PHARMACEUTICAL COMPOUNDS

Abstract: The invention provides novel cryptophycin compounds which can be useful for disrupting the microtubulin system, as antineoplastic 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 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. Such clinical experience has also demonstrated that drugs which disrupt the microtubule system of the cytoskeleton can be effective in inhibiting the proliferation of neoplastic cells. 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 mitotic 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. Further, such agents having the ability to disrupt the microtubule system can be useful for research purposes. Such anti-mitotic agents or poisins may be classified into three groups on the basis of their molecular mechanism of action. The first group consists of agents, including colchicine and colcemid, which inhibit the formation of microtubules by sequestering tubulin. The second group consists of agents, including vinblastine and vincristine, which induce the formation of paracrystalline aggregates of tubulin. Vinblastine and vincristine 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. The present invention provides novel compounds having the desired anti-microtubule action. Such compounds can be prepared using total synthetic methods and are therefore well suited for development as pharmaceutically useful agents. The presently claimed invention provides novel cryptophycin compounds of Formula I wherein Ar is selected from the group consisting of phenyl, unsubstituted aromatic, substituted aromatic, unsubstituted heteroaromatic, and substituted heteroaromatic; R 1 is selected from the group consisting of halogen, SH, amino, monoalkylamino, dialkylamino, trialkylammonium, alkylthio, dialkylsulfonium, sulfate, and phosphate; R 2 is OH or SH; or R 1 and R 2 may be taken together with Ciβ and C 19 to form an epoxide ring, an aziridine ring, an episulfide ring, a sulfate ring, or monoalkylphosphate ring; or R 1 and R 2 may be taken together to form a second bond between Ci8 and C19; R 3 is a lower alkyl group; R 4 is H or H2; R 5 is H or H 2 ; R 4 and R 5 may be taken together to form a second bond between C 13 and C 14 ; R 6 is selected from the group consisting of benzyl, hydroxybenzyl, alkoxybenzyl, halohydroxybenzyl, dihalohydroxybenzyl, haloalkoxybenzyl, and dihaloalkoxybenzyl group; R 7 is H or a lower alkyl group; R 8 is H or a lower alkyl group; R 7 and R 8 may be taken together to form a spiro group; R 9 is H or a lower alkyl group; R 10 is H or a lower alkyl group; R 11 is selected from the group consisting of simple alkyl, OH, phenyl, substituted phenyl, benzyl, and substituted benzyl; X is O, NH or alkylamino; Y is O, NH or alkylamino; or a pharmaceutically acceptable salt or solvate thereof. The present invention provides pharmaceutical formulations, a method for disrupting a microtubulin 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. As used herein, the term "simple alkyl" shall refer to C 1 -C 7 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, 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 "sulfate" shall have the meaning commonly attributed to the term by the artisan. Thus the term refers to a salt of sulfuric acid. For example, the term may refer to, but is not necessarily limited to, -SO 4 or -SO 3 H. The term "phosphate shall have the meaning commonly attributed to the term by the artisan. Thus the term refers to a salt of phosphoric acid. For example, the term may refer to, but is not necessarily limited to, -PO 4 or -PO 4 H 2 . As used herein, the term "substituted phenyl" shall refer to a phenyl group with from one to three substituents which may be independently selected from the group consisting of simple alkyl, OR 12 ' Cl, Br, F, and I, wherein R 12 ' is selected from simple alkyl. Most preferred, R 12 ' is C 1 -C 3 alkyl. It is especially preferred that R 12 ' is CH 3 . As used herein, the term "substituted benzyl" shall refer to a benzyl group with from one to three subεtitutents which may be independently selected from the group consisting of simple alkyl, Cl, Br, F, and I. As used herein, "spiro group" refers to a cycloalkyl. For example, the spiro group is most preferredly cyclopropyl. The term "cycloalkyl" refers to a saturated C 3 - Cg cycloalkyl group. As used herein "Lower alkoxy1 group" means any alkyl group of one to five carbon atoms bonded to an oxygen atom. As used herein "lower alkyl" means an alkyl group of one to five 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 alkyl substitution on it. As used herein, "monoalkylamino" refers to a monoalkylamino group in which the alkyl portion of the group may be straight or branched. Examples of such groups include but are not limited to: methylamino, ethylamino, 2- propylamino, t-butylamino, and the like. As used herein, "dialkylamino" refers to a dialkylamino group in which the alkyl portion of the group may be straight or branched. Examples of such groups include, but are not limited to: dimethylamino, diethylamino, di (n-propyl)amino, di(iso-propyl)amino, methyl-n-propylamino, and the like. Likewise, "trialkylammonium" refers to a trialkylammonium group in which the alkyl portion of the group may be straight or branched. As used herein, "dialkylsulfonium" refers to a dialkylsulfonium group in which the alkyl portion of the group may be straight or branched. 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" meansa 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" menas a three-membered ring whos backbone consists of two carbon and a sulfur atom. As used herein "sulfate group" menas 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, "monalkylphosphate 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, the term "aromatic" refers to aromatic rings having 4n+2 π electrons in a moncyclic conjugated system. It is most preferred that the aromatic ring is an 10 member ring or less, but also having more than 3 members. It is especially preferred that the aromatic ring has from three to six members. As used herein, "unsubstituted aromatic" refers to common aromatic rings having 4n+2 π electrons in a moncyclic conjugated system, for example, but not limited to: furyl, pyrrolyl, thienyl, pyridyl and the like, or a bicyclic conjugated system, for example but not limited to indolyl or naphthyl. As used herein "substituted aromatic" refers to a common aromatic, as defined supra , which is substituted with a single group selected from the group consisting of halogen, -OR 12 ', and lower alkyl group, wherein R 12 ' is defined supra . As used herein, "heteroaromatic" refers to aromatic rings which contain one or more non-carbon substituent selected from the group consisting of oxygen, nitrogen, and sulfur. It is preferred that the heteroaromatic has from one to three non-carbons selected from oxygen, nitrogen, and sulfur. As used herein, "halogen" refers to those members of the group on the periodic table historically known as halogens. Preferred halogens are Br, Cl, F, and I. 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 "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. Some preferred characteristics of this invention are set forth in 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) R 8 is ethyl, propyl, isopropyl, butyl, isobutyl or isopentyl; B) R 7 is ethyl, propyl, isopropyl, butyl, isobutyl, pentyl, or isopentyl; C) R 7 is H, R 8 is methyl, R 3 is methyl, and X and Y are not both O; D) R 3 is ethyl, propyl, isopropyl, butyl, isobutyl, pentyl or isopentyl; E) R 9 is methyl, ethyl, propyl, butyl, isobutyl, pentyl, or isopentyl; F) R 10 is methyl, ethyl, propyl, butyl, isobutyl, pentyl, or isopentyl; G) a cryptophycin compound wherein at least one of the groups selected from the group consisting of C- 3, C-6, C-7, C-10, C-16, C-17, C-18, C-19, and R 11 has R stereochemistry (numbering as set forth in claim 1 infra . ) ; H) a cryptophycin compound wherein at least one of the groups selected from the group consisting of C- 3, C-6, C-7, C-10, C-16, C-17, C-18, C-19, and R 11 has S stereochemistry (numbering as set forth in claim 1 infra . ) ; I) Ar is phenyl with a substituent selected from the group consisting of hydrogen, halogen, simple alkyl, and -OR 12 ' wherein R 12 ' is lower alkyl; J) a compound wherein the C-7 substituent is R configuration; K) a compound wherein the C-7 substituent is S configuration; L) R 7 , R 8 are each hydrogen; M) R 7 and R 8 are each selected from hydrogen or OH; N) R 11 is simple alkyl; 0) R 11 is selected from the group consisting of methyl, ethyl, n-propyl, and phenyl; P) R 1 and R 2 form an epoxide ring; Q) both X and Y are 0; R) R 4 and R 5 form a double bond; S) R 6 is substituted benzyl wherein one substituent is a halogen and one is an OR 12 group wherein R 12 is lower alkyl; T) a compound of Formula I is used for disruption of a microtubulin system; U) a compound of Formula I is used as an anti¬ neoplastic agent; V) a compound of Formula I is used for the treatment of cancer in a mammal; W) Ar is para ethyl substituted phenyl; and X) R 7 and R 8 join to form a spiro group. 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 adenocarcinoma, 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, neuroendocrine which includes thyroid and non-Hodgkin's disease and Hodgkin'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, eleminates, retards, or reverses the neoplastic phenotype of a cell. 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. PharmacoOeia . Vol. XXII and National Formulary vol XVII, U.S. Pharmacopeia Convention. Inc. Rockville, MD (1989). Additional modes of treatment are provided in AHFS Druσ 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 that the pharmaceutical composition used to treat neoplastic disease contains 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, antineoplastic 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- mercaptopurine, cytosine, 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 in no way limited to those selected from the group consisting of cyclophosphamide, mephalan, busulfan, paraplatin, 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 vincristine, vinblastine, taxol, and etoposide. In a further embodiment, the anti-neoplastic agents contemplated are antibiotics which may include, but are in no way limited to those selected from the group consisting of doxorubicin, daunorubicin, mitomycin C, and bleomycin. 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. Alternatively, dosages within these ranges can be administered by constant infusion over an extended period of time, usually exceeding 24 hours, 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. 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, emulsifiers, 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. One such study provided the following results: IC50 (nM) GC3/C1 HT-29 The compounds are screened for minimum inhibitory- concentrations against KB, a human nasopharyngeal carcinoma cell line, LoVo, a human colorectal adenocarcinoma cell line. The Corbett assay, see Corbett, T.H. et al. Cytotoxic Anticancer Druσs: 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 Aσents: Kluwer Academic Publishers, Norwell, 1993. 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 verses mena 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. 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 compounds of this invention can be prepared as illustrated using the following schemes. More specifically, compounds of this invention can be prepared as illustrated in the following schemes: (1) (1) (2) (1) (2) (P) (Q) TMSCI CH2CI2/THF (R) (S) (D) (E) TMSCI CH2CI2/THF MCPBA v TMSCI _-_,. Chlorohydrins The artisan can utilize appropriate starting materials and reagents to prepare desired compounds using the guidance of the previous schemes and following examples. To further illustrate the invention the following examples are provided. The scope of the invention is in no way to be construed as limited to or by the following examples. Preparation 1 Synthesis of (3R) -benzyl-3-aminopropanoic acid (TFA salt) (1) A sample of t-Butyl-3- (R) -benzyl-3-amino-propanoic acid (1) (purchased from oxford Asymmetry, England, >99% e.e) was dissolved in trifluoroacetic acid (herein "TFA") and then let stirred at room temperature for about 4 hours. The trifluoroacetic acid was removed in vacuo to give an oily residue which was then triturated with methanol to give a white solid. TLC: Rf= (CHCI 3 /CH 3 OH/NH 4 OH: 6:3.2:0.8) IR (cm -1 ) : . 1 HN R(300 MHz,DMSO-d6) d: 7.93 (bs, 2H) , 7.32 (m, 5H) , 3.63 (t, J= 7.2 Hz, 1H) , 2.91 (dd, J= 5.9 Hz, J= 13.6 Hz, 2H) , 2.77 (dd, J= 8.1 Hz, J= 13.6 Hz, 2H) Anal: Calcd for C1 2 H 14 NO4 : C, 49.15; H,4.81; N,4.78. Found: C, 48.87; H,4.73, N, 4.70. Preparation 2 Synthesis of (3R) -benzyl-3- (tert-butoxycarbonyl)amino- propanoic acid A sample of (1) was dissolved in 1, 4-dioxane/H 2 θ/2.0 NaOH (2:2:1) at about 0°C (ice bath). To this was then added di-t- butyl-dicarboxylate and the ice bath was removed and the resulting reaction mixture was stirred at room temperature for about 18 hours. The reaction mixture was then concentrated to ca 10 ml and 25 ml of EtOAc was added. To this was then added 0.5 N aHS0 4 to lower the pH of aqueous phase to ca. 2-3. The organic layer was then separated and the aqueous layer was extracted with EtOAc (20 ml x3) . The combined EtOAc layer were then washed with water and brine and dried over NaS0 4 . The solvent was then removed in vacuo to give a pale yellow solid. TLC: Rf= (CHCI 3 /CH 3 OH/NH 4 OH: 6:3.2:0.8) IR (cirr 1 ) : 3361, 2985, 1670, 1686, 1526, 1266, 1168, 700. UV (CH 3 OH) : 258 nm (e= 158) . OR: [a] D = -136.71 (CHCI 3 , c= ) !HNMR(300 MHz,DMSO-d6) d: 7.20 (m, 5H) , 6.75 (d, J= 8.6 Hz, IH) , 3.88 ( , IH) , 2.64 (d, J= 7.0 Hz, 2H) , 2.28 (t, J= 5.1 Hz, 2H)1.27 (s, 9H) . Mass (FAB) : 280 (M++H) . Anal: Calcd for C 15 H 2 1NO 4 : C, 64.50; H,7.58; N,5.01. Found: C, 63.25; H, 7.35, N, 4.99. Preparation 3 Synthesis of Allyl (2S) -2- [3 ' (tert- Butoxycarbonyl )amino-2 • - (R) -benzylpropanoyloxy] -4- methylpentanoate: (2) To a solution of allyl (2S) -2-hydroxy-4-methylpentanoate (1) and (1.2 eq) of (3R) -methyl-3- (tert-butoxycarbonyl)amino- propanoic acid in 10 ml of dry methylene chloride at 0°C (ice bath), was added (4.4mmol) of dicyclohexylcarbodiimide and then followed by (0.2 eq) of DMAP. The reaction mixture was then let stirred at room temperature for about 3 hours (TLC indicated the completion of the reaction) . The reaction mixture was then filtered through a small pad of celite and the filtrate was washed with 5 % NaHCθ 3 , brine and dried over Na 2 S0 4 . The solvent was removed in vacuo and the residue was flash chromatographed on Siθ 2 (15 % EtOAc/hexane) to give the product {%) of (2) as a clear oil. TLC: Rf= (20 % EtOAc/hexane) IR (cm" 1 ) : 2961, 2933, 1742, 1715, 1497, 1366, 1249, 1170, 1127. UV (CH30H) : 258 ran (e= 218) . OR: [a] D = +7.55 (CHC13, c= ) 1 HNMR(300 MHz, CDCI 3 ) d: 7.25 (m, 5H) , 5.89 (m, IH) , 5.20- 5.36 (m, 3H) , 5.10 (dd, J= 3.9 Hz, J= 9.6 Hz, IH) , 4.65 (d, J= 5.4 Hz, 2H) , 4.15 (bs, IH) , 2.87 (m, 2H) , 2.62 (dd, J= 5.6 Hz, J= 15.4 Hz, IH) , 2.50 (dd, J= 5.0 Hz, J= 15.4 Hz, IH) , 1.60-1.85 (m, 3H) , 1.40 (s, 9H) , 0.95 (d, J= 4.3 Hz, 3H) , 0.93 (d, J= 4.3 Hz, 3H) . Mass(FAB) : 434.4 (M + +H) . Anal: Calcd for C 24 H 35 NO 6 : C 66.49; H,8.14; N,3.23. Found: C, 66.32; H, 8.29, N, 3.42. Preparation 4 Synthesis of (2S) -2- [3 ' (tert-Butoxycarbonyl)amino-2 ' - (R) -benzylpropanoyloxy] -4-methylpentanoic acid (2) <D (2) To a sample of 0.98 g (2.26 mmol) of (1), 0.277 g (0.23mmol) of tetrakistriphenyl-palladium in 50 ml of dry THF was added 2.2 ml of anhydrous morpholine. The reaction mixture was then let stirred at room temperature for 1.5 h and TLC showed the disappearance of the starting material. The reaction mixture was then diluted with 30 ml of diethyl ether and washed with 50 ml of 1.0 N HCl (X2) . The organic layer was then extracted with 2 X 50 ml of 5 % NaHC0 3 . The combined aqueous layer was then acidified with 0.5 -1.0 N HCl to pH 3- 4 and then extracted with ether (400 mL) . The ether layer was then washed with brine and dried over Na 2 Sθ 4 and concentrated in vacuo to give a pale yellow solid (2) weight 0.73 g (82 %) . TLC: Rf= (10 % CH30H/CHC13) IR (cm -1 ): 3034, 2962, 2934, 1727, 1709, 1498, 1455, 1393, 1369, 1253, 1198, 1165, 1127. UV (CH30H) : 259 nm (e= 214) . 1 H,MR(300 MHz, CDC13) d: 7.20 (m 5H) , 5.13 (m, 2H) , 4.19 (bs, IH) , 2.84 (m, 2H) , 2.40 -2.65 (m, 2H) ,1.60 -1.85 (m, 3H) , 1.38 (s, 9H) , 1.23 (d, J= 6.8 Hz, 3 H) , 0.96 (d, J= 5.8 Hz, 3H) , 0.93 (d, J= 5.8 Hz, 3H) . OR: [a] D = +12.91 (CHC13, c= ) Mass(FD) : 394.4 (M++H) . Anal: Calcd for C 2 ιH3iN0 6 : C, 64.10; H,7.94; N,3.56. Found: C, 64.16; H, 7.97, N, 3.43. Preparation 5 Synthesis of compound (M) (M) To a flame-dried 100 ml three-neck round bottom flask under argon was added 0.66 g (1.68 mmol, 1.5 eq) of (2S)-2- [3 ' (tert-Butoxycarbonyl)amino-2' - (R)-benzylpropanoyloxy] -4- methylpentanoic acid (1) and 0.66 g (1.13 mmol ) of compound (2) in 15 ml of dry methylene chloride at 0°C. To this solution was then added 34 mg of DMAP and 0.37 g (1.68 mmol, 1.50 eq) of dicyclohexylcarbodiimide and the resulting solution was stirred at Room Temperature for about 18 hours. TLC showed the completion of the reaction after lδhours and the white precipitate was filtered through a short pad of celite and the filtrate was diluted with 500 ml of ether. The organic layer was then washed with 50 ml of 1.0 N HCl followed by 50 ml of 5 % NaHC0 3 . The solvent was then re oved in vacuo to give a crude solid which weight ca. 1.22 g. This crude solid was then flash chromatographed on Siθ 2 (35:65 EtOAc/hexane) to give a total of 0.958 g (88 %) of coupled product (M) as a foamed white solid. TLC: Rf= (1:1 EtOAc/hexane) IR (cm- 1 ) : 1734, 1706, 1679, 1503, 1281, 1259, 1169. UV (95 % EtOH) : 230 ran (e= 21,823), 246 nm (e= 21,462). 1 HNMR(300 MHz,DMSO-d6) d: 7.16-7.34 (m, 11H) , 7.04 (dd, J= 2.0 Hz, J= 8.4 Hz, IH) , 6.79 (d, J= 8.2 Hz, IH) , 6.74 (m, IH) , 6.55 (d, J= 8 Hz, IH) , 6.39 (d, J= 15.9hz, IH) , 5.96 (dd, J= 8.6 Hz, J= 15.8 Hz, IH) , 5.89 (d, J= 15.8 Hz, IH) , 5.04 (m, 3H) , 4.93 (dd, J= 3.8 Hz, J= 8.0Hz, IH) , 4.76 (d, J= 11.9 Hz, IH) , 4.65 (d, J= 11.9 Hz, IH) , 4.12 (m, IH) , 3.82 (S, 3H) , 3.15 (m, IH) , 3.08 (m, IH) , 2.85 (m, 2H),2.50 (m, 5H) , 1.50-1.75 (m, 3H) , 1.38 (s, 9H) , 1.10 (d, J- 6.8 Hz, 3H) , 0.85 (d, J= 6.4 Hz, 3H) , 0.79 (d, J= 6.4 hz, 3H) . OR: [a] D = +4.51 (CHC1 3 , c= ) Mass(FAB) : 965.4 (M + + H) . Anal: Calcd for C 48 H 58 N 2 OιoCi 4 : C, 59.76; H,6.06; N,2.90. Found: C, 59.74; H, 6.19, N, 2.97. Preparation 6 Synthesis of compound (N) A sample of 0. 80 g ( 0 . 83 mmol ) of ( 1 ) was dissolved in 30 ml of glacial acetic acid and to this was added 3 . 0 g of zinc dust. The mixture was then sonicated (room temperature) for about 1.5 hour (TLC showed the complete consumption of the starting material) . The zinc dust was then filtered away and the filtrate was concentrated to give a white solid. This crude solid was then dissolved immediately in 7.5 ml of trifluoroacetic acid and stirred at room temperature for about 2 hours. Trifluoroacetic acid (TFA) was then removed in vacuo and the oily solid was triturated with ether/hexane to give a white solid (0.814 g) . This crude solid looks very good by TLC and IHNMR, and this was then dissolved in ca. 50 ml of distilled water and triturated for 1 hour (with sonication) . The white solid was then collected and dried in vacuo at about 50° C to give 0.55 g (78 % in two steps) of TFA salt of compound (N) (the aqueous filtrate from water trituration, however, did not contain any UV active material) . TLC: Rf= (10 % CH30H/CHCL3) IR (cm -1 ) : 2964, 1729, 1674, 1628, 1501, 1397, 1280, 1258, 1148, 1088. UV (95 %EtOH) : 232 n (e= 23,051), 247 n <e= 24,678) . 1 HNMR(300 MHz,CD40D) d: 7.14-7.37 ( , 12H) , 7.06 (d, J= 1.9 Hz, J= 8.3 Hz, IH) , 6.86 (d, J= 8.4 Hz, IH) , 6.65 (m, IH) , 6.40 (d, J= 15.8 Hz, IH) , 5.92-6.05 (m, 2H) , 4.97 (m, 2H) , 4.51 (m, IH) , 3.73 (s, 3H) , 3.66 (m, 2H) , 2.80-3.15 (m, 4H) , 2.40-2.72 (m, 3H) , 1.40-1.70 (m, 3H) , 1.09 (d, J= 6.7 Hz, 3H) , 0.77 (d, J= 6.4 Hz, 3H) , 0.68 (d, J= 6.4 hz, 3H) . OR: [a] D = -29.80 (CH 3 OH, c= ) Mass(FD) : 733.4 (M++H) . Anal: Calcd for C 1 H 49 2 O 8 CI (TFA salt) : C, 67.17; H,6.69; N,3.82. Found: C, 68.04; H, 6.57, N, 3.47. Preparatign I Synthesis of (3R) -methyl-3-aminopropanoic acid (TFA salt) O £ H 3 TFA, RT O £ H 3 But ^ 0^ ^ ^ -^Λ NH 2 ► HO ^Λ^ NH 2 . TFA ( 1 ) A sample of 750 mg (4.7mmol) of t-Butyl-3- (R) -methyl-3- a ino-propanoic acid (1) (purchased from oxford Asymmetry, England, >99% e.e) was dissolved in 7.0 ml of trifluoroacetic acid (TFA) and then let stirred at room temperature for about 4 hours. The trifluoroacetic acid was removed in vacuo to give an oily residue which was then triturated with methanol to give a white solid, yield: 1.05 g (100%) TLC: Rf=0.15 (CHCI 3 /CH 3 OH/NH 4 OH: 6:3.2:0.8) IR (cm "1 ): 3286, 3092, 2996, 2914, 1714, 1654, 1504, 1448, 1237, 1196, 1143, 723. 1 HNMR(300 MHz,CD30D) d: 3.61 (q, J= 6.6 Hz, IH) , 2.62 (t, J= 6.0 Hz, 2H) , 1.32 (d, J=6.7 Hz, 3H) Anal: Calcd for C 6 H 10 NO 4 F 3 : C, 33.19; H,4.64; N,6.45. Found: C, 33.32; H,4.64, N, 6.46. Preparation 8 Synthesis of (3R) -methyl-3- (tert-butoxycarbonyl)amino¬ propanoic acid HO A sample of 1.0 g (4.6 mmol) of (1) was dissolved in 20 ml of l,4-dioxane/H 2 O/2.0 NNaOH (2:2:1) at 0°C (ice bath) . To this was then added 1.16 ml (5.06 mmol) of di-t-butyl- dicarboxylate and the ice bath was removed and the resulting reaction mixture was let stirred at room temperature for 18h. The reaction mixture was then concentrated to ca 10 ml and 25 ml of EtOAc was added. To this was then added 0.5 N NaHS04 to lower the pH of aqueous phase to ca 2-3. The organic layer was then separated and the aqueous layer was extracted with EtOAc (20 ml x3) . The combined EtOAc layer were then washed with water and brine and dried over NaSθ 4 . The solvent was then removed in vacuo to give a pale yellow solid. Yield: 0.88g (94%) . TLC: Rf=0.52 (CHCI 3 /CH 3 OH/NH 4 OH: 6:3.2:0.8) IR (cirT 1 ) : 2981, 1711, 1504, 1368, 1244, 1166. 1 HNMR(300 MHz,DMSO-d6) d:3.74 ( , IH) , 2.35 (dd, J= 6.3 Hz, J=15.2 Hz, IH) , 2.16 (dd, J=7.6 Hz, J= 15.2Hz, 1H)1.33 (s, 9H) , 0.99 (d, J= 6.6 Hz, 3H) . Mass (FAB) : 204.2 (M++H) , 223.1 (M++Na) . Anal: Calcd for C9H17NO4: C, 53.19; H,8.43; N,6.89. Found: C, 53.42; H, 8.69, N, 6.77. Preparation 9 Synthesis of Allyl (2S) -2- [3 ' (tert- Butoxy carbonyl ) amino -2 ' - (R) -methylpropanoyloxy] -4- methylpentanoate : (2) To a solution of 0.81 g (3.98 mmol) of allyl (2S) -2-hydroxy- 4-methylpentanoate (1) and 0.82 g (4.78 mmol, 1.2 eq) of (3R) -methyl-3- (tert-butoxycarbonyl)amino-propanoic acid in 10 ml of dry methylene chloride at 0°C (ice bath), was added 0.91 g (4.4mmol) of dicyclohexylcarbodiimide and then followed by 0.13 g (0.96 mmol, 0.2 eq) of DMAP. The reaction mixture was then stirred at room temperature for about 3 hours (TLC indicated the completion of the reaction) . The reaction mixture was then filtered and the filtrate was washed with 5 % NaHC0 3 , brine and dried over Na 2 S0 . The solvent was removed in vacuo and the residue was flash chromatographed on Si0 (15 % EtOAc/hexane) to give 1.3 g (91%) of (2) as a clear oil . TLC: Rf=0.48 (20 % EtOAc/hexane) IR (cm "1 ) : 3442, 2963, 2937. 2874, 1738, 1706, 1503, 1469, 1456, 1391, 1368, 1341, 1274, 1239, 1169, 1121, 1104, 1013, 112, 930. 1 HNMR(300 MHz,DMSO-d6) d: 6,76 (d, J=7.7 Hz, IH) , 5.84 (m, IH) , 5.26 (d, J= 17.5Hz, IH) , 5.18 (d, J= 10.4 Hz, IH) , 4.89 (dd, J= 4.0 Hz, J= 9.0 Hz, IH) , 4.56 (d, J=4.9 Hz, 2H) , 3.77 (m, IH) , 2.55 (dd, J= 6.2 Hz, J= 15 Hz, IH) , 2.31 (dd, J= 7.9 Hz, J= 15 Hz, IH) , 1.69 (m, 2H) , 1.54 (m, IH) , 1.33 (s, 9H) , 1.01 (d, J= 6.6Hz, 3H) , 0.87 (d, J= 6.2 Hz, 3H) , 0.84 (d, J= 6.3 Hz, 3H) . Mass (FAB) : 358.2 (M + +H) . Anal: Calcd for C 18 H 3 iN0 6 : C, 60.48; H,8.74; N,3.92. Found: C, 60.50; H, 8.96, N, 3.66. Preparation 10 Synthesis of (2S) -2- [3 ' (tert-Butoxycarbonyl)amino-2 ' - (R) -methylpropanoyloxy] -4-methylpentanoic acid (2): (1) (2) To a sample of 1.23 g (3.44 mmol) of (1), 0.40 g (0.344mmol) of tetrakistriphenyl-palladium in 70 ml of dry THF was added 3.31 ml of anhydrous morpholine. The reaction mixture was then let stirred at room temperature for 1.5 h and TLC showed the disappearance of the starting material. The reaction mixture was then diluted with 30 ml of diethyl ether and washed with 200 ml of 1.0 N HCl. The organic layer was then extracted with 3 X 200 ml of 5 % NaHCθ 3 . The combined aqueous layer was then acidified with 0.5 -1.0 N HCl to pH 3- 4 and then extracted with ether (400 mL) . The ether layer was then washed with brine and dried over Na 2 Sθ 4 and concentrated in vacuo to give a pale yellow solid (2) weight 1.02 g (93 %) . TLC: Rf= IR (cm" 1 ) : 2980, 2963, 2934, 2873, 1727, 1504, 1456, 1411, 1392, 1369, 1342, 1245, 1168, 1128, 1104, 1065. 1 HNMR(300 MHz,DMSO-d6) d: 5.09 (m, IH) , 5.01 (m, IH) , 4.08 (m, IH) , 2.61 (m, 2H) , 1.74 (m, 3H) , 1.44 (s, 9H) , 1.23 (d, J= 6.8 Hz, 3 H) , 0.96 (d, J= 6.2 Hz, 3H) , 0.93 (d, J= 6.2 Hz, 3H) . OR: [a] D = +18.045 (CHCI3, c= ) Mass(FAB) : 318.2 (M++H) , 340.2 (M++ Na) . Anal: Calcd for C 15 H 27 NO 6 : C, 56.77; H,8.57; N,4.41. Found: C, 57.82; H, 9.08, N, 4.11. Ex mple Synthesis of compound (0 ) A sample of 0.295 mmol of (1) was dissolved in 50 ml of dry DMF under argon atmosphere and to this was then added 1.3 eq of pentafluorophenyldiphenylphosphinate (hereinafter "FDPP"). The resulting reaction mixture was then stirred at ambient conditions for about 18h. Another aliquot of FDPP was added after 18 h if there was unreacted starting material. The reaction was completed after stirring at room temperature for another 4 h. Dimethylformamide (hereinafter "DMF") was then removed in vacuo and the residue triturated with hexane to give a crude solid. This solid was then flash chromatographed on Siθ 2 (5 % CH 3 OH/CHCI 3 ) to give cyclized compound (O) as a solid. The product was characterized by NMR, elemental analysis and mass spectrometry to confirm the product identity. Examnle 2 Synthesis of epoxides (P) and (Q) A (0.168 mmol) sample of compound (0) was dissolved in 6 ml of dry methylene chloride, to this was added 2.0 eq of m- chloroperoxybenzoic acid (hereinafter referred to as "MCPBA" ) at room temperature. This is stirred under argon for 18 about hours. The reaction mixture was checked by HPLC (2X4.6mmX15cm Novapak C-18 column, 75/25 CH3CN:H20, 1.0 ml/min, monitored at 254 nm) . An additional equivalent of MCPBA was added to utilize unreacted starting material and the reaction again monitored by HPLC. About 1.5 ml of the sample (ca 30 % eq) was removed and used immediately for the chlorohydrin reaction for the next step (see Example 3). Of the remaining 70 % reaction mixture, it was then diluted with methylene chloride, washed with 5%NaHCθ 3 (20mlX2) to remove the unreacted MCPBA and metachlorobenzoic acid. The organic layer was dried and concentrated in vacuo. Example 3 Synthesis of chlorohydrin compounds (R) and (S) (P) (Q) TMSCI CH2C12/THF (R) (S) To the 1.5 ml methylene chloride solution obtained from the previous MCPBA reaction of Example 2 is added another 1.0 ml of dry THF. The reaction mixture is kept under argon atmosphere and cooled to -60 C. To this is added 5.0 eq of trimethylchlorosilane. The reaction is followed by TLC (5 % CH30H/CHC13) . The reaction is stirred at -60 C for about 3 h before another lOOul of TMSCI is added. The reaction is then terminated after stirring at -40 C for about another 3 h. The solvent is then removed in vacuo to give a solid which is further purified by preparative reversed phase HPLC using conditions specified below: solvent: CH3CN/H20: flow rate: 5.0 ml/min UV: 254 nm This gives the two chlorohydrins (R) and (S) : Structures are verified using NMR, elemental analysis, and mass spectometry. Example 4 Synthesis of compound (A) (A) To a flame-dried 100 ml three-neck round bottom flask under argon was added 0.80 g (2.52 mmol, 1.5 eq) of (2S)-2- [ ' (tert-Butoxycarbonyl)amino-2 '- (R) -methylpropanoyloxy]-4- methylpentanoic acid (1) and 0.99 g (1.68 mmol ) of compound (2) in 50 ml of dry methylene chloride at 0°C. To this solution was then added 50 mg of DMAP and 0.52 g (2.52 mmol, 1.50 eq) of dicyclohexylcarbodiimide and the resulting solution was let stirred at RT for 4 h. TLC showed the completion of the reaction after 4h and the white precipitate was filtered through a short pad of celite and the filtrate was diluted with 500 ml of ether. The organic layer was then washed with 50 ml of 1.0 N HCl followed by 50 ml of 5 % NaHC03. The solvent was then removed in vacuo to give a crude solid which weight ca. 1.70 g. This crude solid was then flash chromatographed on Si02 (5 % EtOAc/methylene chloride) to give a total of 1.06 g (72 %) of coupled product (A) as a foamed solid. TLC: Rf= 0.52 (1:1 EtOAc/hexane) IR (cm -1 ): 2964, 1743, 1713, 1677, 1642, 1504, 1367, 1281, 1259, 1170, 1127, 1066. UV (CH30H) : 246 nm (e= 21,047) . iH MROOO MHz,DMSO-d6) d: 7.31 (m, 5H) , 7.19 (d, J= 2.0 Hz, IH) , 7.06 (dd, J= 2.0Hz, J= 8.4 Hz, IH) , 6.82 (d, J= 8.4 Hz, IH) , 6.65-6.78 (m, 2H) , 6.38 (d, J= 15.9 Hz, IH) , 6.00 (dd, J= 8.7 Hz, J= 15.9 Hz, IH) , 5.89 (d, J= 15.6 Hz, IH) , 5.03 (m, 2H) , 4.90 (dd, J= 3.7 Hz, j= 9.9Hz, IH) , 4.79 (d, J= 11.9 Hz, IH) , 4.69 (d, J= 11.9 Hz, IH) , 4.00 (m, IH) , 3.85 (S, 3H) , 3.14 ( , 2H) , 2.52 (m, 6H) , 1.50-1.75 (m, 3H) , 1.41 (s, 9H) , 1.18 (d, J= 6.7 Hz, 3H) , 1.10 (d, J= 6.8 Hz, 3H) , 0.84 (d, J= 6.4 Hz, 3H) , 0.79 (d, j= 6.4 hz, 3H) . OR: [a] D = +0.792 (CHC13 , c= ) Mass(FD) : 888.8 (M+) . Anal: Calcd for C42H54N2O10C14: C, 56.76; H,6.12; N,3.15. Found: C, 56.54; H, 6.16, N, 3.11. Example 5 Synthesis of compound (B) A sample of 0.98 g (1.12 mmol) of (1) in 40 ml of glacial acetic acid and to this was added 3.9 g of zinc dust. The mixture was then sonicated (room temperature) fro 45 min (TLC showed the completion of the starting material) . The zinc dust was then filtered away through a short pad of celite and the filtrate was concentrated to give a white solid. This crude solid was then dissolved immediately in 50 ml of trifluoroacetic acid and let stirred at room temperature for 2 h. TFA was then removed in vacuo and the oily solid was triturated with ether/hexane to give a white solid (1.30 g) . This crude solid ws then flash chromatographed on Si02 (10 % CH30H/CHC13) to give a white solid (1.0 g) . This solid was then dissolved in ca. 50 ml of distilled water and triturated for i h. The white solid was then collected and dried in vacuo at 50° C to give 0.62 g (72 % in two steps) of TFA salt of compound (B) as a white solid. TLC: Rf= 0.19 (10 % CH30H/CHCL3 ) IR (cm "1 ) : 2961, 2935,1741, 1674, 1621, 1503, 1442, 1393, 1281, 1258, 1202, 1144, 1127, 1066, 970. UV (CH30H) : 230 nm (e= 24,055), 247 nm (e= 24,515) . 1 HNMR(300 MHz,CD40D) d: 7.30 (m, 5H) , 7.17 (d, J= 2.1 Hz, IH) , 7.09 (dd, J= 1.9Hz, J= 8.4 Hz, IH) , 6.90 (d, J= 8.4 Hz, IH), 6.65 (m, IH), 6.40 (d, J= 15.8 Hz, IH) , 6.02 (m, 2H) , 4.93 (m, 2H) , 4.53 (m, IH) , 3.79 (s, 3H) , 3.85 (m, IH) , 3.57 (m, IH) , 3.14 (m, 2H) , 2.90 (m, 2H) , 2.62 (m, 4H) , 1.42-1.70 (m, 3H) , 1.27 (d, J= 6.7 Hz, 3H) , 1.10 (d, J= 6.8 Hz, 3H) , 0.77 (d, J= 6.4 Hz, 3H) , 0.70 (d, J= 6.4 hz, 3H) . OR: [a] D = (CHC13, c= ) Mass(FD) : 785.4 (M + ) . Anal: Calcd for C38H48N2O10F3C1 (TFA salt): C, 58.12; H,6.16; N,3.57. Found: C, 57.92; H, 6.11, N, 3.91. Example 6 Synthesis of compound (C) A sample of 0.30 g (0.39 mmol) of (1) was dissolved in 50 ml of dry DMF under argon atmosphere and to this was then added 0.19 g (0.51 mmol, 1.3 eq) of pentafluorophenyl- diphenylphosphinate (FDPP) in 8 ml of dry DMF. The resulting reaction mixture was then let stirred at room temperature for 5h. DMF was then removed in vacuo and the residue was triturated with ether/hexane to give a crude solid. This solid was then flash chromatographed on Si02 (5 % CH30H/CHC13) to give 0.18 g (72 %) of cyclized compound (C) as white solid. TLC: Rf= 0.21 (10 % CH30H/CHCL3) IR (cm -1 ) : 3394, 3290, 2960, 1744, 1728, 1676, 1659, 1539, 1521, 1503, 1442, 1204, 1173, 751. UV (CH30H) : 247 nm (e= 21,980). 1 HNMR(300 MHz,CDCl3) d: 7.85 (d, J=9.6 Hz, IH) , 7.31 (m, 5H) , 7.19 (d, J= 2.0 Hz, IH) , 7.03 (dd, J= 2.0 Hz, J= 8.3 Hz, IH) , 6.83 (d, J= 8.2 Hz, IH) , 6.79 (m, IH) , 6.38 (d, J= 15.9 Hz, IH) , 6.00 (dd, J= 8.8 Hz, J= 15.3 H, IH) , 5.74 (d, J= 14.7hz, IH) , 5.39 (d, J= 8 Hz, IH) , 5.12 (m, IH) , 4.77 (m, 2H) , 4.25 ( , IH) , 3.88 (ε, 3H) , 3.26 (dd, J= 5.8 Hz, J= 13.2 Hz, IH) , 2.94 (dd, J= 5.2 Hz, J= 14.3 Hz, IH) , 2.42 - 2.70 (m, 4H) , 1.64 (m, 2H) , 1.31 (m, IH) , 1.14 (d, J= 4.4 Hz, 3H) , 1.12 (d, J= 4.4 Hz, 3H) , 0.74 (d, J= 2.9 Hz, 3H) , 0.71 (d, J= 2.9 Hz, 3H) . OR: [a] D = (CHC13, c= ) Mass(FAB) : 639.4 (M + ) . Anal: Calcd for C35H43N207C1: C, 66.20; H,6.94; N,4.29. Found: C, 66.04; H, 6.82, N, 4.38. Example 7 The synthesis of epoxides (D) and (E) A sample of mg ( mmol) of compound (C) was dissolved in ml of dry methylene chloride, to this was then added mg (2.0 eq) of MCPBA at room temperature. This was then let stirred under argon for 18 h. The reaction mixture was checked by HPLC (2X4.6mmXl5cm Novapak C-18 column, 75/25 CH3CN:H20, 1.0 ml/ in, monitored at 254 nm) and it was found that ca % of starting material still remain after 18 h. Another mg (1.0 eq) of MCPBA was added and reaction again monitored by HPLC. After stirring overnight (ca 41 h total) reaction, mg of MCPBA was added (total: mg, eq) to the reaction mixture. The reaction was let stirred for another 6 h until HPLC showed only 1.3 % of starting material remained unreacted. HPLC also indicated the two epoxides (D) and (E) existed in a ratio of ca. :1.0. Of the 5.5ml of the reaction mixture, 1.5 ml of the sample (ca 30 % eq) was removed and used immediately for the chlorohydrin reaction for the next step (see next experimental) . Of the remaining 70 % (ca. 4.0 ml) reaction mixture, it was then diluted with methylene chloride (50 ml) and washed with 5%NaHC03 (20mlX2) to remove the unreacted MCPBA and metachlorobenzoic acid. The organic layer was then dried over Na2S04 and concentrated in vacuo to give mg of pale yellow solid which was purified and separated on a semiprep reversed phase C-18 HPLC column with the following condition: column: solvent: CH3CN/H20: flow rate: 5.0 ml/min UV: 254 nm This gives the two epoxides (D) and (E) : Examnle 8 Synthesis of chlorohydrin compounds (F) and (G) (D) (E) TMSCI CH2C12/THF (F) (G) To the ml methylene chloride solution obtained from the previous MCPBA reaction ( % eq, ca mg of epoxides in theory, mmol) was added another 1.0 ml of dry THF. The reaction mixture was then kept under argon atmosphere and cooled to -60 C. To this was added ul ( mg, 5.0 eq) of trimethylchlorosilane. The reaction was then followed by TLC (5 % CH30H/CHC13) , the reaction was let stirred at -60° C for 3 h before another ul of TMSCI was added. TLC showed the appearance of two more polar spots with ratio of ca 2:1. The reaction was then terminated after stirring at -40 C for another 3 h (TLC still showed some unreacted material) . The solvent was then removed in vacuo to give a white solid which was further purified by preparative reversed phase HPLC using condition specified below: column: solvent: CH3CN/H20: flow rate: 5.0 ml/min UV: 254 nm This gives the two chlorohydrins (F) and (G) :

Claims: C laims 1. The presently claimed invention provides novel cryptophycin compounds of Formula II wherein Ar is selected from the group consisting of phenyl, unsubstituted aromatic, substituted aromatic, unsubstituted heteroaromatic, and substituted heteroaromatic; R 1 is selected from the group consisting of halogen, SH, amino, monoalkylamino, dialkylamino, trialkylammonium, alkylthio, dialkylsulfonium, sulfate, and phosphate; R 2 is OH or SH; or R 1 and R 2 may be taken together with C Q and C 19 to form an epoxide ring, an aziridine ring, an episulfide ring, a sulfate ring, or monoalkylphosphate ring; or R 1 and R 2 may be taken together to form a second bond between C 18 and C 1 9; R 3 is a lower alkyl group; R 4 is H or H 2 ; R 5 is H or H2; R 4 and R 5 may be taken together to form a second bond between C 13 and C 14 ; R 6 is selected from the group consisting of benzyl, hydroxybenzyl, alkoxybenzyl, halohydroxybenzyl, dihalohydroxybenzyl, haloalkoxybenzyl, and dihaloalkoxybenzyl group; R 7 is H or a lower alkyl group; R 8 is H or a lower alkyl group; R 7 and R 8 may be taken together to form a spiro group; R 9 is H or a lower alkyl group; R 10 is H or a lower alkyl group; R 11 is selected from the group consisting of simple alkyl, OH, phenyl, substituted phenyl, benzyl, and substituted benzyl; X is 0, NH or alkylamino; Y is 0, NH or alkylamino; or a pharmaceutically acceptable salt or solvate thereof. 2. A compound of Claim 1 wherein Ar is phenyl or substituted phenyl wherein in the phenyl substituents are selected from the group consisting of hydrogen, simple alkyl, and halogen. 3. A compound of Claim 2 wherein R 7 and R 8 are each hydrogen. 4. A compound of Claim 3 wherein R 1 and R 2 form an epoxide ring. 5. A compound of Claim 4 wherein R 11 is selected from the group consisting of saturated simple alkyl. 6. A compound of Claim 4 wherein R 11 is selected from the group consisting of unsaturated simple alkyl. 7. A compound of Claim 4 wherein R 11 is selected from the group consisting of benzyl and substituted benzyl wherein the benzyl substituents are selected from the group consisting of simple alkyl and halogen. 8. A compound of Claim 2 wherein R 7 is hydrogen and R 8 is OH. 9. A compound of Claim 8 wherein R 1 and R 2 form an epoxide ring. 10. A compound of Claim 9 wherein R 11 is selected from the group consisting of saturated simple alkyl. 11. A compound of Claim 9 wherein R 11 is selected from the group consisting of unsaturated simple alkyl. 12. A compound of Claim 9 wherein R 11 is selected from the group consisting of benzyl and substituted benzyl wherein the benzyl substituents are selected from the group consisting of simple alkyl and halogen. 13. A method for disrupting a microtubulin system comprising contacting an effective amount of a compound of Claim 1 to a mammalian cell. 14. A method for inhibiting the proliferation of a hyperproliferative mammalian cell comprising administering an effective amound of a compound of Claim 1 together with a pharmaceutically acceptable carrier or excipient therefor. 15. A formulation comprising a compound of Claim 1 together with one or more pharmaceutically acceptable excipients, diluents, or carriers therefor. 16. A formulation comprising an effective amount of a compound of Claim 1 together with one or more pharmaceutically acceptable excipients, diluents, or carriers therefor. 17. A use of a compound as claimed by any one of Claims 1 through 12 for use in the manufacture of a medicament. 18. A compound of Claim 1 wherein the compound is selected from the group consisting of

Inventors: SHIH CHUAN, SHIH, CHUAN
Applicants: LILLY CO ELI, SHIH CHUAN, UNIV HAWAII, UNIV WAYNE STATE, ELI LILLY AND COMPANY, SHIH, CHUAN, UNIVERSITY OF HAWAII, WAYNE STATE UNIVERSITY
Application year: 1996
Main classification: A61K31
IPC codes: A61K31, A61P35, C07D273, C07D413