PHARMACEUTICAL COMPOUNDS

COMPOSES PHARMACEUTIQUES

English 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.

French Abstract

L'invention concerne de nouveaux composés de cryptophycine, lesquels peuvent être utiles pour interrompre le système de la microtubuline en tant qu'agents anti-néoplasiques et dans le traitement du cancer. L'invention concerne également une formulation destinée à l'administration des nouveaux composés de cryptophycine.

Claims

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Claims

1. A compound of Formula I

Image

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'

Image;


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, C1-C6
alkyl, simple aromatic, phenyl, COOR57, PO3H, SO3H, SO2R58,
NR59R60, NHOR61, NHCHR61', CN, NO2, halogen, OR62, and SR63;


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R55 is selected from the group consisting of hydrogen, C1-C6
alkyl, simple aromatic, phenyl, COOR57, PO3H, SO3H, SO2R58,
NR59R60, NHOR61, NHCHR61', CN, NO2, 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, NO2, 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,
(C1-C6) alkyl and fluorenylmethoxycarbonyl (FMOC);
R60 is selected from the group consisting of hydrogen and
(C1-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 C1-C6 alkyl;
R64 is selected from the group consisting of hydrogen,
(C1-C6) alkyl, CH2NR66R67
R65 is selected from the group consisting of hydrogen and
C1-C6 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
C1-C6 alkyl;
R1 and R2 are each independently selected from the group
consisting of halogen, OH, SH, amino, mono(C1-C6)alkylamino,
di(C1-C6)alkylamlno, tri(C1-C6)alkylammonlum,



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(C1-C6)alkylthio, di(C1-C6)alkylsulfonium, sulfate, phosphate,
OR31, and SR31;
provided that one selected from the group consisting of
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 C18 and C17 to form a
second bond between C18 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)C1-C6)alkylR38,
and

Image;

R34 is (C1-C4) alkyl;
R35 is hydrogen or (C1-C3)alkyl;
R36 is OH, halo, (C1-C3)alkyl, OR34, NO2, NH2 and
heteromatic;


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Image
R38 is COOR39, , NH2, and amino acid;
R39 is H or (C1-C6)alkyl;
R40, R41, and R42 are each independently selected from the
group consisting of hydrogen, OR43, halo, NH2, NO2,
OPO3(R46)2, -O(C1-C6)alkylphenyl, 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.

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.


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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.

Description

10152025_3o35CA 02264297 1999-02-222 WO 98108829 PCT/US97/15236-1-PHARMACEUTICAL COMPOUNDSThis invention relates to the fields ofpharmaceutical and organic chemistry and provides novelcryptophycin compounds useful as anti-microtubule agents.Neoplastic diseases, characterized by the proliferationof 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 demonstratedthat new and more effective drugs are desirable to treatthese diseases.The microtubule system of eucaryotic cells is amajor component of the cytoskeleton and is a dynamicassembly and disassembly; this is heterodimers of tubulinare polymerized and form microtubule. Microtubules play akey role in the regulation of cell architecture, metabolism,and division. The dynamic state of microtubules is criticalto their normal function. With respect to cell division,tubulin is polymerized into microtubles that form themitotic spindle. The microtubules are then depolymerizedwhen the mitotic spindle's use has been fulfilled.Accordingly, agents which disrupt the polymerization ordepolymerization of microtubules, and thereby inhibitmitosis, comprise some of the most effective cancerchemotherapeutic agents inclinical use.Additionally, the compounds claimed herein possessfungicidal properties as well. Further, such agents havingthe ability to disrupt the microtubule system can be usefulfor research purposes.Certain cryptophycin compounds are known in theliterature; however, cryptophycin compounds having evengreater solubility and stability are desired. Further, abroader library of cryptophycin compounds could provide101520CA 02264297 1999-02-22~ WO 98108829 PCT/US97/15236_2..additional treatment options for the patient suffering fromcancer. Applicants have now discovered novel cryptophycincompounds which can be prepared using total syntheticmethods and are therefore well suited for development aspharmaceutically useful agents. Conveniently, commerciallyavailable amino acids are utilized to complete thecryptophycin ring system.The presently claimed invention provides novelcryptophycin compounds of Formula ICl C) C) I{DJ “~whereinAr is selected from the group consisting of phenyl or anysimple unsubstituted, substituted aromatic, heteroaromaticgroup, C1-C12 alkyl, C2-C12 alkene, C2—C12 alkyne, NR51R52,OR53, and Formula Ar‘R51 is selected from the group consisting of hydrogen andC1-C3 alkyl;R52 is selected from the group consisting of hydrogen andC1-C3 alkyl;1015202530CA 02264297 1999-02-22- WO 98/08829 PCT/US97I15236-3-R53 is selected from the group consisting of C1-C12 alkyl;R54 is selected from the group consisting of hydrogen, C1-C5alkyl, simple aromatic, phenyl, COOR57, PO3H, SO3H, SO2R55,NR59R60, NHOR61, NHcHR61', CN, N02, halogen, oR62, and SR63;R55 is selected from the group consisting of hydrogen, C1-C5alkyl, 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-C5alkyl, simple aromatic, phenyl, COOR57, PO3H, SO3H, SO2R53,NR59R60, NHOR51, NHCHR61‘, CN, N02, halogen, OR52, and SR63:R57 is selected from the group consisting of hydrogen andC1-C12 alkyl;R58 is selected from the group consisting of hydrogen andC1-C1; alkyl;R59 is selected from the group consisting of hydrogen, (C1-C5$ alkyl and fluorenylmethoxycarbonyl (FMOC);R50 is selected from the group consisting of hydrogen and(C1-C5) alkyl;R61 is selected from the group consisting of hydrogen, OR64,CHZNHR55, NHR55 and fluorenylmethoxycarbonyl (FMOC);-R51' is selected from the group consisting of hydrogen, OR64,CHgNHR55, NHR65 and fluorenylmethoxycarbonyl (FMOC);R62 is selected from hydrogen and C1-C5 alkyl;R63 is selected from hydrogen and C1-C5 alkyl;R64 is selected from the group consisting of hydrogen, (C1-C5) alkyl, cH2NR56R67R55 is selected from the group consisting of hydrogen andC1-C5 alkyl, NH2, and fluorenylmethoxycarbonyl (FMOC);R65 is selected from the group consisting of hydrogen andC1-C5 alkyl and fluorenylmethoxycarbonyl (FMOC);10152025CA 02264297 1999-02-22' WO 98/08829 PCT/US97/15236_4_R67 is selected from the group consisting of hydrogen andC1-C6 alkyl;R1 and R2 are each independently selected from the groupconsisting of halogen, OH, SH, amino, mono(Cy%k)alkylamino,di(C1-C6)alkylamino, tri(C1—C6)alkylammonium, (C1-C6)alkylthio, di(C1-C6)alkylsulfonium, sulfate, phosphate,OR31, and SR31;provided that one selected from the group consisting of R1and R2 is selected from the group consisting of OH, SH, OR31and SR31; orR1 and R2 may be taken together to form an epoxide ring, anaziridine ring, an episulfide ring, a sulfate ring, acyclopropyl ring or monoalkylphosphate ring; orR1 and R2 may be taken together with C13 and C17 to form asecond bond between C13 and C17;R3 is a lower alkyl group;R7 is selected from the group consisting of H, a lower alkylgroup, 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, R32r and Si;R32 is selected from the group consisting of amino acid,carbohydrate, amino sugar, {saccharide)q, C(O)C1—C5)alkylR38,W 2NAss .R34 is (C1-C4) alkyl;R35 is hydrogen or (C1-C3)alkyl;101520.25CA 02264297 1999-02-224 WO 98/08829 PCT/US97/15236_ 5 _R36 is OH, halo, (C1-C3)alkyl, OR34, NO2, NH2 andheteromatic;_.|I_ R40L /. £g\;>\R“ . .R35 is COOR39, R , NH2, and amino acid;R39 is H or (C1—C5)alkyl;R40, R41, and R42 are each independently selected from thegroup consisting of hydrogen, OR43, halo, NH2, N02,OPO3(R45)2, -O(C1—C5)alkylphenyl, and R45R43 is C1-C6 alkyl;R45 is selected from the group consisting of an aromaticgroup and a substituted aromatic group;R45 is selected from the group consisting of H, Na, and -C(CH3)3;q is 2, 3, or 4; ora pharmaceutically acceptable salt thereof.The present invention provides pharmaceuticalformulations, a method for disrupting a microtubule systemusing an effective amount of a compound of Formula I, amethod for inhibiting the proliferation of mammalian cellscomprising administering an effective amount of a compoundof Formula I, and a method for treating neoplasia in amammal comprising administering an effective amount of acompound of Formula I. Also, provided is a method forcontrolling a mycotic infection comprising administering toan animal infected with or susceptible to infection with afungi, an antifungally effective amount of a compound ofFormula I.1015202530CA 02264297 1999-02-22» WO 98/08829 PCT/US97/15236-6-As used herein, the term "simple alkyl" shallrefer 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 groupsand 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 definedabove, having at least one triple bond. It is especiallypreferred that alkynyl has only one triple bond. The termC1-Cnv alkyl; wherein n‘ is an integer from 1 to 12 means analkyl group having from one to the indicated number ofcarbon atoms. The C1-Cnv alkyl can be straight or branchedchain.As used herein, the term "D— and L- amino acid"refers to and includes both the D— and L— forms of thefollowing amino acids: alanine, leucine, isoleucine, valine,serine, glutamate, glutamine, aspartate, tryptophan, lysine,arginine, tyrosine, histidine, methionine, phenylalanine,asparagine, cysteine, glycine, proline, and threonine.Accordingly, the term “side chains of all D- and L— aminoacids” means the group attached to the carbon that isattached to both the organic acid and the amino group. Forexample, but not limited to —CH3 for alanine, CHgCH(CHg2 forleucine, and so on.As used herein the term "amino acid" means anorganic acid containing an amino group. The term includesboth naturally occurring and synthetic amino acids,therefore, the amino group can be, but is not required tobe, attached to the carbon next to the acid. The amino acidgroup is attached to the parent molecule via the acidfunctionality. The term shall refer to, but is in no way10152025R30CA 02264297 1999-02-22A WO 98108829 PCT/US97/15236-7-limited to (CH2)2NH2COOH, CH2CH(NH2)CH2COOH,CH3CHg(NH2)CH2COOH, CH3SCH2CH2(NH2)CHCOOH and the like.As used herein, the term "carbohydrate" refers toa class of substituents made up of carbon, hydrogen, andoxygen wherein hydrogen and oxygen are in the sameproportions as in water or nearly the proportions as water.The term "carbohydrate" further refers to an aldehyde orketone alcohol or a compound which on hydrolysis producesand aldehyde or ketone. The term "carbohydrate" is ascommonly understood by the skilled artisan. For example,the term refers to, but is in no way limited to, C12H22O11and C5H1oO5_As used herein, the term "amino sugar" refers to acarbohydrate group containing from one to three aminosubstituents at any available position on the carbohydratemolecule.As used herein, the term "saccharide" refers tocarbohydrate subunits to form disaccharides orpolysaccharides. The term means for example, but in no waylimited to, lactose, maltose, sucrose, fructose, starch, andthe 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 selectedfrom 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 independentlyselected from the group consisting of simple alkyl, Cl, Br,F, and I wherein such substituents may be attached at anyavailable carbon atom.As used herein "cycloalkyl" refers to a saturatedC3-C9 cycloalkyl group wherein such group may include fromzero to three substituents selected from the group1015202530CA 02264297 1999-02-22- WO 98/08829 PCT/US97/15236._8_Consisting of C1-C3 alkyl, halo, and OR22 wherein R22 isselected from hydrogen and C1-C3 alkyl. Such substituentsmay be attached at any available carbon atom. It isespecially preferred that cycloalkyl refers to substitutedor unsubstituted cyclohexyl.As used herein "Lower alkoxyl group" means anyalkyl group of one to five carbon atoms bonded to an oxygenatom. As used herein "lower alkyl group" means an alkylgroup of one to six carbons and includes linear and non-linear hydrocarbon chains, including for example, but notlimited to, methyl, ethyl, propyl, isopropyl, butyl,isobutyl, tert-butyl, sec-butyl, methylated butyl groups,pentyl, tert pentyl, sec-pentyl, and methylated pentylgroups. As used herein "allylically substituted alkene"means any alkene having from one to seven carbon atoms whichcontains an allyl substitution on it. As used herein theterm "unsaturated lower alkyl" means a lower alkyl group asdefined supra., except that C, is obviously not envisionedin this instance, wherein from one to two double bonds arepresent in the unsaturated lower alkyl substituent. Apreferred unsaturated lower alkyl is —CH2—CH=CH2. The term"lower alkyl—C3—C5 cycloalkyl" refers to C1-C6 alkylsubstituted with a C3-Cscycloalkyl group. A preferred loweralkyl-C3-C5 cycloalkyl group is -CH2—cyclopropyl; whereinthe group is attached to the cryptophycin core structure atR9 via the CH2_As used herein "epoxide ring" means a three-membered ring whose backbone consists of two carbons and anoxygen atom. As used herein, "aziridine ring" means athree—membered ring whose backbone consists of two carbonatoms and a nitrogen atom. As used herein "sulfide ring"means a three—membered ring whose backbone consists of twocarbon atoms and a sulfur atom. As used herein "episulfidering" means a three—membered ring whose backbone consists of10152025‘30CA 02264297 1999-02-22— W0 98l08829 PCT/US97ll5236_9_two carbon atoms and a sulfur atom. As used herein "sulfategroup" means a five membered ring consisting of a carbon-carbon—oxygen—sulfur-oxygen backbone with two additionaloxygen atoms connected to the sulfur atom. As used herein"cyclopropyl ring" means a three member ring whose backboneconsists of three carbon atoms. As used herein,"monoalkylphosphate ring" means a five membered ringconsisting of a carbon—carbon—oxygen—phosphorous-oxygenbackbone with two additional oxygen atoms, one of whichbears a lower alkyl group, connected to the phosphorousatom.As used herein, "simple unsubstituted aromaticgroup" refers to common aromatic rings having 4n+2 electronsin a monocyclic conjugated system, for example, but notlimited to: phenyl, furyl, pyrrolyl, thienyl, pyridyl andthe like, or a bicyclic conjugated system, for example butnot limited to indolyl or naphthyl.As used herein "simple substituted aromatic group"refers to a phenyl group substituted with a single groupselected from the group consisting of halogen and loweralkyl group.As used herein, "heteroaromatic group" refers toaromatic rings which contain one or more non—carbonsubstituent selected from the group consisting of oxygen,nitrogen, and sulfur. It is most preferred, but not limitedto, an aromatic ring having from three to eight memberswherein at least one member of the ring system is aheteroatom and the remaining members of the ring system arecarbon.As used herein, "halogen" or "halo" refers tothose members of the group on the periodic tablehistorically known as halogens. Methods of halogenationinclude, but are not limited to, the addition of hydrogenhalides, substitution at high temperature,1015202530CA 02264297 1999-02-22-WO 98/08829 PCTIUS97/15236_.lO_photohalogenation, etc., and such methods are known to theskilled artisan.As used herein, the term "mammal" shall refer tothe 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 isnot limited to, a human. The term "treating" as used hereinincludes prophylaxis of the named condition or ameliorationor elimination of the condition once it has beenestablished. The cryptophycin compounds claimed herein canbe useful for veterinary health purposes as well as for thetreatment of a human patient.The processes to prepare the compounds of thisinvention most preferably are completed in the presence of asolvent. The skilled artisan can select appropriatesolvents using standard methodologies. Suitable inertorganic solvents include those known to the skilled artisan,for example, but not limited to, tetrahydrofuran (THF) anddimethylformamide (DMF). DMF is especially preferred.Aqueous based solvents may be appropriate for some of theprocesses utilized herein. The pH of such aqueous solventsmay be adjusted as desired to facilitate the process.Some preferred characteristics of this inventionare set forth in the following tabular form wherein thefeatures may be independently selected to provide preferredembodiments of this invention. The invention is in no waylimited to the features described below:A) R5 is chloro methoxy phenyl;B) R7 is ethyl, propyl, isopropyl, butyl,isobutyl, pentyl, or isopentyl;C) R7 is isopropyl;D) R3 is methyl;1015202530” WO 98/08829CA 02264297 1999-02-22PCT/US97llS236-11..E) Ar‘ is phenyl with substituent selected fromthe group consisting of NR59R6°, NHOR61, andNHCHR51' ,-F) R1 and R2 form an epoxide ring;G) R7 is methylH) R2 is a glycinate;I) R2 is an acylate;J) a compound of Formula I is used for disruptionof 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 thetreatment of cancer in a mammal;M) a compound of Formula I is used as anantifungal agent;N) a compound of Formula I is used as anantibacterial agent;0) Ar is phenyl;P) Ar is phenyl substituted with one or two fromthe group consisting of OH, OCH3, halo, andmethyl;Q) R2 is selected from the group consisting ofhalogen, amino, monoalkylamino, dialkylamino,trialkylammonium, alkylthio,dialkylsulfonium, sulfate, phosphate, OR31,and sR31;;R) a compound of Formula I is used for thetreatment of a fungal infection;8) Ar’ is para ethyl substituted phenyl; andT) Ar’ is para methyl substituted phenyl.The present invention provides a method ofalleviating a pathological condition caused byhyperproliferating mammalian cells comprising administering1015202530CA 02264297 1999-02-22' WO 98/08829 PCT/US97/ 15236_l2_to a subject an effective amount of a pharmaceutical orVeterinary composition disclosed herein to inhibitproliferation of the cells. In a preferred embodiment ofthis invention, the method further comprises administeringto the subject at least one additional therapy directed toalleviating the pathological condition. In a preferredembodiment of the present invention, the pathologicalcondition is characterized by the formation of neoplasms.In a further preferred embodiment of the present invention,the neoplasms are selected from the group consisting ofmammary, small—cell lung, non-small-cell lung, colorectal,leukemia, melanoma, pancreatic adenocarcinoma, centralnervous system (CNS), ovarian, prostate, sarcoma of softtissue or bone, head and neck, gastric which includespancreatic and esophageal, stomach, myeloma, bladder, renal,neuroendocrine which includes thyroid and non—Hodgkin‘sdisease and Hodgkin's disease neoplasms.As used herein "neoplastic" refers to a neoplasm,which is an abnormal growth, such growth occurring becauseof a proliferation of cells not subject to the usuallimitations of growth. As used herein, "anti-neoplasticagent" is any compound, composition, admixture, co-mixture,or blend which inhibits, eliminates, retards, or reversesthe neoplastic phenotype of a cell.Anti—mitotic agents may be classified into threegroups on the basis of their molecular mechanism of action.The first group consists of agents, including colchicine andcolcemid, which inhibit the formation of microtubules bysequestering tubulin. The second group consists of agents,including vinblastine and vincristine, which induce theformation of paracrystalline aggregates of tubulin.Vinblastine and vincristine are well known anticancer drugs:their action of disrupting mitotic spindle microtubulespreferentially inhibits hyperproliferative cells. The third10152025‘30CA 02264297 1999-02-22’ WO 98108829 PCT/US97/15236_l3_.group consists of agents, including taxol, which promote thepolymerization of tubulin and thus stabilizes microtubules.The exhibition of drug resistance and multiple-drug resistance phenotype by many tumor cells and theclinically proven mode of action of anti-microtubule agentsagainst neoplastic cells necessitates the development ofanti-microtubule agents cytotoxic to non-drug resistantneoplastic cells as well as cytotoxic to neoplastic cellswith a drug resistant phenotype.Chemotherapy, surgery, radiation therpy, therapywith biological response modifiers, and immunotherapy arecurrently used in the treatment of cancer. Each mode oftherapy has specific indications which are known to those ofordinary skill in the art, and one or all may be employed inan attempt to achieve total destruction of neoplastic cells.Moreover, combination chemotherapy, chemotherapy utilizingcompounds of Formula I in combination with other neoplasticagents, is also provided by the subject invention ascombination therapy is generally more effective than the useof a single anti-neoplastic agent. Thus, a further aspectof the present invention provides compositions containing atherapeutically effective amount of at least one compound ofFormula I, including the non-toxic addition salts thereof,which serve to provide the above recited benefits. Suchcompositions can also be provided together withphysiologically tolerable liquid, gel, or solid carriers,diluents, adjuvants and excipients. Such carriers,adjuvants, and excipients may be found in the g;§;Pharmacopeia, Vol. XXII and National Formulary vol XVII,U.S. Pharmacopeia Convention, Inc. Rockville, MD (1989).Additional modes of treatment are provided in AHFS DrugInformation, 1993 e. by the American Hospital FormularyService, pp. 522-660. Each of these references are wellknown and readily available to the skilled artisan.1015202530CA 02264297 1999-02-22’ WO 98108829 PCT/US97I15236_l4_The present invention further provides apharmaceutical composition used to treat neoplastic diseasecontaining at least one compound of Formula I and at leastone additional anti~neoplastic agent. Anti-neoplasticagents which may be utilized in combination with Formula Icompounds include those provided in the Merck Index 11, pp16-17, Merck & Co., Inc. (1989). The Merck Index is widelyrecognized and readily available to the skilled artisan.In a further embodiment of this invention,antineoplastic agents may be antimetabolites which mayinclude but are in no way limited to those selected fromthe group consisting of methotrexate, 5—fluorouracil, 6-mercaptopurine, cytosine, arabinoside, hydroxyurea, and 2-chlorodeoxyadenosine. In another embodiment of the presentinvention, the anti—neoplastic agents contemplated arealkylating agents which may include but are in no waylimited to those selected from the group consisting ofcyclophosphamide, mephalan, busulfan, paraplatin,chlorambucil, and nitrogen mustard. In a furtherembodiment, the anti—neoplastic agents are plant alkaloidswhich may include but are in no way limited to thoseselected from the group consisting of vincristine,vinblastine, taxol, and etoposide. In a further embodiment,the anti-neoplastic agents contemplated are antibioticswhich may include, but are in no way limited to thoseselected from the group consisting of doxorubicin,daunorubicin, mitomycin C, and bleomycin. In a furtherembodiment, the anti—neoplastic agents contemplated arehormones which may include, but are in no way limited tothose selected from the group consisting of calusterone,diomostavolone, propionate, epitiostanol, mepitiostane,testolactone, tamoxifen, polyestradiol phosphate, megesterolacetate, flutamide, nilutamide, and trilotane.1015202530CA 02264297 1999-02-22‘ WO 98/08829 PCT/US97/15236-15-In a further embodiment, the anti-neoplasticagents contemplated include enzymes which may include, butare in no way limited to those selected from the groupconsisting of L—Asparginase and aminoacridine derivativessuch as, but not limited to, amsacrine. Additional anti-neoplastic agents include those provided by Skeel, RolandT., "Antineoplastic Drugs and Biologic Response Modifier:Classification, Use and Toxicity of Clinically UsefulAgents" Handbook of Cancer Chemotherapy (3rd ed.), LittleBrown & Co. (1991).These compounds and compositions can beadministered to mammals for veterinary use. For example,domestic animals can be treated in much the same way as ahuman clinical patient. In general, the dosage required fortherapeutic effect will vary according to the type of use,mode of administration, as well as the particularizedrequirements of the individual hosts. Typically, dosageswill range from about 0.001 to 1000 mg/kg, and more usually0.01 to 10 mg/kg of the host body weight. Or,alternatively, dosages within these ranges can beadministered as a bolus or IV injection, until the desiredtherapeutic benefits are obtained. Indeed, drug dosage, aswell as route of administration, must be selected on thebasis of relative effectiveness, relative toxicity, growthcharacteristics of tumor and effect of Formula I compound oncell cycle, drug pharmacokinetics, age, sex, physicalcondition of the patient and prior treatment, which can bedetermined by the skilled artisan.The compound of Formula I, with or withoutadditional anti-neoplastic agents, may be formulated intotherapeutic compositions as natural or salt forms.Pharmaceutically acceptable non—toxic salts include baseaddition salts which may be derived from inorganic basessuch as for example, sodium, potassium, ammonium, calcium,1015202530CA 02264297 1999-02-22- WO 98/08829 PCTIUS97/15236_16_or ferric hydroxides, and such organic bases asisopropylamine, trimethylamine, 2—ethylamino ethanol,histidine, procaine, and the like. Such salts may also beformed as acid addition salts with any free cationic groupsand will generally be formed with inorganic acids such asfor example, hydrochloric or phosphoric acids or organicacids such as acetic, oxalic, tartaric, mandelic, and thelike. Additional excipients which further the invention areprovided to the skilled artisan for example in the_E;§;Pharmacopeia.The suitability of particular carriers forinclusion in a given therapeutic composition depends on thepreferred route of administration. For example, anti-neoplastic compositions may be formulated for oraladministration. Such compositions are typically prepared asliquid solution or suspensions or in solid forms. Oralformulation usually include such additives as binders,fillers, carriers, preservatives, stabilizing agents,emulsifiers, buffers, mannitol, lactose, starch, magnesiumstearate, sodium saccharin, cellulose, magnesium carbonate,and the like. These compositions may take the form ofsolutions, suspensions, tablets, pills, capsules, sustainedrelsease formulations, or powders, and typically contain 1%to 95% of active ingedient. More preferably, thecomposition contains from about 2% to about 70% activeingredient.Compositions of the present invention may beprepared as injectables, either as liquid solutions,suspensions, or emulsions; solid forms suitable for solutionin or suspension in liquid prior to injection. Suchinjectables may be administered subcutaneously,intravenously, intraperitoneally, intramuscularly,intrathecally, or intrapleurally. The active ingredient oringredients are often mixed with diluents, carriers, or10152025‘3OCA 02264297 1999-02-227 W0 98l08829 PCT/US97/15236..l7_excipients which are physiologically tolerable andSuitable diluentsand excipients are for example, water, saline, dextrose,compatible with the active ingredient(s).glycerol, or the like and combinations thereof. Inaddition, if desired, the compositions may contain minoramounts of auxilary substances such as wetting oremulsifying agents, stabilizing or pH buffering agents.The invention further provides methods for usingFormula I compounds to inhibit the proliferation ofmammalian cells by contacting these cells with a Formula Icompound in an amount sufficient to inhibit theproliferation of the mammalian cell. A preferred embodimentis a method to inhibit the proliferation ofhyperproliferative mammalian cells. For purposes of thisinvention "hyperproliferative mammalian cells" are mammaliancells which are not subject to the characteristiclimitations of growth (programmed cell death for example).A further preferred embodiment is when the mammalian cell ishuman. The invention further provides contacting themammalian cell with at least one Formula I compound and atleast one anti—neoplastic agent. The types of anti-neoplastic agents contemplated are discussed supra.The invention further provides methods for using acompound of Formula I to inhibit the proliferation ofhyperproliferative cells with drug—resistant phenotypes,including those with multiple drug—resistant phenotypes, bycontacting said cell with a compound of Formula I in anamount sufficient to inhibit the proliferation of ahyperproliferative mammalian cell. A preferred embodimentis when the mammalian cell is human. The invention furtherprovides contacting a Formula I compound and at least oneadditional anti-neoplastic agent, discussed supra.The invention provides a method for alleviatingpathological conditions caused by hyperproliferating1015202530CA 02264297 1999-02-22* W0 98l08829..18_mammalian cells for example, neoplasia, by administering toa subject an effective amount of a pharmaceuticalcomposition containing Formula I compound to inhibit theproliferation of the hyperproliferating cells. As usedherein "pathological condition" refers to any pathologyarising from the proliferation of mammalian cells that arenot subject to the normal limitations of growth. Suchproliferation of cells may be due to neoplasms as discussedsupra.In a further preferred embodiment the neoplasticcells are human. The present invention provides methods ofalleviating such pathological conditions utilizing acompound of Formula I in combination with other therapies,as well as other anti-neoplastic agents.The effectiveness of the claimed compounds can beassessed using standard methods known to the skilledartisan.Examples of such methods are as follows:Compounds of this invention have been found to beuseful against pathogenic fungi. For example, theusefulness for treating Cryptococcus neoformans can beillustrated with test results against Cryptococcusneoformans employing yeast nitrogen base detrose agarmedium. In carrying out the assay, a compound of thisinvention is solubilized in dimethyl sulfoxide supplementedwith Tween 20. Twofold dilutions are made with steriledistilled water/10 percent DMSO to obtain final drugconcentrations in the agar dilution assay plates rangingfrom 0.008 ug/ml to 16.0 ug/ml against an expanded panel of84 Cryptococcus neoformans strains. The minimum inhibitoryconcentration against the panel of 84 Cryptococcusneoformans isolates is determined to illustrate the desiredantifungal activity.PCTIUS97/152361015202530CA 02264297 1999-02-22’ WO 98/08829 PCT/US97/15236-19..The compounds are screened for minimum inhibitoryconcentrations against KB, a human nasopharyngeal carcinomacell line, LoVo, a human colorectal adenocarcinoma cell lineusing The Corbett assay, see Corbett, T.H. et al. CytotoxicAnticancer Drugs: Models and Concepts for Drug Discovery andDevelopment, pp 35-87,1992. see also, Valeriote, et al. Discovery and DevelopmentKluwer Academic Publishers: Norwell,of Anticancer Agents; Kluwer Academic Publishers, Norwell,1993 is used for the evaluation of compounds.The most active compounds are further evaluatedfor cytotoxicity against four different cell types, forexample a murine leukemia, a murine solid tumor, a humansolid tumor, and a low malignancy fibroblast using theCorbett assay.The compounds are further evaluated against abroad spectrum of murine and human tumors implanted in mice,including drug resistant tumors.Tumor burden (T/C) (mean tumor burden in treatedanimals versus mean tumor burden in untreated animals) areused as a further assessment. T/C values that are less than42% are considered to be active by National Cancer InstituteStandards; T/C values less than 10% are considered to haveexcellent activity and potential clinical activity byNational Cancer Institute standards.MaterialsVinblastine, cytochalasin B, tetramethylrhodamineisothiocyanate (TRITC)—phalloidin, sulforhodamine B (SRB)and antibodies against fi—tubulin and vimentin arecommercially available from recognized commercial vendors.Basal Medium Eagle containing Earle's salts (BME) and FetalBovine Serum (FBS) are also commercially available.1015202530CA 02264297 1999-02-22' WO 98108829 PCT/US97I 15236_20_Cell LinesThe Jurkat T cell leukemia line and A-10 rat aorticsmooth muscle cells are obtained from the American TypeCulture Collection and are cultured in BME containing 10%FBS and 50ug/mL gentamycin sulfate. Human ovarian carcinomacells (SKOV3) and a sub-line which has been selected froresistance to vinblastine (SKVLB1) were a generous gift fromDr. Victor Ling of the Ontario Cancer Institute. Both celllines are maintained in BME containing 10% FBS and 50ug/mLgentamycin sulfate. Vinblastine is added to a finalconcentration of lug/mL to SKVLB1 cells 24 hours afterpassage to maintain selection pressure for P—glycoprotein—overexpressing cells.Cell Proliferation and Cycle Arrest AssaysCell proliferation assays are performed as described bySkehan et al. For Jurkat cells, cultures are treated withthe indicated drugs as described in Skehan and total cellnumbers are determined by counting the cells in ahemacytometer. The percentage of cells in mitosis aredetermined by staining with 0.4% Giemsa in PBS followed byrapid washes with PBS. At least 1000 cells per treatmentare scored for the presence of mitotic figures and themitotic index is calculated as the ration of the cells withmitotic figures to the total number of cells counted.Immunofluorescence AssaysA—l0 cells are grown to near—confluency on glasscoverslips in BME/10% FBS. Compounds in PBS are added tothe indicated final concentrations and cells are incubatedfor an additional 24 hours. For the staining ofmicrotubules and intermediate filaments, the cells are fixedwith cold methanol and incubated with PBS containing 10%calf serum to block nonspecific binding sites. Cells are10152025‘3OCA 02264297 1999-02-22~ WO 98/08829 PCT/US97/ 15236-21-then incubated at 37_C for 60 min. with either monoclonalanti-B-tubulin or with monoclonal anti—vimentin at dilutionsrecommended by the manufacturer. Bound primary antibodiesare subsequently visualized by a 45-minute incubation withfluorescein—conjugated rabbit antimouse IgG. The coverslipsare mounted on microscope slides and the fluorescencepatterns are examined and photographed using a ZeissPhotomicroscope Ill equipped with epifluorescence optics forfluorescein. For staining of microfilaments, cells arefixed with 3% paraformaldehyde, permeabilized with 0.2%Triton X-100 and chemically reduced with sodium borohydride(lmg/ML). PBS containing lOOnM TRITC-phalloidin is thenadded and the mixture is allowed to incubate for 45 min. at37_C. The cells are washed rapidly with PBS before thecoverslips are mounted and immediately photographed asdescribed above.Effects of cryptophycins and vinblastine on Jurkat cellproliferation and cell cycleDose—response curves for the effects of cryptophycincompounds and vinblastine on cell proliferation and thepercentage of cells in mitosis are determined.Effects of cytochalasin B, vinblastine and cryptophycins onthe cytoskeletonAortic smooth muscle (A-10) cells are grown on glasscoverslips and treated with PBS, 2uM cytochalasin B, lOOnMAfter 24hours, microtubules and Vimentin intermediate filaments arevinblastine or lOnM cryptophycin compoundsvisualized by indirect immunofluorescence and microfilamentsare stained using TRITC - phalloidin. The morphologicaleffects of each drug is examined. Untreated cells displayedextensive microtubule networks complete with perinuclearmicrotubule organizing centers. Vimentin intermediate1015202530CA 02264297 1999-02-22—W0 98l08829 PCT/US97/15236_22_filaments were also evenly distributed throughout thecytoplasm, while bundles of microfilaments were concentratedalong the major axis of the cell. Cytochalasin B causedcomplete depolymerization of microfilaments along with theaccumulation of paracrystalline remnants. This compound didnot affect the distribution of either microtubules orintermediate filaments. The cryptophycin treatedmicrotubules and vimentin intermediates are observed fordepletion of microtubules, and collapse of rimentinintermediate filaments.Effects of cryptophycins and vinblastine on taxol—stabilizedmicrotubulesA719 cells are treated for 3 hours with D or lOuM taxolbefore the addition of PBS, l00nM vinblastine or lOnMcryptophycin compound. After 24 hours, microtubuleorganization is examined by immunofluorescence as describedabove. Compared with those in control cells, microtubulesin taxol—treated cells were extensively bundled, especiallyin the cell polar regions. As before, vinblastine causedcomplete depolymerization of microtubules non—pretreatedcells. However, pretreatment with taxol preventedmicrotubule depolymerization in response to vinblastine.Similarly, microtubules pretreated with taxol are observedwith cryptophycin treatment.Reversibility of microtubule depolymerization by vinblastineand cryptophycinA—lO cells are treated with either 100nM vinblastine orlOnM cryptophycins for 24 hr., resulting in completemicrotubule depolymerization. The cells are then washed andincubated in drug-free medium for periods of 1 hour or 24hours. Microtubules repolymerized rapidly after the removalof vinblastine, showing significant levels of microtubules10152025'30CA 02264297 1999-02-22‘ W0 98/118829 PCT/US97ll5236_23..after 1 hour and complete morphological recovery by 24 hour.Cells are visualized for microtubule state after treatmentwith a cryptophycin compound of this invention at either 1hour or 24 hours after removal of the cryptophycincompounds.Effects of combinations of Vinblastine and cryptophycins oncell proliferationSKOV3 cells are treated with combinations ofcryptophycins and Vinblastine for 48 hours. The percentagesof surviving cells are then determined and the IC50s foreach combination is calculated.Toxicity of cryptophycins, Vinblastine and taxol towardSKOV3 and SKVLB1 cellsSKVLB1 cells are resistant to natural productanticancer drugs because of their over expression of P—glycoprotein. The abilities of taxol, Vinblastine andcryptophycin compounds to inhibit the growth of SKOV3 andSKVLB1 cells are observed. Taxol caused dose—dependentinhibition of the proliferation of both cell lines withIC50s for SKOV3 and SKVLB1 cells of l and 8000nM,respectively. Vinblastine also inhibited the growth of bothcell lines, with IC5os of 0.35 and 4200nM for SKOV3 andSKVLB1 cells, respectively. Cryptophycins compounds of thisinvention demonstrate activity with an IC5os of from about 1to about l000pm for SKOV3 and SKVLB1 cells.Thus it can be demonstrated that the present inventionprovides novel cryptophycin compounds which are potentinhibitors of cell proliferation, acting by disruption ofthe microtubule network and inhibition of mitosis. Thesestudies can illustrate that cryptophycin compounds disruptmicrotubule organization and thus normal cellular functions,including those of mitosis.1015202530CA 02264297 1999-02-22- WO 98/08829 PCTIUS97I15236-24..Classic anti—microtubule agents, such as colchicine andVinca alkaloids, arrest cell division at mitosis. It seemsappropriate to compare the effect of one of these agents oncell proliferation with the cryptophycin compounds. Forthis purpose, the Vinca alkaloid vinblastine was selected asrepresentative of the classic anti-microtubule agents.Accordingly, the effect of cryptophycin compounds andvinblastine on the proliferation and cell cycle progressionof the Jurkat T—cell leukemia cell line is compared.Since antimitotic effects are commonly mediated bydisruption of microtubules in the mitotic spindles, theeffects of cryptophycin compounds on cytoskeletal structuresare characterized by fluorescence microscopy.Immunofluorescence staining of cells treated with either acryptophycin compound or vinblastine demonstrate that bothcompounds cause the complete loss of microtubules. Similarstudies with SKOV3 cells can show that the anti—microtubuleeffects of cryptophycin compounds are not unique to thesmooth muscle cell line.GC3 human Colon Carcinoma ScreenSelected wells of a 96 well plate were seeded withGC3 human colon carcinoma cells (lxlO cells in lOOul assaymedium/well) twenty four hours prior to test compoundaddition. Cell free assay medium was added to other selectwells of the 96 well plate. The assay medium (RPMI—164O wasthe medium used; however, any medium that will allow thecells to survive would be acceptable) was supplemented with10% dialyzed fetal bovine serum and 25 mM HEPES buffer.The test compound was stored in an amber bottleprior to testing. Fresh dimethylsulfoxide stock solution(200ug/ml) was prepared immediately prior to preparation oftest sample dilutions in phosphate—buffered saline (PBS). Adilution of l:2O dimethylsulfoxide solution in PBS was1015202530CA 02264297 1999-02-22~ WO 98/08829 PCTlUS97/ 15236-25-prepared such that the final concentration was 10 ug/ml.Serial 1:3 dilutions using PBS (.5ml previous sample of lmlPBS) were prepared. Falcon 2054 tubes were used for theassay.A lOul sample of each dilution of test compoundwas added in triplicate to wells of GC3 plates. The plateswere incubated for 72 hours at about 37 C. A 10 ul sampleof stock 3-[4,5—dimethyl—2—yl]-2,5—diphenyltetrazoliumbromide salt ("MTT" 5 mg/ml in PBS) was added to each well.The plates were incubated for about an hour at 37 C. Theplates were centrifuged, media was decanted from the wellsand l00ul acid—isopropanol (0.04 N HCl in isopropanol) wasadded to each well. The plate was read within one hourusing a test wavelength of 570nm (SpectraMax reader).Evaluation of compounds of Formula I suggest thatthe compounds can be useful in the treatment methods claimedherein. Further, the compounds will be useful fordisrupting the microtubule system.The preparation of the compounds of thisinvention can be completed using several protocolsinvolving an activated ester followed by chromatographyand acid induced deblocking where necessary.Preparation of any ester wherein R1 or R2 isderived from a carboxylic acid includes a variety oftechnologies employing acid chlorides, anhydrides, andcommon activating reagents (eg., carbodiimides).2 Anysolvent other than participating alcohols can be used. Anymild bases and/or catalysts (amines, carbonates) can be usedto aid in esterification.The conversion of carbamates to the correspondingsalts could be effected with any strong acid, namely,mineral acids comprised of hydrogen halides, hydrogen10152025CA 02264297 1999-02-22- WO 98/08829 PCT/US97/15236_26_sulfates, hydrogen phosphates, hydrogen nitrates, hydrogenperchlorates, or strong organic acids such astrifluoroacetic, p—toluenesulfonic, and methanesulfonic.The same acids could be used to produce new salts from thecorresponding free base.Compounds of this invention have been tested in the Gc3assay and have IC50 values ranging from less than one toabout 700 nM; however, the most typical values are less thanlOOnM.The processes described herein are most preferablycompleted in the presence of a solvent. The artisan canselect an appropriate solvent for the above describedprocess. Inert organic solvents are particularly preferred;however, under certain conditions an aqueous solvent can beappropriate. For example, if R27 is hydrogen and and R13 isBOC an aqueous base as solvent will be effective.The product of the schemes provided herein can befurther derivatized using standard methods to providefurther cryptophycin compounds.The artisan can utilize appropriate startingmaterials and reagents to prepare desired compounds usingthe guidance of the previous schemes and following examples.The ester starting material can be prepared, forexample, as follows:CA 02264297 1999-02-22- WO 98/08829-27- / CH0Step 1HEWTMGV / C02MeStep 2DIBALPCT/US97/15236CA 02264297 1999-02-22- wo 98/08829 PCT/US97/15236-28-Me/ | OTs\ (SHStep 6TBS-OTfEt3NV OE6TBsStep 7NBS/AIBN\ {masStep 8 y/\DBU/ACN Y/\)e\/\X I \ E OTS\ (-DTBSStep 9KCN Step 10DIBAL;HEW1015CA 02264297 1999-02-22» WO 98/08829 PCT/US97/15236_29_R6 has the meaning defined supra.The scheme for preparing the ester is furtherexplained by the Preparation Section herein which providesone specific application of the scheme for the convenienceof the skilled artisan.The Scheme for preparing the ester is applicableto the Ar substituents claimed herein. The schemeillustration is not intended to limited the synthesis schemeonly to the phenyl ring illustrated. Rather, the artisancan broadly apply this process to provide desired startingmaterials for the compounds claimed herein.The necessary reaction time is related to thestarting materials and operating temperature. The optimumreaction time for a given process is, as always, acompromise which is determined by considering the competinggoals of throughput, which is favored by short reactiontimes, and maximum yield, which is favored by long reactiontimes.CA 02264297 1999-02-22- WO 98/08829 PCT/US97l 15236._30_Z 4/ Z 4/morpholincTHFCH: 7gm (4) WI‘ 3 /cl 9 1g T(3) R ODMAPDCC/ oo ,,u"1)Zn,AcOH \f‘ 2) TFA H<M Y (5) ‘\‘<9% : :°(6)FDPP H |DMPCA 02264297 1999-02-22- W0 98l08829 PCT/U S97! 15236..3l_CH, ggpjgiiif(7)m—CPBACH2c|,, RT 1015202530CA 02264297 1999-02-22-WO 98108829 PCTIUS97/15236-32-To further illustrate the invention the followingexamples are provided. The scope of the invention is in noway to be construed as limited to or by the followingexamples.Preparation 1Step 1. Methyl 5—Phenylpent-2(E)-enoate. A solution oftrimethyl phosphonoacetate (376 g, 417 mL, 2.07 mol) in THF(750 mL) was stirred at 0 °C in a 3L 3—neck round bottomflask equipped with a mechanical stirrer and N2 inlet. Tothe chilled solution, neat tetramethyl guanidine (239 g, 260mL, 2.07 mol) was added dropwise via an addition funnel.The chilled clear pale yellow solution was stirred for 25minutes at 0 °C.253 g, 248 mL, 1.9 mol) in THF (125 mL) was added dropwiseA solution of hydrocinnamaldehyde (90%,to the reaction solution slowly. Upon completion ofaddition, the reaction was stirred for 10 h rising to roomtemperature. GC indicated a 95:5 ratio of product tostarting material. 500ml of water was added to the reactionvessel and the reaction stirred overnight separating intotwo layers. The organic layer was isolated and the aqueouslayer was extracted with t—BuOMe. The organic layers werecombined and dried over MgSO4, then concentrated in vacuo toyield an orange oil. The crude product was distilled at 129°C/0.3mm Hg yielding 360.5g, 91.7% yield, of a clearslightly yellow oil.EIMS m/z l90(13; M+), 159(410, 158(39), 131(90), 130(62),117(22), 104(12), 95(57), 91(100), 77(21), 65(59): HREIMSm/z 190.0998 (C12H1402 D -0.4 mnu): UV lmax (e) 210 (8400),260 (230) nm; IR nmax 3027, 2949, 1723, 1658, 1454, 1319,1203, 978, 700 cm‘1; 1H NMR d (CDCl3) 7.15-7.3 (Ph—H5;bm),7.00 (3-H;dt, 15.6/0.6), 5.84 (2-H;dt, 15.6/1.2), 3.701015202530CA 02264297 1999-02-22» WO 98108829 PCT/US97/15236..33_(OMe;S), 2.76 (5-H2;t, 7.2), 2.51 (4-H2; bdt, 6.6/7.2); 13CNMR d (CDCl3) 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—1—ol. To a 12L 4-neck roundbottom flask equipped with a thermocouple, mechanicalstirrer 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 °Cvia a i—PrOH/CO2 bath. To the reaction vessel, was addeddropwise DIBAL (2.5 L, 1.5 M in toluene, 3.75 mol) at a rateto maintain the reaction temperature < -50 °C. Uponcomplete addition, the reaction was stirred overnight withthe reaction temperature < -50 °C. TLC (3:1 Hexanes:EtOAc,SiO2) indicated absence of starting material after 16 h.The reaction temperature was allowed to raise to —15OC. Thereaction was quenched slowly with1N HC1 (150 mL). At thispoint the reaction setup into a gelatinous solid. A.spatulawas employed to breakup the the semi—solid and 1N HC1 (200mL) was added making the mixture more fluid. ConcentratedHC1 (625 mL) was charged to form a two phase system. Thelayers were separated and the product extracted with t-BuOMe. The organic layer was dried over MgSO4 andconcentrated in vacuo to yield a clear pale yellow oil,247.8g. The crude product was distilled at 145 °C/O.25mm Hgyielding 209.7g, 86.29.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); IRnmax 3356, 2924, 1603, 1496, 1454, 970, 746, 700 cm'1; 1HNMR 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 (OH;bs), 2.36(4-H2; dt, 6.0/7.2); 13c NMR d141.6 (Ph1015202530CA 02264297 1999-02-22- WO 98/08829 PCT/US97ll5236-34-1'), 131.8(3), 129.5 (2), 128.3/128.2 (Ph 2'/3'/5‘/6'),125.7 (Ph 4'), 63.3 (1), 35.4/33.8 (4/5).Step 3. To a 1L 3(2S,3S)-2,3-Epoxy—5-phenyl-1—pentanol.neck round bottom flask equipped with a mechanical stirrer,thermocouple and nitrogen inlet was added CH2Cl2 (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 inCH2Cl2, 182 mL, 0.78 mol) at a rate to maintain thetemperature 2 -20 °C. Upon complete addition, the reactionmixture was stirred for another 30 min, and then treatedwith a solution of the allylic alcohol (50 g, 0.31 mol) inCH2Cl2 (30 mL) at a rate to maintain the temperature 2 -20°C. The reaction was stirred at the same temperature for 5h, then filtered into a solution of ferrous sulfateheptahydrate (132 g) and tartaric acid (40 g) in water (400mL) at 0 °C. The mixture was stirred for 20 min, thentransferred to a separatory funnel and extracted with t-BuOMe (2x200 mL). The combined organic phase was stirredwith 30% NaOH solution containing NaCl, for l h at 0 °C.The layers were again separated, and the aqueous phaseextracted with t—BuOMe. The combined organic phase waswashed with brine, dried over MgSO4 and concentrated toyield 52.8 g as an amber oil.Step 4. (2R, 3R)—2—hydroxy—3—methyl—5—phenylpentan-1—olTo a 5L 3 neck round bottom flask equipped with a mechanicalstirrer, thermocouple and nitrogen inlet was added hexanes(lL) and cooled to 0 °C. A 2.0M solution of Me3Al inhexanes (800 mL, 1.6 mol) was added, followed by a solutionof the epoxide (120 g, 0.677 mol) in hexanes (250 mL)/CH2Cl21015202530CA 02264297 1999-02-22-WO 98/08829 PCT/US97/15236__35_(50 mL) maintaining the temperature below 20 °C. Uponcomplete addition, the cloudy reaction mixture was stirredat 5 °C for 35 min, whereupon a solution of 10% HC1 (300 mL)was added dropwise, followed by the addition of concd HCl(350 mL). The layers were separated, and the organic phasewas washed with brine and dried over MgSO4. After removalof the volatiles in vacuo, 122.1 gram of an oil wasobtained.Step 5. (2R, 3R)—2—hydroxy—3-methyl-5-phenylpent-1-ylTosylate. To a 2L 3 neck round bottom flask equipped with amechanical stirrer and nitrogen inlet was added the diol (58g, 0.30 mol), dibutyltin oxide (1.5 g, 0.006 mol, 2 mol%),toluenesulfonyl chloride (57.5 g, 0.30 mol), CH2Cl2 (580 mL)and triethylamine (42.0 mL, 0.30 mol). The resultingmixture was stirred at room temperature for 2 h (althoughthe reaction was complete within 1 h), filtered, washed withwater and dried over MgSO4. Concentration of the volatilesin Vacuo afforded 104.1 gram of a slightly amber oil.Step 6. (2R, 3R)-2-[(tert-Butyldimethylsilyl)oxy]—3—methyl-5-phenylpent—1—yl Tosylate. A solution of the tosylate (100g, 0.29 mol) and triethylamine (81.0 mL, 0.58 mol) in CH2Cl2(1200 mL) was treated with neat TBS—OTf (99 mL, 0.43 mol)dropwise with continued stirring for another 20 min. Thereaction was washed twice with brine, dried over MgSO4 andconcentrated to dryness. The oil was dissolved in a minimalamount of hexanes and filtered over a silica pad, elutingwith hexanes:EtOAc (9:1) to yield a slightly amber oil, 134g.1015202530CA 02264297 1999-02-22- WO 98/088259 PCT/US97l 15236_36_Step 7. (2R, 3R,5RS)—2-[(tert-Butyldimethylsilyl)oxy]-3-methyl-5-bromo—5-phenylpent—1-yl Tosylate. To a 5L 3 neckround bottom flask equipped with a mechanical stirrer,reflux condenser and nitrogen inlet was added CCl4 (1680mL), TBS Ts (140 g, 0.30 mol), NBS (65g, 0.365 mol) and AIBN(16.5 g, 0.10 mol).under full vacuum with stirring, and backfilling withThe mixture was degassed by evacuationThe reaction mixture was then heated toAfter 15 minnitrogen (3x).reflux, whereupon the color became dark brown.at vigorous reflux, the reaction mixture became lightyellow, and chromatographic analysis indicated the reactionwas complete. After cooling to room temperature, thereaction was filtered and the filtrate concentrated todryness. The residue was redissolved in hexanes andfiltered again, and concentrated to dryness to afford 170.3gram as an amber oil.Step 8. (2R, 3R)—2—[(tert-Butyldimethylsilyl)oxy]-3-methyl-5—phenylpent-4(E)-en—1—y1 Tosylate. To a 2L 3 neck roundbottom flask equipped with a mechanical stirrer, refluxcondenser and nitrogen inlet was added a solution of thebromide (100 g, 0.186 mol) in acetonitrile (700 mL). DBU(83.6 mL, 0.557 mol) was added and the resulting dark brownsolution was stirred at reflux for 15 min. After cooling toroom temperature, the solvent was removed in vacuo, and theresidue digested in CH2Cl2 (200 mL) and filtered through asilica pad. The volatiles were again evaporated, and theresidue dissolved in EtOAc and washed with water, brine anddried over MgSO4 and concentrated to dryness. Preparativemplc (Prep 500) chromatography afforded the desiredunsaturated compound (50.3 g, 60% yield over 4 steps).1015202530CA 02264297 1999-02-22- WO 98/08829 PCT/US97ll5236-37..Step 9. (38, 4R)—3-[(tert—Butyldimethylsilyl)oxy]-4—methyl-6-phenylhex-5(E)-en—1—nitrile. The tosylate (50 g, 0.11mol) was dissolved in DMSO (1 L) and treated with KCN (14.2g, 0.22 mol) and water (25 mL), and the resulting mixturewas stirred at 60 °C under nitrogen for 18 h. After coolingto room temperature, the reaction mixture was partitionedbetween EtOAc (1 L) and water (1 L). The aqueous phase wasextracted with EtOAc (500 mL), and the combined organicphase was washed with brine and dried over Na2SO4. Flashchromatography over silica with CH2Cl2 afforded the desirednitrile in 92% yield.Step 10. Methyl (SS, 6R)—5-[(tert-Butyldimethylsilyl)oxy]-6—methyl-8~phenylocta-2(E),7(E)-dienoate. The nitrile(14.67 g, 46.5 mmol) was dissolved in toluene (200 mL) andcooled to -78 °C under nitrogen. A 1.5M solution of DIBALin toluene (37.2 mL, 55.8 mmol) was added dropwise withvigorous stirring. Upon complete addition, the cooling bathwas removed and the reaction was stirred at room temperaturefor 1 h. The reaction mixture was carefully poured into 1NHCl and the mixture stirred at room temperature for 30 min.The layers were separated, and the organic phase was washedwith a saturated aqueous solution of sodium potassiumtartrate (2x), brine and dried over Na2SO4. The volatileswere removed in vacuo, and the crude pale yellow oil wasused directly in the subsequent condensation.The crude aldehyde from above was dissolved in THF (90 mL)and treated with trimethyl phosphonoacetate (9.03 mL, 55.8mmol) and tetramethylguanidine (7.0 mL, 55.8 mmol) at roomtemperature under nitrogen. The reaction mixture wasstirred for 16 h, then partitioned between EtOAc (200 mL)and water (100 mL). The aqueous phase was back extractedwith EtOAc (100 mL), and the combined organic phase waswashed with water, brine and dried over Na2SO4. The10152025CA 02264297 1999-02-22-W098l08829-38..volatiles were removed in vacuo, and the crude yellow oil(17.0 g) was chromatographed over silica gel with CH2Cl2cyclohexane (1 1 to 2 : 1) to afford 13.67 grams of thedesired ester, 78.5%.Exam le 1Synthesis of Allyl (2S)—2-[2'(tert—Butoxycarbonyl)amino—1’—(S)—methyl1-4-methylpentanoate: (2a)0 + K} O DMAP DCC o 3OH H0 Y I o \|/0 o(23)(1)846 mg (5.02 mmol, 0.95 eq) of allyl (2S)—2-hydroxy-4-methylpentanoate (1), 1g (5.29 mmol) N—(tert-Butoxycarbonyl)-1-alanine and 122 mg (1.06 mmol, 0.2 eq) of4-dimethylaminopyridine (DMAP) were combined in 8 ml of drymethylene chloride and stirred under a nitrogen atmosphereat 00C (ice bath). 1.2 g (5.82 mmol, 1.1 eq) of 1,3-dicyclohexylcarbodiimide (DCC) was added in one portion andreaction stirred at room temperature overnight. Thereaction was filtered through filter paper and extractiveworkup with 5% NaHCO3 and brine was performed. Themethylene chloride was dried over NaSO4 then removed inVacuo. The crude oil was flash chromatographed on SiO2 (10%Etoac/hexane) to yield 1.45 g (80%) of (2a) as a colorlessoil.TLC: Rf=0.309 (20% EtOAc/Hex)PCT/US97/ 15236101520.25CA 02264297 1999-02-22* WO 98/08829 PCT/US97/15236-39-IR (Cmd) (CHC13): 3442, 2982, 2963, 2937, 1745, 1711, 1503,1369, 1163.1H NMR(300MHZ, CDCl3) d: 5.84-5.93 (m, 1H), 5.28 (dd, 2H,J=17.1 and 5.5 HZ), 5.09-5.13 (m, 1H), 5.01 (br s, 1H), 4.62(d, 2H, J=5.7 HZ), 4.35-4.41 (m, 1H), 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 C1fib9NO6: C, 59.46; H, 8.51; N, 4.08. Found:C, 59.63; H, 8.48; N, 4.11.gynthesis of Allyl (2S)—2—j2'(tert-Butoxycarbonyl)amino-1’—(S)—isopropyl]-4-methylpentanoate: (2b)? ?Compound (2b) was prepared acccording to the procedure for(2a) (86%).TLC: Rf=O.447 (20% EtOAc/Hex)1) (CHCl3): 2966, 1743, 1712, 1503, 1368, 1173, 1158.IR (cm'1H NMR(300MHZ, CDCl3) d: 5.85-5.94 (m, 1H), 5.28 (dd, 2H,J=l6 and 8 HZ), 5.06-5.10 (m, 1H), 5.01 (br 5, 1H), 4.62 (d,2H, J=5.6 HZ), 4.29-4.33 (m, 1H), 2.25-2.29 (m, 1H), 1.64-1.85 (m, 3H), 1.44 (s, 9H), 0.85-1.06 (m, 12H).Mass (FD): 372 (M++H).10152025CA 02264297 1999-02-22‘WO 98/08829 PCT/US97IlS236-40..Anal: Calcd for C1fi£BNO6: 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)Z . 4/.. . 4/pu(pn,P).fi.::phonne 0 O%/ 3 O“' it 1“an an827 mg (2.41 mmol) of Allyl (2S)-2—[2’(tert—Butoxycarbony1)amino-1'-(S)—methy1]-4—methylpentanoate (2a)was dissolved in 50 ml of dry tetrahydrofuran (THF) andstirred under a nitrogen atmosphere at room temperature.279 mg (0.241 mmol, 0.1 eq) oftetrakis(triphenylphosphine)pa11adium(0) was added followedby 2.31 ml (26.5 mmol, 11 eq) of dry morpholine. TLC (10%MeOH/CHC13) after 1 hr indicated the reaction was complete.The reaction was diluted with diethyl ether (200 ml) thenextracted with 1N HC1 followed by 5% NaHCO3. The combined5% NaHCO3 aqueous layers were then acidified with 1N HCl topH 2 and extracted with diethyl ether (600 ml). Thecombined ether extracts were then washed with brine anddried over NaSO4. The ether was removed in Vacuo yielding ayellow solid (3a) weight 714 mg (98%).TLC: Rf=O.114 (10% MeOH/CHClfl1IR (Cm_) (CHCl3): 2982, 2963, 1746, 1713, 1503, 1369, 1163.101520‘25CA 02264297 1999-02-22. WO 98/08829 PCT/U S97/ 15236-41-1H NMR(300MHz, CDCl3) d: 7.70 (br s, 1H), 5.13-5.06 (m, 2H),4.41-4.34 (m, 1H), 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 C1fib5NO6: C, 55.43; H, 8.31; N, 4.62. Found:0, 55.65; H, 8.37; N, 4.68.Synthesis of (2S)-2-[2’(tert-Butoxycarbonyl)amino—1'—(S)-isopropyl]-4-methylpentanoatez (3b)0 N o 0 ‘J oP0 Y W 0 Y0 0(2b) (3b)Compound (3b) was prepared acccording to the procedure for(3a) (81%).TLC: Rf=O.183 (10% MeOH/CHClfl1IR (cm' 2966, 2936, 1727, 1714, 1504, 1393, 1369,1159.) 31H NMR(300MHz, CDCI3) d: 5.10 (dd, 1H, J=9.l and 3.7 Hz),5.03 (d, 1H, J=9.l Hz), 4.29 (dd, 1H, J=9.l and 4.1 Hz),2.26-2.30 (m, 1H), 1.68-1.88 (m, 3H), 1.44 (s, 9H), 0.91-1.02 (m, 12H).Mass (FD): 332 (M++H).Anal: Calcd for CIHQENO6: C, 57.99; H, 8.82; N, 4.22. Found:C, 58.22; H, 8.72; N, 4.39.10152025CA 02264297 1999-02-22- WO 98/08829 PCT/US97/15236_42_Synthesis of compound (5a) .10 “III;)<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 DMAPwere dissolved in 3 ml methylene chloride in a flame—driedround bottom flask under a nitrogen atmosphere. 118 mg(0.572 mmol, 1.1 eq) of DCC was then added in one portionand the reaction was stirred overnight. TLC (50%EtOAc/hexane) indicated that the reaction was complete. Thereaction was filtered through filter paper and extractiveworkup with 5% NaHCO3 and brine was performed. Themethylene chloride was dried over NaSO4 and then removed invacuo. The crude solid was then flash chromatographed onSiO2 (35% EtOAc/hexane) to yield 259 mg (82%) of a whitesolid (5a).TLC: Rf=IR (cm*) (KBr):.UV (95% EtOH):1H NMR(300MHz, CDCl3) d: 7.18-7.37 (m, 7H), 7.05 (dd,lH,J=6.0 and 1.7 Hz), 6.82 (d, 1H, J=7 4 Hz), 6.65-6.78 (m,1H), 6.40 (d, 1H, J=l5.7 Hz), 6.01 (dd, 1H, J=l5.8 and 8.8Hz), 5.86 (d, 1H, J=15.8 Hz), 5.35 (dd, 1H, J=8.0 and 2.3Hz), 5.01 (m, 2H), 4.96 (dd, 1H, J=9.4 and 3.6 Hz), 4.80 (d,1H, J=ll.9 Hz), 4.74 (d, 1H, J=ll.9 Hz), 4.42 (m, 1H), 3.85101520'25CA 02264297 1999-02-22. WO 98/08829 PCT/US97/15236_43...(s, 3H), 3.20 (dd, 1H, J=l4.0 and 5.9 Hz), 3.06 (dd, 1H,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=6.4Hz), 0.81 (d, 3H, J=6.6 Hz).Mass (FD):.Anal: Calcd for c4gg2c1m@o10gynthesis of compound (5b)CH in, 15111,)O 0 Y DMAP I OH O DCC0am am °\F:500 mg (0.848 mmol) of (4), 267 mg (0.806 mmol, 1.05 eq)(25)-2-[2’(tert—Butoxycarbonyl)amino-1'-(S)-methyl]—4—methylpentanoate (3b) and 21 mg (0.172 mmol, 0.2 eq) of DMAPwere dissolved in 5 ml methylene chloride in a flame-dried192 mg(0.933 mmol, 1.1 eq) of DCC was then added in one portionround bottom flask under a nitrogen atmosphere.and the reaction was stirred overnight. TLC (50%EtOAc/hexane) indicated that the reaction was complete. Thereaction was filtered through filter paper and extractiveworkup with 5% NaHCO3 and brine was performed. Themethylene chloride was dried over NaSO4 and then removed invacuo. The crude solid was then flash chromatographed onSiO2 (30% EtOAc/hexane) to yield 682 mg (89%) of a whitesolid (Sb).TLC: Rf=0.447 (50% EtOAC/hexane)10152025CA 02264297 1999-02-22- W0 98I08829 PCT/US97/ 15236-44..IR (cm*) (KBr): 2965, 2934, 1752, 1716, 1680, 1645, 1504,1368, 1282, 1158, 1067.UV (95% EtOH): 232 nm (e= 22672), 247 nm (e= 21889).1H NMR(300MHz, CDCI3) d: 7.16-7.32 (m, 7H), 7.05 (d, 1H,J=8.3 Hz), 6.66-6.84 (m, 2H), 6.40 (d, 1H, J=15.9 Hz), 6.01(dd, 1H, J=8.8 and 15.9 Hz), 5.86 (d, 1H, J=l5.7 Hz), 5.47(d, 1H, J=9.9 Hz), 5.03-5.14 (m, 2H), 4.91 (dd, 1H, J=9.9and 3.8 Hz), 4.76 (s, 2H), 4.38 (dd, 1H, J=9.5 and 4.1 Hz),3.84 (s, 3H), 3.20 (dd, 1H, J=14.0 and 5.7 Hz), 3.05 (dd,1H, J=l4.0 and 7.3 Hz), 2.46-2.59 (m, 3H), 2.26-2.32 (m,1H), 1.44-1.78 (m, 3H), 1.42 (s, 9H), 1.11 (d, 3H, J=6.7Hz), 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 C43H56Cl4N2O10: C, H, N, 3.10.Found: C, 58.09; H, 7.04; N, 3.26.Synthesis of compound (6aLI1)2m.AcoH “HOJW/f ° T 2) TFA oJ\r "° ° i‘%w/W .G¢am(m “\F: (W249 mg (0.285 mmol) of (5a) and 1 g of zinc dust were dissolved in 10 ml glacial acetic acid. The reaction wasthen sonicated for 1 hr. TLC (10% MeOH/ CHCl3) showed nomore starting material, so zinc was filtered through a padof celite and the filtrate concentrated in vacuo leaving anoff—white solid. The crude solid was then dissolved in 1210152025CA 02264297 1999-02-22»WO 98/08829 PCT/US97/15236-45-ml of trifluoroacetic acid (TFA) and stirred at roomtemperature for 1 hr. TFA was removed in Vacuo andremaining oil was triturated with MeOH until white solidformed. This solid was collected and dried under highvacuum yielding 150 mg(70%) of the TFA salt of compound(6a).TLC: Rf=0.169 (10% MeOH/CHC1fiIR (cm%) (KBr): 2960, 1768, 1745, 1640, 1504, 1391, 1258,1198.UV (95%EtOH): 232 nm (e= 24795), 247 nm (e= 26763).1H NMR(300MHz, CD3OD) d: 7.15-7.34 (m, 7H), 7.07 (dd, 1H,J=8.3 and 1.5 Hz), 6.90 (d, 1H, J=8.4 Hz), 6.55-6.61 (m,1H), 6.43 (d, 1H, J=15.8 Hz), 5.94-6.08 (m, 2H), 4.86-5.02(m, 4H), 4.46-4.50 (m, 1H), 4.01-4.06 (m, 1H), 3.80 (s, 3H),3.09 (dd, 1H, J=13.6 and 5.4 Hz), 2.88 (dd, 1H, J=13.6 and7.0 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 (ED): 643 (M++H).Anal: Calcd for C3Jh3ClNgO8 (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) E- H ' CI0NHO HO O T,CF3COOl-I(6b) 10152025CA 02264297 1999-02-22. WO 98/08829 PCT/US97I15236-46....Compound (6b) was prepared acccording to the procedure for(6a) (72%). »TLC: Rf=O.369 (20% MeOH/CHCI3)IR (cm*) (KBr): 2962, 2935, 1747, 1675, 1646, 1606, 1502,1390, 1257, 1196, 1065.UV (95% EtOH): 232 nm (e= 22882), 251 nm (e= 25493).1H NMR(3OOMHz, CDCl3) d: 8.20 (d, 1H, 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, 1H,J=5.9 Hz), 6.40 (d, 1H, J=l5.7 Hz), 5.86-6.05 (m, 2H), 5.11-5.15 (m, 1H), 4.87 (dd, 1H, J=9.5 and 2.8 Hz), 4.60-4.62 (m,1H), 3.85 (br s, 3H), 3.59 (br s, 1H), 3.05-3.21 (m, 1H),2.46-2.60 (m, 3H), 2.27-2.32 (m, 1H), 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 C3ah7ClNg% (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) H ,1 CI H E; Cl0 H O Oo H rc1;:ooH(m) 0”133 mg (0.176 mmol) of (6a) was dissolved in 40 ml of dryN,N—dimethylformamide (DMF) and stirred under an argon1O152025CA 02264297 1999-02-22- WO 98/08829 PCT/US97/15236_47_atmosphere at room temperature. 88 mg (0.229 mmol, 1.3 eq)of pentafluorophenyldiphenylphosphinate (FDPP) was thenadded and reaction stirred for 16 hrs. TLC (20% MeOH/CHClQindicated the reaction was complete, so DMF was removed invacuo. The resulting oil was triturated with hexanes toyield a solid which was flash chromatographed on SiO2 (5%MeOH/CHCl3) yielding 60 mg (55%, based on TFA salt as thestarting material) of cyclized product (7a) as a whitesolid.TLC: Rf=0.677 (5% MeOH/CHClfi1IR (Cm_) (KBr): 3322, 3286, 2950, 1756, 1735, 1663, 1643,1539, 1503, 1258, 1214, 1066, 971, 746, 693.UV (95%EtOH): 249 nm (e= 19840).1H NMR(300MHz, CDCl3) d: 7.20-7.34 (m, 7H), 7.11 (dd, 1H,J=8.3 and 1.9 Hz), 6.83 (d, 1H, J=8.4 Hz), 6.70-6.77 (m,1H), 6.34-6.43 (m, 2H), 6.02 (dd, 1H, J=15.8 and 8.8 Hz),5:76 (d, 1H, J=l5 Hz), 5.64 (d, 1H, J=10.1 Hz), 5.11-5.18(m, 1H), 4.78-4.86 (m 2H), 4.62-4.68 (m, 1H), 3.86 (s, 3H),3.18 (dd, 1H, J=14.2 and 8.0 Hz), 2.82 (dd, 1H, J=14.2 and7.1 Hz), 2.45-2.63 (m, 2H), 2.37-2.42 (m, 1H), 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 C3Ah1ClN2O7: C, 65.32; H, 6.61; N, 4.48.Found: C, 65.12; H, 6.44; N, 4.47.10152025CA 02264297 1999-02-22- WO 98/08829 PCT/US97ll5236._ 4 8 __Example 2Synthesis of compound (7bLCH 05mm mmCompound (7b) was prepared acccording to the procedure for(7a) (48%).TLC: Rf=0.351 (50% EtOAc/hexane)IR (cmfl) (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).1H NMR(3OOMHz, CDC13) d: 7.19-7.39 (m, 7H), 7.12 (d, 1H,J=8.0 Hz), 6.83 (d, 1H, J=8.5 Hz), 6.71 (m, 1H), 6.54 (d,1H, J=10.5 Hz), 6.40 (d, 1H, J=l6.0 Hz), 5.95-6.05 (m, 1H),5.76 (d, 1H, J=15.0 Hz), 5.54 (d, 1H, J=9.9 Hz), 5.20 (m,1H), 4.78-4.89 (m, 2H), 4.41 (dd, 1H, J=10.6 and 4.9 Hz),3.87 (s, 3H), 3.19 (dd, 1H, J=l4.6 and 6.9 Hz), 2.85 (dd,1H, 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 (m, 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 C3&h5ClNfiL: C, 66.20; H, 6.94; N, 4.29.Found: C, 65.94; H, 6.86; N, 4.23.HN ,| Cl ’ CI0 0NI-11 H ] ____.’X FDPP, DMF ’\lH O O O H 0 Ocg:ooH )101520CA 02264297 1999-02-229 W0 98l08829 PCT/US97/15236-49-Example 3§ynthesis of epoxides (8a) and (9a) IInOin CI (83)Om-CPBA0 CHC|,, RT I0 H(7a) loI- H ‘I; Cl0 ( H 0 T(9a)50 mg (0.08 mmol) of (7a) was dissolved in 3 ml drymethylene chloride and stirred under a nitrogen atmosphereat room temperature. 28 mg (0.16 mmol, 2 eq) of meta-chloroperoxybenzoic acid (m-CPBA) was then added andreaction monitored by HPLC (2X3.9mmXl50mm Novapak Cmcolumns, 80% CH3CN/H20, 1.0 ml/min, l= 256 nm). Thereaction was stirred 4.5 hrs and HPLC shows reaction only50% complete so 28 mg (2 eq) more m-CPBA was added. Afterstirring overnight (24 hrs) HPLC shows no starting materialand the two epoxides (8a) and (9a) formed in a 2/1 ratio.The reaction was diluted with methylene chloride and washedwith 5% NaHCO3 and brine. The methylene chloride was thendried over NaSO4 and concentrated in vacuo yielding 73 mg ofcrude white solid which was purified on a semi—prep reversephase Cm HPLC column using the following conditions:column: Rainin 2.5 cmX3O cm semi-prep reverse phase Cm HPLCcolumn101520CA 02264297 1999-02-22— W0 98/118829 PCTIU S97] 15236-50..solvent: 68% CH3CN/H20 isocraticflow rate 20 ml/minl= 220 nmepoxide (Ba): beta—isomer, retention time (2X3.9mmXl50mmNovapak Cm columns, 80% CH3CN/H20, 1.0 ml/min, l= 220 nm)7.51 min.epoxide (9a): alpha—isomer, retention time (2X3.9mmXl50mmNovapak Cw columns, 80% CH3CN/H20, 1.0 ml/min, l= 220 nm)8.33 min.Example 4Synthesis of epoxides (8b) and (9b) Io H ’. CI (Sb)0..__..'£’§’L., - .RT 2° ~ ° r “G” E1”0(7h) O H o T(915)Epoxides (8b) and (9b) were prepared according to theprocedure for (8a) and (9a) except a different column wasused for the purification.column: Technikrom kromasil 2 cmX25 cm semi—prep reversephase Cw HPLC column (5m)solvent: gradient 50% CH3CN/H20-70% CH3CN/H20 over 2 hrsCA 02264297 1999-02-22- WO 98108829 PCT/US97/15236-51-flow rate 28 ml/minl= 220 nmepoxide (Bb): beta—isomer, retention time (2X3.9mmX150mmNovapak C58 columns, 70% CH3CN/H20, 1.0 ml/min, l= 220 nm)14.6 min.epoxide (9b): alpha—isomer, retention time (2X3.9mmXl50mmNovapak C18 columns, 70% CH3CN/H20, 1.0 ml/min, l= 220 nm)16.6 min.