WO2006055578A2 - Asymmetric synthesis of (-)-agelastatin a - Google Patents

Abstract

The total asymmetric synthesis of the cytotoxic marine metabolite (-)-agelastatin A (I) has been achieved from a 4,5-diamino-cyclopenten-2-one derived intermediate. This key intermediate was efficiently prepared from a sulfinimine-derived alpha-beta-diamino ester using ring closing metathesis.

Description

ASYMMETRIC SYNTHESIS OF (-)-AGELASTATIN A

Cross Reference

[0001] This is a nonprovisional application that claims the benefit of U.S. Provisional Application No. 60/628,456 filed November 16, 2004, which is incorporated herein by reference in its entirety.

Reference to Government Grant

[0002] The present invention was made at least partly with government funds under grant no. 57870 from the National Institute of General Medical Sciences. The U.S. government may have certain rights in the invention.

Background of the Invention

[0003] (-)-Agelastatin A is an architecturally unique cytotoxic tetracyclic alkaloid isolated from the axinellid marine sponge Agelas dendromorpha in 1993 as reported by D'Ambrosio et al., J. Chem. Soc, Chem. Comm. 1993 and D'Ambrosio et al., HeN. CHm. Acta 1994, 77, 1895. Molinski et al., J. Nat. Prod. 1998, 61 158, recently reported the isolation of (-)-agelastatin A, along with two minor congeners, from the West Australian sponge Cymbastela sp. This alkaloid exhibits potent cytotoxicity against L1210 in mice and human KB nasopharyngeal tumor cell lines at low concentrations as disclosed by D'Ambrosio et al. HeIv. CHm. Acta 1996, 79, 727. To date the mechanism of antitumor activity has not been elucidated. As disclosed by Meijer et al. Chem. Biol. 2000, 7, 51, (-)-agelastatin A is also reported to selectively inhibit glycogen synthase kinase- 3β (GSK-3β) at low concentrations and could play a role in preventing Alzheimer's disease (AD), inhibiting neuronal apoptosis after stroke, and can function as an insulin mimetic. Potent insecticidal activity against beet army worm larvae and corn rootworm has also been reported by Molinski et al., J. Nat. Prod. 1998, 61 158.

[0004] The scarcity of this biologically significant alkaloid isolated from natural sources makes a total enantioselective synthesis of (-)-agelastatin A of prime importance for further biological evaluation as well as for analogue synthesis. The first total synthesis of agelastatin A was a racemic synthesis (not optically active) as reported by Weinreb et al., J. Am. Chem. Soc. 1999, 121, 9574. This synthesis employed a hetero Diels-Alder cycloaddition reaction and a Sharpless/Kresze allylic amination protocol in the preparation of this alkaloid and required 14 steps, resulting in an overall yield of 7%. The key step in asymmetric synthesis of (-)-agelastatin A was vinylcarbene C-H insertion sequence for preparation of the C-ring core as reported by Feldman et al., J. Org. Chem. 2002, 67, 7096. This synthesis required more than thirteen steps with an overall yield of 3.8%. A formal asymmetric synthesis of (-)-agelastatin A was accomplished as reported by Hale et al., Org. Lett. 2003, 5, 2927 in an enantioselective synthesis of Weinreb's C-ring intermediate from a Hough-Richardson aziridine. The total synthesis of (-)-agelastatin A from a chiral bicyclic cyclopentene oxazolidinone intermediate was recently reported by Domostoj et al., Org. Lett. 2004, 6, 2615. In this synthesis more than twenty-six steps were necessary with an overall yield of 0.5%. In each of these syntheses, construction of a central C-ring core from a bicyclic cyclopentene oxazolidinone proved to be the critical step. Another previous asymmetric synthesis of a C-ring intermediate was reported by Baron et al., Tetrahedron Lett. 2002, 43, 723.

[0005] Sulfinimine based methodologies for the asymmetric synthesis of biorelevant nitrogen compounds have been developed as reported by Zhou et al., Tetrahedron 2004, 60, 8003. For example, as reported by Davis et al., Org. Lett. 2004, 6, 1269, the asymmetric synthesis of (+)-4-aminocyclopentenone, which is an important chiral building block for the synthesis of antitumor carbocyclic nucleosides, can be accomplished from a sulfinimine derived N-sulfmyl amino β-ketodiene using ring closing metathesis (Figure 1). Another procedure reported by Davis et al., Org. Lett. 2004, 6, 2789, concerns a new method for the asymmetric synthesis of syn- and anti-α,β-diamino acids via the addition of N-protected glycine enolates to enantiopure sulfinimines (Figure 1). However, these methodologies have not been applied to the synthesis of intermediates of (-)-agelastatin A, and it was heretofore unclear whether synthesis of this complex tetracyclic alkaloid could be completed from such intermediates.

[0006] What is needed, therefore, is a more efficient method of synthesizing the tetracyclic alkaloid (-)-agelastatin A, which method results in a higher yield of product.

Summary of the Invention

[0007] For ease of discussion, certain chemical species are referred to by a reference number which indicates the corresponding chemical structure set forth in the figures. For example, (-)-2 refers to the 4,5-diamino cyclopent-2-enone C-ring intermediate as shown in Fig. 2.

[0008] Asymmetric synthesis of the cytotoxic tetracyclic marine alkaloid (-)-agelastatin A can be been accomplished using new sulfinimine based methodologies in approximately ten or eleven steps under eight operations (about 9% over all yield), from sulfinimine (-)-6 and readily available materials. The synthetic method comprises the sulfinimine-mediated enantioselective synthesis of syn-α,β-diamino ester (-)-4, ring closing metathesis of diamino ketodiene (-)-3 to give the C-ring intermediate (-)-2 and the D-ring formation by the addition of methyl isocyanate to (-)-2 under reductive conditions.

[0009] The invention thus provides a method for the asymmetric synthesis of (-)-agelastatin A, comprising:

(1) adding the acrolein-derived sulfinimine (R)-(-)-6 to a preformed lithium enolate of ethyl (dibenzylamino)acetate (5), thereby forming an unsaturated a, /3-dramino ester (-)-4 comprising a major diastereoisomer of syn diastereoisomer (-)-4, and isolating the major syn diastereoisomer (-) -4 thus formed;

(2) treating the ester (-)-4 with lithium N,0-dimethylhydroxylamine, thereby forming Weinreb amide (-)-7, and isolating the Weinreb amide (-)-7;

(3) deprotecting of the N-sulfinyl amino group (TFA/MeOH) of the Weinreb amide (-)-7 to give an amine;

(4) reacting the amine from step 3 with pyrrole-2-carboxylic acid, the coupling reagent HBTU, and DIPEA (Hunig's base) to give amide (+)-8, and isolating the amide (+)-8;

(5) treating the amide with allylmagnesium bromide at 0 ⁰C to give the γ,β-unsaturated ester intermediate;

(6) isomerizing the γ,β-unsaturated ester intermediate with Et₃Ν/EtOH to give the diamino ketodiene (-)-3 and isolating the diamino ketodiene (-)-3;

(7) refluxing (-)-3 in DCM with Grubb's second generation catalyst to give a C-ring intermediate 4, 5 -diamino cyclopenten-2-enone (-)-2;

(8) treating (-)-2 with Cs₂CO₃MeOH to give a tricyclic ring system (-)-10 (the B-ring intermediate);

(9) deprotecting the tricyclic ring system (-)-10;

(10) treating the free amine thus formed with methyl isocyanate to give a D-ring intermediate; and

(11) brominating the D-ring intermediate to complete the synthesis of (-)-agelastatin A

(1).

[0010] The invention also provides a method for the synthesis of the C-ring intermediate 4,5-diamino cyclopenten-2-enone (-)-2, the B-ring intermediate tricyclic ring system (-)-10 and the D-ring intermediate according to steps set forth above.

[0011] The invention also provides the C-ring intermediate 4,5-diamino cyclopenten-2-enone (-)-2, the B-ring intermediate tricyclic ring system (-)-10 and the D-ring intermediate. [0012] The invention further provides a method for treating disorders such as cancer,

Alzheimer's disease, diabetes or stroke comprising optionally synthesizing (-)-agelastatin A according to the present method, and administering an effective amount of (-)-agelastatin A to a subject in need of such treatment.

[0013] The invention yet further provides the use of (-)-agelastatin A in the preparation of a pharmaceutical composition (a.k.a. a medicament) for the treatment of disorders such as cancer, Alzheimer's disease, diabetes or stroke.

[0014] The invention yet further provides a method of identifying compounds with anti-cancer or other therapeutic activity, comprising synthesizing (-)-agelastatin A according to the present method, modifying the (-)-agelastatin A to produce analogs of (-)-agelastatin A according to the present method, and testing said analogs for anti-cancer or other therapeutic activity.

[0015] The invention yet further provides analogs of (-)-agelastatin A and methods of making such analogs.

Brief Description of the Drawings

[0016] Fig. 1 is a synthetic scheme showing the asymmetric synthesis of carbocyclic nucleosides from sulfinimine derived from N-sulfmyl amino β-ketodiene using ring-closing metathesis, and the asymmetric synthesis of syn- and anti-α,β-diamino acids via the addition of N-protected glycine enolates to enantiopure sulfinimines.

[0017] Fig. 2 is a synthetic scheme showing the retrosynthetic route to (-)-agelastatin A (1) from the diamino ester (-)-4 (Scheme 1).

[0018] Fig. 3 is a synthetic scheme showing the synthesis of the C-ring core intermediate (-)-2 (Scheme 2).

[0019] Fig. 4 is a synthetic scheme showing the conversion of the C-ring core intermediate (-)-2 into the tricyclic intermediate (-)-10, deprotection of the tricyclic intermediate, formation of the D-ring intermediate debromoagelastatin (-)-13, and bromination of the D-ring intermediate to give (-)-agelastatin A (1) (Scheme 3).

[0020] Fig. 5 is a general foπnula representing analogs of (-)-agelastatin A (1) according to the invention.

Detailed Description of the Invention

[0021] The total asymmetric synthesis of (-)-l, which differs from previous syntheses of (-)-l mainly in the synthesis of the C-ring cyclopentene, is presented below. [0022] In the present method, a 4,5-diamino cyclopent-2-enone (-)-2 was constructed as the

C-ring intermediate (Scheme 1; see Fig. 2). It was found that the methodology devised for the synthesis of 4-amino cyclopentenone (Figure 1) could be employed to prepare (-)-2 from diamino ketodiene (-)-3 using ring-closing metathesis. Addition of the enolate of (dibenzylamino)acetate to an acrolein-derived sulfinimine furnished (-)- 4. The conversion of (-)-2 to (-)-l could be accomplished using chemistry similar to that reported by Weinreb et al., J. Am. Chem. Soc. 1999, 121, 951 A and Weinreb et al., J. Org. Chem. 1998, 63, 7594.) in their racemic synthesis of 1.

[0023] The present synthesis of (-)-l begins with the preparation of the requisite unsaturated α,β-diamino ester (-)-4, obtained by addition of the acrolein-derived sulfinimine (i?)-(-)-6 to 5.0 equivalents of the preformed lithium enolate of ethyl (diben2ylamino)acetate (5). Three of the four possible diastereoisomers were detected in a ratio of about 18:1:5 with the major syn diastereoisomer (-)-4 being isolated in about 77% yield (Scheme 1). Treatment of ester (-)-4 with about 5.0 equivalents of lithium N,0-dimethylhydroxylamine gave the corresponding Weinreb amide (-)-7 in about 89% isolated yield. Deprotection of the N-sulfmyl amino group (TFA/MeOH) gave the amine, which was not isolated, but was immediately reacted with pyrrole- 2-carboxylic acid, the coupling reagent HBTU, and DIPEA (Hunig's base) to afford amide (+)-8 in about 88% isolated yield for the two steps.

[0024] Next the amide was treated with 2 equivalents of allylmagnesium bromide at about O⁰C to give the presumed γ,β-unsaturated ester intermediate, which was isomerized with Et₃N/EtOH affording the diamino ketodiene (-)-3 in about 85% yield for the two-step sequence (Scheme 2; see Fig. 3). (Thin Layer Chromatography (TLC) indicated the presence of a new spot that was converted to (-)-3 on exposure to Et₃N.) Refluxing (-)-3 in dichloromethane (DCM) with about 10 mol% to about 20 mol%, preferably about 10 mol%, of Grubb's second generation catalyst 9 for about 12 hours resulted in 4,5-diamino cyclopenten-2-enone (-)-2 in about 82% to about 87% yield, the present C-ring intermediate.

[0025] The B-ring intermediate of (-)-l was constructed from the C-ring intermediate (-)-2 by an intramolecular Michael cyclization, in a fashion similar to that described in earlier syntheses of (-)-agelastatin A (1) by Weinreb et al., J. Am. Chem. Soc. 1999, 121, 9574; Weinreb et al., S. M. J. Org. Chem. 1998, 63, 7594 and Domostoj et al., Org. Lett. 2004, 6, 2615. Indeed, with an intermediate similar to (-)-2, Weinreb et al. reported that the cyclization occurs in about 61-64% yield in the presence of Cs₂CO₃MeOH. On the other hand, Hale et al., Org. Lett. 2004, 6, 2615, also with a species similar to (-)-3, was unable to affect the cyclization using the conditions of Weinreb et al. Nevertheless it was found in the present method that treatment of (-)-2 with about 10 equivalents of Cs₂CO₃/MeOH for about 16 minutes resulted in about a 68% yield of the desired tricyclic ring system (-)-10 along with trace amounts of cyclopentenone (-)-ll. Longer reaction times (e.g., about 2 hours) produce the eneone as the major product. However, use of 2,4,7- trinitro-9-fluorenone (TNF) in place of MeOH for the Michael addition with Cs₂CO₃ avoids the formation of the (-)-ll side product even after about 2 hours of reaction. Hale et al., Org. Lett. 2004, 6, 2615, isolated a similar product in their attempts to affect the Michael cyclization.

[0026] Next, the N-benzyl protecting groups were removed and the resulting free amine was treated with methyl isocyanate to give the D-ring intermediate, which was brominated to complete the synthesis of (-)-agelastatin A (1). Hydrogenation under a variety of conditions (Pd(OH)₂; Pd/C; solvents ) failed to produce characterizable products. Hydrogenation followed by treatment of the crude reaction mixture methyl isocyanate gave similar results. It was discovered that if (-)- 10 and methylisocynate were hydrogenated together in one "pot," N-benzyl debromoagelastatin A (-)-12 and debromoagelastatin (-)-13 could be isolated in about 32% and about 47% yields, respectively (Scheme 3; see Fig. 4). Alternatively, using a large excess (about 2 equivalents) of the Pd catalyst, debromoagelastatin (-)-13 could be isolated in about 70% yield with only trace amounts of N-benzyl debromoagelastatin A (-)-12 being formed (Scheme 3; see Fig. 4).

[0027] To date all attempts to debenzylate (-)-12 have been unsuccessful and suggest that (-)-12 is not an intermediate in the formation of (-)-13. Bromination of (-)-13 with ΝBS in THF according the Feldman protocol (Feldman et al., J. Am. Chem. Soc. 2002, 124, 9060 and Feldman et al., J. Org. Chem. 2002, 67, 7096 the entire disclosures of which are herein incorporated by reference) for about 12 hours afforded (-)-agelastatin A (1) in about 69% isolated yield.

[0028] The (-)-agelastatin A synthesized by the present method can be used to treat a variety of disorders. For example, (-)-agelastatin A is known to have anticancer activity, and therefore can be used to treat cancers such as leukemia, epithelial tumors and nasopharyngeal cancer. Other cancers which can be treated with (-)-agelastatin A include breast, prostate, ovarian, lung, colorectal, brain (e.g., gliomas, glioblastomas and astrocytomas), renal, pancreatic, lung (small cell and non-small cell), skin (e.g., melanomas), ovarian, lymphomas (particularly Burkets lymphoma and acute lymphocytic lymphoma), uterine sarcomas; squamous cell carcinomas and papillomas, basal cell carcinomas and papillomas, and epidermoid cancers.

[0029] The (-)-agelastatin A synthesized by the present method also inhibits glycogensynthase kinase-3β (GSK-3β), and thus is useful in preventing the onset of Alzheimer's disease and in inhibiting neural apoptosis after stroke. The (-)-agelastatin A synthesized by the present method can also function as an insulin mimetic. The present invention therefore provides methods of treating disorders such as cancer, Alzheimer's disease, diabetes and stroke by administering an effective amount of (-)-agelastatin A synthesized by the present method to a subject in need of such treatment.

[0030] As used herein, an "effective amount" of the (-)-agelastatin A synthesized by the present method is any amount which delays the onset of, improves, inhibits or in any way ameliorates one or more symptoms of the disorder being treated. The ordinarily skilled physician is readily able to determine both when a subject is in need of treatment for a particular disorder, and when one or more symptoms of a given disorder are delayed, inhibited or in any way ameliorated.

[0031] The effective amount of the (-)-agelastatin A synthesized by the present method can depend on absorption, inactivation and excretion rates of the drug, as well as other factors known to those of skill in the art. The effective amount can also vary with the penetration of the disorder, or the severity of the symptoms to be alleviated. It is also understood that for any particular subject, specific dosage regimens can be adjusted over time according to individual need, and that the concentration ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed methods. The effective amount of the (-)-agelastatin A synthesized by the present method can be administered in a single dose, or can be divided into a number of smaller doses to be administered at varying time intervals.

[0032] As used herein, a "subject" includes any mammal, for example a rodent or primate mammal, and is preferably a human being. However, it is understood that veterinary applications, in certain indications, are included in the present methods.

[0033] The (-)-agelastatin A synthesized by the present method can be formulated into a pharmaceutical compositions for administration to a subject. As used herein, "pharmaceutical compositions" comprise an amount of the (-)-agelastatin A synthesized by the present method, for example about 0.01% to about 90% w/w, in a pharmaceutically acceptable carrier or excipient. As used herein, "pharmaceutical compositions" include compositions for human and veterinary use.

[0034] Pharmaceutical compositions of the invention are also known as "medicaments." Thus, the invention specifically contemplates the use of the (-)-agelastatin A synthesized by the present method for the production of a medicament for the treatment of disorders such as cancer, Alzheimer's disease, diabetes and stroke. In treating the disorders the pharmaceutical composition is administered to a subject, such that the pharmaceutical composition delivers an effective amount of (-)-agelastatin A. [0035] Methods for preparing the pharmaceutical compositions of the invention are within the skill in the art, for example as described in Remingtons 's Pharmaceutical Science, 17^th Ed., Mack Publishing Co., Easton, PA, the entire disclosure of which is herein incorporated by reference.

[0036] Pharmaceutically acceptable carriers or excipients are known in the art, and include any substance which is used in the formulation of a drug dosage form, as are described below.

[0037] For example, pharmaceutical compositions for oral administration generally comprise an inert excipient or an edible carrier, and can be formulated into tablets, troches or capsules (e.g., gelatin capsule). Binding agents and/or adjuvant materials can be included as part of the oral pharmaceutical composition. For purposes of the present invention, such binding agents or adjuvant materials are considered pharmaceutically acceptable carriers or excipients. Preferred oral dosage forms include liquids, gelatin capsules and tablets.

[0038] The tablets, pills, capsules, troches and the like of the invention can contain any of the following ingredients, or compounds of a similar nature, all of which are considered pharmaceutically acceptable carriers: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring. When the oral dosage form is a capsule, it can contain, in addition to material of the above type, a liquid carrier such as a fatty oil. In addition, oral dosage forms of the invention can contain various other materials which modify the physical form of the dosage unit, for example, coatings of sugar, shellac, or other enteric agents.

[0039] Liquid oral dosage forms of the invention can comprise an elixir, solution, suspension, syrup, wafer, chewing gum or the like. A syrup can further comprise a sweetening agent (such as sucrose or other sugar, or artificial sweetener such as aspartame or xylitol or Splenda® (available from McNeil Nutritionals LLC, Fort Washington, PA)) and certain preservatives, dyes and colorings and flavors, all of which are considered pharmaceutically acceptable carriers or excipients.

[0040] Gelatin capsules can contain the (-)-agelastatin A synthesized by the present method and suitable pharmaceutically acceptable carriers or excipients, such as lactose, sucrose, mannitol, starch, cellulose derivatives, magnesium stearate and steric acid. Similar pharmaceutically acceptable carriers or excipients can be used to make compressed tablets. Both tablets and capsules can be manufactured for the sustained release of the (-)-agelastatin A synthesized by the present method over a period -of time; e.g., minutes to hours. Compressed tablets can be sugar coated or film coated to mask any unpleasant taste and protect the tablet from the atmosphere, or they can be enteric coated for selective disintegration in the gastrointestinal tract.

[0041] Suppositories can contain the (-)-agelastatin A synthesized by the present method in an oleaginous or water-soluble base. Suitable oleaginous bases include cocoa butter and other fats with similar properties. Suitable water-soluble bases include the polyethylene glycols.

[0042] Solutions or suspensions used for parenteral, intradermal, or subcutaneous application are characterized as being at least sterile and pyrogen free, and can include the following pharmaceutically acceptable carriers or excipients: a sterile excipient such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. Pharmaceutical compositions for parenteral administration can be enclosed in a vessel for storage and use; for example in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

[0043] Topical compositions of the invention preferably comprise suitable creams, ointments, lotions, gels, pastes, foams, powders, aerosols and sprays or the like, all of which comprise well known pharmaceutically acceptable carriers or excipients. Topical pharmaceutical compositions may be prepared according to procedures well known in the art, and may contain a variety of ingredients such as, for example, certain preservatives, dyes, colorings and flavors. Suitable carriers and excipients for topical compositions include mixtures of animal sterols with petrolatum, such as Hydrophilic Petrolatum, U.S.P. Other suitable carriers and excipients for topical pharmaceutical compositions include Eucerin and Aquaphor (Beiersdorf) and Polysorb (Fougera).

[0044] Liquid pharmaceutical compositions of the invention for parenteral administration preferably comprise suitable stabilizing or preservative agents, such as sodium bisulfite, sodium sulfite and ascorbic acid, citric acid and its salts, ethylenediaminetetraacetic acid, benzalkonium chloride, methyl- or propylparaben chlorobutanol, and combinations thereof, all of which are considered pharmaceutically acceptable excipients.

[0045] If administered intravenously, preferred carriers for pharmaceutical compositions of the invention are physiological saline or phosphate buffered saline (PBS). [0046] In a preferred embodiment, the (-)-agelastatin A synthesized by the present method is formulated with pharmaceutically acceptable carriers that protect the (-)-agelastatin A against rapid elimination from the body, such as a controlled release formulation (e.g., liposomes, implants and microencapsulated delivery systems), and biodegradable and/or biocompatible polymers (e.g., ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters and polylactic acid).

[0047] Liposomes (including liposomes targeted to particular cells, for example with monoclonal antibodies) are also preferred as pharmaceutically acceptable carriers. Liposomes can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811, the entire disclosure of which is incorporated herein by reference.

[0048] For example, liposome formulations can be prepared by dissolving appropriate lipid(s) (such as stearoyl phosphatidyl ethanolamine, stearoyl phosphatidyl choline, arachadoyl phosphatidyl choline, and cholesterol) in an inorganic solvent that is then evaporated, leaving behind a thin film of dried lipid on the surface of the container. An aqueous solution of the (-)-agelastatin A synthesized by the present method can be introduced into the container. The container is then agitated to free the lipid material from the sides and disperse the lipid aggregates, which contain the (-)-agelastatin A, to form a liposomal suspension.

[0049] The (-)-agelastatin A synthesized by the present method, or pharmaceutical composition thereof, can be administered to a subject by any appropriate enteral or parenteral route; for example orally (including rectally and intranasally), intravascularly (e.g., intraarterially or intravenously), intradermally, subcutaneously, intramuscularly, intraperitioneally, by direct injection into or near a tumor or other affected site in a subject, or topically. Nonlimiting examples of dosage forms include in both immediate release and extended-release or controlled release oral formulations, transdermal drug delivery as either a patch, gel, or cream, and sterile formulations for intravenous, intraarterial or peri- or intra-tumoral injection. Suitable transdermal carriers and excipients include poly(N-vinyl pyrrolidone), poly(methyl methacrylate), polylactides, and polyglycolides. The permeation of (-)-agelastatin A through the skin can also be enhanced by physical methods such as iontophoresis (i.e., application of low level electric current), phonophoresis (i.e., use of ultra sound energy) and by chemical penetration enhancers; e.g., fatty acids, fatty alcohols and terpenes.

[0050] The (-)-agelastatin A synthesized by the present method can also be used to construct analogs which have anti-cancer, anti-stroke, anti-diabetes, anti-Alheimer's or other therapeutic activity. Analogs of the (-)-agelastatin A synthesized by the present method can be synthesized and isolated by one skilled in the art by standard chemical techniques. As shown in Fig. 5, analogs could include those formed by substitution of various ring positions of (-)-agelastatin A, wherein Ri, R₂, and R₃ are the same or different, and are a hydrogen, halogen, hydroxyl group, alkyl group or aryl group; R₄, is an alkyl group, hydrogen or benzyl group; and R₅ and R₆ are the same or different, and are an alkyl group, aryl group or hydrogen. The fluoro, chloro and iodo analogs of can be prepared by reaction of (-)-13 with various active halogen compounds including, but not limited to, N-chlorosuccinimide, N-iodosuccinimide, l,3-dichloro-5,5-dimethylhydantion, and N- fluorobenzenesulfonimide. For example treatment of (-)-13 with 3,3-dichloro-5,5- dimethylhydantion results in a 40% yield of the chloro analog of agelastatin A (Br replaced by Cl). Thus, analogs may be formed by introducing halogens at R₁, R_2> and/or R₃ by halogenation of (-)-13 with I, Br or Cl. Other analogs can be prepared by use of substituted pyrrole carboxylates (compound (-)-7 to (+)-8) in the synthesis where the pyrrole carboxylate is substituted with an alkyl, F, hydroxyl and/or aryl substituents. Substitutent R₄ can be introduced into compound (+)-8 or (-)-2 by alkylation with an alkyl or benzyl halide. Substituents R₅ and R₆ can be introduced by alkylation, while R₅ can also be introduced by way of the isocyanate in the formation of the D-ring.

[0051] Such analogs can then be tested for therapeutic activity by standard assays, for example by the mouse L1210 or human KB nasopharyngeal cell line cytotoxicity assays disclosed in D'Ambrosio et al., HeIv. Chim. Acta 1996, 79, 727, the entire disclosure of which is herein incorporated by reference. Antitumor screening of (-)-agelastatin against various human tumor cell lines is disclosed by Hale et al., Strategies and Tactics in Organic Synthesis 2005, 6, 352, the entire disclosure of which is herein incorporated by reference.

[0052] All documents referred to herein are incorporated by reference. While the present invention has been described in connection with the preferred embodiments of the various figures, it is to be understood that other similar embodiments can be used or modifications and additions made to the described embodiments for performing the same function of the present invention without deviating therefrom. Therefore, the present invention should not be limited to any single embodiment described herein.

Examples

[0053] General Procedures. Column chromatography was performed on silica gel, Merck grade 60 (230-400 mesh). TLC plates were visualized with UV, in an iodine chamber, or with phosphomolybdic acid, unless noted otherwise. ¹H ΝMR and ¹³C ΝMR spectra were recorded on a Bruker 400, operating at 400 and 100 MHz, respectively. Ethyl (dibenzylamino)acetate (5) was prepared by a literature method as reported by Scolasstico et al., Synthesis 1985, 850. Example 1

[0054] In a 50-mL, one-necked, round-bottomed flask equipped with a magnetic stirring bar, rubber septum and argon balloon was placed />-toluenesulfinamide (0.618 g, 5.0 mmol), DCM (10 mL), acrolein (0.380 mL, 5.5 mmol), and Ti(OEt)₄ (5.2 mL, 25.0 mmol) respectively. The solution was stirred for 5-8 h at rt, quenched with ice-H₂O (5 mL), and filtered. The white predicate was washed with DCM (3 X 10 mL), and the aqueous phase was separated and extracted with DCM (3 X 5 mL). The combined organic phases were washed with brine (15 mL), dried (Na₂SO₄), and concentrated. Chromatography (10% EtOAc/hexane) afforded 0.687 g (86%) of a yellow oil; [α]²⁰ _D = -796 (c 1.1, CHCl₃), (as reported by Davis et al., J. Org. Chan. 1997, 62, 2555, [α]²⁰D = +729, for the (^-isomer); IR (neat): 2923, 1577, 1096, 810 cm-¹; ¹H NMR (CHCl₃) δ 2.43 (s, 3H), 6.03 (S, 1 H), 6.07 (d, J= 6.9 Hz, 1 H), 6.68 (m, 1 H), 7.33 (d, J= 7.8 Hz, 2 H), 7.59 (d, J= 7.8 Hz, 2 H), 8.39 (d, J= 9.3 Hz, 1 H); ¹³C NMR δ 21.4, 124.6, 129.9, 132.2, 134.6, 141.4, 141.9, 162.0. The thus formed (R) -(-)-N-(Propylidme)-/?-toluenesulfmamide (6) has the formula:

p-Tolyl "^S"Ν

Example 2

[0055] In a 25-mL, one-necked, round-bottomed flask equipped with a magnetic stirring bar, rubber septum, and argon balloon was placed 5 (3.3 g, 11.6 mmol), Et₂O (8.0 mL). The solution was cooled to O⁰C. In a second 100-mL, one-necked, round-bottomed flask equipped with a magnetic stirring bar, rubber septum, and argon balloon was placed LDA (5.8 mL, 2.0 M in cyclohexane/heptane^enzene from Aldrich) and Et₂O (30.0 mL). The solution of 5 was added via a double-end needle at -78⁰C. The yellow-orange solution was stirred for 30 minutes at this temperature and sulfinimine (-)-6 (0.45Og, 2.33 mmol) in Et₂O (6.0 mL) was added dropwise at - 78⁰C. The solution was stirred at this temperature, monitored by TLC for completion (about 30 min), and quenched by addition of sat. aq. NH₄Cl (10 mL). The solution mixture was warmed to room temperature (hereinafter referred to as "rt") and the aqueous phase was separated and extracted with EtOAc (3 X 10 mL). The combined organic phases were washed with brine (15 mL), dried (Na₂SO₄), and concentrated. Chromatography (5% and then 10% EtOAc/hexane) afforded 0.810 g (73%) of a yellow oil; [α]²⁰ _D = -121.4 (c 1.7, CHCl₃); IR (neat): 3229, 1734, 1465, 1094 cm^"1; ¹H NMR (CHCl₃) δ 1.35 (t, J= 7.0 Hz, 3 H), 2.48 (s, 3 H), 3.17 (d, J= 10.5 Hz, 1 H), 3.30 (d, J= 13.0 Hz, 2 H), 3.95 (d, J= i3.0 Hz, 2 H), 4.27 (m, 2 H), 4.38 (dd, J= 7.5 Hz, J=

10.5 Hz, 1 H), 5.15 (s, 1 H), 5.30 (m, 1 H), 5.45-5.57 (m, 2 H), 7.12-7.23 (m, 10 H), 7.32 (d, J = 8.0 Hz , 2 H), 7.56 (d, J = 8.0 Hz , 2 H); ¹³C NMR δ 14.8, 21.7, 53.4, 54.4, 60.8, 64.6, 122.0, 125.4, 127.5, 128.6, 129.6, 134.7, 138.2, 141.4, 143.2, 168.8 (one carbon miss due to the overlap in the aromatic region). HRMS calculated for C₂₈H₃₂N₂O₃SNa (M + Na): 499.2031. Found: 499.2041. The thus formed (5_R,25,3i?)-(-)-Ethyl-2-(N,N-dibenzylamino)-3-N-(p- toluenesulfinyl)amino-pent-4-enoate (4) has the formula:

Example 3

[0056] In a 50-mL, one-necked, round-bottom flask equipped with a magnetic stirring bar, rubber septum, and argon balloon was placed (-)-4 (0.530 g, 1.1 mmol) in THF (10 ml) and the solution was cooled to -78⁰C. In a separate 100-mL, one-necked, round-bottomed flask equipped with a magnetic stirring bar, rubber septum and argon balloon was placed N₅O- dimethylhydroxylamine hydrochloride (0.542 g, 5.56 mmol) in THF (35 mL). The solution was cooled to -78⁰C and «-BuLi (7.OmL, 1.6 M in hexane) was added via syringe. The reaction mixture was warmed to rt, stirred for 20 min, and cooled to -78⁰C. At this time the -78⁰C solution of (-)-4 was slowly added via a double-end needle to the lithium N,O-dimethylhydroxylamine solution. The reaction mixture was stirred, monitored for completion by TLC (10 min), and quenched at -78⁰C by addition of sat. aqueous NH₄Cl (1OmL). The solution was warmed to rt and the aqueous phase was separated and extracted with EtOAc (3 X 1OmL). The combined organic phases were washed with brine (15mL), dried (Na₂SO₄), and concentrated. Chromatography (30% EtOAc/hexane) afforded 0.488 g (89%) of a yellow oil; [α]²⁰ _D = -18.6 (c 0.8, CHCl₃); IR (neat): 3229, 3028, 1653, 1453, 1092 cm^"1; ¹H NMR (CHCl₃) δ 2.48 (s, 3 H), 3.26 (s, 3 H), 3.30 (d, J = 14.0 Hz, 2 H), 3.46 (s, 3 H), 3.73 (d, J= 10.5 Hz, 1 H), 4.04 (d, J= 14.0 Hz, 2 H), 4.79 (d, J= 8.0 Hz, J= 10.5 Hz, 2 H), 5.32 (m, 2 H), 5.49 (m, 1 H), 5.59 (m, 1 H), 7.17 (m, 10 H)₃ 7.35 (d, J= 8.0 Hz , 2 H), 7.61 (d, J = 8.0 Hz, 2 H); ¹³C NMR δ 21.7, 31.8, 53.9, 54.2, 60.0, 61.6, 121.8, 125.4, 127.2, 128.4, 129.4, 129.6, 135.0, 139.2, 141.4, 143.4, 169.5. HRMS calculated for C₂₈H₃₄N₃O₃S(M + H): 492.2321. Found 492.2314. The thus formed ($_R,2S,3R)-(-)-3-N-(p- Toluenesulfinyl)amino-2-(dibenzylamino)-N-methoxy- N-methylpent-4-enamide (7) has the formula:

Example 4

[0057] In a 100-mL, one-necked, round-bottomed flask equipped with a magnetic stirring bar, rubber septum, and argon balloon was placed MeOH (30 mL), (-)-7 (0.440 g, 0.9 mmol), and TFA (0.345 mL, 4.5 mmol) was added at O⁰C. The reaction mixture was stirred for 2 to 4 h at rt and concentrated. The residue was dissolved in DCM (30 mL), and sat aqueous NaHCO₃ was used to adjust solution to pH to about 7. The aqueous phase was separated and extracted with EtOAc (3 X 8 mL) and the organic phases were combined, washed with brine (15 mL), dried (Na₂SO₄), and concentrated. The residue was loaded into a short-pad column, chromatography (35% EtOAc/hexane) was used to elute the sulfmyl by-products, and MeOH (50 mL) was used to elute the amine product. The solvent was concentrated in a 100-mL, one-necked, round-bottomed flask that was then equipped with a magnetic stirring bar, rubber septum, and argon balloon. To the flask was added CH₃CN (20 ml), 2-pyrrole carboxylate acid (0.110 g, 0.99 mmol), followed by HBTU (0.390 g, 1.03 mmol), and DIPEA (0.611 mL, 4.5 mmol). The reaction was stirred at rt for 8 h, diluted with EtOAc (40 mL), and the organic phase was washed with Na₂CO₃ (15 mL), H₂O (10 mL), and 1 N HCl (10 mL). The organic phase was then washed with brine (15 mL), dried (Na₂SO₄), and concentrated. Chromatography (20% EtOAc/hexane) afforded 0.357 g (88%) of a white solid, mp 74.5 °C-75.5°C; [α]²⁰ _D = +131.6 (c 0.6, CD₃OD); IR (neat): 3406, 3253, 1646, 742 cm⁴; ¹H NMR (CHCl₃) δ 3.34 (s, 3 H), 3.44 (d, J= 14.0 Hz, 2 H), 3.53 (s, 3 H), 3.86 (d, J= 10.5 Hz, 1 H), 4.05 (d, J= 14.0 Hz, 2 H), 5.02 (m, 1 H), 5.16 (m, 1 H), 5.35 (m, 1 H), 5.68 (m, 1 H) 6.35 (m, 1 H), 6.42 (m, 1 H), 6.64 (s, 1 H), 6.98 (s, 1 H), 7.26 (m, 10H), 9.82 (b, 1 H); ¹³C NMR δ 31.9, 51.1, 54.4, 59.8, 61.6, 109.1, 109.9, 117.8, 122.1, 126.2, 127.4, 128.6, 129.4, 136.2, 139.8, 161.4, 169.8. HRMS calculated for C₂₆H₃₀N₄O₃Na (M + Na): 469.2216. Found 469.2227. The thus formed N-[(2S,3R)-(+)-l -(N-Methoxy-N-methylcarbamoyl)- 1 -(dibenzylamino)but-3 -en-2- yi)]-lH-pyrrole-2-carboxamide (8) has the formula:

Example 5

[0058] In a 50-mL, one-necked, round-bottomed flask equipped with a magnetic stirring bar, rubber septum, and argon balloon was placed compound (+)-8 (0.350 g, 0.78 mmol) in THF (20 niL) and allylmagnesium bromide (3.14 mL, 1.0 M in THF) was added at O⁰C. The reaction mixture was stirred for 20 min and quenched with sat. aqueous NH₄Cl (5 mL) at -78⁰C. The solution was warmed to rt and the aqueous phase was separated and extracted with EtOAc (3 X 6 mL). The organic phase was washed with brine (10 mL), dried (Na₂SO₄), and concentrated. The residue was dissolved in EtOH (30 mL), placed in a 50-mL, one-necked, round-bottomed flask equipped with a magnetic stirring bar, rubber septum, and argon balloon. To the solution was added Et₃N (4 mL), and the reaction mixture was stirred for 12 h and concentrated. Chromatography (10% EtOAc/hexane) afforded 0.285 g (85%) of a yellow solid, mp 124.5- 125.5⁰C; [α]²⁰D = -30.3 (c 1.1, CHCl₃); IR (neat): 3365, 3232, 1650, 1562, 742 cm^"1; ¹H NMR (CHCl₃) δ 1.99 (d, J= 6.5 Hz, 3 H), 3.49 (d, J- 14.0 Hz, 2 H), 3.67 (d, J= 10.5 Hz, 1 H), 3.98 (d, J= 14.0 Hz, 2 H), 5.15 (m, 2 H), 5.30 (m, 1 H), 5.72 (m, 1 H), 6.33 (m, 3 H), 6.62 (m, 1 H), 6.82 (m, 1 H), 6.98 (m, 1 H), 7.23 (m, 10 H), 9.98 (b, 1 H); ¹³C NMR δ 18.8, 50.0, 54.6, 65.7, 109.0, 110.0, 118.1, 122.0, 126.3, 127.7, 128.8, 129.3, 132.8, 136.0, 139.2, 144.7, 161.0, 198.0. HRMS calculated for C₂₇H₂₉N₃O₂Na(M + Na): 450.2157. Found 450.2165. The thus formed N-(-)- [(£',3i?,45)-4-(dibenzylamino)-5-oxoocta-l,6-dien-3-yl)]-lH-pyrrole-2-carboxamide (3) has the formula:

Example 6

[0059] In a 250-mL, one-necked, round-bottomed flask equipped with a magnetic stirring bar, rubber septum, and argon balloon was placed (-)~3 (0.150 g, 0.35 mmol) and Grubbs 2^nd generation catalyst (0.059 g, 20 mol%) in DCM (70 mL). The reaction mixture was refluxed for 12 h and concentrated. Chromatography (5% EtOAc/hexane) afforded 0.116 g (87%) of a yellow solid, mp 85.5-86.5⁰C; [α]²⁰ _D = -147.4 (c 0.25, CHCl₃); IR (neat): 3265, 1717, 1646, 1333 cm^"1; ¹H NMR (CHCl₃) δ 3.45 (d, J= 4.0 Hz, 1 H), 3.46 (d, J= 14.0 Hz, 2 H), 3.88 (d, J= 10.5 Hz, 1 H),

5.48 (m, 1 H), 6.07 (d, J= 8.7 Hz, 1 H),^"6.20 (d, J= 6.0 Hz, 1 H), 6.29 (s, 1 H), 6.52 (s, 1 H), 7.02 (s, 1 H), 7.27 (m, 5 H), 7.38 (m, 5 H); 10.22 (b, 1 H); ¹³C NMR δ 51.5, 52.3, 70.3, 110.1, 110.3, 122.7, 125.3, 127.5, 128.6, 129.1, 134.8, 139.2, 160.4, 161.1, 205.4. HRMS calculated for C₂₄H₂₃N₃O₂Na(M + Na): 408.1688. Found 408.1699. The thus formed N-(-)-[(lR,5S)-5- (dibenzylamino)-4-oxocyclopent-2-enyl)]-lH-pyrrole-2-carboxamide (2) has the formula:

Example 7

[0060] In a 100-mL, one-necked, round-bottomed flask equipped with a magnetic stirring bar, rubber septum, and argon balloon was placed (-)-2 (0.062 g, 0.160 mmol) in MeOH (40 mL) and Cs₂CO₃ (0.524 g, 1.60 mmol) was added in one portion. The reaction mixture was carefully monitored by TLC for completion (15 min). It has been found that if THF is used the reaction time can be extended to 2 hours with an improved yield up to 80%. At this time the reaction mixture was transferred to a premixed solution of EtOAc (150 mL) and brine (30 mL) in a 500-mL separated funnel and shaken vigorously for 1 minute. The organic phase was separated, washed with brine (2 X 30 mL), dried (Na₂SO₄), and concentrated. Chromatography (30% EtOAc/hexane) afforded 0.042 g (68%) of a solid, mp 195⁰C dec; [α]²⁰ _D = -10.2 (c 0.28, CHCl₃); IR (neat): 3854, 1653, 1350 cm-¹; ¹HNMR (CHCl₃) δ 2.63 (dd, J= 6.0 Hz, J= 19.2 Hz, 1 H), 2.97 (d, J= 19.2 Hz, 1 H), 3.51 (d, J= 11.7 Hz, 1 H), 3.89 (m, 1 H), 3.94 (s, 4 H), 4.68 (t, J= 6.0 Hz, 1 H), 6.26 (m, 1 H), 6.48 (d, J= 3.3 Hz, 1 H), 6.71 (m, 1 H), 6.91 (m, 1 H), 7.27 (m, 10 H); ¹³C NMR δ 43.0, 50.3, 54.4, 56.4, 69.7, 111.2, 115.3, 122.4, 123.7, 127.9, 128.9, 138.7, 158.9, 211.2 (one carbon is absent due to the ring strain or overlap in aromatic region). HRMS calculated for C₂₄H₂₃N₃O₂Na(M + Na): 408.1688. Found 408.1699. The thus formed (-)-Pyrrole (10) has the formula:

Example 8

[0061] The procedure for preparing compound 10 was followed except that the reaction time was 2 h. Chromatography (30% EtOAc/hexane) afforded 0.031 g (46%) of an oil; [α]²⁰ _D = -95.3 (c 0.4, CHCl₃); IR (neat): 3344, 3189, 1708, 1652, 1604, 1454 cm ¹; ¹H NMR (CHCl₃) δ 2.32 (dd, J= 1.1 Hz, J= 19.2 Hz, 1 H), 3.23 (dd, J= 6.0 Hz, J= 19.2 Hz, 1 H), 4.55 (s, 4 H), 5.48 (b, 2 H), 5.89 (d, J= 3.3 Hz, 1 H), 5.97 (t, J= 3.3 Hz, 1 H), 6.28 (m, 2 H), 6.60 (dd, J= 0.9 Hz, J= 3.6 Hz, 1 H), 7.27 (m, 10 H); ¹³C NMR δ 46.0, 52.9, 54.0, 108.0, 114.1, 123.9, 124.2, 126.6, 127.6, 128.0, 128.8, 138.0, 148.0, 163.5, 202.6. HRMS calculated for C₂₄H₂₃N₃O₂Na(M + Na): 408.1688. Found 408.1699. The thus formed (-)-l-(i?)-3-(dibenzylamino)-4-oxocyclopent-2-enyl)-lH- pyrrole-2-carboxamide (11) has the formula:

Example 9

[0062] In a 10-mL, two-necked, round-bottomed flask equipped with a magnetic stirring bar, an outlet and inlet stopcock equipped with a H₂ filled balloon, and a rubber septum was placed THF (4 mL), (-)-10 (0.022 g, 0.057 mmol), and Pd/C (ca. 0.005 g). The solution was evacuated and then filled with H₂, and this sequence was repeated 5 times. The mixture was stirred for 5 min prior to addition of niethylisocynate (0.034 mL, 0.57 mmol). The reaction mixture was stirred for 12 h at rt, the catalyst was removed by filtration through Celite and the filtrate concentrated. Chromatography (5% MeOH/DCM) afforded first 12 (see below) and then 0.007 g (47%) of 13 as an off-white solid, mp 243.5-244.5⁰C [as reported by Feldman et al., J. Org. Chem. 2002, 67, 7096, mp 244-245⁰C]; [α]²⁰ _D = -66.2 (c 0.21, MeOH) [as reported by Feldman et al., J. Org. Chem. 2002, 67, 7096, [α]²⁰ _D = -68.4 (c 0.4, MeOH)]; IR (neat): 3281, 2849, 1653, 1559 cm^"1; ¹H NMR (CHCl₃) δ 2.27 (dd, J= 13.0 Hz, J= 10.5 Hz, 1 H), 2.62 (dd, J= 6.0 Hz, J= 13.0 Hz, 1 H), 2.78 (s, 3 H), 3.79 (d, J= 1.0 Hz, 1 H), 3.99 (dd, J= 1.0 Hz, J= 6.0 Hz, 1 H), 4.63 (m, 1 H), 6.21 (dd, J= 2.5 Hz, J= 4.0 Hz, 1 H), 6.87 (dd, J= 1.5 Hz, J= 4.0 Hz, 1 H), 7.01 (dd, J= 2.5 Hz, J = 1.5 Hz, 1 H); ¹³C NMR δ 24.2, 41.6, 55.6, 62.8, 68.0, 95.8, 111.0, 115.4, 122.9, 125.6, 161.3, 162.0.

[0063] It has been found that if excess {e.g., 2 equivalents of the catalysts) is used in the hydrogen step the yield of (13) improves to 70%.

[0064] The thus formed (-)-Debromoagelastatin A (13) has the formula:

Example 10

[0065] Chromatography (5% MeOH/DCM) afforded 0.0068 g (32%) of an off-white solid, mp 105.0⁰C dec; [α]²⁰ _D = -57.9 (c 0.29, CHCl₃); IR (neat): 3279, 2925, 1653, 1525 cm^"1; ¹H NMR (CHCl₃) δ 2.26 (dd, J= 13.0 Hz, J= 10.5 Hz, 1 H), 2.62 (dd, J= 6.0 Hz, J= 13.0 Hz, 1 H), 2.84

(s, 3H), 3.67 (d, J= 1.0 Hz, 1 H), 3.94 (dd, J= 1.0 Hz, J= 6.0 Hz, 1 H), 4.39 (d, J= 15.0 Hz, 1 H), 4.58 (m, 1 H), 4.59 (d, J= 15.0 Hz, 1 H), 6.19 (m, 1 H), 6.83 (m, 1 H), 6.97 (m, 1 H), 7.26 (m, 1 H), 7.32 (m, 4 H); ¹³C NMR δ 25.5, 41.8, 48.4, 55.1, 59.4, 72.2, 93.1, 111.4, 116.2, 121.6, 124.9, 128.6, 129.0, 129.6, 137.8, 159.0, 161.0. HRMS calculated for C₁₉H₂₁N₄O₃(M + H): 353.1614. Found 353.1629. The thus formed (-)-3-N-Benzyl-Debromoagelastatin A (12) has the formula:

Example 11

[0066] A literature procedure was employed to prepare (-)-l, as reported in Feldman et al., J. Org. Chem. 2002, 67, 7096. In a 10-mL, one-necked, round-bottomed flask equipped with a magnetic stirring bar, rubber septum and argon balloon was placed MeOH (1.0 mL), THF (2.0 mL), (-)-12 (0.004 g, 0.0152 mmol), and NBS (0.0025 g, 0.0145 mmol). The reaction solution was stirred at rt for 8 to 12 h and concentrated. Preparative TLC (2% MeOH/DCM) afforded 0.0036 g (69%) of an off-white solid. [α]²⁰ _D = -62.2 (c 0.18, MeOH) [as reported by Feldman et al.,. J. Org. Chem. 2002, 67, 7096, -65.5 (c 0.5, MeOH); as reported by Hong et al., J. Nat. Prod. 1998, 61, 158 -59.3 (c 0.13, MeOH)]; IR (neat): 3289, 2917, 1657, 1564 cm⁴; ¹H NMR (CHCl₃) δ 2.08 (dd, J= 12.5 Hz, J= 12.5 Hz, 1 H), 2.62 (dd, J= 6.0 Hz, J= 13.5 Hz, 1 H), 2.79 (s, 3H), 3.87 (s, 1 H), 4.07 ( J= 5.5 Hz, 1 H), 4.58 (m, 1 H), 6.30 (J= 4.5 Hz, 1 H), 6.89 (J= 4.5 Hz, 1 H); ¹³C NMR D 24.6, 40.4, 54.8, 62.6, 67.8, 96.1, 107.6, 114.2, 116.4, 124.6, 161.5, 161.8. The thus formed (-)-agelastatin A (1) has the formula:

Claims

ClaimsWhat is claimed is:

1. A method of asymmetric synthesis of (-)-agelastatin A comprising:

(1) adding an acrolein-derived sulfinimine (i?)-(-)-6 to a preformed lithium enolate of ethyl (dibelzylamino) acetate (5), thereby forming an unsaturated a, /3-dramino ester (-)-4 comprising a major diastereoisomer of syn diastereoisomer (-)-4, and isolating the major syn diastereoisomer (-)-4;

(2) treating the unsaturated a, /3-diamino ester (-)-4 with lithium N, O- dimethylhydroxylamine, thereby resulting in a Weinreb amide (-)-7, and isolating the Weinreb amide (-)-7;

(3) deprotecting the N-sulfmyl amino group of the Weinreb amide to give an amine;

(4) reacting the amine of step 3 with pyrrole-2-carboxylic acid, a coupling reagent HBTU and DIPEA to give amide (+)-8;

(5) treating amide (+)-8 with allylmagnesium bromide at 0⁰C to give a γ,/3-unsaturated ester intermediate;

(6) isomerizing the γ,/3-unsaturated ester intermediate with Et₃Ν/EtOH to give diamino ketodiene (-)-3, and isolating the diamino ketodiene (-)-3;

(7) refluxing diamino ketodiene (-)-3 in DCM with Grubb's second generation catalyst to give a C-ring intermediate 4, 5-diamino cyclopenten-2-enone (-)-2;

(8) treating the C-ring intermediate with Cs₂CO₃ZMeOH to give B-ring intermediate tricyclic ring system (-)-10;

(9) deprotecting the B-ring intermediate tricyclic ring system (-)-10 to give a free amine;

(10) treating the free amine with methyl isocyarate to give a D-ring intermediate (-)-13; and

(11) brominating the D-ring intermediate (-)-13 to give (-)-agelastatin A(I).

2. A method for synthesizing C-ring intermediate 4,5-diamino cyclopenten-2-enone (-)-2 comprising:

(1) adding an acrolein-derived sulfinimine (i?)-(-)-6 to a preformed lithium enolate of ethyl (dibelzylamino) acetate (5), thereby forming an unsaturated a, /3-dramino ester (-)-4 comprising a major diastereoisomer of syn diastereoisomer (-)-4, and isolating the major syn diastereoisomer (-)-4; (2) treating the unsaturated a, jδ-diamino ester (-)-4 with lithium N, O- dimethylhydroxylamine, thereby resulting in a Weinreb amide (-)-7, and isolating the Weinreb amide (-)-7;

(3) deprotecting the N-sulfinyl amino group of the Weinreb amide to give an amine;

(4) reacting the amine of step 3 with pyrrole-2-carboxylic acid, a coupling reagent HBTU and DIPEA to give amide (+)-8;

(5) treating amide (+)-8 with allylmagnesium bromide at about 0⁰C to give a γ,/3- unsaturated ester intermediate;

(6) isomerizing the γ,/3-unsaturated ester intermediate with Et₃Ν/EtOH to give diamino ketodiene (-)-3, and isolating the diamino ketodiene (-)-3; and

(7) refluxing diamino ketodiene (-)-3 in DCM with Grubb's second generation catalyst to give a C-ring intermediate 4, 5-diamino cyclopenten-2-enone (-)-2.

3. A C-ring intermediate produced according to the method of claim 2.

4. A C-ring intermediate 4,5-diamino cyclopenten-2-enone (-)-2 of the formula:

5. A method for synthesizing a D-ring intermediate (-)-13 comprising:

(1) adding an acrolein-derived sulfinimine (i?)-(-)-6 to a preformed lithium enolate of ethyl (dibelizylamino) acetate (5), thereby forming an unsaturated a, /?-dramino ester (-)-4 comprising a major diastereoisomer of syn diastereoisomer (-)-4, and isolating the major syn diastereoisomer (-)-4;

(2) treating the unsaturated α, /3-diamino ester (-)-4 with lithium N, O- dimethylhydroxylamine, thereby resulting in a Weinreb amide (-)-7, and isolating the Weinreb amide (-)-7;

(3) deprotecting the N-sulfinyl amino group of the Weinreb amide to give an amine; (4) reacting the amine of step 3 with pyrrole-2-carboxylic acid, a coupling reagent

HBTU and DIPEA to give amide (+)-8; ^'

(5) treating amide (+)-8 with allylmagnesium bromide at about 0⁰C to give a y,β- unsaturated ester intermediate;

(6) isomerizing the γ,/3-unsaturated ester intermediate with Et₃N/EtOH to give diamino ketodiene (-)-3, and isolating the diamino ketodiene (-)-3;

(7) refluxing diamino ketodiene (-)-3 in DCM with Grubb's second generation catalyst to give a C-ring intermediate 4, 5-diamino cyclopenten-2-enone (-)-2;

(8) treating the C-ring intermediate with Cs₂CO₃ZMeOH to give B-ring intermediate tricyclic ring system (-)-10;

(9) deprotecting the B-ring intermediate tricyclic ring system (-)-10 to give a free amine; and

(10) treating the free amine with methyl isocyarate to give a D-ring intermediate (-)-13.

6. A D-ring intermediate (-)-13 produced according to the method of claim 5.

7. A D-ring intermediate (-)-13 of the formula:

o

8. A method for synthesizing a B-ring intermediate (-)-10 comprising:

(1) adding an acrolein-derived sulfinimine (i?)-(-)-6 to a preformed lithium enolate of ethyl (dibelzylamino) acetate (5), thereby forming an unsaturated a, /3-dramino ester (-)-4 comprising a major diastereoisomer of syn diastereoisomer (-)-4, and isolating the major syn diastereoisomer (-)-4;

(2) treating the unsaturated a, /3-diamino ester (-)-4 with lithium N, O- dimethylhydroxylamine, thereby resulting in a Weinreb amide (-)-7, and isolating the Weinreb amide (-)-7;

(3) deprotecting the N-sulfϊnyl amino group of the Weinreb amide to give an amine;

(4) reacting the amine of step 3 with pyrrole-2-carboxylic acid, a coupling reagent HBTU and DIPEA to give amide (+)-8;

(5) treating amide (+)-8 with allylmagnesium bromide at about 0⁰C to give a γ,/3- unsaturated ester intermediate;

(6) isomerizing the γ,/3-unsaturated ester intermediate with Et₃NTEtOH to give diamino ketodiene (-)-3, and isolating the diamino ketodiene (-)-3;

(7) refluxing diamino ketodiene (-)-3 in DCM with Grubb's second generation catalyst to give a C-ring intermediate 4, 5-diamino cyclopenten-2-enone (-)-2; and

(8) treating the C-ring intermediate with Cs₂CO₃/MeOH to give a B-ring intermediate tricyclic ring system (-)-10.

9. A B-ring intermediate (-)-10 produced according to the method of claim 8.

10. A B-ring intermediate (-)-10 of the formula:

ΝBno

11. A method for treating a disorder, comprising: synthesizing the (-)-agelastatin A according to claim 1 ; and administering an effective amount of the (-)-agelastatin A to a subject.

12. The method according to claim 11, wherein the effective amount is administered as a single dose.

13. The method according to claim 11, wherein the effective amount is administered as a number of smaller doses at varying time intervals.

14. The method according to claim 11, wherein the disorder is cancer, Alzheimer's, diabetes or stroke.

15. The method according to claim 11 , wherein the subject is a human being.

16. The method according to claim 11, wherein the effective amount of (-)-agelastatin A is administered by an enteral or parenteral route.

17. The method according to claim 16, wherein the effective amount of (-)-agelastatin A is administered orally, intravascularly, intradermally, subcutaneously, intramuscularly, intraperitioneally, by direct injection or topically.

18. The method according to claim 17, wherein the effective amount of (-)-agelastatin A is administered rectally, intranasally, intraarterially, or intravenously.

19. A pharmaceutical composition comprising an effective amount of the (-)-agelastatin A synthesized according to Claim 1 in a pharmaceutically acceptable carrier or excipient.

20. The pharmaceutical composition according to claim 19, wherein the (-)-agelastatin A is present in an amount ranging from about 0.01% to about 90% w/w.

21. The pharmaceutical composition according to claim 19, wherein the pharmaceutical composition is in a dosage form selected from the group consisting of liquids, tablets, pills, capsules, troches or suppositories.

22. The pharmaceutical composition according to claim 19, wherein the pharmaceutically acceptable carrier includes controlled release formulations, biodegradable polymers, biocompatible polymers or biodegradable and biocompatible polymers.

23. The pharmaceutical composition according to claim 21, wherein the dosage form comprises immediate release, extended release or controlled release oral formulation, transdermal drug delivery or sterile formulations for intravenous, intra-arterial or peri- or intra-tormoral injection.

24. A method for treating a disorder comprising administering the pharmaceutical composition according to claim 19 to a subject, such that the pharmaceutical composition delivers an effective amount of (-)-agelastatin A.

25. The method according to claim 24, wherein the disorder is cancer, Alzheimer's, diabetes or stroke.

26. The method according to claim 24, wherein the effective amount of (-)-agelastatin A is administered by an enteral or parenteral route.

27. The method according to claim 26, wherein the effective amount of (-)-agelastatin A is administered orally, intravascularly, intradermally, subcutaneously, intramuscularly, intraperitioneally, by direct injection or topically.

28. The method according to claim 27, wherein the effective amount of (-)-agelastatin A is administered rectally, intranasally, intraarterially, or intravenously.

29. A method of identifying compounds with therapeutic activity comprising: synthesizing (-)-agelastatin A according to the method of claim 1; modifying the (-)-agelastatin A by substituting a alkyl group, aryl group, hydroxyl group, halogen, or amine group at various ring positions to produce an analog of (-)-agelastatin A; and testing said analogs for therapeutic activity.

30. The method according to claim 29, wherein the therapeutic activity is anti-cancer, anti- stroke, anti-diabetes or anti-Alzheimer's.

31. An (-)-agelastatin A analog of the formula:

wherein R₁, R₂₎ and R₃ are the same or different, and are a hydrogen, halogen, hydroxyl group, alkyl group or aryl group; R₄, is an alkyl group, hydrogen or benzyl group; and R₅ and R₆ are the same or different, and are an alkyl group, aryl group or hydrogen.

32. A method of asymmetric synthesis of an (-)-agelastatin A analog comprising:

(1) adding an acrolein-derived sulfinimine (i?)-(-)-6 to a preformed lithium enolate of ethyl (dibelzylamino) acetate (5), thereby forming an unsaturated a, /3-dramino ester (-)-4 comprising a major diastereoisomer of syn diastereoisomer (-)-4, and isolating the major syn diastereoisomer (-)-4;

(2) treating the unsaturated a, β-diamino ester (-)-4 with lithium N, O- dimethylhydroxylamine, thereby resulting in a Weinreb amide (-)-7, and isolating the Weinreb amide (-)-7;

(3) deprotecting the N-sulfinyl amino group of the Weinreb amide to give an amine;

(4) reacting the amine of step 3 with pyrrole-2-carboxylic acid, a coupling reagent HBTU and DIPEA to give amide (+)-8;

(5) treating amide (+)-8 with allylmagnesium bromide at O⁰C to give a γ,/3-unsaturated ester intermediate; (6) isomerizing the γ,/3-unsaturated ester intermediate with Et₃NZEtOH to give diamino ketodiene (-)-3, and isolating the diamino ketodiene (-)-3;

(7) refluxing diamino ketodiene (-)-3 in DCM with Grubb's second generation catalyst to give a C-ring intermediate 4, 5-diamino cyclopenten-2-enone (-)-2;

(8) treating the C-ring intermediate with Cs₂CO₃/MeOH to give B-ring intermediate tricyclic ring system (-)-10;

(9) deprotecting the B-ring intermediate tricyclic ring system (-)-10 to give a free amine;

(10) treating the free amine with methyl isocyarate to give a D-ring intermediate (-)-13; and

(11) halogenating the D-ring intermediate (-)-13 with I, Br and/or Cl to give an (-)- agelastatin A(I) analog.

33. An (-)-agelastatin A(I) analog produced according to claim 32.

34. A method of asymmetric synthesis of an (-)-agelastatin A analog comprising:

(1) adding an acrolein-derived sulfinimine (i?)-(-)-6 to a preformed lithium enolate of ethyl (dibelzylamino) acetate (5), thereby forming an unsaturated ot, /3-dramino ester (-)-4 comprising a major diastereoisomer of syn diastereoisomer (-)-4, and isolating the major syn diastereoisomer (-)-4;

(2) treating the unsaturated a, /3-diamino ester (-)-4 with lithium N, O- dimethylhydroxylamine, thereby resulting in a Weinreb amide (-)-7, and isolating the Weinreb amide (-)-7;

(3) deprotecting the N-sulfinyl amino group of the Weinreb amide to give an amine;

(4) reacting the amine of step 3 with a substituted pyrrole carboxylate, a coupling reagent HBTU and DIPEA to give amide (+)-8, wherein the pyrrole carboxylate is substituted with an alkyl group, aryl group, F and/or hydroxyl group;

(5) treating amide (+)-8 with allylmagnesium bromide at O⁰C to give a γ,j8-unsaturated ester intermediate;

(6) isomerizing the γ,/3-unsaturated ester intermediate with Et₃Ν/EtOH to give diamino ketodiene (-)-3, and isolating the diamino ketodiene (-)-3;

(7) refluxing diamino ketodiene (-)-3 in DCM with Grubb's second generation catalyst to give a C-ring intermediate 4, 5-diamino cyclopenten-2-enone (-)-2; (8) treating the C-ring intermediate with Cs₂CO₃/MeOH to give B-ring intermediate tricyclic ring system (-)-10;

(9) deprotecting the B-ring intermediate tricyclic ring system (-)-10 to give a free amine;

(10) treating the free amine with methyl isocyarate to give a D-ring intermediate (-)-13; and

(11) brominating the D-ring intermediate (-)-13 to give an (-)-agelastatin A(I) analog.

35. An (-)-agelastatin A(I) analog produced according to claim 34.

36. A method of asymmetric synthesis of an (-)-agelastatin A analog comprising:

(1) adding an acrolein-derived sulfmimine (R)-(-)-6 to a preformed lithium enolate of ethyl (dibelzylamino) acetate (5), thereby forming an unsaturated a, /3-dramino ester (-)-4 comprising a major diastereoisomer of syn diastereoisomer (-)-4, and isolating the major syn diastereoisomer (-)-4;

(2) treating the unsaturated a, /3-diamino ester (-)-4 with lithium N, O- dimethylhydroxylamine, thereby resulting in a Weinreb amide (-)-7, and isolating the Weinreb amide (-)-7;

(3) deprotecting the N-sulfinyl amino group of the Weinreb amide to give an amine;

(4) reacting the amine of step 3 with pyrrole-2-carboxylic acid, a coupling reagent HBTU and DIPEA to give amide (+)-8;

(5) treating amide (+)-8 with allylmagnesium bromide at O⁰C to give a γ,/3-unsaturated ester intermediate;

(6) isomerizing the γ,(3-unsaturated ester intermediate with Et₃NZEtOH to give diamino ketodiene (-)-3, and isolating the diamino ketodiene (-)-3;

(7) refluxing diamino ketodiene (-)-3 in DCM with Grubb's second generation catalyst to give a C-ring intermediate 4, 5-diamino cyclopenten-2-enone (-)-2;

(8) treating the C-ring intermediate with Cs₂CO₃/MeOH to give B-ring intermediate tricyclic ring system (-)-10;

(9) deprotecting the B-ring intermediate tricyclic ring system (-)-10 to give a free amine;

(10) treating the free amine with methyl isocyarate to give a D-ring intermediate (-)-13; and (11) alkylation the D-ring intermediate (-)-13 to give an (-)-agelastatin A(I) analog, wherein the D-ring intermediate is alkylated with an alkyl group and/or aryl group.

37. An (-)-agelastatin A(I) analog produced according to the method of claim 36.

38. A method of asymmetric synthesis of an (-)-agelastatin A analog comprising:

(1) adding an acrolein-derived sulfinimine (i?)-(-)-6 to a preformed lithium enolate of ethyl (dibelzylamino) acetate (5), thereby forming an unsaturated a, /3-dramino ester (-)-4 comprising a major diastereoisomer of syn diastereoisomer (-)-4, and isolating the major syn diastereoisomer (-)-4;

(2) treating the unsaturated a, /3-diamino ester (-)-4 with lithium N, O- dimethylhydroxylamine, thereby resulting in a Weinreb amide (-)-7, and isolating the Weinreb amide (-)-7;

(3) deprotecting the N-sulfinyl amino group of the Weinreb amide to give an amine;

(4) reacting the amine of step 3 with pyrrole-2-carboxylic acid, a coupling reagent HBTU and DIPEA to give amide (+)-8;

(5) treating amide (+)-8 with allylmagnesium bromide at O⁰C to give a γ,j8-unsaturated ester intermediate;

(6) isomerizing the γ,β-unsaturated ester intermediate with Et₃Ν/EtOH to give diamino ketodiene (-)-3, and isolating the diamino ketodiene (-)-3;

(7) refiuxing diamino ketodiene (-)-3 in DCM with Grubb's second generation catalyst to give a C-ring intermediate 4, 5-diamino cyclopenten-2-enone (-)-2;

(8) treating the C-ring intermediate with Cs₂CO₃/MeOH to give B-ring intermediate tricyclic ring system (-)-10;

(9) deprotecting the B-ring intermediate tricyclic ring system (-)-10 to give a free amine;

(10) treating the free amine with a substituted isocyanate to give a D-ring intermediate (- )-13, wherein the isocyanate is substituted with an alkyl group or aryl group; and

(11) brominating the D-ring intermediate (-)-13 to give an (-)-agelastatin A(I) analog.

39. An (-)-agelastatin A(I) analog produced according to the method of claim 38.

40. A method of asymmetric synthesis of an (-)-agelastatin A analog comprising: (1) adding an acrolein-derived sulfinimine (i?)-(-)-6 to a preformed lithium enolate of ethyl (dibelzylamino) acetate (5), thereby forming an unsaturated a, j3-dramino ester (-)-4 comprising a major diastereoisomer of syn diastereoisomer (-)-4, and isolating the major syn diastereoisomer (-)-4;

(2) treating the unsaturated a, /3-diamino ester (-)-4 with lithium N, O- dimethylhydroxylamine, thereby resulting in a Weinreb amide (-)-7, and isolating the Weinreb amide (-)-7;

(3) deprotecting the N-sulfinyl amino group of the Weinreb amide to give an amine;

(4) reacting the amine of step 3 with pyrrole-2-carboxylic acid, a coupling reagent HBTU and DIPEA to give amide (+)-8, wherein an alkyl or benzyl halide is introduced into (+)-8 by alkylation;

(5) treating amide (+)-8 with allylmagnesium bromide at 0⁰C to give a γ,/3-unsaturated ester intermediate;

(6) isomerizing the γ,/3-unsaturated ester intermediate with Et₃Ν/EtOH to give diamino ketodiene (-)-3, and isolating the diamino ketodiene (-)-3;

(7) refluxing diamino ketodiene (-)-3 in DCM with Grubb's second generation catalyst to give a C-ring intermediate 4, 5-diamino cyclopenten-2-enone (-)-2;

(8) treating the C-ring intermediate with Cs₂CO₃ZMeOH to give B-ring intermediate tricyclic ring system (-)-10;

(9) deprotecting the B-ring intermediate tricyclic ring system (-)-10 to give a free amine;

(10) treating the free amine with methyl isocyarate to give a D-ring intermediate (-)-13; and

(11) brominating the D-ring intermediate (-)-13 to give an (-)-agelastatin A(I) analog.

41. An (-)-agelastatin A(I) analog produced according to the method of claim 40.

42. A method of asymmetric synthesis of an (-)-agelastatin A analog comprising:

(1) adding an acrolein-derived sulfinimine (i?)-(-)-6 to a preformed lithium enolate of ethyl (dibelzylamino) acetate (5), thereby forming an unsaturated a, /3-dramino ester (-)-4 comprising a major diastereoisomer of syn diastereoisomer (-)-4, and isolating the major syn diastereoisomer (-)-4; (2) treating the unsaturated a, /3-diamino ester (-)-4 with lithium N, O- dimethylhydroxylamine, thereby resulting in a Weinreb amide (-)-7, and isolating the Weinreb amide (-)-7;

(3) deprotecting the N-sulflnyl amino group of the Weinreb amide to give an amine;

(4) reacting the amine of step 3 with pyrrole-2-carboxylic acid, a coupling reagent HBTU and DIPEA to give amide (+)-8;

(5) treating amide (+)-8 with allylmagnesium bromide at O⁰C to give a γ,/3-unsaturated ester intermediate;

(6) isomerizing the γ,/3-unsaturated ester intermediate with Et₃Ν/EtOH to give diamino ketodiene (-)-3, and isolating the diamino ketodiene (-)-3;

(7) refluxing diamino ketodiene (-)-3 in DCM with Grabb's second generation catalyst to give a C-ring intermediate 4, 5-diamino cyclopenten-2-enone (-)-2, wherein an alkyl group or benzyl halide is introduced into (-)-2 by alkylation;

(8) treating the C-ring intermediate with Cs₂CO₃/MeOH to give B-ring intermediate tricyclic ring system (-)-10;

(9) deprotecting the B-ring intermediate tricyclic ring system (-)-10 to give a free amine;

(10) treating the free amine with methyl isocyarate to give a D-ring intermediate (-)-13; and

(11) brominating the D-ring intermediate (-)-13 to give (-)-agelastatin A(I).

43. An (-)-agelastatin A(I) analog produced according to the method of claim 42.

44. A method of asymmetric synthesis of (-)-agelastatin A comprising:

(1) adding an acrolein-derived sulfinimine (i?)-(-)-6 to about 5.0 equivalents of a preformed lithium enolate of ethyl (dibelzylamino) acetate (5), thereby forming an unsaturated a, ι8-dramino ester (-)-4 comprising a major diastereoisomer of syn diastereoisomer (-)-4, and isolating the major syn diastereoisomer (-)-4;

(2) treating the unsaturated a, /3-diamino ester (-)-4 with about 5.0 equivalents of lithium N, O-dimethylhydroxylamine, thereby resulting in a Weinreb amide (-)-7, and isolating the Weinreb amide (-)-7;

(3) deprotecting the N-sulfinyl amino group of the Weinreb amide to give an amine;

(4) reacting the amine of step 3 with pyrrole-2-carboxylic acid, a coupling agent and DIPEA to give amide (+)-8; (5) treating amide (+)-8 with about 2.0 equivalents of allylniagnesium bromide at 0°C to give a γ,/3-unsaturated ester intermediate;

(6) isomerizing the γ,/3-unsaturated ester intermediate with Et₃NZEtOH to give diamino ketodiene (-)-3, and isolating the diamino ketodiene (-)-3;

(7) refluxing diamino ketodiene (-)-3 in DCM with about 10 mol%-20 mol% Grubb's second generation catalyst for about 12 hours to give a C-ring intermediate 4, 5-diamino cyclopenten-2-enone (-)-2;

(8) treating the C-ring intermediate with about 10 equivalents of Cs₂CO₃/MeOH to give B-ring intermediate tricyclic ring system (-)-10;

(9) deprotecting the B-ring intermediate tricyclic ring system (-)-10 to give a free amine;

(10) treating the free amine with methyl isocyarate to give a D-ring intermediate (-)-13; and

(11) brominating the D-ring intermediate (-)-13 to give an (-)-agelastatin A(l)analog.