-alanine and 122 mg (1.06 mmol, 0.2 eq) of 4-dimethylaminopyridine (DMAP) were combined in 8 ml of dry methylene chloride and stirred under a nitrogen atmosphere at 0°C (ice bath). 1.2 g (5.82 mmol, 1.1 eq) of 1,3- dicyclohexylcarbodiimide (DCC) was added in one portion and reaction stirred at room temperature overnight. The reaction was filtered through filter paper and extractive workup with 5% NaHC0₃ and brine was performed. The methylene chloride was dried over NaS0 then removed in vacuo. The crude oil was flash chromatographed on SiO., (10% EtOac/hexane) to yield 1.45 g (80%) of (2a) as a colorless oil .

TLC: Rf-0.309 (20% EtOAc/Hex) IR (cm^-1) (CHCI₃) : 3442, 2982, 2963, 2937, 1745, 1711, 1503, 1369, 1163. ^lE NMR (300MHz, CDClj) d: 5.84-5.93 (m, IH) , 5.28 (dd, 2H, J=17.1 and 5.5 Hz), 5.09-5.13 (m, IH) , 5.01 (br s, IH) , 4.62 (d, 2H, J=5.7 Hz), 4.35-4.41 (m, IH) , 1.65-1.85 (m, 3H) , 1.45 (d, 3H, J=8 Hz), 1.44 (s, 9H) , 0.95 (d, 3H, J=6.6 Hz), 0.93 (d, 3H, J=6.6 Hz) .

Mass (FD) : 344 (M⁺+H) .

Anal: Calcd for C₁₇H,₉N0₆: C, 59.46; H, 8.51; N, 4.08. Found:

C, 59.63; H, 8.48; N, 4.11.

Synthesis of Allyl (2S) -2- [2^/ (tert-Butoxycarbonyl) amino-l ' - (S) -isopropyl ] -4-methylpentanoate: (2b)

Compound (2b) was prepared acccording to the procedure for (2a) (86%).

TLC: Rf=0.447 (20% EtOAc/Hex)

IR (cm^"1) (CHCI₃) : 2966, 1743, 1712, 1503, 1368, 1173, 1158. ^lH NMR (300MHz, CDC1₃) d: 5.85-5.94 (m, IH) , 5.28 (dd, 2H, J=16 and 8 Hz), 5.06-5.10 (m, IH) , 5.01 (br s, IH) , 4.62 (d, 2H, J=5.6 Hz), 4.29-4.33 (m, IH) , 2.25-2.29 (m, IH) , 1.64- 1.85 (m, 3H) , 1.44 (s, 9H) , 0.85-1.06 (m, 12H) .

Mass (FD) : 372 (M⁺+H) . Anal: Calcd for C₁₉H₃₃N0₆: C, 61.43; H, 8.95; N, 3.77. Found: C, 61.71; H, 9.12; N, 3.88.

Synthesis of (2S) -2- [2' (tert-Butoxycarbonyl) amino-1' - (S) - methyl ] -4-methylpentanoate : (3a)

827 mg (2.41 mmol) of Allyl (2S) -2- [2^f ( tert- Butoxycarbonyl) amino-1' - (S) -methyl] -4-methylpentanoate (2a) was dissolved in 50 ml of dry tetrahydrofuran (THF) and stirred under a nitrogen atmosphere at room temperature. 279 mg (0.241 mmol, 0.1 eq) of tetrakis (triphenylphosphine) palladium (0) was added followed by 2.31 ml (26.5 mmol, 11 eq) of dry morpholme. TLC (10% MeOH/CHCl₃) after 1 hr indicated the reaction was complete. The reaction was diluted with diethyl ether (200 ml) then extracted with IN HCl followed by 5% NaHC0₃. The combined 5% NaHC0₃ aqueous layers were then acidified with IN HCl to pH 2 and extracted with diethyl ether (600 ml). The combined ether extracts were then washed with brine and dried over NaS0 . The ether was removed in vacuo yielding a yellow solid (3a) weight 714 mg (98%) .

TLC: Rf=0.114 (10% MeOH/CHCl₃)

IR (cm^"!) (CHC1₃) : 2982, 2963, 1746, 1713, 1503, 1369, 1163. ²H NMR(300MHz, CDCl₃) d: 7.70 (br s, IH) , 5.13-5.06 (m, 2H) , 4.41-4.34 (m, IH) , 1.70-1.87 (m, 3H) , 1.44 (br s, 12H) , 0.96 (d, 3H, J=6.2 Hz), 0.93(d, 3H, J=6.2 Hz).

Mass (FD) : 304 (M⁺+H) .

Anal: Calcd for C₁₄H₂₅N0₆: C, 55.43; H, 8.31; N, 4.62. Found:

C, 55.65; H, 8.37; N, 4.68.

Synthesis of (2S) -2-[2' (tert-Butoxycarbonyl) amino-1' - (S) - isopropyl ] -4-methylpentanoate : (3b)

Compound (3b) was prepared acccording to the procedure for (3a) (81%) .

TLC: Rf=0.183 (10% MeOH/CHCl₃)

IR (cm^"1) (CHC1₃) : 2966, 2936, 1727, 1714, 1504, 1393, 1369, 1159. E NMR(300MHz, CDC1₃) d: 5.10 (dd, IH, J=9.1 and 3.7 Hz),

5.03 (d, IH, J=9.1 Hz), 4.29 (dd, IH, J=9.1 and 4.1 Hz),

2.26-2.30 (m, IH) , 1.68-1.88 (m, 3H) , 1.44 (s, 9H) , 0.91- 1.02 ( , 12H) .

Mass (FD) : 332 (M⁺+H) .

Anal: Calcd for Cι₆H₂9N0₆: C, 57.99; H, 8.82; N, 4.22. Found:

C, 58.22; H, 8.72; N, 4.39. Synthesis of compound (5a)

213 mg (0.362 mmol) of (4), 177 mg (0.536 mmol, 1.5 eq) (2S)-2-[2' (tert-Butoxycarbonyl) amino-1' - (S) -methyl] -4- methylpentanoate (3a) and 11 mg (0.09 mmol, 0.2 eq) of DMAP were dissolved in 3 ml methylene chloride in a flame-dried round bottom flask under a nitrogen atmosphere. 118 mg (0.572 mmol, 1.1 eq) of DCC was then added in one portion and the reaction was stirred overnight. TLC (50%

EtOAc/hexane) indicated that the reaction was complete. The reaction was filtered through filter paper and extractive workup with 5% NaHC0₃ and brine was performed. The methylene chloride was dried over NaS0₄ and then removed in vacuo . The crude solid was then flash chromatographed on SiO_? (35% EtOAc/hexane) to yield 259 mg (82%) of a white solid (5a) .

TLC: Rf= IR (cm^-1) (KBr) : . UV (95% EtOH) : H NMR(300MHz, CDC1₃) d: 7.18-7.37 (m, 7H) , 7.05 (dd, IH, J=6.0 and 1.7 Hz), 6.82 (d, IH, J=7.4 Hz), 6.65-6.78 ( , IH) , 6.40 (d, IH, J=15.7 Hz), 6.01 (dd, IH, J=15.8 and 8.8 Hz), 5.86 (d, IH, J=15.8 Hz), 5.35 (dd, IH, J=8.0 and 2.3

Hz), 5.01 (m, 2H) , 4.96 (dd, IH, J=9.4 and 3.6 Hz), 4.80 (d, IH, J=11.9 Hz), 4.74 (d, IH, J=11.9 Hz), 4.42 (m, IH) , 3.85 (s, 3H) , 3.20 (dd, IH, J=14.0 and 5.9 Hz), 3.06 (dd, IH,

J=14.0 and 7.8 Hz), 2.47-2.59 (m, 3H) , 1.43-1.79 (m, 6H) ,

1.41 (s, 9H) , 1.11 (d, 3H, J=6.8 Hz), 0.88 (d, 3H, J=6.4

Hz), 0.81 (d, 3H, J=6.6 Hz) .

Mass (FD) : .

Anal: Calcd for CιH₅₂Cl₄N₂O₁₀

Synthesis of compound (5b)

500 mg (0.848 mmol) of (4), 267 mg (0.806 mmol, 1.05 eq) (2S) -2- [2' (tert-Butoxycarbonyl) amino-1' - (S) -methyl] -4- methylpentanoate (3b) and 21 mg (0.172 mmol, 0.2 eq) of DMAP were dissolved in 5 ml methylene chloride in a flame-dried round bottom flask under a nitrogen atmosphere. 192 mg (0.933 mmol, 1.1 eq) of DCC was then added in one portion and the reaction was stirred overnight. TLC (50% EtOAc/hexane) indicated that the reaction was complete. The reaction was filtered through filter paper and extractive workup with 5% NaHC0₃ and brine was performed. The methylene chloride was dried over NaS0 and then removed in vacuo. The crude solid was then flash chromatographed on Si0₂ (30% EtOAc/hexane) to yield 682 mg (89%) of a white solid (5b) .

TLC: Rf=0.447 (50% EtOAc/hexane) IR (cm^-1) (KBr) : 2965, 2934, 1752, 1716, 1680, 1645, 1504,

1368, 1282, 1158, 1067.

UV (95% EtOH) : 232 nm (e= 22672), 247 nm (e= 21889).

^XH NMR (300MHz, CDC1₃) d: 7.16-7.32 (m, 7H) , 7.05 (d, IH, J=8.3 Hz), 6.66-6.84 (m, 2H) , 6.40 (d, IH, J=15.9 Hz), 6.01 (dd, IH, J=8.8 and 15.9 Hz), 5.86 (d, IH, J=15.7 Hz), 5.47 (d, IH, J=9.9 Hz), 5.03-5.14 ( , 2H) , 4.91 (dd, IH, J=9.9 and 3.8 Hz), 4.76 (s, 2H) , 4.38 (dd, IH, J=9.5 and 4.1 Hz), 3.84 (s, 3H) , 3.20 (dd, IH, J=14.0 and 5.7 Hz), 3.05 (dd, IH, J=14.0 and 7.3 Hz), 2.46-2.59 (m, 3H) , 2.26-2.32 (m, IH), 1.44-1.78 ( , 3H) , 1.42 (s, 9H) , 1.11 (d, 3H, J=6.7 Hz), 1.03 (d, 3H, J=6.8 Hz), 0.95 (d, 3H, J=6.8 Hz), 0.87 (d, 3H, J=6.4 Hz), 0.80 (d, 3H, J=6.5 Hz).

Mass (FD) : 902 (M⁺) .

Anal: Calcd for C₄₃H₅₆Cl₄N_?O₁₀: C, 57.21; H, 6.25; N, 3.10.

Found: C, 58.09; H, 7.04; N, 3.26.

Synthesis of compound (6a)

249 mg (0.285 mmol) of (5a) and 1 g of zinc dust were dissolved in 10 ml glacial acetic acid. The reaction was then sonicated for 1 hr . TLC (10% MeOH/ CHC1₃) showed no more starting material, so zinc was filtered through a pad of celite and the filtrate concentrated in vacuo leaving an off-white solid. The crude solid was then dissolved in 12 ml of trifluoroacetic acid (TFA) and stirred at room temperature for 1 hr . TFA was removed in vacuo and remaining oil was triturated with MeOH until white solid formed. This solid was collected and dried under high vacuum yielding 150 mg(70%) of the TFA salt of compound (6a) .

TLC: Rf=0.169 (10% MeOH/CHCl₃)

IR (cm^-1) (KBr) : 2960, 1768, 1745, 1640, 1504, 1391, 1258, 1198.

UV (95%EtOH) : 232 nm (e= 24795), 247 nm (e= 26763). ^lE NMR (300MHz, CD₃0D) d: 7.15-7.34 (m, 7H) , 7.07 (dd, IH, J=8.3 and 1.5 Hz), 6.90 (d, IH, J=8. Hz) , 6.55-6.61 (m, IH), 6.43 (d, IH, J=15.8 Hz), 5.94-6.08 (m, 2H) , 4.86-5.02 (m, 4H) , 4.46-4.50 (m, IH) , 4.01-4.06 (m, IH) , 3.80 (s, 3H) ,

3.09 (dd, IH, J=13.6 and 5.4 Hz), 2.88 (dd, IH, J=13.6 and 7.0 Hz), 2.42-2.65 (m, 3H) , 1.49-1.66 (m, 7H) , 1.12 (d, 3H, J=6.7 Hz), 0.78 (d, 3H, J=6.4 Hz), 0.69 (d, 3H, J=6.5 Hz) .

Mass (FD) : 643 (M⁺+H) . Anal: Calcd for C₃₄H₄₃C1N₂0₈ (TFA salt) : C, 57.10; H, 5.86; N,

3.70. Found: C, 62.97; H, 6.49; N, 4.42.

Synthesis of compound (6b)

Compound (6b) was prepared acccordmg to the procedure for (6a) (72%) .

TLC: Rf=0.369 (20% MeOH/CHCl₃)

IR (cm^-1) (KBr) : 2962, 2935, 1747, 1675, 1646, 1606, 1502,

1390, 1257, 1196, 1065.

UV (95% EtOH) : 232 nm (e= 22882), 251 nm (e= 25493). ^λK NMR(300MHz, CDCl₃) d: 8.20 (d, IH, J=5.2 Hz), 7.17-7.37 (m, 7H) , 6.98-7.13 (m, 3H) , 6.70-6.86 (m, 2H) , 6.60 (d, IH, J=5.9 Hz), 6.40 (d, IH, J=15.7 Hz), 5.86-6.05 (m, 2H) , 5.11- 5.15 (m, IH) , 4.87 (dd, IH, J=9.5 and 2.8 Hz), 4.60-4.62 (m, IH) , 3.85 (br s, 3H) , 3.59 (br s, IH) , 3.05-3.21 (m, IH) , 2.46-2.60 ( , 3H) , 2.27-2.32 (m, IH) , 1.45-1.76 (m, 3H) , 1.11 (d, 3H, J=6.6 Hz), 0.94-1.06 (m, 6H) , 0.86 (d, 3H, J=6.2 Hz) , 0.79 (d, 3H, J=6.3 Hz) .

Mass (FD) : 672 (M⁺+H) .

Anal: Calcd for C₃₆H₄₇C1N₂0₈ (TFA salt) : C, 58.12; H, 6.16; N,

3.57. Found: C, 63.35; H, 7.02; N, 4.73.

Synthesis of compound (7a)

(6a) (7a)

133 mg (0.176 mmol) of (6a) was dissolved in 40 ml of dry

N,N-dιmethylformamιde (DMF) and stirred under an argon atmosphere at room temperature. 88 mg (0.229 mmol, 1.3 eq) of pentafluorophenyldiphenylphosphinate (FDPP) was then added and reaction stirred for 16 hrs . TLC (20% MeOH/CHCl₃) indicated the reaction was complete, so DMF was removed in vacuo . The resulting oil was triturated with hexanes to yield a solid which was flash chromatographed on Si0₂ (5% MeOH/CHCl₃) yielding 60 mg (55%, based on TFA salt as the starting material) of cyclized product (7a) as a white solid.

TLC: Rf=0.677 (5% MeOH/CHCl₃)

IR (cm^-1) (KBr) : 3322, 3286, 2950, 1756, 1735, 1663, 1643, 1539, 1503, 1258, 1214, 1066, 971, 746, 693. UV (95%EtOH) : 249 nm (e= 19840) . ^:H NMR (300MHz, CDC1₃) d: 7.20-7.34 (m, 7H) , 7.11 (dd, IH, J=8.3 and 1.9 Hz), 6.83 (d, IH, J=8.4 Hz), 6.70-6.77 (m, IH) , 6.34-6.43 (m, 2H) , 6.02 (dd, IH, J=15.8 and 8.8 Hz), 5.76 (d, IH, J=15 Hz), 5.64 (d, IH, J=10.1 Hz), 5.11-5.18 (m, IH), 4.78-4.86 (m 2H) , 4.62-4.68 (m, IH) , 3.86 (s, 3H) , 3.18 (dd, IH, J=14.2 and 8.0 Hz), 2.82 (dd, IH, J=14.2 and

7.1 Hz), 2.45-2.63 (m, 2H) , 2.37-2.42 (m, IH) , 1.55-1.65 (m, 2H) , 1.40 (d, 3H, J=7.3 Hz), 1.14 (d, 3H, J=6.8 Hz), 0.71- 0.74 (m, 6H) .

Mass ( FD) : 624 (M⁺-H) . Anal : Calcd for C₃₄H₄₁C1N₂0₇ : C, 65 . 32 ; H, 6 . 61 ; N, 4 . 48 .

Found : C , 65 . 12 ; H, 6 . 44 ; N, 4 . 47 . Example 2

Synthesis of compound (7b)

(6b) (7b)

Compound (7b) was prepared acccording to the procedure for (7a) (48%) .

TLC: Rf=0.351 (50% EtOAc/hexane)

IR (cm^-1) (KBr) : 3349, 3290, 2968, 2958, 1752, 1732, 1657,

1633, 1535, 1506, 1255_/ 1175, 1067, 1016, 691.

UV (95% EtOH) : 230 nm (e= 21399), 249 nm (e= 25158). ^lE NMR (300MHz, CDC1₃) d: 7.19-7.39 (m, 7H) , 7.12 (d, IH, J=8.0 Hz), 6.83 (d, IH, J=8.5 Hz), 6.71 (m, IH) , 6.54 (d, IH, J=10.5 Hz), 6.40 (d, IH, J=16.0 Hz), 5.95-6.05 ( , IH) , 5.76 (d, IH, J=15.0 Hz), 5.54 (d, IH, J=9.9 Hz), 5.20 (m, IH) , 4.78-4.89 (m, 2H) , 4.41 (dd, IH, J=10.6 and 4.9 Hz), 3.87 (s, 3H) , 3.19 (dd, IH, J=14.6 and 6.9 Hz), 2.85 (dd, IH, J=14.6 and 7.9 Hz), 2.30-2.63 (m, 4H) , 1.37-1.66 (m, 2H) , 1.13 (d, 3H, J=6.8 Hz), 0.96-0.99 ( , 6H) , 0.74 (d, 3H, J=6.2 Hz), 0.70 (d, 3H, J=6.2 Hz).

Mass (FD) : 652 (M⁺-H) .

Anal: Calcd for C₃₆H₄c,ClN₂0₇ : C, 66.20; H, 6.94; N, 4.29.

Found: C, 65.94; H, 6.86; N, 4.23. Example 3 Synthesis of epoxides (8a) and (9a)

50 mg (0.08 mmol) of (7a) was dissolved in 3 ml dry methylene chloride and stirred under a nitrogen atmosphere at room temperature. 28 mg (0.16 mmol, 2 eq) of meta- chloroperoxybenzoic acid (m-CPBA) was then added and reaction monitored by HPLC (2X3.9mmXl50mm Novapak C₁₈ columns, 80% CH₃CN/H₂0, 1.0 ml/min, 1= 256 nm) . The reaction was stirred 4.5 hrs and HPLC shows reaction only 50% complete so 28 mg (2 eq) more m-CPBA was added. After stirring overnight (24 hrs) HPLC shows no starting material and the two epoxides (8a) and (9a) formed in a 2/1 ratio. The reaction was diluted with methylene chloride and washed with 5% NaHC0₃ and brine. The methylene chloride was then dried over NaS0₄ and concentrated in vacuo yielding 73 mg of crude white solid which was purified on a semi-prep reverse phase C₁₈ HPLC column using the following conditions:

column: Rainin 2.5 cmX30 cm semi-prep reverse phase C_ιe HPLC column solvent: 68% CH₃CN/H₂0 isocratic flow rate 20 ml/min 1- 220 nm

epoxide (8a): beta-iso er, retention time (2X3.9mmX150rnm Novapak C₁₈ columns, 80% CH₃CN/H₂0, 1.0 ml/min, 1= 220 nm) 7.51 min. epoxide (9a): alpha-isomer, retention time (2X3.9mmX150mm Novapak C₁₈ columns, 80% CH₃CN/H₂0, 1.0 ml/mm, 1= 220 nm) 8.33 min.

Example 4 Synthesis of epoxides (8b) and (9b)

<«.

Epoxides (8b) and (9b) were prepared according to the procedure for (8a) and (9a) except a different column was used for the purification.

column: Technikrom kromasil 2 cmX25 cm semi-prep reverse phase C₁₈ HPLC column (5m) solvent: gradient 50% CH₃CN/H₂O-70% CH₃CN/H0 over 2 hrs flow rate 28 ml/min 1= 220 nm

epoxide (8b): beta-isomer, retention time (2X3.9mmX150mm Novapak C₁₈ columns, 70% CH₃CN/H₂0, 1.0 ml/min, 1= 220 nm) 14.6 min. epoxide (9b): alpha-isomer, retention time (2X3.9mmX150mm Novapak C₁₈ columns, 70% CH₃CN/H_?0, 1.0 ml/min, 1= 220 nm) 16.6 min.

Claims

Claims

1. A compound of Formula I

wherein

Ar is selected from the group consisting of phenyl or any simple unsubstituted, substituted aromatic, heteroaromatic group, C₁-C₁₂ alkyl, C₂-C1₂ alkene, C₂-C₁₂ alkyne, NR⁵¹R⁵², OR⁵³, and Formula Ar'

R⁵¹ is selected from the group consisting of hydrogen and C₁-C₃ alkyl; R⁵² is selected from the group consisting of hydrogen and

C1-C3 alkyl;

R⁵³ is selected from the group consisting of C₁-C₁₂ alkyl;

R⁵⁴ is selected from the group consisting of hydrogen, Ci-Cg alkyl, simple aromatic, phenyl, COOR⁵⁷, PO₃H, SO₃H, SO₂R⁵⁸, NR⁵⁹R⁶⁰, NHOR⁶¹, NHCHR⁶¹', CN, N0₂, halogen, OR⁶², and SR⁶³; R⁵⁵ is selected from the group consisting of hydrogen, Ci-Cβ alkyl, simple aromatic, phenyl, COOR⁵⁷, PO₃H, SO₃H, SO₂R⁵⁸,

NR⁵⁹R⁶⁰, NHOR⁶¹, NHCHR⁶¹', CN, N0 , halogen, OR⁶², and SR⁶³;

R⁵⁶ is selected from the group consisting of hydrogen, C1-C₆ alkyl, simple aromatic, phenyl, COOR⁵⁷, PO₃H, SO₃H, S0₂R⁵⁸,

NR⁵⁹R⁶⁰, NHOR⁶¹, NHCHR⁶¹', CN, N0₂, halogen, OR⁶², and SR⁶³;

R⁵⁷ is selected from the group consisting of hydrogen and

C1-C12 alkyl;

R⁵⁸ is selected from the group consisting of hydrogen and C1-C12 alkyl;

R⁵⁹ is selected from the group consisting of hydrogen, (Ci-

Cβ ) alkyl and fluorenylmethoxycarbonyl (FMOC) ;

R⁶⁰ is selected from the group consisting of hydrogen and

(Cι-C₆) alkyl; R⁶¹ is selected from the group consisting of hydrogen, OR⁶⁴,

CH₂NHR⁶⁵, NHR⁶⁵ and fluorenylmethoxycarbonyl (FMOC) ;

R⁶¹' is selected from the group consisting of hydrogen, OR⁶⁴,

CH₂NHR⁶⁵, NHR⁶⁵ and fluorenylmethoxycarbonyl (FMOC) ;

R⁶² is selected from hydrogen and C1-C₆ alkyl; R⁶³ is selected from hydrogen and Ci-Cβ alkyl;

R⁶⁴ is selected from the group consisting of hydrogen, (Ci-

Cβ) alkyl, CH₂NR⁶⁶R⁶⁷

R⁶⁵ is selected from the group consisting of hydrogen and Ci-C_δ alkyl, NH₂, and fluorenylmethoxycarbonyl (FMOC) ; R⁶⁶ is selected from the group consisting of hydrogen and C1-C₆ alkyl and fluorenylmethoxycarbonyl (FMOC) ;

R⁶⁷ is selected from the group consisting of hydrogen and Ci-Cβ alkyl;

R¹ and R² are each independently selected from the group consisting of halogen, OH, SH, amino, mono (C]-C.) alkylamino, di (C-C₆) alkylamino, tri (Cι~C₆) alkylam onium, (Cι~ C₆) alkylthio, di (C]-C₆) alkylsulfonium, sulfate, phosphate, OR³¹, and SR³¹; provided that one selected from the group consisting of R¹ and R² is selected from the group consisting of OH, SH, OR³¹ and SR³¹; or

R¹ and R² may be taken together to form an epoxide ring, an aziridine ring, an episulfide ring, a sulfate ring, a cyclopropyl ring or monoalkylphosphate ring; or R¹ and R² may be taken together with Ciβ and C η to form a second bond between Cχ₈ and C₁₇; R³ is a lower alkyl group;

R⁷ is selected from the group consisting of H, a lower alkyl group, and the side chains of all D- and L- amino acids; R³¹ is selected from the group consisting of P, S, (C₁-C₁₂) alkyl, B, R³²' and Si;

R³² is selected from the group consisting of amino acid, carbohydrate, amino sugar, (saccharide) _q, C (O) Cχ-C₆) alkylR³⁸, and

R³⁴ is (C₁-C₄) alkyl;

R³⁵ is hydrogen or (C₁-C₃) alkyl;

R³⁶ is OH, halo, (C₁-C3) alkyl, OR³⁴, N0₂, NH₂ and heteromatic; R³⁸ is COOR³⁹,

, NH₂, and amino acid;

R³⁹ is H or (Cι-C₆) alkyl;

R⁴⁰, R⁴¹, and R⁴² are each independently selected from the group consisting of hydrogen, OR⁴³, halo, NH₂, NO₂, OP0₃(R⁴⁶)₂, -0(Ci-C₆)alkylphenyl, and R⁴⁵ ; R⁴³ is Ci-Cβ alkyl;

R⁴⁵ is selected from the group consisting of an aromatic group and a substituted aromatic group;

R⁴⁶ is selected from the group consisting of H, Na, and - C(CH₃)₃; q is 2, 3, or 4; or a pharmaceutically acceptable salt thereof.

2. A compound of Claim 1 wherein R⁷ is isopropyl.

3. A compound of Claim 1 wherein R⁷ is isobutyl.

4. A compound of Claim 1 wherein R⁷ is selected from a side chain of a D- and L- amino acid.

5. A compound of Claim 4 wherein Ar is paramethyl substituted phenyl.

6. A compound of Claim 4 wherein R² is OR³¹.

7. A compound of Claim 4 wherein R² is SR³¹.

8. A compound of Claim 7 wherein R³ is methyl.

9. A method for disrupting microtubule binding in a mammal comprising administering an effective amount of a compound of Claim 1.

10. A method for disrupting microtubule binding in vitro comprising administering an effective amount of a compound of Claim 1.

11. A method for treating a neoplasm in a mammal comprising administering an effective amount of a compound of Claim 1 to a patient in need thereof.

12. A formulation comprising a compound of Claim 1 and one or more pharmaceutically acceptable diluents or carriers therefor.

13. A method for treating an animal infected with or susceptible to infection with a fungi, an antifungally effective amount of a compound of Claim 1.

14. A method of Claim 18 wherein the animal is a mammal .

15. A method of Claim 18 wherein the animal is infected with a fungi.