WO2006124358A2

WO2006124358A2 - Method of treating a coccidioides infection by administering an ambruticin compound

Info

Publication number: WO2006124358A2
Application number: PCT/US2006/017671
Authority: WO
Inventors: Leonard Katz; C. Richard Hutchinson; Zong-Qiang Tian; David C. Myles; Zhan Wang
Original assignee: Kosan Biosciences Incorporated
Priority date: 2005-05-19
Filing date: 2006-05-08
Publication date: 2006-11-23
Also published as: WO2006124358A3

Abstract

A method of treating or reducing the probability of a Coccidioides infection in a subject in need of such treatment or reduction of probability, comprising administering to the subject a pharmaceutical composition comprising a compound represented by formula (I) where R1 R2, R3 R4, R10 R11 X1 X2, and X3 are as defined herein.

Description

METHOD OF TREATING A COCCIDIOIDES INFECTION BY ADMINISTERING AN

AMBRUTICIN COMPOUND

TECHNICAL FIELD OF THE INVENTION [0001] This invention relates to the treatment of diseases and conditions caused by Coccidioides spp. infection by the administration of ambruticin compounds.

BACKGROUND OF THE INVENTION

[0002] Coccidioidomycosis (also known as Valley Fever and Desert Fever) is a fungal disease caused by inhaling arthroconidia (spores) of the soil saprophytes Coccidioides immitis and Coccidioides posadasii and is endemic to the southwestern United States and areas of South America. Approximately 100,000 infections occur annually in the United States (Chiller et ah, (2003) Coccidioidomycosis. Infect. Dis. Clin. N. Am. 17, 41-57, viii). Coccidioidomycosis has a high morbidity rate as about 40% of infected individuals become symptomatic. About 10% of those infected can progress to diffuse pulmonary involvement (pneumonia) and, in the severest cases (~1%), to disseminated disease involving all organs including the central nervous system (Chiller et ah, supra; Galgiani, Ann. Intern. Med. 130:293-300 (1999)). Coccidioides spores have been cited as a possible biological weapon due to their ease and low cost of production and ease of dissemination and the resulting disease's potential to overwhelm a medical system by causing large numbers of casualties (Deresinski, Sem. Resp. Infect. 18:216-219 (2003)).

[0003] Current treatment for coccidioidomycosis includes administration of amphotericin B, ketoconazole, and fluconazole. However, the renal toxicity of amphotericin B limits its use to patients with the severest cases of the disease. In addition, it requires intravenous delivery. Ketoconazole and fluconazole are somewhat effective, albeit not fungicidal, but also have the disadvantage of requiring a long-term administration of 8-12 months. They also have high relapse rates (Deresinski, Curr. Opin. Infect. Dis. 14:693-696 (2001)). Clinical failure during antifungal therapy is not uncommon (Drutz, Drugs 26:337-46 (1983)).

[0004] Ambruticin S (also referred to as Acid S, W 7783, (5S, <5i?)-5,6-dihydroxypoly- angioic acid, or, sometimes, simply as ambruticin) is an antifungal compound isolated from cultures of Polyangium cellulosum var.fulvum and has the structure shown below. See Strandtmann et al, US 3,804,948 (1974); Barnes et al, Tetrahedron Letters 22 (18), 1751- 1754 (1981); Kende et al, J. Am. Chem. Soc. 112 (26), 9645-9646 (1990).

Ambruticin S

[0005] Subsequently, another research group isolated from cultures of Sorangium cellulosum strain Se eel 0 a series of six structurally closely related compounds having at C5 an amino group instead of a hydroxyl group. Bedorf et al, WO 91/00860 (1991); Hofle et al., Liebigs Ann. Chem. 1991, 941-945. These compounds have been named ambruticin VS- 1, VS-2, VS-3 (or (J5',(5i?)-5-(dimethylamino)-6-hydroxypolyangioic acid), and so on, and have the structures shown below. (Herein, ambruticin S and the VS-series compounds and their analogs and derivatives are collectively referred to as "the ambruticins.")

VS-I: R = NMe₃ ⁺ of VS-I

VS-3 N-oxide: R = N⁺(Me₂)O^'

[0006] Other disclosures relating to the chemistry or mechanism of action of the ambruticins include: Connor et al, US 3,932,620 (1976); Connor et al, US 3,932,621 (1976); Connor et al, US 4,001,398 (1977); Connor et al, US 4,009,261 (1977); Connor et al, US 4,016,257 (1977); Connor et al, US 4,098,998 (1978); Connor et al, US 4,107,429 (1978); Connor et al, US 4,138,550 (1979); Connor et al, US 4,191,825 (1979); Connor et al, US RE 30,339 (1980); Connor e al, DE 2,659,575 (1978) (Chem. Abs. 89:109030); Connor et al, J. Med. Chem. 22 (9), 1055-1059 (1979); Connor et al, J. Med. Chem. 22 (9), 1144-1147 (1979); Knauth et al, J. Antibiotics 53 (10), 1182-1190 (2000); and Tian et al, US Application Ser. No. 11/305,802, filed Dec. 16, 2005.

[0007] Various researchers have published on the antifungal activities of the ambruticins. Ambruticin VS-3 has been reported to be active against the yeast Hansenula anomala (Knauth et al, J. Antibiotics 53:1182-1190 (2000)). Ambruticin S is reportedly orally effective in the treatment of experimental acute pulmonary coccidioidomycosis in mice| (Ringel, Antimicrob. Agents Chemother. 13:762-769 91978); Levine et al. Chest 73:202-206 (1978)). Ambruticin S is also reportedly active in vitro against both forms of C. immitis, with retention of activity in the presence of serum or plasma (Levine et al. (1978); Shadomy et al., Antimicrob. Agents Chemother 14:99-104 (1978)).

[0008] The disclosures of the foregoing documents and the other documents cited in this BACKGROUND OF THE INVENTION section are incorporated herein by reference.

[0009] The present invention provides methods for treating a Coccidioides infection using an ambruticin.

BRIEF SUMMARY OF THE INVENTION

[0010] This invention provides a method for treating or reducing the probability of a Coccidioides infection in a subject in need of such treatment or reduction of probability, comprising administering to such subject a therapeutically effective amount of a compound represented by formula I:

and the pharmaceutically acceptable salts, solvates, hydrates, and prodrug forms thereof, wherein

R¹⁰ and R¹¹ are independently H or CH₃; X¹ is either a bond or O; X² and X³ are each H or together are a bond;

R⁵ N \

R² and R³ are independently H, C₁-C₅ alkyl, C₂-C₅ alkenyl, C₂-C₅ alkynyl, cycloalkyl,

3TyI(C₁-C₅ alkyl), aryl(C₂-C₅ alkenyl), aryl(C₂-C₅ alkynyl), cycloaLkyl(Ci-C₅ alkyl), cycloalkyl(C₂-C₅ alkenyl), cycloalkyl(C₂-C₅ alkynyl),

R⁴ is H, ^{^}Y , or ^{^}Y ^% R⁹ ; or R³ and R⁴ combine to form \^ ; O O O

R⁵ is, independently for each occurrence thereof, H, C₁-C₅ alkyl, C₂-C₅ alkenyl, C₂-C₅ alkynyl, cycloalkyl, or aryl; R⁶ and R⁷ are independently H, C₁-C₅ alkyl, C₂-C₅ alkenyl, C₂-C₅ alkynyl, cycloalkyl, or aryl; or R⁶ and R⁷ and the nitrogen to which they are commonly bonded combine to form an aziridinyl, azetidinyl, pyrrolidinyl, or piperidinyl ring;

H

R is R or R⁵ Y ; O

R⁹ is, independently for each occurrence thereof, H, C₁-C₅ alkyl, C₂-C₅ alkenyl, C₂-C₅ alkynyl, cycloalkyl, 8TyI(C₁-C₅ alkyl), aryl(C₂-C₅ alkenyl), aryl(C₂-C₅ alkynyl), cycloalkyl(Ci-C₅ alkyl), cycloalkyl(C₂-C₅ alkenyl), or cycloalkyl(C₂-C₅ alkynyl), provided that R⁹ is not H when Z is O; R¹² and R¹³ together are O, or R¹² is H and R¹³ is R⁵; Y is O or N-OR⁵; and Z is, independently for each occurrence thereof, O or NH. [0011 ] This invention also provides for the use of the compound of formula I for the preparation of a medicament for treating a Coccidioides infection.

[0012] This invention also provides for the method such that the growth of the Coccidioides spp. is inhibited. The inhibition of the Coccidioides spp. includes the reduction in the growth of the Coccidioides spp. The reduction of growth includes one or more of the following: a decrease in the growth of individual Coccidioides cells, a decrease in the rate of cell division of individual Coccidioides cells, and the killing of individual Coccidioides cells. This invention also provides for the method such that the subject is cleared of a Coccidioides infection, or is relieved of a symptom caused by a Coccidioides infection. This invention also provides for the method such that the subject, who but for the administering of the pharmaceutical composition to the subject avoids a Coccidioides infection. BRIEF DESCRIPTION OF THE DRAWING(S)

[0013] Fig. 1 is an alignment map for two different ambrutin polyketide synthase (PKS) acyltransferase (AT) domains, used in homologous recombination experiments described in Example 23 hereinbelow. DETAILED DESCRIPTION OF THE INVENTION

Definitions

[0014] "Alkyl" means an optionally substituted straight or branched chain hydrocarbon moiety having the specified number of carbon atoms in its longest chain portion (e.g., as in "C₃ alkyl," "C₁-C₅ alkyl," or "C₁ to C₅ alkyl," the latter two phrases referring to an alkyl group having from 1 to 5 carbon atoms in the longest chain portion) or, where the number of carbon atoms is not specified, from 1 to 4 carbon atoms in the longest chain portion.

[0015] "Alkenyl" means an optionally substituted straight or branched chain hydrocarbon moiety having at least one carbon-carbon double bond and the specified number of carbon atoms in its longest chain portion (e.g., as in "C₃ alkenyl," "C₂-C₅ alkenyl," or "C₂ to C₅ alkenyl," the latter two phrases referring to an alkenyl group having from 2 to 5 carbon atoms in the longest chain portion) or, where the number of carbon atoms is not specified, from 2 to 4 carbon atoms in the longest chain portion.

[0016] "Alkynyl" means an optionally substituted straight or branched chain hydrocarbon moiety having at least one carbon-carbon triple bond and the specified number of carbon atoms in its longest chain portion (e.g., as in "C₃ alkenyl," "C₂-C₅ alkynyl," or "C₂ to C₅ alkynyl," the latter two phrases referring to an alkynyl group having from 2 to 5 carbon atoms in the longest chain portion) or, where the number of carbon atoms is not specified, from 2 to 4 carbon atoms in the longest chain portion.

[0017] "Aryl" means an aromatic monocyclic, fused bicyclic, or fused polycyclic hydrocarbon or heterocyclic group having 1 to 20 carbon atoms in the ring portion(s), such as phenyl, napthyl, pyrrolyl, indolyl, pyrazolyl, pyrazolinyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, thiadazolyl, isothiazolyl, furyl, thienyl, oxadiazolyl, pyridinyl, N-oxo-pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, tetrazinyl, triazinyl, triazolyl, benzothiazolyl, benzoxazolyl, benzothienyl, quinolinyl, quinolinyl-N-oxide, isoquinolinyl, benzimidazolyl, benzofuryl, chromonyl, coumarinyl, cinnolinyl, quinoxalinyl, indazolyl, benzisothiazolyl, benzisoxazolyl, benzodiazinyl, tetrazolyl, benzofurazanyl, benzothiopyranyl, benzpyrazolyl, indolinyl, isochromanyl, isoindolinyl, naphthyridinyl, phthalazinyl, purinyl, quinazolinyl, and the like. Aryl groups may be optionally substituted.

[0018] "Arylalkyl,"(cycloalkyl)alkyl," "arylalkenyl," "arylalkynyl," "biarylalkyl," and the like mean an aryl, cycloalkyl, or biaryl group, as the case may be, bonded directly to an alkyl, alkenyl, or alkynyl moiety, as the case may be, with the open (unsatisfied) valence at the alkyl, alkenyl, or alkynyl group, for example as in benzyl, phenethyl, N-imidazoylethyl, N-morpholinoethyl, and the like.

[0019] "Cycloalkyl" means an optionally substituted, saturated or unsaturated, non- aromatic cyclic hydrocarbon ring system, preferably containing 1 to 3 rings and 3 to 7 carbons per ring which may be further fused with a saturated or unsaturated C₃-C₇ carbocyclic ring. Exemplary cycloalkyl ring systems include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclodecyl, cyclododecyl, and adamantyl, especially the first four listed.

[0020] "Halogen" or "halo" means fluorine, chlorine, bromine or iodine. [0021] Pharmaceutically acceptable ester" means an ester that hydrolyzes in vivo (for example in the human body) to produce the parent compound or a salt thereof or has per se activity similar to that of the parent compound. Suitable ester groups include, without limitation, those derived from pharmaceutically acceptable aliphatic carboxylic acids, particularly alkanoic, alkenoic, cycloalkanoic and alkanedioic acids, in which each alkyl or alkenyl moiety preferably has no more than six carbon atoms. Illustrative esters include formates, acetates, propionates, butyrates, acrylates, citrates, succinates, and ethylsuccinates.

[0022] "Pharmaceutically acceptable salt" means a salt of a compound suitable for the pharmaceutical formulation. Where a compound has one or more basic functionalities, the salt can be an acid addition salt, such as a sulfate, hydrobromide, tartrate, mesylate, maleate, citrate, phosphate, acetate, pamoate (embonate), hydroiodide, nitrate, hydrochloride, lactate, methylsulfate, fumarate, benzoate, succinate, mesylate, lactobionate, suberate, tosylate, and the like. Where a compound has one or more acidic moieties, the salt can be a salt such as a calcium salt, potassium salt, magnesium salt, meglumine salt, ammonium salt, zinc salt, piperazine salt, tromethamine salt, lithium salt, choline salt, diethylamine salt, 4-phenyl- cyclohexylamine salt, benzathine salt, sodium salt, tetramethylammoniuni salt, and the like. [0023] Where it is indicated that a group may be substituted, for example by use of "substituted or unsubstituted" or "optionally substituted" phrasing, such group may have one or more independently selected substituents, preferably one to five in number, more preferably one or two in number. It is understood that substituents and substitution patterns can be selected by one of ordinary skill in the art to provide compounds that are chemically stable and that can be synthesized by techniques known in the art as well as the methods set forth herein. Examples of suitable substituents include alkyl, alkenyl, alkynyl, aryl, halo, trifluoro- methoxy, trifluoromethyl, hydroxy, alkoxy, cycloalkyloxy, heterocyclooxy, alkanoyl, alkanoyloxy, amino, alkylamino quarternary ammonium, aralkylamino, cycloalkylamino, heterocycloamino, dialkylamino, alkanoylamino, thio, alkylthio, cycloalkylthio, heterocyclo- thio, ureido, nitro, cyano, carboxy, caroboxylalkyl, carbamyl, alkoxycarbonyl, alkylthiono, arylthiono, alkylsulfonyl, sulfonamindo, aryloxy, and the like, in addition to those specified herein. Preferably, the substituent(s) for alkyl, alkenyl, and alkynyl moieties are from one to three in number and are independently selected from N-pyrrolidinyl, N-morpholinyl, N- azetidinyl, hydroxyl, halo, alkoxyl, cyano, amino, alkylamino, and dialkylamino, especially hydroxyl, halo, amino, and alkoxyl. Preferably, the substituent(s) for aryl, cycloalkyl, and heterocycloalkyl moieties are from one to three in number and are independently selected from alkyl, alkenyl, alkynyl, hydroxyalkyl, haloalkyl, hydroxyl, halo, alkoxyl, cyano, amino- alkyl, alkylaminoalkyl, dialkylaminoalkyl, amino, alkylamino, and dialkylamino. [0024] "Therapeutically effective amount" means that amount of active compound(s) or pharmaceutical agent(s) that elicit the biological or medicinal response in a tissue system, animal or human sought by a researcher, veterinarian, medical doctor or other clinician, which response includes alleviation of the symptoms of the disease or disorder being treated. The specific amount of active compound(s) or pharmaceutical agent(s) needed to elicit the biological or medicinal response will depend on a number of factors, including but not limited to the disease or disorder being treated, the active compound(s) or pharmaceutical agent(s) being administered, the method of administration, and the condition of the patient.

[0025] Where a range is stated, as in "C₁-C₅ alkyl" or "5 to 10%," such range includes the end points of the range. Compounds

[0026] Preferably, in formula I, the stereochemistry at C5 is S. In a preferred embodiment, R¹ is

R⁴ is H, and C5 has S stereochemistry, corresponding to a compound represented by formula II

INVENTION section hereinabove.

[0027] In another preferred embodiment, R¹ is

R⁴ is H, and C5 has S stereochemistry, corresponding to a compound represented by formula III:

where R², R\ R , 1ⁱ0^υ, _T R, 1¹1¹, X vl¹, X _V2^z and X³ are as defined in the BRIEF SUMMARY OF THE INVENTION section hereinabove. In a preferred embodiment of compounds of formula III, R² and R³ are each CH₃. [0028] In another preferred embodiment, R¹ is

and C5 has S stereochemistry, corresponding to a compound represented by formula IV

where R², R³, R⁴, R¹⁰, R¹¹, X¹, X² and X³ are as defined in the BRIEF SUMMARY OF THE INVENTION section hereinabove.

[0029] In one embodiment, in formula I, R¹⁰ and R¹¹ are each CH₃, X¹ is a bond, and X² and X³ together are a bond, corresponding to a compound represented by formula I-A

[0030] Preferably, in formula I-A, the stereochemistry at C5 is S.

[0031] In a preferred embodiment, in formula I-A, R¹ is

R⁴ is H, and C5 has S stereochemistry, corresponding to a compound represented by formula H-A

where R² and R³ are as defined in the BRIEF SUMMARY OF THE INVENTION section hereinabove. [0032] In another preferred embodiment, in formula I-A, R¹ is

R⁴ is H, and C5 has S stereochemistry, corresponding to a compound represented by formula iπ-A:

where R² and R³ are as defined in the BRIEF SUMMARY OF THE INVENTION section hereinabove. In a preferred embodiment of compounds of formula III- A, R² and R³ are each CH₃.

[0033] In another preferred embodiment, in formula I- A, R¹ is

and C5 has S stereochemistry, corresponding to a compound represented by formula IV-A

where R², R³ and R⁴ are as defined in the BRIEF SUMMARY OF THE INVENTION section hereinabove.

[0034] In one embodiment, in formula I, R¹⁰ is H, R¹¹ is CH₃, X¹ is a bond, and X² and X³ together are a bond, corresponding to a compound represented by formula I-B

[0035] Preferably, in formula I-B, the stereochemistry at C5 is S. [0036] In a preferred embodiment, in formula I-B, R¹ is

R⁴ is H, and C5 has S stereochemistry, corresponding to a compound represented by formula H-B

where R² and R³ are as defined in the BRIEF SUMMARY OF THE INVENTION section hereinabove.

[0037] In another preferred embodiment, in formula I-B, R¹ is

HO-^V

R⁴ is H, and C5 has S stereochemistry, corresponding to a compound represented by formula III-B:

where R² and R^J are as defined in the BRIEF SUMMARY OF THE INVENTION section hereinabove. In a preferred embodiment of compounds of formula III-B, R and R are each CH₃.

[0038] In another preferred embodiment, in formula I-B, R¹ is

H₂N^ X

O and C5 has S stereochemistry, corresponding to a compound represented by formula TV-B

[0039] In one embodiment, in formula I, R¹⁰ is CH₃, R¹¹ is H, X¹ is a bond, and X² and X³ together are a bond, corresponding to a compound represented by formula I-C

[0040] Preferably, in formula I-C, the stereochemistry at C5 is S.

[0041] In a preferred embodiment, in formula I-C, R¹ is

^HV o" ^■

R is H, and C5 has S stereochemistry, corresponding to a compound represented by formula II-C

[0042] hi another preferred embodiment, in formula I-C, R¹ is

R⁴ is H, and C5 has S stereochemistry, corresponding to a compound represented by formula III-C:

where R² and R³ are as defined in the BRIEF SUMMARY OF THE INVENTION section hereinabove. In a preferred embodiment of compounds of formula III-C, R² and R³ are each CH₃.

[0043] In another preferred embodiment, in formula I-C R¹ is H₂N_^ A

O and C5 has S stereochemistry, corresponding to a compound represented by formula IV-C

[0044] In one embodiment, in formula I, R¹⁰ and R¹¹ are both CH₃, X¹ is a bond, and X² and X³ are each H, corresponding to a compound represented by formula I-D

[0045] Preferably, in formula I-D, the stereochemistry at C5 is S. [0046] In a preferred embodiment, in formula I-D, R¹ is

R⁴ is H, and C5 has S stereochemistry, corresponding to a compound represented by formula H-D

[0047] In another preferred embodiment, in formula I-D, R¹ is

HCX >^ R⁴ is H, and C5 has S stereochemistry, corresponding to a compound represented by formula III-D:

where R² and R³ are as defined in the BRIEF SUMMARY OF THE INVENTION section hereinabove. In a preferred embodiment of compounds of formula III-D, R² and R³ are each CH₃.

[0048] In another preferred embodiment, in formula I-D, R¹ is

H₂N_x A.

O and C5 has S stereochemistry, corresponding to a compound represented by formula IV-D

[0049] In one embodiment, in formula I, R¹⁰ and R¹¹ are both CH₃, X¹ is O, and X² and X³ together are a bond, corresponding to a compound represented by formula I-E

[0050] Preferably, in formula I-E, the stereochemistry at C5 is S. [0051] In a preferred embodiment, in formula I-E, R¹ is

R is H, and C5 has S stereochemistry, corresponding to a compound represented by formula H-E

[0052] In another preferred embodiment, in formula I-E, R¹ is

R⁴ is H, and C5 has S stereochemistry, corresponding to a compound represented by formula III-E:

where R² and R³ are as defined in the BRIEF SUMMARY OF THE INVENTION section hereinabove. hi a preferred embodiment of compounds of formula III-E, R² and R³ are each CH₃.

[0053] In another preferred embodiment, in formula I-E, R¹ is

O and C5 has S stereochemistry, corresponding to a compound represented by formula IV-E

where R², R³ and R⁴ are as defined in the BRIEF SUMMARY OF THE INVENTION section hereinabove. [0054] In a preferred embodiment of compounds represented by formula II, R³ is other than H or CH₃ - that is, a non-naturally occurring ambruticin VS analog.

[0055] In another preferred embodiment of compounds represented by formula π, R is CH₃, X¹ is a bond, X² and X³ form a bond, and NR²R³ is N(CH₃)₂, NH(CH₃), or NH₂, corresponding to the naturally occurring ambruticins VS-3, VS-4, and VS-5, respectively.

[0056] Some other preferred embodiments of R and R in the formulae above are now disclosed.

[0057] In one preferred embodiment, R¹ is

,

[0058] Some other preferred embodiments of R² and R³ in formulae I, II, III, VI, I- A, II-

A, πi-A, ΓV-A, I-B, II-B, III-B, ΓV-B, I-C, II-C, III-C, ΓV-C, I-D, Π-D, IΠ-D, ΓV-D, I-E, II-E, III-E, and/or IV-E are now disclosed. In one embodiment, R² is H, CH₃, aryl(CH₂), cycloalkyl(CH₂), or cycloalkyl; and R³ is C₂-C₅ alkyl, aryl(CH₂), cycloalkyl(CH₂), or cycloalkyl.

[0059] In another preferred embodiment, R² is H, CH₃, CH₃CH₂, HOCH₂CH₂,

[0060] In another preferred embodiment, R³ is CH₃CH₂, CH₂CH₂OH, (CH₃)₂CH, CH₃CH₂CH₂, CH₃CH₂CH₂CH₂, COCF₃, CH₂CH₂F₉CH₂CHF₂, CH₂CF₃,

[0061] In yet another preferred embodiment, R³ is CH₃CH₂, CH₂CH₂OH, (CH₃)₂CH, CH₃CH₂CH₂, CH₃CH₂CH₂CH₂,

[0062] In one embodiment, R² and R³ together are CH₂CH₂CH₂.

[0063] In another preferred embodiment, R² and R³ are the same but each is

HO. JN, other than H or CH₃ when R¹ is Jf , R¹⁰ and R¹¹ are both CH₃, X¹ is a bond, X²

O and X³ are a bond, and R² is H or CH₃, then R³ is other than H or CH₃.

[0064] hi another preferred embodiment, R¹ is CO₂H, R² is CH₃ or CH₃CH₂, R⁴ is H, and R³ is selected from the group consisting OfCH₃CH₂, HOCH₂CH₂, (CH₃)₂CH,

[0065] The present invention includes within its scope prodrugs of the compounds of this invention. Such prodrugs are in general functional derivatives of the compounds that are readily convertible in vivo into the required compound. Thus, in the methods of treatment of the present invention, the term "administering" shall encompass the treatment of the various disorders described with the compound specifically disclosed or with a compound which may not be specifically disclosed, but which converts to the specified compound in vivo after administration to a subject in need thereof. Conventional procedures for the selection and preparation of suitable prodrug derivatives are described, for example, in Wermuth, "Designing Prodrugs and Bioprecursors," in Wermuth, ed., The Practice of Medicinal Chemistry, 2nd Ed., pp. 561-586 (Academic Press 2003). Prodrugs include esters that hydrolyze in vivo (for example in the human body) to produce a compound of this invention or a salt thereof. Suitable ester groups include, without limitation, those derived from pharmaceutically acceptable aliphatic carboxylic acids, particularly alkanoic, alkenoic, cycloalkanoic and alkanedioic acids, in which each alkyl or alkenyl moiety preferably has no more than six carbon atoms. Illustrative esters include formates, acetates, propionates, butyrates, acrylates, citrates, succinates, and ethylsuccinates.

[0066] Unless particular stereoisomers are specifically indicated (e.g., by a bolded or dashed bond at a relevant stereocenter in a structural formula, by depiction of a double bond as having E or Z configuration in a structural formula, or by use stereochemistry-designating nomenclature), all stereoisomers are included within the scope of the invention, as pure compounds as well as mixtures thereof. Unless otherwise indicated, individual enantiomers, diastereomers, geometrical isomers, and combinations and mixtures thereof are all encompassed by the present invention. Polymorphic crystalline forms and solvates are also encompassed within the scope of this invention. It is understood that compounds of formula I having both a carboxylic acid and an amine group may exist in a zwitterionic form. Formula I is intended to embrace such zwitterionic forms.

[0067] Exemplary compounds of this invention, according to Formula I- A, are shown in Table A (the stereochemistry at C5 being S, except for compound I-kkk which is a mixture of C5 R and S epimers): Table A (Part 1) — Compounds According to Formula I-A

Compound R¹ R² R³ R⁴

Table A (Part 3)

Compound R¹ R² R³ R⁴

Table A (Part 4)

Compound R¹ R² R³ R⁴

Table A (Part 5)

Compound R¹ R² R³ R⁴

I-hhh H H

I-iii

H CH₂CF₃ H i-jjj

H CH₂CHF₂ H

I-kkk

R² and R³ together are CH₂CH₂CH₂ H

1-111 CH₃ CH₃ H

I-mmm CH₃ CH₃ H

I-nnn CH₃ CH₃ H

I-ooo CH₃ CH₃ H

[0068] Illustrative compounds according to Formula I-B are shown in Table B:

Table B — Compounds According to Formula I-B

Compound R¹ R² R³ R⁴

II-a CH₃ CH₃ H

II-b

CH₃ CH₃ H

[0069] An illustrative compound according to Formula I-C is shown in Table C.

Table C — Compound According to Formula I-C

Compound R¹ R² R³ R⁴

III-a CH₃ CH₃ H

[0070] Illustrative compounds according to Formula I-D are shown in Table D. Table D — Compounds According to Formula I-D

Compound R¹ R² R³ R⁴

IV-a H H H

O

IV-b CH₃ CH₃ H

O

IV-c CH₃ CH₃ H

IV-d T CH(CH₃)₂ H H

O

[0071] Illustrative compounds according to Formula I-E are shown in Table E.

Coccidioides infection

[0072] A Coccidioides infection treatable in accordance with this invention is caused by a fungus of the genus Coccidioides, especially C. immitis or C. posadasii. C. immitis strains include Silveira, 46, ATCC 7366, K9-71X, 98-449, 98-571, Kr, DA, Ma, Mc, Co, Si, hi, La, Sy, and Ro (Gonzalez et al. Antimicrob. Agents Chemother. 45(6):1854-1859 (2001); Rifkind et al., Antimicrob. Agents Chemother. 6(6):783-784 (1974); Ward et al., Infect Immun. 12(5):1093-1097 (1975)). The Coccidioides infection can be coccidioidomycosis (also known as Valley Fever or Desert Fever). The site of Coccidioides infection can be in the subject's respiratory system (especially the lungs), kidneys, spleen, lymph nodes, brain, blood, and/or thyroid gland.

[0073] The subject can be suffering from coccidioidomycosis that is asymptomatic, acute symptomatic, or chronic pulmonary. Acute symptomatic coccidioidomycosis can have one or more of the following symptoms: pulmonary syndrome combined with cough, chest pain, shortness of breath, fever, and/or fatigue; diffuse pneumonia; skin manifestations (such as fine papular rash, erythema nodosum, and erythema multiforme); migratory arthralgias; and, fever. Chronic pulmonary coccidioidomycosis can have one or more of the following symptoms: pulmonary nodules and peripheral thin-walled cavities. [0074] The subject can be suffering from coccidioidomycosis that is extrapulmonary or disseminated. Coccidioidomycosis that is extrapulmonary or disseminated has one or more of the following symptoms: keratotic ulcers; verrucose ulcers; subcutaneous fluctuant abscesses; synovitis and effusion affecting the knees, wrists, feet, ankles, and/or pelvis; lytic lesions affecting the axial skeleton; meningeal disease; and, infection of the thyroid, gastrointestinal tract, adrenal glands, genitourinary tract, pericardium, and/or peritoneum. The subject can be suffering from coccidioidal meningitis.

[0075] Especially susceptible subject are those who are immunocompromised, such as patients infected with HIV, organ transplant recipients, cancer patients undergoing chemotherapy, and patients on high dosages of corticosteroids. The subject can also be a person who has a high chance of acquiring Coccidioides infection, such as a person planning to travel to or through an area where coccidioidomycosis is endemic.

[0076] The subject is typically a human, although the methods of the invention can be practiced for veterinary purposes, with suitable adjustment of the unit dose for the particular mammal of interest (including cats, cattle, dogs, horses, and the like). Modes of Administration and Pharmaceutical Formulations

[0077] Suitable modes of administration of the pharmaceutical composition include, but are not limited to, oral, topical, aerosol, inhalation by spray, parenteral, subcutaneous, intravenous, intramuscular, interperitoneal, rectal, and vaginal administration. The term parenteral, as used herein, includes subcutaneous injections, and intravenous, intrathecal, intramuscular, and intrasternal injection or infusion techniques. A preferred mode of administration is one that brings the compound of formula I to the actual or potential site(s) of Coccidioides infection in the subject. The pharmaceutical composition can be in a solid, semi-solid, or liquid form

[0078] The pharmaceutically acceptable carriers include, vehicles, adjuvants, excipients, and diluents, are well known to those who are skilled in the art and are readily available. Preferably, the carrier is chemically inert to ambruticins and has no detrimental side effects or toxicity under the conditions of use. Preferably, the pharmaceutically acceptable carrier is free of pyrogen. The pharmaceutically acceptable carriers which can be used include, but are not limited to, water, glucose, lactose, gum acacia, gelatin, mannitol, starch paste, magnesium trisilicate, talc, corn starch, keratin, colloidal silica, potato starch, and urea. [0079] The amount of the compound of formula I that may be combined with the pharmaceutically acceptable carrier to produce a single dosage form will vary depending upon the subject treated and the particular mode of administration. Suitable dosage levels of the active ingredient are on the order from about 0.01 mg to about 100 mg per kg body weight per day, preferably from about 0.1 mg to about 50 mg per kg body weight per day. Dosage unit forms will generally contain from about 0.1 mg to about 500 mg of the active ingredient. For external administration, the active ingredient may be formulated within the range of, for example, 0.00001% to 60% by weight, and preferably from 0.001% to 10% by weight, hi addition, the pharmaceutical composition can be administered on an intermittent basis, i.e., at daily, semi-weekly, or weekly intervals. It will be understood, however, that the specific dose level for a particular subject will depend on a variety of factors. These factors include the activity of the specific compound employed; the age, body weight, general health, sex, and diet of the subject; the time and route of administration and the rate of excretion of the drug; whether a drug combination is employed in the treatment; and, the severity of the particular disease or condition for which therapy is sought. [0080] The pharmaceutical compositions for oral administration include (a) liquid formulations; (b) capsules, sachets, tablets, lozenges, and troches, each containing a predetermined amount of the active ingredient, as solids or granules; (c) powders; (d) suspensions; and (e) emulsions. Liquid formulations may include diluents, such as water and alcohols, and optionally a pharmaceutically acceptable surfactant. Capsule forms can be of the ordinary hard- or soft-shelled gelatin type containing, for example, surfactants, lubricants, and inert fillers. Tablet forms can include one or more of lactose, sucrose, mannitol, corn starch, potato starch, alginic acid, microcrystalline cellulose, acacia, gelatin, guar gum, colloidal silicon dioxide, croscarmellose sodium, talc, magnesium stearate, calcium stearate, zinc stearate, stearic acid, and the like. The tablet can further comprise one or more colorants, diluents, buffering agents, disintegrants, moistening agents, preservatives, or flavoring agents.

[0081] The pharmaceutical composition, alone or in combination with other suitable components, can be made into aerosol formulations to be administered via inhalation. These aerosol formulations can be placed into pressurized acceptable propellants (such as dichlorodifluoromethane, propane, nitrogen, and the like) or non-pressured preparations (such as in a nebulizer or an atomizer). When the site(s) of infection of a subject is the lungs, a preferred mode of administration is inhalation of an aerosol formulation either orally or nasally. Preferably, the aerosol formulation comprises particles of a respirable size, including, but not limited to, mean particle sizes of 5 μm to 500 μm.

[0082] The pharmaceutical composition can be an injectable formulation. The requirements for effective carriers for injectable compositions are well known to those of ordinary skill in the art (see, e.g., Pharmaceutics and Pharmacy Practice, J. B. Lippincott Company, Philadelphia, Pa., Banker and Chalmers, eds., pages 238-250 (1982), and ASHP Handbook on Injectable Drugs, Toissel, 4th ed., pages 622-630 (1986)). Preferably, injectable compositions are administered intravenously. Formulations suitable for parenteral administration include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives.

[0083] The pharmaceutical composition can further comprise an excipient. Excipients that may be used include one or more carriers, surface active agents, thickening or emulsifying agents, solid binders, dispersion or suspension aids, solubilizers, colorants, flavoring agents, coatings, disintegrating agents, lubricants, sweeteners, preservatives, isotonic agents, and combinations thereof. The selection and use of suitable excipients is taught in Gennaro, ed., Remington: The Science and Practice of Pharmacy, 20th Ed. (Lippincott Williams & Wilkins 2003), the disclosure of which is incorporated herein by reference.

Production of ambruticins by fermentation [0084] Certain compounds of this invention (or their precursors) can be made using an isolated or recombinant cell comprising the genes of the ambruticin biosynthetic gene cluster and producing one or more ambruticins, wherein the activity the ambP, ambO, ωnbS, or ambM gene product is reduced or disrupted. (The ambruticin gene cluster is described in Reeeves et al, US 2005/0266434 Al (2005).) The reduction of activity can be due to the reduced expression of the gene encoding a gene product, or the gene can be modified so that the resulting gene product has less or no activity. The gene of interest can be disrupted or deleted by transposon insertion, homologous recombination, mutagenesis using a mutagen, or the like. The ambruticin biosynthetic gene cluster is described in

[0085] An isolated or recombinant cell wherein the activity of the ambM gene product is reduced or disrupted can be used to produce an ambruticin in which R¹⁰ is H. Or, an isolated or recombinant cell wherein the activity of the ambP and/or ambO gene product(s) is/are reduced or disrupted can be used to produce an ambruticin analog in which X² and X³ are each H. Or, an isolated or recombinant cell wherein the activity of the ambS gene product is reduced or disrupted can be used to produces elevated amounts of ambruticin VS-5 and ambruticin S and does not produce ambruticin VS-I, ambruticin VS-2, ambruticin VS-3 and ambruticin VS-4.

[0086] In one embodiment, the isolated or recombinant cell is disrupted for the ambP or ambO gene, or both genes; and, when cultured, the cell produces 20,21-dihydro analogs of the ambruticin, for example, compounds IV-a and IV-b, and compound H-D wherein R² is CH₃ and R³ is H. [0087] Where the isolated or recombinant cell is disrupted for the ambM gene and when cultured, the cell produces ambruticin lacking the C27 methyl group, for example, compound III-a and compound H-B wherein R² is H or CH₃ and R³ is H.

[0088] Where the cell has the ambM gene deleted and the malonate specific AT domain from module 7 is replaced or engineered into a loading domain, the cell produces ambruticin S that lacks the C24 and C27 methyl groups. Such compounds are represented by compound (II), wherein R¹⁰ and R¹¹ are each H, X¹ is a bond, X² and X³ are together a bond, and R² and R³ are independtly H or CH₃.

[0089] In one aspect, the cell is native to the ambruticin biosynthetic gene cluster. Alternatively, the cell is a host cell that is heterologous to the ambruticin gene cluster, wherein the ambruticin biosynthetic genes are present either on a vector or integrated into the chromosome of the cell. A cell native to the ambruticin biosynthetic gene cluster is a cell of the genus Sorangium. Preferably, the cell is a Sorangium cellulosum, more preferably of the So celO, NCIMB 12601 or So ce307 strains. A host cell heterologous to the ambruticin gene cluster includes, but is not limited to, eubacterial cells such as Escherichia coli, yeast cells such as Saccharomyces cerevisiae, or myxobacterial cells such as Myxococcus xanthus. Reeves et al, US 2005/0266434 Al (2005), incorporated herein by reference, disclose a method for expressing ambruticin using a M. xanthus host cell.

[0090] Compounds IV-a (20,21 -dihydro ambruticin VS-5), IV-b (20,21 -dihydro ambruticin VS-3) and IV-e (20,21 -dihydro ambruticin VS-4) are produced by the cell described above that lack the activity of the ambO and/or ambP gene product(s). Preferably, the cell is deleted for the ambO and/or ambP genes. More preferably, the cell is Sorangium cellulosum So ce 10. In one embodiment the cell is cultured and the compounds of interest are isolated or purified using methods previously described (see Examples 22 and 23 hereinbelow and Reeves et al., cited supra. These methods can be used to produce and purify other ambruticin compounds and analogs disclosed in this specification. [0091] In another embodiment, the cell is deleted for the ambO and/or ambP genes, and is also deleted for the ambS gene. The cell produces compound IV-a and 20,21 -dihydro ambruticin S.

[0092] In another embodiment, the cell has the ambO and/or ambP genes and the ambM gene deleted. The cell produces 20,21 -dihydro ambruticin S which lacks the C27 methyl group, and the compounds as represented by compound (II), wherein R¹⁰ is H, R¹¹ is CH₃, X¹ is a bond, X² and X³ are each H, and R² and R³ are independently H or CH₃.

[0093] hi another embodiment, the cell is deleted for the ambO and/or ambP genes, and the malonate specific AT domain from module 7 is replaced or engineered into a loading domain. The cell produces 20,21 -dihydro ambruticin S which lacks the C24 methyl, and the compounds as represented by compound (II), wherein R¹⁰ is CH₃, R¹¹ is H, X¹ is a bond, X² and X³ are each H, and R² and R³ are independently H or CH₃.

[0094] hi another embodiment, the cell is deleted for the ambM and ambO and/or ambP genes, and the malonate specific AT domain from module 7 is replaced or engineered into a loading domain. The cell produces 20,21 -dihydro ambruticin S which lacks the C24 methyl, and the compounds as represented by compound (II), wherein R¹⁰ and R¹¹ are each H, X¹ is a bond, X² and X³ are each H, and R² and R³ are independtly H or CH₃. [0095] Additional details on the preparation of ambruticin compounds are disclosed in Julien et al, WO 2005/086907 Al (2005) and Tian et al, US Pat. Appl'n No. 11/305,802, filed Dec. 16, 2005, the disclosures of which are incorporated herein by reference.

[0096] The practice of this invention can be further understood by reference to the following examples, which are provided by way of illustration and not of limitation.

Example 1

[0097] Compounds I wherein R¹ is CO₂H; R² is CH₃; R³ is alkyl, cycloalkyl, etc.; and R⁴ is H were prepared from ambruticin VS-4 per the following equation:

Ambruticin VS-4 R³ = alkyl, cycloalkyl, etc. [0098] This general procedure was used: To a solution of ambruticin VS-4 ((5S, 6R)S- (methylamino)-6-hydroxypolyangioic acid, 0.1 mmol) in methanol (1 niL) was added the aldehyde or ketone (0.2 mmol) and acetic acid (0.4 mmol), followed by sodium cyanoboro- hydride (0.2 mmol). The solution was stirred at 20 to 25 ⁰C (for reactive aldehydes) or 50 to 60 ⁰C (for less reactive aldehydes and ketones) until all of the ambruticin VS-4 was con- sumed. The reaction mixture was concentrated on a rotary evaporator, re-dissolved in a mixture of water and acetonitrile (AcCN), filtered through a one-gram plug of C-18 silica gel, and purified by reversed-phase HPLC, eluted using a gradient of AcCN in water containing 0.1% acetic acid. The product was obtained as a white solid after lyophilization of desired fractions. [0099] Compound I-a ((55',(5i?)-5-(Λ'^"-ethyl-methylamino)-6-hydroxypolyangioic acid) was synthesized using acetaldehyde at room temperature. ESI-TOF-MS m/z 516.3701, calcd for C₃₁H₅₀NO₅ ([M + H]⁺) 516.3684.

[0100] Compound I-b (( 5S, <5i?)-5-(iV-cyclopropylmethyl-methylamino)-6-hydroxy- polyangioic acid) was synthesized using cyclopropanecarboxaldehyde at room temperature. ESI-TOF-MS m/z 542.3828, calcd for C₃₃H₅₂NO₅ ([M + H]⁺) 542.3840. [0101] Compound I-c ((5S, (5i?)-5-(N-cyclopentyl-methylammo)-6-hydroxypolyangioic acid) was synthesized using cyclopentanone at 50 ⁰C. ESI-TOF-MS m/z 556.3981, calcd for C₃₄H₅₄NO₅ ([M + H]⁺) 556.3996.

[0102] Compound I-f ((5S, (5i?)-5-(iV-(2-naρhthyl)metliyl-methylamino)-6-hydroxy- polyangioic acid) was synthesized using 2-naphthaldehyde at room temperature. ESI-TOF- MS m/z 628.3967, calcd for C₄₀H₅₄NO₅ ([M + H]⁺) 628.3996.

[0103] Compound I-h ((5S, (5i?)-5-(iV-(4-imidazolyl)methyl-methylamino)-6-hydro- xypolyangioic acid) was synthesized using 4-imidazolecarboxaldehyde at room temperature.

[0104] Compound I-n ((5S, (5i?)-5-(N-(2-hydroxyethyl)-methylammo)-6-hydroxy- polyangioic acid) was synthesized using glycolaldehyde dimer at room temperature. ESI- TOF-MS m/z 532.3638, calcd for C₃₁H₅₀NO₆ ([M + H]⁺) 532.3633.

[0105] Compound I-t ((5S, (5i?)-5-(iV-propyl-methylamino)-6-hydroxypolyangioic acid) was synthesized using propionaldehyde at room temperature. ESI-TOF-MS m/z 530.3838, calcd for C₃₂H₅₂NO₅ ([M + H]⁺) 530.3840. [0106] Compound I-u ((5iS,(5i?)-5-(N-butyl-methylamino)-6-hydroxypolyangioic acid) was synthesized using butyraldehyde at room temperature. ESI-TOF-MS m/z 544.3969, calcd for C₃₃H₅₄NO₅ ([M + H]⁺) 544.3997.

[0107] Compound I-v ((5S, 6/?)-5-(N-isoproyl-methylamino)-6-hydroxypolyangioic acid) was synthesized using acetone at 50 ⁰C. ESI-TOF-MS m/z 530.3841, calcd for C₃₂H₅₂NO₅ ([M + H]⁺) 530.3840.

[0108] Compound I-w ((5S, (5i?)-5-(N-(3-pyridyl)methyl-methylamino)-6-hydroxy- polyangioic acid) was synthesized using nicotinaldehyde at 50 ⁰C. ESI-TOF-MS m/z 579.3827, calcd for C₃₅H₅₁N₂O₅ ([M + H]⁺) 579.3893.

[0109] Compound I-x ((5S, 672)-5-(iV-(2-thiazolyl)methyl-methylamino)-6-hydroxy- polyangioic acid) was synthesized using 2-thiazolecarboxaldehyde at 60 ⁰C. ESI-TOF-MS m/z 585.3341, calcd for C₃₃H₄₉N₂O₅S ([M + H]⁺) 585.3357.

[0110] Compound I-y ((5S, d^)-5-(N-(2-imidazolyl)methyl-methylamino)-6-hydroxy- polyangioic acid) was synthesized using 2-imidazolecarboxaldehyde at 60 ⁰C. ESI-TOF-MS m/z 568.3719, calcd for C₃₃H₅₀N₃O₅ ([M + H]⁺) 568.3745. Example 2

[0111] Compounds I wherein R¹ is CO₂H; R² and R³ are H, alkyl, etc.; and R⁴ is H were prepared from ambruticin VS-5 per the following equation:

Ambruticin VS-5 R² = H, alkyl, etc.

R³ = alkyl [0112] The following general procedure was used: To a solution of ambruticin VS-5 ((5S,6R)-5-amino-6-hydroxypolyangioic acid, 0.1 mmol) in methanol (1 mL) was added the aldehyde or ketone (0.2 mmol) and acetic acid (0.4 mmol), followed by sodium cyanoboro- hydride (0.2 mmol). The solution was stirred at 20 to 25 ⁰C until all of the ambruticin VS-5 was consumed. The reaction mixture was concentrated on a rotary evaporator, re-dissolved in a mixture of water- AcCN, filtered through a one-gram plug of C- 18 silica gel, and purified by reversed-phase HPLC, eluted using a gradient of AcCN in water containing 0.1% acetic acid. The product was obtained as a white solid after lyophilization of desired fractions.

[0113] Compound I-d ((5S, d/?)-5-(cyclopentylamino)-6-hydroxypolyangioic acid) was synthesized using cyclop entanone at room temperature. ESI-TOF-MS m/z 542.3848, calcd for C₃₃H₅₂NO₅ ([M + H]⁺) 542.3840.

[0114] Compound I-e ((5S, 6/?)-5-[di(cyclopropyhnethyl)amino]-6-hydroxypolyangioic acid) was synthesized using cyclopropanecarboxaldehyde. ESI-TOF-MS m/z 582.4163, calcd for C₃₆H₅₆NO₅ ([M + H]⁺) 582.4153.

[0115] Compound I-g ((J5^r,ά/?)-5-{di[(4-imidazolyl)methyl]amino}-6-hydroxypoly- angioic acid) was synthesized using 4-imidazolecarboxaldehyde. ESI-TOF-MS m/z 634.3966, calcd for C₃₆H₅₂N₅O₅ ([M + H]⁺) 634.3963.

[0116] Compound I-z ((5S, d/?)-5-(diethylamino)-6-hydroxypolyangioic acid) was synthesized using acetaldehyde. ESI-TOF-MS m/z 530.3842, calcd for C₃₂H₅₂NO₅ ([M + H]⁺) 530.3840. [0117] Compound I-aa ((5S, (5i?)-5-[di(2-hydroxyethyl)amino]-6-hydroxypolyangioic acid) was synthesized using glycoaldehyde dimer. [0118] Compound I-ggg ((5S, 6i-)-5-(2,2-difluoroethyl)amino-6-hydroxypolyangioic acid) was synthesized using difluoroacetaldehyde ethyl hemiacetal. ESI-TOF-MS m/z 538.3337, calcd for C₃₀H₄₆F₂NO₅ ([M + H]⁺) 538.3339.

[0119] Compound I-hhli ((5S, 6i?)-5-(3,4-dimethoxybenzyl)amino-6-hydroxypolyangioic acid) was synthesized using 3,4-dimethoxybenzaldehyde. ESI-TOF-MS m/z 624.3887, calcd for C₃₇H₅₄NO₇ ([M + H]⁺) 542.3452.

Example 3

[0120] Compounds I wherein R¹ is CO₂H, R² is CH₃, R³ is acyl and R⁴ is H were prepared from ambruticin VS-4 per the following equation:

Ambruticin VS-4 R³ = R⁵CO

[0121] The following general procedure was used: To a solution of ambruticin VS-4 (0.1 mmol) in methanol (1 rnL) was added the anhydride (1 mmol). After stirred at 20 to 25 ⁰C for 20 h, the reaction mixture was concentrated on a rotary evaporator. The residue was re- dissolved EtOAc (EtOAc). The solution was washed with water and brine and dried over Na₂SO₄. The Na₂SO₄ was removed by filtration and the filtrate was evaporated to dryness. The crude product was purified by reversed-phase HPLC, eluted using a gradient of AcCN in water containing 0.1% acetic acid. The product was obtained as a white solid after lyophilization of desired fractions.

[0122] Compound I-i ((5S, 6i?)-5-[iV-methyl(acetamido)]-6-hydroxypolyangioic acid) was synthesized using acetic anhydride. ESI-TOF-MS m/z 530.3500, calcd for C₃₁H₄₈NO₆ ([M + H]⁺) 530.3476.

[0123] Compound I-k (methyl (5S, (5i?)-5-[iV-methyl(acetamido)]-6-hydroxypolyangioate) was a side product in the preparation of compound I-i. ESI-TOF-MS m/z 544.3658, calcd for C₃₂H₅₀NO₆ ([M + H]⁺) 544.3633. [0124] Compound I-m ((56',(5i-)-5-[iV-methyl(propionamido)]-6-hydroxypolyangioic acid) was synthesized using propionic anhydride. ESI-TOF-MS m/z 544.3644, calcd for C₃₂H₅₀NO₆ ([M + H]⁺) 544.3633. Example 4

[0125] Compounds I wherein R¹ is CO₂H, R² is H, R³ is acyl and R⁴ is H were prepared from ambruticin VS-5 per the following equation:

Ambruticin VS-5 R³ = R⁵CO [0126] The general procedure of Example 3 was followed, except that ambruticin VS-5 was used instead of ambruticin VS-4.

[0127] Compound I-j ((J^tfφ-S-acetarmdo-ό-hydroxypolyangioic acid) was synthesized using acetic anhydride. ESI-TOF-MS m/z 516.3339, calcd for C₃₀H₄₆NO₆ ([M + H]⁺) 516.3320. [0128] Compound 1-1 ((5S, <5i?)-5-propionamido-6-hydroxypolyangioic acid) was synthesized using propionic anhydride. ESI-TOF-MS m/z 530.3457, calcd for C₃₁H₄₈NO₆ ([M + H]⁺) 530.3476.

[0129] Compound I-fff ((5S, 6i?)-5-trifluroacetamido-6-hydroxypolyangioic acid) was synthesized using trifluoroacetic anhydride in dichloromethane (DCM). ESI-TOF-MS m/z 592.2862, calcd for C₃₀H₄₂F₃NO₆Na ([M + Na]⁺) 592.2856.

Example 5

[0130] Compounds I wherein R¹ is CO₂H, R² is H or CH₃, R³ is R^bOCO and R⁴ is H were prepared from ambruticin VS-4 or VS-5 per the following equation:

Ambruticin VS-4 or R³ = R⁹OCO ambruticin VS-5 [0131] This general procedure was used: To a suspension of ambruticin VS-4 or VS-5 (0.1 mmol) in dry tetrahydrofuran (THF, 1 mL) was added ΛζN-diisopropylethylamine (DIEA, 0.3 mmol), followed by the alkyl chloroformate (0.2 mmol). After stirred at 20 to 25 ⁰C for 20 h, the reaction mixture was concentrated on a rotary evaporator. The residue was re- dissolved in EtOAc. The solution was washed with 0.1 M HCl (aq) and brine, and dried over Na₂SO₄. The salt was removed by filtration and the filtrate was evaporated to dryness. The crude product was purified by reversed-phase HPLC, eluted using a gradient of AcCN in water containing 0.1% acetic acid. The product was obtained as a white solid after lyophilization of desired fractions.

[0132] Compound I-o ((J^ό^-S-fN-memy^methoxycarbonylamino) was synthesized from ambruticin VS-4 and methyl chloroformate. ESI-TOF-MS m/z 568.3227, calcd for C₃₁H₄₇NO₇Na ([M + Na]⁺) 568.3248. [0133] Compound I-bb ((J»S^r,di-)-5-methoxycarbonylamino-6-hydroxypolyangioic acid) was synthesized from ambruticin VS-5 and methyl chloroformate. ESI-TOF-MS m/z 554.3094, calcd for C₃₀H₄₅NO₇Na ([M + Na]⁺) 554.3088.

[0134] Compound I-r ((5S, di?)-5-[N-methyl(isobutoxycarbonylamino)]-6- hydroxypolyangioic acid) was synthesized from ambruticin VS-4 and isobutyl chloroformate. ESI-TOF-MS m/z 610.3707, calcd for C₃₄H₅₃NO₇Na ([M + Na]⁺) 610.3714.

Example 6

[0135] Compounds I wherein R¹ is CO₂H, R² is H or CH₃, R³ is R^CNHCO and R⁴ is H or R⁰NHCO were prepared from ambruticin VS-4 or VS-5 per the following equation:

Ambruticin VS-4 or R³ = R⁹NHCO ambruticin VS-5 R⁴ = H or R⁹NHCO [0136] The following general procedure was used: To a suspension of ambruticin VS-4 or ambruticin VS-5 (0.1 mmol) in dry THF (1 mL) was added the isocyanate (0.5 mmol). After the mixture was stirred at 50 ⁰C for 20 h, 300 mg OfPS-TsNHNH₂ resin (Argonaut, CA) was added, and the mixture was stirred at room temperature overnight. The mixture was then diluted in methanol and filtered to remove the resin. The filtrate was concentrated on a rotary evaporator. The residue was re-dissolved EtOAc. The solution was washed with 0.1 M HCl (aq) and brine, and dried over Na₂SO₄. The salt was removed by filtration and the filtrate was evaporated to dryness. The crude product was purified by reversed-phase HPLC, eluted using a gradient of AcCN in water containing 0.1% acetic acid. The product was obtained as a white solid after lyophilization of desired fractions.

[0137] Compound I-q ((J5',(5i?)-5-(3-allyl-l-methylureido)-6-hydroxypolyangioic acid) was synthesized from ambruticin VS-4 and allyl isocyanate. ESI-TOF-MS m/z 593.3582, calcd for C₃₃H₅₀N₂O₆Na ([M + Na]⁺) 593.3561.

[0138] Compound I-cc ((5<S',(5i-)-5-(3-allyl-l-methylureido)-6-(allylcarbamoyl)poly- angioic acid) was a side product from the preparation of compound I-q, above. ESI-TOF-MS m/z 676.3905, calcd for C₃₇H₅₅N₃O₇Na ([M + Na]⁺) 676.3932.

[0139] Compound I-p ((5₎S',(5i?)-5-(3-benzyl-l-methylureido)-6-hydroxypolyangioic acid) was synthesized from ambruticin VS-4 and benzyl isocyanate. ESI-TOF-MS m/z 643.3746, calcd for C₃₇H₅₂N₂O₆Na ([M + Na]⁺) 643.3718.

[0140] Compound I-dd ((5S, <5i?)-5-(3-benzyl-l-methylureido)-6-(benzylcarbamoyl)- polyangioic acid) was a side product from synthesis of compound I-p, above. ESI-TOF-MS m/z 776.4254, calcd for C₄₅H₅₉N₃O₇Na ([M + Na]⁺) 776.4245. [0141] Compound I-s ((5iS'₎di?)-5-(3-benzylureido)-6-hydroxypolyangioic acid) was synthesized from ambruticin VS-5 and benzyl isocyanate. ESI-TOF-MS m/z 629.3567, calcd for C₃₆H₅₀N₂O₆Na ([M + Na]⁺) 629.3561.

Example 7

[0142] Compounds I wherein R¹ is CH₂OH, R² and R³ are H or CH₃, and R⁴ is H were prepared from ambruticin VS-3, VS-4, or VS-5 or compound I-fff or I-ggg per the following equation:

Ambruticin VS-3, VS-4, or VS-5 or compound I-fff or I-ggg

[0143] The following general procedure was used: To a suspension of ambruticin VS compound (0.1 mmol) in dry THF (10 mL) was added a solution of 1 M lithium aluminum hydride in THF (1 mL). After the mixture was heated to reflux under nitrogen atmosphere for 3 h, it was cooled in an ice-bath, and a few drops of water was added, followed by MgSO₄ (25 mg). The precipitate was removed by filtration and thoroughly washed with EtOAc. The combined filtrate was evaporated to give an oil, which was purified by reversed-phase HPLC to give the product as a colorless oil.

[0144] Compound I-ee ((5S, <5i?)-5-(methylamiiio)polyangi- 1 ,6-diol) was synthesized from ambruticin VS-4. ESI-TOF-MS m/z 474.3570, calcd for C₂₉H₄₈NO₄ ([M + H]⁺) 474.3578.

[0145] Compound I-ff ((5S, (5i?)-5-(dimethylamino)polyangi- 1 ,6-diol) was synthesized from ambruticin VS-3. ESI-TOF-MS m/z 488.3737, calcd for C₃₀H₅₀NO₄ ([M + H]⁺) 488.3734. [0146] Compound I-iii ((5»S^r,6i?)-5-(2,2,2-trifluroethyl)aminopolyangi-l,6-diol) was synthesized from compound I-fff. ESI-TOF-MS m/z 542.3430, calcd for C₃₀H₄₇F₃NO₄ ([M + H]⁺) 542.2452.

[0147] Compound I-jjj ((5S, 6i?)-5-(2,2-difluroethyl)aminopolyangi- 1 ,6-diol) was synthesized from compound I-ggg. ESI-TOF-MS m/z 524.3557, calcd for C₃₀H₄₈F₂NO₄ ([M + H]⁺) 524.3546.

Example 8

[0148] Polyangiamide (ambruticin amide) compounds can be prepared according to the following illustrative procedure for compound I-hh ((5iS',6i-)-5-(dimethylamino)poly- angiamide). [0149] To a solution of ambruticin VS-3 (35 mg) in dry THF (0.1 mL) cooled at 0 ⁰C was added triethylamine (13 μL), followed by ethyl chloroformate (9 μL). After the mixture was stirred at 0 ⁰C for 30 min., a solution of 28% aqueous ammonia (90 μL) in THF (0.5 mL) was added. The mixture was allowed to warm to room temperature over 1 h with stirring. Water and EtOAc were added. The organic layer was washed with saturated NaCl (aq), dried over anhydrous Na₂SO₄, filtered, and evaporated in vacuo. The crude product was purified by reversed-phase HPLC, eluted using a gradient of AcCN in water containing 0.1% acetic acid. Compound I-hh was obtained as a white solid (17 mg) after lyophilization of desired fractions. ESI-TOF-MS m/z 501.3691, calcd for C₃₀H₄₉N₂O₄ ([M + H]⁺) 501.3687. [0150] Compound I-ii ((5iS',6i?)-5-(dimethylaiiimo)-6-etlioxycarbonyloxy-polyangiamide) was obtained as a by-product in the preparation of compound I-hh, isolated as a white solid (5 mg). ESI-TOF-MS m/z 573.3903, calcd for C₃₃H₅₃N₂O₆ ([M + H]⁺) 573.3898.

Example 9

[0151] Ambruticin VS compounds having an inverted (Ji?) stereochemistry at position C5 can be made from ambruticin S. In one approach, ambruticin S is oxidized directly to 5- keto ambruticin S using Dess-Martin periodinane, although it appears that the yield is rather low. Reductive amination followed by separation of epimers affords 5R ambruticin VS compound.

Ambruticin S

[0152] Alternatively, and perhaps preferably, ambruticin S is first converted to the methyl ester and then oxidized to 5-keto ambruticin S methyl ester, as disclosed in Conner et al, US RE 30,339 (1980). The keto ester is then reductively aminated, the epimers are separated, and the 5R ester is hydrolyzed to afford the 5R ambruticin VS compound.

Separate epimers

methyl ester

[0153] The 5i?-ambruticin VS compounds so produced can then be used to make further ambruticin derivatives, by methods analogous to those described in the examples herein. Example 10

[0154] Compounds I wherein R¹ is CO₂H; R² is H; R³ is alkyl, cycloalkyl, etc.; and R⁴ is H ((55^r,6i?)-5-(Alkylamino)-6-hydroxypolyangioic acid) were prepared from ambraticin VS-5 per the following series of equations:

Ambruticin VS-5 R³ = alkyl, cycloalkyl, etc.

[0155] To a solution of ambruticin VS-5 (0.1 mmol) in methanol (1 mL) was added the aldehyde or ketone (0.1 mmol) and acetic acid (0.4 mmol), followed by sodium cyanoboro- hydride (0.2 mmol). The solution was stirred at 20 to 25 ⁰C until all of the ambruticin VS-5 was consumed. The reaction mixture was concentrated on a rotary evaporator, re-dissolved in water- AcCN, filtered through a one-gram plug of C-18 silica gel, and purified by reversed- phase HPLC, eluted using a gradient of AcCN in water containing 0.1% acetic acid. The product was obtained as a white solid after lyophilization of desired fractions.

[0156] Compound I-jj ((5£,6i?)-5-(cyclobutylamino)-6-hydroxypolyangioic acid) was synthesized using cyclobutanone. ESI-TOF-MS m/z 528.3686, calcd for C₃₂H₅₀NO₅ ([M + H]⁺) 528.3684.

[0157] Compound I-kk ((55^r,6i?)-5-(isopropylamino)-6-hydroxypolyangioic acid) was using acetone. ESI-TOF-MS m/z 516.3682, calcd for C₃₁H₅₀NO₅ ([M + H]⁺) 516.3684.

[0158] Compound 1-11 ((55',6i?)-5-[(2-hydroxyethyl)amino]-6-hydroxypolyangioic acid) was synthesized using glycolaldehyde dimer. ESI-TOF-MS m/z 518.3494, calcd for C₃₀H₄₈NO₆ ([M + H]⁺) 518.3476.

[0159] Compound I-mm ((5S, 6i?)-5-(ethylamino)-6-hydroxypolyangioic acid) was synthesized using acetaldehyde. ESI-TOF-MS m/z 502.3533, calcd for C₃₀H₄₈NO₅ ([M + H]⁺) 502.3527.

Example 11 [0160] Compounds I wherein R¹ is CH₂OH; R² is CH₃; R³ is alkyl, etc.; and R⁴ is H ((5<S,6i?)-5-(alkylamino)polyangi-l,6-diol) were prepared from ambruticin VS-4 per the following series of equations:

Ambruticin VS-4 Compound l-ee R³ = alkyl, etc.

[0161 ] Compound I-ee was prepared using the method of Example 7. To a solution of compound I-ee (0.1 mmol) in methanol (1 mL) was added the aldehyde or ketone (0.2 mmol) and acetic acid (0.4 mmol), followed by sodium cyanoborohydride (0.2 mmol). The solution was stirred at 20 to 25 ⁰C (for reactive aldehydes) or 50 to 60 ⁰C (for less reactive aldehydes and ketones) until all of Compound I-ee was consumed. The reaction mixture was concentrated on a rotary evaporator, re-dissolved in a mixture of water- AcCN, filtered through a one-gram plug of C- 18 silica gel, and purified by reversed-phase HPLC, eluted using a gradient of AcCN in water containing 0.1% acetic acid. The product was obtained as a white solid after lyophilization of desired fractions.

[0162] Compound I-nn (5,S',6i?)-5-[N-(2-hydroxyethyl)methylamino]polyangi-l,6-diol) was synthesized using glycolaldehyde dimer at room temperature. ESI-TOF-MS m/z 518.3839, calcd for C₃₁H₅₂NO₅ ([M + H]⁺) 518.3840.

[0163] Compound I-oo ((5(S, 6i?)-5-(7V-isoproyl-methylamino)polyangi- 1 ,6-diol) was synthesized using acetone at 50 ⁰C. ESI-TOF-MS m/z 516.4038, calcd for C₃₂H₅₄NO₄ ([M + H]⁺) 516.4047.

Example 12

[0164] Compounds I wherein R¹ is

(R⁷)(R⁶)N. X o ; R² is CH₃; R³ is CH₃; and R⁴ is H were prepared from ambruticin VS-3 per the following scheme:

^*" . DMF

Ambruticin VS-3 [0165] To a solution of ambruticin VS-3 (0.1 mmol) in iV^-dimethylformamide (DMF, 1 mL) was added the amine (0.2 mmol) and DIEA (0.3 mmol), followed by (9-(7-azabenzo- triazol-l-y^-ΛζΛ^iV'.iV'-tetramethyluronium hexafluorophosphate (HATU, 0.12 mmol). After being stirred at 20 to 25 ⁰C for 20 h, the reaction mixture was diluted with EtOAc (ca. 30 mL). The solution was then washed with saturated aqueous NaHCO₃ (30 mL), brine (30 mL), and dried over anhydrous Na₂SO₄. The salt was removed by filtration and the filtrate was evaporated to dryness. The crude product was purified by reversed-phase HPLC, eluted using a gradient of AcCN in water containing 0.1% acetic acid. The product was obtained as a white solid after lyophilization of desired fractions as determined by HPLC/MS. [0166] Compound I-pp (5S, 6R)- 1 -(azetidin- 1 -yl)- 5-dimethylamino-6-hydroxypolyangi- 1-one was synthesized using ambruticin VS-3 and azetidine. ESI-TOF-MS m/z 541.3984, calcd for C₃₃H₅₃N₂O₄ ([M + H]⁺) 541.4000.

[0167] Compound I-qq (5S, 6R)- 5-dimethylamino-6-hydroxy-iV-[(2-dimethyl- amino)ethyl]polyangiamide was synthesized using ambruticin VS-3 and AζiV-dimethyl- ethylenediamine. ESI-TOF-MS m/z 572.4408, calcd for C₃₄H₅₈N₃O₄ ([M + H]⁺) 572.4422.

[0168] Compound I-rr (5S, 6i?)-5-dimethylamino-6-hydroxy-iV-(2-hydroxyethyl)- polyangiamide was synthesized using ambruticin VS-3 and ethanolamine. ESI-TOF-MS m/z 545.3938, calcd for C₃₂H₅₃N₂O₅ ([M + H]⁺) 545.3949.

[0169] Compound I-ss (5S, 6i?)-5-dimethylamino-6-hydroxy-N-(methoxycarbonyl- methyl)polyangiamide was synthesized using ambruticin VS-3 and methyl glycinate hydrochloride. ESI-TOF-MS m/z 573.3907, calcd for C₃₃H₅₃N₂O₆ ([M + H]⁺) 573.3898.

[0170] Compound I-tt (5S, ό^-S-dimethylamino-ό-hydroxy-N-methoxy-N-methyl- poryangiamide was synthesized using ambruticin VS-3 and N, O-dimethylhydroxylamine. ESI-TOF-MS m/z 545.3928, calcd for C₃₂H₅₃N₂O₅ ([M + H]⁺) 545.3949. Example 13

[0171 ] Compounds I wherein R¹ is "R^X-O~N=CH-" (R^x is H or an alkyl), R² is CH₃ or H; R³ is CH₃; and R⁴ is H were prepared from ambruticin VS-3 per the following scheme: Ambruticin

VS-3

[0172] To a solution of ambruticin VS-3 (0.45 g, 0.9 mmol) in methanol (10 mL) cooled in an ice bath was added thionyl chloride (0.08 mL, 1.1 mmol). The mixture was allowed to warm to room temperature with stirring over 4.5 h. The mixture was concentrated on a rotary evaporator and re-dissolved in EtOAc. The solution was washed with saturated aqueous NaHCO₃ and then brine and dried over anhydrous Na₂SO₄. The drying agent was removed by filtration. The filtrate was evaporated to dryness in vacuo. Methyl (5S,6R)-5-dimeth.yl- amino-6-hydroxypolyangiate (ambruticin VS-3 methyl ester) was obtained as a yellow solid (0.42 g). To a solution of methyl (5»S',6i?)-5-dimethylamino-6-hydroxypolyangiate (0.35 g, 0.7 mmol) in dry toluene (10 mL) cooled at -78 ⁰C under nitrogen atmosphere was added 1.0 M solution of diisobutylaluminum hydride in toluene (2.5 mL, 2.5 mmol). The mixture was stirred at -78 ⁰C for 5 minutes, water (0.2 mL) was added, followed by EtOAc. The mixture was stirred at room temperature for 20 minutes, then dried with anhydrous Na₂SO₄. The drying agent was removed by filtration. The filtrate was evaporated to dryness in vacuo. (5S, 6i?)-5-Dimethylamino-6-hydroxypolyangial was obtained in quantitative yield as a light yellow solid.

[0173] To a solution of (5S, 6i?)-5-dimethylamino-6-hydroxypolyangial (0.05 mmol) in 2- propanol (0.2 mL) was added the alkoxylamine (0.25 mmol, the hydrochloride salt is neutralized with aq. NaOH before addition if applicable), followed by acetic acid (0.2 mmol). The mixture was heated at 50 ⁰C overnight. The mixture was concentrated in vacuo, re- dissolved in 1 : 1 water/AcCN, filtered, and purified by reversed-phase HPLC on a Varian Metasil Basic column, eluted using a gradient of AcCN in water containing 0.1% acetic acid. The product (about 1 : 1 mixture of E/Z isomers) was obtained as a white solid after lyophilization of desired fractions as determined by HPLC/MS. [0174] Compound I-uu (5S, 6i?)-5-dimethylamino-6-hydroxypolyangial oxime (R = H) was synthesized using (5«S',6i-)-5-dimethylammo-6-hydroxypolyangial and hydroxylamine. ESI-TOF-MS m/z 501.3665, calcd for C₃₀H₄₉N₂O₄ ([M + H]⁺) 501.3687. [0175] Compound I-w (5S, 6i?)-5-dimethylamino-6-hydroxyporyangial O-methyl oxime (R = Me) was synthesized using (55,6i?)-5-dimethylamino-6-hydroxyporyangial and methoxylamine. ESI-TOF-MS m/z 515.3827, calcd for C₃₁H₅₁N₂O₄ ([M + H]⁺) 515.3843.

[0176] Compound I-ww (5S, 6i?)-5-dimethylamino-6-hydroxyporyangial O- carboxymethyl oxime (R = CH₂CO₂H) was synthesized using (55!, 6i?)-5-dimethylamino-6- hydroxypolyangial and carboxymethoxylamine. ESI-TOF-MS m/z 559.3721, calcd for C₃₂H₅₁N₂O₆ ([M + H]⁺) 559.3742.

[0177] Compound I-xx (5_JS',6i-)-5-dimethylamino-6-hydroxypolyangial O-tørt-butyl oxime (R = ¹Bu) was synthesized using (5£6i?)-5-dimethylamino-6-hydroxyporyangial and fert-butoxylamine. ESI-TOF-MS m/z 557.4271 , calcd for C₃₄H₅₇N₂O₄ ([M + H]⁺) 557.4313.

[0178] Compound I-yy (5S, 6i?)-5-dimethylammo-6-hydroxyporyangial O-4-nitrobenzyl oxime (R = P-NO₂-C₆H₄-CH₂) was synthesized using (5_<S',6i?)-5-dimethylamino-6- hydroxypolyangial and 4-nitrobenzyloxylamine.

[0179] Compound I-zz (5S, 6i?)-5-methylamino-6-hydroxypolyangial oxime was synthesized from ambruticin VS-4 following the following scheme. ESI-TOF-MS m/z 487.3513, calcd for C₂₉H₄₇N₂O₄ ([M + H]⁺) 487.3530.

I) SOCI₂₁ MeOH Ambruti- ₊. cin VS-4 2) BoC₂O, NaHCO₃

THRH₂O

Example 14

[0180] Compounds I wherein R¹ is R⁶R⁷NCH₂; R² and R³ are CH₃; and R⁴ is H were prepared from ambruticin VS-3 aldehyde per the following scheme:

[0181 ] To a solution of (5S, 6i-)-5-dimethylamino-6-hydroxypolyangial (0.05 mmol) in methanol (0.5 mL) was added the amine (0.25 mmol), acetic acid (0.2 mmol), and sodium cyanoborohydride (0.1 mmol). After stirred at room temperature overnight, the mixture was concentrated in vacuo, re-dissolved in 1 : 1 water/AcCN, filtered, and purified by reversed- phase HPLC, eluted using a gradient of AcCN in water containing 0.1% acetic acid. The product was obtained as a white solid after lyophilization of desired fractions as determined by HPLC/MS. [0182] Compound I-aaa (5_iS',6i?)-l-(azetidin-l-yl)-5-(dimethylammo)polyangi-6-ol was synthesized using (5£,6i?)-5-dimethylamino-6-hydroxypolyangial and azetidine. ESI-TOF- MS m/z 527.4185, calcd for C₃₃H₅₅N₂O₃ ([M + H]⁺) 527.4207.

[0183] Compound I-bbb (55^r,6i?)-l-amino-5-(dimethylamino)polyangi-6-ol was synthesized using (5£6i?)-5-dimethylamino-6-hydroxyporyangial and ammonium acetate. ESI-TOF-MS m/z 487.3896, calcd for C₃₀H₅₁N₂O₃ ([M + H]⁺) 487.3894.

[0184] Compound I-ccc (55^r,6i?)-l-[(5)-2-carboxypyrrolidin-l-yl]-5-(dimethylamino)- polyangi-6-ol was synthesized using (5£,6i?)-5-dimethylamino-6-hydroxypolyangial and L- proline. ESI-TOF-MS m/z 585.4234, calcd for C₃₅H₅₇N₂O₅ ([M + H]⁺) 585.4262.

[0185] Compound I-ddd (5)S',6i?)-l-[(i?)-2-carboxypyrrolidm-l-yl]-5-(dimethylamino)- polyangi-6-ol was synthesized using (5S,6R)-5 -dimethylamino-6-hydroxypolyangial and D- proline. ESI-TOF-MS m/z 585.4231, calcd for C₃₅H₅₇N₂O₅ ([M + H]⁺) 585.4262.

[0186] Compound I-eee (55,6i?)-l-(azetidin-l-yl)-5-aminopolyangi-6-ol was synthesized using ambruticin VS-5 per the scheme below. Its analytical characteristics were: ESI-TOF- MS m/z 499.3878, calcd for C₃iH₅₁N₂O₃ ([M + H]⁺) 499.3894.

Example 15

[0187] A mixture of 5R and 5S isomers (Compound I-fckk; (6i?)-5-azetidinyl-6- hydroxypolyangioic acid) was synthesized using the following procedure

-1 :1 mixture of 5-epimers

[0188] To a solution of ambruticin S (48 mg, 0.1 mmol) in DCM was added Dess-Martin periodinane (85 mg, 0.2 mmol). After the solution was stirred at 20 to 25 ⁰C for 16 h, 0.04 mL of 1 M Na₂S₂O₃ (aq) was added. The mixture was stirred for 20 min. Ethyl acetate was added. The organic phase was separated and washed with saturated aqueous NaHCO₃, brine, and dried over anhydrous Na₂SO₄. The drying agent was removed by filtration and the filtrate was evaporated to dryness. The crude product was purified by reversed-phase HPLC, eluted using a 30 min-gradient of 25 to 75% AcCN in water containing 0.1% acetic acid. (6i?)-5-Oxo-6-hydroxypolyangioic acid was obtained as a white solid after lyophilization of desired fractions as determined by HPLC/MS. Yield -10%.

[0189] To a solution of (6i?)-5-oxo-6-hydroxypolyangioic acid (~10 mg) in methanol was added azetidine (7 μL), acetic acid (10 μL), and sodium cyanoborohydride (6 mg). After stirred at room temperature overnight, the mixture was concentrated in vacuo, re-dissolved in 1:1 water/AcCN, filtered, and purified by reversed-phase HPLC on a Varian Metasil Basic column, eluted using a gradient of AcCN in water containing 0.1% acetic acid. The product (~1:1 mixture of epimers) was obtained as a white solid (1.2 mg) after lyophilization of desired fractions as determined by HPLC/MS. ESI-TOF-MS m/z 514.3507, calcd for C₃₁H₄₈NO₅ ([M + H]⁺) 514.3527. One skilled in the art can isolate/separate the 5R and 5S isomers of compound I-kkk using standard procedures in the art. Example 16

[0190] Compound II-b ((55,6i?)-5-dimethylamino-15-desmethylpolyangi-l,6-diol) was synthesized using the following procedure:

[0191 ] To a suspension of compound II-a ((5S, 6i?)-5-dimethylamino-6-hydroxy- 15- desmethylpolyangioic acid (15-desmethyl ambruticin VS-3)) (8 mg, 0.016 mmol) in dry THF (0.8 mL) was added a solution of 1 M lithium aluminum hydride in THF (0.16 mL). After the mixture was heated at 50 ⁰C under nitrogen atmosphere for 1 h, it was cooled in an ice- bath, and a few drops of water was added, followed by MgSO₄ (25 mg). The precipitate was removed by filtration and thoroughly washed with THF. The combined THF solutions were evaporated to dryness. The residue was re-dissolved in 1 :3 water/ AcCN and filtered through a 0.2 μm PTFE filter. The filtrate was purified by reversed-phase HPLC on a Varian Metasil Basic column, eluted using a gradient of AcCN in water containing 0.1% acetic acid. The product was obtained product as a light yellow gel after lyophilization of desired fractions as determined by HPLC/MS. ESI-TOF-MS m/z 474.3545, calcd for C₂₉H₄₈NO₄ ([M + H]⁺) 474.3578.

Example 17

[0192] The following procedure was used to synthesize 16,17-epoxy analogs.

[0193] To a solution of ambruticin VS-5 (0.12 g, 0.25 mmol) in methanol (6 mL) was added thionyl chloride (0.04 mL, 0.5 mmol). After the mixture was stirred at -40 to 25 ⁰C overnight, HPLC analysis showed that the reaction was complete. The reaction mixture was stirred with aqueous NaHCO₃ (20 mL) for 10 minutes. The mixture was concentrated on a rotary evaporator to remove methanol and extracted with EtOAc. The combined extracts were washed with water, aqueous NaHCO₃, and brine, and dried over anhydrous Na₂SO₄. The drying agent was removed by filtration and the filtrate was evaporated to dryness. Methyl (5S,6R)-5-amino-6-hydroxypolyangiate (ambruticin VS-5 methyl ester) was obtained as a yellow solid (0.12 g). [0194] To a solution of methyl (5S,6R)-5-amino-6-hydroxypolyangiate (95 mg, 0.2 mmol) in THF (6 mL) was added di-tert-butyl dicarbonate (85mg, 0.4 mmol) at 0 ⁰C. The mixture was allowed to warm to 25 ⁰C in 1.5 h, when TLC indicated that the reaction was complete. After the solvent was removed, the crude product was purified by flash chromatography on silica gel eluted with 0-30% EtOAc in hexane. Methyl (5S,6R)-5-tert- butoxycarbonylamino-6-hydroxyporyangiate was obtained as a light yellow gel (60 mg). ESI- TOF-MS m/z 610.3696, calcd for C₃₄H₅₃NO₇Na ([M + Na]⁺) 610.3669.

[0195] To a solution of methyl (5S,6R)-5-tert-butoxycarbonylamino-6-hydroxypoly- angiate (0.3 g, 0.5 mmol) in DCM (12 mL) cooled at -20 ⁰C was added 3-chloroperbenzoic acid (mCPBA) in portions. The reaction was monitored by TLC. Three portions of mCPBA (130 mg, 45 mg, and 45 mg, total 1.25 mmol) were added over a 5 h period to consume most of the starting material. TLC showed two major products and several minor products. The mixture was stirred with aqueous sodium thiosulfate for 20 minutes and was extracted with EtOAc. The combined extracts were washed with water, aqueous NaHCO₃, and brine, and dried over anhydrous Na₂SO₄. The drying agent was removed by filtration and the filtrate was evaporated to dryness. Flash chromatography of the crude product on silica gel column eluted with 0-50% EtOAc in hexane gave a mixture of the 16,17-epoxy derivatives, which was purified by reversed-phase HPLC on a Varian Metasil Basic column, eluted using a gradient of AcCN in water containing 0.1% acetic acid. Two products, methyl (5S,6R, 16R, 17i?)-5-tert-butoxycarbonylamino-6-hydroxy- 16,17-epxoy- 16,17-dihydro- polyangiate and methyl (5S,6R,l 6S,17_lS)-5-tert-butoxycarbonylamino-6-hydroxy- 16,17- epxoy-16,17-dihydropolyangiate, were obtained as white solids.

[0196] Compound V-a methyl ((5S,6R, 16R, 17i?)-5-tert-butoxycarbonylamino-6-hydroxy- 16,17-eρxoy-16,17-dihydroρolyangiate). ESI-TOF-MS m/z 626.3651, calcd for C₃₄H₅₃NO₈Na ([M + Na]⁺) 626.3663.

[0197] Compound V-b methyl ((methyl (5£,6i?,16S,17,S)-5-tert-butoxycarbonylammo-6- hydroxy- 16,17-epxoy- 16, 17-dihydroρolyangiate). ESI-TOF-MS m/z 626.3664, calcd for

C₃₄H₅₃NO₈Na ([M + Na]⁺) 626.3663.

[0198] Attempts to remove the Boc protecting groups under acidic conditions resulted in rearrangement of the epoxide and/or decomposition. An alternative route was used to obtain the unprotected 16,17-epoxy compounds.

Ambruticin VS-5

[0199] To a mixture of ambruticin VS-5 (0.5 g, 1 mmol) in mixed THF (6 mL) and 1 M aqueous sodium carbonate (2 mL) was slowly added a solution of Fmoc-OSu (0.5g, 1.5 mmol) in THF. The mixture was stirred overnight, maintaining the pH at ~9. The mixture was concentrated to remove THF and washed with ethyl ether. The aqueous layer (turbid) was acidified with 0.1 M aqueous HCl to pH ~5 and extracted with EtOAc. The combined EtOAc extracts were evaporated to dryness. The crude product was purified by flash chromatography on silica gel eluted with 20-30% EtOAc in hexane. A solid of 0.13 g of(5S,6R)- 5-(9-fluorenyl)methyloxycarbonylamino-6-hydroxypolyangioic acid (1) was obtained.

[0200] To a solution of 1 (0.13 g, 0.19 mmol) in THF (3 mL) cooled in an ice bath was added dropwisely a 2.0 M solution of (trimethylsilyl)diazomethane in hexanes (1 mL, 2 mmol) under nitrogen atmosphere. The mixture was allowed to warm to room temperature in 2 h. Water was added and the mixture was concentrated to remove the THF. The mixture was extracted with EtOAc. The combined EtOAc extracts were washed with 0.1 M aqueous HCl, saturated aqueous NaHCO₃, and brine, and dried over anhydrous Na₂SO₄. The drying agent was removed by filtration and the filtrate was evaporated to dryness. Methyl (5S,6i?)- 5-(9-fluorenyl)methyloxycarbonylamino-6-hydroxypolyangioate (2) was obtained as a solid (0.11 g). [0201 ] To a solution of 2 (0.11 g, 0.15 mmol) in DCM (2 mL) cooled at 0 ⁰C was added 3-chloroperbenzoic acid (røCPBA) in three 26 mg portions over 5 h. HPLC analysis showed that most of the starting material had been consumed. The mixture was stirred with aqueous sodium thiosulfate for 20 minutes and was extracted with DCM. The combined extracts were washed with water, aqueous NaHCO₃, and brine, and dried over anhydrous Na₂SO₄. The drying agent was removed by filtration and the filtrate was evaporated to dryness. Flash chromatography of the crude product on silica gel column eluted with 10-30% EtOAc in hexane gave a mixture of the 16,17-epoxy derivatives (3), 13 mg.

[0202] To a solution of 3 (13 mg) in THF (0.4 mL) was added 1 M aqueous lithium hydroxide (0.8 mL). After the mixture was stirred at room temperature for 3 h, it was concentrated on a rotary evaporator to remove THF. The crude mixture was purified by reverse- phase HPLC on a Varian Metasil Basic column, eluted using a gradient of 25 - 50% AcCN in water containing 0.1% acetic acid. Two products, (5S,6R,16R,l7R)-5-amino-6-h.yoxoxy- 16,17-epxoy-16,17-dihydropolyangioic acid and (5S,6R,l6S,17S)-5-annno-6-hydroxy-16,l7- epxoy-16,17-dihydropolyangioic acid, were obtained as white solids (3.7 mg and 2.5 mg).

[0203] Compound V-c methyl ((5S,6R, 16R, 17i?)-5-amino-6-hydroxy- 16, 17-epxoy- 16,17- dihydropolyangioic acid). ESI-TOF-MS m/z 512.2964, calcd for C₂₈H₄₃NO₆ ([M + H]⁺) 512.2983.

[0204] Compound V-d methyl ((5S,6R, 16S, 17,S)-5-amino-6-hydroxy- 16, 17-epxoy- 16,17- dihydropolyangioic acid). ESI-TOF-MS m/z 512.2958, calcd for C₂₈H₄₃NO₆ ([M + H]⁺) 512.2983.

Example 18

[0205] Several 20,21-dihydro analogs were synthesized from compound IV-a using the following procedure:

N ¹⁰aB ^eqH-₃ ^CC^HN^s,° HO ^(aA^qc^-), MeOH r "OH

Compound IV-a Compounc IV-b

2 eq. acetone LiAIH₄ NaBH₃CN THF HOAc, MeOH 80 ⁰C, 1 h 75% yield 62% yield

Compound IV-c

Compound IV-e

[0206] Compound IV-b was synthesized from compound IV-a using the scheme above. ESI-TOF-MS m/z 504.3676, calcd for C₃₀H₅₀NO₅ ([M + H]⁺) 504.3684.

[0207] Compound IV-c was synthesized from compound IV-b using the scheme above. ESI-TOF-MS m/z 490.3885, calcd for C₃₀H₅₂NO₄ ([M + H]⁺) 490.3891.

[0208] Compound IV-d was synthesized from compound IV-a using the scheme above. ESI-TOF-MS m/z 518.3838, calcd for C₃₁H₅₂NO₅([M + H]⁺) 518.3840.

[0209] Compound IV-e was synthesized from compound IV-d using the scheme above. ESI-TOF-MS m/z 532.3998, calcd for C₃₂H₅₄NO₅ ([M + H]⁺) 532.3997. [0210] Compound IV-b (20,21-Dihydroambruticin VS-3) is also isolated from a side stream in a large scale production of ambruticin VS-3. A mixture obtained from the wild- type strain that produces ambruticins VS-3, VS-4, and VS-5 are treated with excess formaldehyde, sodium cyanoborohydride, and acetic acid in methanol to convert all NH₂ and MeNH groups to Me₂N groups. Conversion a 19-g mixture yields ~14 g of purified ambruticin VS-3 and 280 mg of 20,21 -dihydroambruticin VS-3, together with other compounds. Example 19

[0211] C-I secondary alcohol analogs can be synthesized from ambruticin VS-3 using the following procedure.

[0212] Specifically, compound I-nnn ((5S, 6i?)-l-methyl-5-(dimethylammo)polyangi-l,6- diol, a mixture of Ii? and IS isomers) was synthesized from ambruticin VS-3 using the following procedure. To a suspension of ambruticin VS-3 (25 mg, 0.05 mmol) in ethyl ether (3 mL) was added 1.5 M methyllithium lithium bromide solution in ether (0.8 niL, 1.2 mmol). After the mixture was stirred at 20 ⁰C for 16 h, it was poured on ice- water. The mixture was extracted with 2 X 20 mL of ether and 2 X 20 mL of EtOAc. The combined organic solutions were dried over anhydrous MgSO₄. The drying agent was removed by filtration. The filtrate was evaporated to dryness in vacuo, giving (5»S',6i?)-l-methyl-5- dimethylamino-6-hydroxypolyangi-l-one (compound I-mmm) as a colorless gel (25 mg). One skilled in the art can isolate compound I-mmm using standard methods of the art. To a solution of this product in methanol (2 mL) was added sodium borohydride (19 mg, 0.5 mmol). The reaction was complete as indicated by HPLC analysis after mixture was stirred at 20 ⁰C for 3 h. The mixture was concentrated on a rotary evaporator, re-suspended in 1:3 water/AcCN, and filtered through a 0.2 μm PTFE filter. The crude product was purified by reversed-phase HPLC on a Varian Metasil Basic column, eluted using a gradient of AcCN in water containing 0.1% acetic acid. The product was obtained as a light yellow gel (15 mg) after lyophilization of desired fractions as determined by HPLC/MS. One skilled in the art can isolate and separate the Ii? and IS isomers using standard methods of the art. ESI-TOF- MS m/z 502.3852, calcd for C₃₁H₅₂NO₄ ([M + H]⁺) 502.3891.

Example 20 [0213] C-I tertiary alcohol analogs can be synthesized from ambruticin VS-3 using the following procedure. MeMgBr

^THF/ether

[0214] Specifically, compound I-ooo ((5S, 6i?)-l,l-dimethyl-5-(dimethylamino)polyangi- 1,6-diol) was synthesized from ambruticin VS-3 methyl ester using the following procedure.

[0215] To a solution of ambruticin VS-3 methyl ester (52 mg, 0.1 mmol) in THF was added 3 M methyl magesium bromide in ether (0.2 mL, 0.6 mmol). The mixture was stirred at room temperature for 30 min and concentrated on a rotary evaporator to dryness. The crude mixture was re-dissolved in 1 : 1 water/ AcCN, filtered, and purified by reversed-phase HPLC on a Varian Metasil Basic column, eluted using a gradient of AcCN in water containing 0.1% acetic acid. The product (compound I-ooo) was obtained as a white solid (15 mg) after lyophilization of desired fractions as determined by HPLC. ESI-TOF-MS m/z 516.4044, calcd for C₃₂H₅₄NO₄ ([M + H]⁺) 516.4047.

Example 21

[0216] Compound 1-111 ((5S, 6i?)-2-ethoxycarbonylamino-5-(dimethylamino)-l- norpolyangi-6-ol) can be synthesized from ambruticin VS-3 using the following procedure.

[0217] To a solution of ambruticin VS-3 (0.15 mmol) in benzene (5 mL) was added diphenylphosphoryl azide (0.17mmol) and triethylamine (0.17 mmol). The solution was stirred at 80 ⁰C for 1 h, then was added ethanol (3 mmol) to be stired at 80 ⁰C for 16 h. The reaction mixture was concentrated on a rotary evaporator, re-dissolved in a mixture of water- AcCN, filtered through a one-gram plug of C- 18 silica gel, and purified by reversed-phase HPLC, eluted using a gradient of AcCN in water containing 0.1% acetic acid. The product (compound 1-111) was obtained as a white solid after lyophilization of desired fractions.ESI- TOF-MS m/z 545.3933, calcd for C₃₂H₅₃N₂O₅ ([M + H]⁺) 545.3949. Example 22

[0218] The following describes the construction of the ambS, ambO, ambP and ambM mutants in Sorangium cellulosum So ce 10 and the analysis of ambruticin compounds so produced. [0219] The construction of the ambS, ambO, ambP and ambM mutants in Sorangium cellulosum So celO was peformed using the following method. The nucleotide sequence of the ambO, ambP, and ambM genes are disclosed in Reeves et al, US 2005/0266434 Al (2005), incorporated herein by reference. Gene regions were amplified by PCR and each amplicon was cloned into the EcoRV site of pKOS175-178, a plasmid that carries the oriT of R6K for conjugal transfer and the phleomycin resistance marker for selection in Sorangium. Primer sequences and plasmid names were as follows: ambM, TGATACAACGACGCTTACACG (SEQ ID NO:1) and CTAGCGGAACGACATGGTGAA (SEQ ID NO:2) to give pKOS546-28M; ambS, TAGGCCAGGTTGAGCCATGAG (SEQ ID NO:3) and CTATTGCTCTCTGGCCAGGAG (SEQ ID NO:4) to give pKOS375-155; ambO, TGAGCGGTCGGCGCCAGCTGG (SEQ ID NO:5) and TCACGTGAAGCGCGCCGCGTC (SEQ ID NO:6) to give pKOS375-189O ; ambP, TGACACCCGGTACTCCTCAGC (SEQ ID NO:7) and TCAGCGCTTGTCCGCCAGACG (SEQ ID NO:8) to give ρKOS375-189P. [0220] Each resulting plasmid was introduced by transformation into E. coli strain C- 2420 containing the helper conjugative plasmid pKOS 111-47. Development of a mariner- based transposon for use in Sorangium cellulosum. Appl Environ Microbiol 69, 6299-6301). The procedure for conjugation of the plasmid from E. coli to So celO was previously described (Jaoua et al. (1992) Plasmid 28, 157-165, "Transfer of mobilizable plasmids to Soran- gium cellulosum and evidence for their integration into the chromosome") and transconju- gants were selected on S42 agar containing kanamycin (50 μg/mL) and phleomycin (50 μg/mL) to give strains K546-40M2, K375-167.4, K546-32O2, and K546-5P3, respectively. The Sorangium cellulosum cells were maintained using the method of Hofle et al. (1991, Liebigs Ann Chem 1991, 941-945) and Gerth et al. (1996, JAntibiot (Tokyo) 49, 71-75. [0221 ] The analyses of anibruticin compounds produced by the various mutant strains were performed using the following method. Colonies were inoculated into seed medium (10 g/L maltodextrin, 4 g/L nonfat dry milk, 4 g/L soy peptone, 4 mL/L glycerol, 1 g/L CaCl₂-2H₂O, 1 g/L MgSO₄-7H₂0, 15 mg/L FeCl₃*6H₂O, 25 mM HEPES, pH 7.6) and grown for 2-3 days at 32°C. Production medium (10 g/L maltodextrin, 5 g/L Pharmamedia, 4 g/L nonfat dry milk, 4 g/L soy peptone, 4 mL/L glycerol, 1 g/L CaCl₂-2H₂O, 1 g/L MgSO₄-7H₂O, 120 mg/L FeCl₃ ^»6H₂O, 50 mM HEPES, pH 7.6) containing 40 g/L XADl 180 was inoculated with 10% seed culture and incubated at 32°C for 8 days. After washing the XAD resin twice with water, the ambruticin compounds were eluted with a volume of methanol equal to half the original culture volume.

[0222] To resolve ambraticins containing an amino group, they were chromatographed using the VS method: Agilent Nucleosil Cl 8 column (4 x 125 mm;), isocratic, 78% methanol, 10 mM ammonium acetate, pH 8.2, 1 rnLnrurT¹, detection at 220 nm. The VS compounds were quantitated from the area under the peak compared to purified standards. Ambruticin S and other ambruticins not containing an amino group were quantitated by the S method: Agilent Eclipse XDB-C8 column (4.6 x 150 mm), isocratic, 64% AcCN, 0.1% acetic acid, 1 mLnrώT¹, detection at 220 nm. For LC/MS analysis the separation method used a MetaChem Inertsil ODS-3 column (4 x 100 mm) with a gradient from 30% to 100% AcCN in 0.1% acetic acid at 1 mL'rnirT¹ on an Agilent 1100 system with a diode array detector connected to a Perseptive Biosystems Mariner biospectrometry workstation.

[0223] Analysis of the ambS mutant. Disruption of ambS, which encodes a methyl transferase homologue in the ambruticin cluster, gave a strain that no longer produced the N- methylated ambruticins VS-4, VS-3 or VS-I. Fig. 1 shows HPLC-UV analysis of extracts from the ambS^~ mutant and the wild type strain. The extracts were also analyzed by LC-MS, which verified the absence of the VS-4, VS-3 and VS-I compounds, and indicated that the small peak eluting after VS-5 had the mass of VS-5 plus two hydrogens. The amount of ambruticin VS-5 produced by the mutant was approximately equal to the sum of ambruticins VS-I, VS-3, VS-4 and VS-5 produced by the wild type strain under the same conditions, indicating that the AmbS protein catalyzes, as the final steps in the pathway, sequential N- methylations of VS-5 to give VS-4, VS-3 and VS-I. Ambruticin VS-5 and the compound two hydrogen atoms heavier were purified from a large scale fermentation of the ambST strain and the heavier compound was shown by NMR analysis to be 20,21-dihydroambruticin VS-5. Upon careful inspection of LC-MS data from cultures of the wild-type strain, very small amounts of all the 20,21-dihydroambruticins could be detected.

[0224] Analysis of the ambO and ambP mutant. The ambruticin gene clusters has a pair of adjacent genes in the same operon encoding a flavin monooxygenase (ambO) and a Rieske iron-sulfur cluster protein (ambP). When either the ambP or ambO genes of Sorangium cellulosum So eel 0 was disrupted, the resulting strains produced a set of ambruticins at similar levels to those produced by the wild type strain, except that each eluted later from the reverse-phase HPLC column and had a mass two hydrogen atoms heavier. LC- MS analysis verified that compounds having the mass of each of the ambruticins produced by the wild type were not detected in either mutant. The putative 20,21 -dihydroarnbruticin VS-5 peak co-eluted with a purified standard of this compound. Disruption of either ambP or ambO prevents formation of the 20,21-double bond. The relative level (and estimated absolute level) of each 20,21-dihydroambruticin was similar to that of each corresponding ambruticin produced by the wild type. [0225] Analysis of the arnbM mutant. An ambM mutant was constructed using the same procedure described above. The extract from four 500ml cultures of K546-40M2 was adjusted to 50% methanol, 50 mM ammonium acetate and loaded onto a 2.5 x 28 cm column of BakerBond C18. After washing with 50% methanol, 50 mM ammonium acetate, fractions were collected during elution with 80% methanol, 50 mM ammonium acetate at 6 mL/min. Fractions containing 27-norambruticin VS-3 were identified using the analytical HPLC method described above, pooled, and the solvent was exchanged over a 0.5 x 26 cm Baker- Bond Cl 8 column to remove the ammonium acetate. The material was dried, dissolved in CD₃OD and analyzed on a Bruker 400 MHz instrument. LC-MS analysis of the extracts indicated that each compound eluting earlier than each of the known ambruticins produced by the wild type had a mass that was 14 atomic mass units lighter, consistent with the loss of a methyl group. The most prevalent compound from the ambM mutant was purified and NMR spectroscopic analysis showed that it is 27-norambruticin VS-3 (compound III-a).

[0226] The AmbM protein is a C-methyltransferase, and the ambM mutant produces the set of ambruticins corresponding to those produced by the wild type strain, except that each is missing the C27 methyl group (for example, 27-norambruticin VS-3, 27-norambruticin VS-4, and 27-norambruticin VS-5). Example 23

[0227] The following is a method for constructing a strain of Sorangium cellulosum So celO that produces compound III-a (24-norambruticin VS-3).

[0228] Replacement of the ambruticin loading AT with the one from module 7. The nucleotide sequence of the ambruticin loading acyltransferase (AT) and the AT of module 7 of the ambruticin PKS gene cluster is disclosed in Reeves et al, US 2005/0266424 Al (2005). The loading module was targeted for engineering to make compound III-a (24- norambruticin VS-3). To alter this AT, ambruticin modules containing malonate specific ATs were examined for similarities in reductive domains to those found in the loading module. The most similar was module 7.

[0229] Fig. 1 shows the boundaries at the amino and carboxy terminal of the 2 ATs. Alignment of the boundaries between the KS and AT domains of modules 0 and 7 from the ambruticin PKS. The top box shows the KS domain and the bottom box shows the AT domain. The arrows show the boundaries chosen for AT swaps: #1 is between KS and AT domains, and #2 is at the end of the ATs.

[0230] Plasmid pKOS396- 185A contains the malonate specific AT from module 7 engineered into the loading module. The plasmid was integrated by homologous recombination that creates an inactive native ambA that allows for expression of the downstream ambruticin genes and expresses the engineered ambA. [0231] Plasmid pKOS396-185A was constructed in several steps. To engineer the AT from module 7 into the loading module, 2 PCR fragments were generated to produce the right and left boundaries of the swap; the left contains the KS-AT boundary and the right contains the AT-ACP boundary. The right fragment was amplified using plasmid pKOS344-l 12E and the oligo pair 5'-TTTTAATTAAGAGGAGCATATGGATCCGCAGC (SEQ ID NO: 9) (Pad restriction sites underlined) and 5'-GCCCGCGGCGGTTCCGGGGCCTCCTCGGACACCACATGC (SEQ ID NO: 10).

[0232] The left fragment was amplified using the same plasmid and oligo pair 5'-GCCATGTGGTGCTCGAGGAGGCCCCGGAACCGCCGCGGGC (SEQ ID NO: 11) and 5 '-TTTCTAGACCTAGGGCCATTGAGCGCCG (SEQ ID NO: 12)

(A vrll restriction site underlined).

[0233] The PCR products from these two PCR reactions were joined together using the two products as the template and the two oligos containing the Pad and _^4vrII restriction sites as primers. The left fragment was amplified in an identical manner. First, two PCR reactions were performed using plasmid pKOS344-l 12E and the oligo pair 5'-AATGGCCCTAGGCAGACCGTCGTCAG (SEQ ID NO: 13) (Avrll restriction sites underlined) and 5'-TAGCGCTGGCGCTGGAATGCGTAGGTCGGCAGCTCCACCC (SEQ ID NO: 14); and the other reaction with the oligo pair

5'-GGGTGGAGCTGCCGACCTACGCATTCCAGCGCCAGCGCTA (SEQ ID NO: 15) and

5'-TTTCTAGAGATCTAGACGAGCGCATCGATG (SEQ ID NO: 16) (BgIR restriction site underlined) and they were joined together using the products as templates and the oligos with the restriction sites as primers. The final PCR products were ligated into pCRscript vector (Stratagene) and the DNA sequence was confirmed. The right fragment cleaved with Pαcl-^lvrll and the left fragment with AvrTL-BglR were ligated to pKOS249-51 cleaved with Pacl-Bgϊil to give pKOS396-185A. This plasmid contains the oriT, which is required for conjugative transfer from E. coli to S. cellulosum. It also contains the Mx9 integrase gene and attP site for site specific recombination in M. xanthus but appears not to function in So eel O, likely due to an inadequate Mx9 attB site in the chromosome. Besides having the engineered AT, pKOS396- 185A also contains truncation in the 5' region of ambA and has the promoter for the epothilone biosynthetic gene positioned just upstream of the engineered ambA. [0234] Integration of pKOS396-185A into So eel O. Transformation of Sorangium cellulosum So celO with pKOS396-185A was performed as described using E. coli donor cells C2420 containing the helper plasmid pKOSl 11-47. Selection was done on S42 medium containing hygromycin (60 μg/mL) and kanamycin (50 μg/mL).

[0235] Production of compound III-a (24-norambruticin VS-3). Ten independent isolates were tested for production. Seed cultures were grown in 25 mL of C307 (per liter 1Og potato starch-soluble (Sigma), 1 g glucose, 5 g select Soytone (Difco), 2 g yeast extract (Fisher), 1 g MgSO₄-7H₂0, 1 g CaCl₂-2H₂O and 0.008g Fe citrate) in unbaffled 250 mL flasks for two days at 32 ⁰C. A 10% v/v inoculum was diluted into 50 mL production media (per liter 5 g maltodextrin DEl 8 (Cerestar), 2.5 g soy peptone (Marcor), 0.5 g MgSO₄ ^»7H₂O, 0.25 g K₂HPO₄, 50 mM HEPES pH 7.6, 1 g ferric citrate and 10 g XAD 1180) seven days at 32°C. After the fermentation, products were eluted from the XAD using 5 mL methanol. [0236] A method for producing and purifying compound III-a is as follows: Seed cultures were inoculated from cells spread on S42 plates containing 200 mg/L hygromycin. A 25-mL tube with five mL of CF9 medium (Fructose 6 g/L, Casitone (Difco) 9 g/L, MgSO₄-7H₂O g/L, CaCl₂-2H₂O 0.5 g/L, and HEPES (1.0 M, pH 7.6, KOH) 25 mL/L) containing hygromycin (200 μg/mL) was inoculated with a 1 cm² patch from S42 plates. A ten percent inoculum was used to expand the seed into a 250-mL unbaffled Erlenmeyer flask containing 50 mL of CF9 medium with hygromycin. The flasks were incubated at 32°C and 190 rpm on a 2-inch throw shaker for three days. The secondary seed culture was transferred (10% v/v) into a 2.8-L unbaffled Fernbach flask containing 500 mL of CF9-H medium. The Fernbach flasks were incubated at 32°C and 190 rpm on a 2-inch throw shaker for three days. The cultures at all seed stages grew as dispersed cultures.

[0237] Production Culture. Two seed cultures were prepared as described above. Each 1-L seed culture was inoculated into a 20-L BiofloIV bioreactor containing 11.0 L of production medium SF-IP (Fructose g/L, Soy Peptone (Marcor) 3 g/L, MgSO₄-7H₂O 1 g/L, CaCl₂-2H₂O 1 g/L, FeCl₃-6H₂O(14.6 g/L in 10 mL/L cone. H₂SO₄) 8 mL/L, XAD-4 20 g/L). The pH of the fermentation was maintained at 7.1 with 2.5 N KOH or 2.5 N H₂SO₄. Airflow was set at 4 L/min, agitation rate at 100 rpm, and overhead pressure at 3 psi. Dissolved oxygen was controlled at 40%. Temperature was controlled at 32.O⁰C. Cognis Clerol FBA 5059 antifoam was added to prevent foam formation as needed. The culture was fed 3.0 g/L/D fructose and 1.5 g/L/D soytone starting at 48 hours after inoculation and continuing until the end of the fermentation.

[0238] Isolation. The XAD-1180 resin (200 mL) was removed from the whole broth by sedimentation. The resin was packed in a glass column (4.5 cm diameter, 28 cm long) and washed with 10 column volumes of water, then with five column volumes of 40%(v/v) methanol: water. The XAD-1180 column was eluted with five column volumes of 100% methanol. The methanol concentration of the eluted fraction was adjusted to 40% (v/v) with water. This solution was loaded at 15 mL/min onto a preconditioned C18 column (2.5 cm diameter, 20 cm long), washed with three column volumes of 40% (v/v) methanol:water, and eluted with 78% (v/v) methanol:50mM ammonium acetate buffer pH 8.2. Fractions (25 mL) were collected and analyzed by mass spectrometry, and those containing the desired compound were combined. Fractions 7-17 from the Cl 8 chromatography were pooled (275 mL). The methanol concentration of the pooled fractions was adjusted to 40% (v/v) with water and loaded onto a preconditioned HP20SS column (2.5 cm diameter, 20 cm long). The column was washed with two column volumes of 40% (v/v) methanol, then 10 column volumes of water. The product was eluted with 100% methanol (200 mL). The material was then dried resulting in 7 mg of a yellow solid. [0239] Final purification was performed by preparative HPLC using a Metachem Polaris column (2.12 cm diameter, 25 cm long) eluted with 78% (v/v) methanol:50mM NH₄OAc buffer pH 8.2. Fractions (10 mL) were collected and analyzed by LC/MS. A final desalting step was performed using a preconditioned HP20SS column (1 cm diameter, 3 cm long). The methanol concentration of the pooled fractions (11-14) from the preparative HPLC was adjusted to 40% (v/v) with water and loaded onto the HP20SS column. The column was washed with 10 column volumes of water and eluted with 10 column volumes of methanol. The product-containing eluate was dried yielding 4.7 mg of solids with a purity of 96% (Figure 11.20).

[0240] 24-norambruticin VS-3 analysis. Samples (50 mL) were taken from the bioreactors using 50-mL conical tubes. Medium in the tube was decanted and The resin was stored at -2O⁰C. To elute the compounds, the resin was first washed with 50 mL of water, and then five mL of methanol were added. The XAD extracts were assayed in a Hewlett Packard 1090 HPLC with UV detection at 210 nm. Twenty-five microliters of the supernatant were injected across a 4.6 x 150 mm, 3.5 Nucleosil column (Agilent) with 78:22 (v/v) methanol:50 niM ammonium acetate, pH 8.2 as the solvent. Under these conditions 24- norambruticin VS-3 was detected at 3.9 min. Compound πi-a (24-norambruticin VS-3) was successfully isolated to purity greater than 95%. The isolation involved Cl 8 chromatography followed by HP20SS chromatography then preparative HPLC. Preparative HPLC was needed to separate impurities that could not be resolved with low-pressure chromatography. The overall yield of the purification was 57%. Alternately, the HP20SS chromatography can be performed before the C 18, thereby eliminating the need for the Cl 8 step. Example 24

[0241] The antifungal activity of several compounds of this invention against Coccidioides immitis (and, for comparison purposes, against several other species of fungus) to compounds of this invention were evaluated at the Fungal Testing Laboratory (FTL) of the University of Texas Health Sciences Center at San Antonio, Texas.

[0242] Testing involved a total of 14 clinical isolates, distributed as follows: Candida albicans (1), Aspergillus fumigatus (2), A.flavus (2), Blastomyces derniatididis (3), Histoplasma capsulatum (3), and (3). The methods outlined in the National Committee for Clinical Laboratory Standards (NCCLS), M-27A2, "Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts; Approved Standard," and M38-A, "Reference Method for Broth Dilution Antifungal Susceptibility Testing of Conidium-Forming Filamentous Fungi; Approved Standard." The methodology included testing in RPMI- 1640 with glutamine and without bicarbonate, an inoculum size of 0.5-2.5 x 10³ for yeasts and 0.4-5 x 10⁴ for moulds, and incubation at 35 °C for 48 h for most isolates. (B. dermatitidis and H. capsulatum were incubated at 30 °C for 96 h.) The minimum inhibitory concentration (MIC) was defined as the lowest concentration that resulted in an 80% reduction in turbidity as compared to a drug-free control tube. Drug concentrations were 0.15-10 μg/mL for all compounds. The test medium was the NCCLS-recommended medium of RPMI-1640 (Hardy Diagnostics, Santa Monica, CA). [0243] Sample preparations were as follows: For Ambruticin VS-4, a 5 mg sample was weighed out and added to 0.5 mL of dimethylsulfoxide (DMSO) containing 0.5% trifluoroacetic acid (TFA). The resulting solution, having a concentration of 10,000 μg/mL, was diluted to a working concentration of 1,000 μg/mL in the same DMSO/TFA solvent. Subsequent dilutions were then made in the same solvent. Final testing concentrations ranged from 0.15 to 10 g/mL. For compound IV-a, a 4.3 mg sample was weighed out and added to 0.43 mL of the same DMSO/TFA solvent. Dilution as before again yielded final testing concentrations of 0.15-10 μg/mL.

[0244] MICs were determined at the first 24 h interval where growth could be determined in the drug-free control tube and again 24 h following the first reading, with the second reading being the reported one, as prescribed by NCCLS. The MIC endpoint used was the lowest concentration that exhibited an 80% reduction in turbidity as compared to the drug- free, diluent-positive, growth control. Results are presented in Table F. Table F — Antifungal MICs for Ambruticins

Ambruticin VS-4 Compound IV-a

Fungus (isolate no.) (μg/mL) (μg/mL)

Aspergillus flavus (05-1174) <0.15 <0.15

Aspergillus flavus (05-1326) >10 >10

Aspergillus fumigatus (05-1465) <0.15 <0.15

Aspergillus fumigatus (05-1484) <0.15 <0.15

Candida albicans (05-1422) <0.15 <0.15

Blastomyces dermatitidis(05-l 06) <0.15 <0.15

Blastomyces dermatitidis(05- 186) <0.15 <0.15

Blastomyces dermatitidis(05 -1291) <0.15 <0.15

Histoplasma capsulatum (05-959) <0.15 <0.15

Histoplasma capsulatum (05-1097) ≤0.15 <0.15

Histoplasma capsulatum (05-1159) <0.15 <0.15

Coccidioides immitis (05-469) <0.15 <0.15

Coccidioides immitis (05-955) <0.15 <0.15

Coccidioides immitis (05-1387) <0.15 <0.15

[0245] Using related methodology, the MIC results shown in Table G were obtained at the University of Arizona.

Table G — Additional MIC Data

C. posadasii strain C. intimitis strain

Drug RMSCC

Silveira C735 RS S46 204305

2127

Amphotericin B 1 1 1 1 1 1 (comparative)

Ambruticin S 2 4 2 2 4 2 (comparative)

Fluconazole 4 16 16 16 16 16 (comparative)

Compound I-d 0.25 0.25 0.125 0.25 0.125 0.25

Compound I-z 0.25 0.125 0.25 0.25 0.25 0.125

Compound I-v 0.25 0.125 0.125 0.25 0.125 0.125

Compound I-t 0.25 0.25 0.125 0.25 0.125 0.125

Compound I-ff 0.5 0.5 0.25 0.25 0.25 0.125

Compound I-oo 0.5 0.5 0.5 0.25 0.25 0.25

Compound I-mm O.0625 O.0625 O.0625 O.0625 <0.0625 O.0625

Example 25

[0246] This example describes in vivo experiments performed at the University of Arizona on the effectiveness of two compounds of this invention (I-v and I-ff) in treating C. posadasii infections in mice.

[0247] Female mice (C57BL/6 females, 8-10 weeks old, from Harlan-Sprague-Dawley) were each infected intranasally with 54 arthrocondia of C. posadasii (strain Silveira), under anesthesia with ketamine-xylazine. The target dose was 50 arthrospores, which is a lethal dose for this strain of mice. [0248] Treatment with the compounds began on day 6 after infection. The compounds were administered in a vehicle of 10% 2-hydroxypropyl-β-cyclodextrin in ethanol. The mice were gavaged with 0.2 mL of compound solution (or vehicle as control) twice daily from Monday through Friday and once on weekends for a total of 19 days of treatment. The mice were observed for an additional 24 days after completion of the treatment cycle and then sacrificed, making it a total of 49 days from infection to sacrifice. A total of 48 mice were used, grouped as shown in Table H:

[0249] None of the mice treated with compound I-v or I-ff died while receiving treatment (except two that died from the gavaging procedure). Weights of mice treated with compound I-v were stable during the treatment period but began to fall when the drug was withdrawn. These mice began to die within 10 days of discontinuation of treatment, with 40% of the mice treated with a 20 mg/kg dose and 50% of the mice treated with a 50 mg/kg dose dieing before the scheduled sacrifice on day 49. All the mice treated with compound I-ff survived until the scheduled sacrifice. Of the control mice, only two survived to the scheduled sacrifice.

[0250] At sacrifice, most mice exhibited gross evidence of disease. Two control mice, two compound I-ff treated mice (at 50 mg/kg), and one compound I-v treated mouse (at 20 mg/kg) had no obvious granulomas and their organ cultures were negative, indicating that they had not been infected. These mice were removed from the data set for organ culture and survival analysis.

[0251] Statistical survival analysis confirmed that treated animals had improved survival compared to control animals, though some treated animals succumbed to infection after withdrawal of treatment, as noted above. Organ culture statistical analysis showed that treatment with compound I-ff at 50 mg/kg appeared to have effected a fungal cure. Treatment with compound I-ff at 20 mg/kg or with compound I-v at 20 or 50 mg/kg prolonged survival and prevented death during treatment, but did not appear to effect a fungal cure. [0252] The foregoing detailed description of the invention includes passages that are chiefly or exclusively concerned with particular parts or aspects of the invention. It is to be understood that this is for clarity and convenience, that a particular feature may be relevant in more than just the passage in which it is disclosed, and that the disclosure herein includes all the appropriate combinations of information found in the different passages. Similarly, although the various figures and descriptions herein relate to specific embodiments of the invention, it is to be understood that where a specific feature is disclosed in the context of a particular figure or embodiment, such feature can also be used, to the extent appropriate, in the context of another figure or embodiment, in combination with another feature, or in the invention in general.

[0253] Further, while the present invention has been particularly described in terms of certain preferred embodiments, the invention is not limited to such preferred embodiments. Rather, the scope of the invention is defined by the appended claims.

Claims

CLAIMSWhat is claimed is:

1. A method for treating or reducing the probability of a Coccidioides infection in a subject in need of such treatment or reduction of probability, comprising administering to such subject a pharmaceutically effective amount of a compound represented by formula I

R¹⁰ and R¹¹ are independently H or CH₃; X¹ is either a bond or O;

X² and X³ are each H or together are a bond;

_R R_l i.s

, , or

; R² and R³ are independently H, C₁-C₅ alkyl, C₂-C₅ alkenyl, C₂-C₅ alkynyl, cycloalkyl, arylCCi-Cs alkyl), aryl(C₂-C₅ alkenyl), aryl(C₂-C₅ alkynyl), cycloa]kyl(Ci-C₅ alkyl), cycloalkyl(C₂-C₅ alkenyl), cycloalkyl(C₂-C₅ alkynyl), , or ;

R⁴ is H₅ , or ; or R³ and R⁴ combine to form ;

R⁹ is, independently for each occurrence thereof, H, C₁-C₅ alkyl, C₂-C₅ alkenyl, C₂-C₅ alkynyl, cycloalkyl, 8TyI(C₁-C₅ alkyl), aryl(C₂-C₅ alkenyl), 8TyI(C₂-C₅ alkynyl), cycloalky^Q-Cs alkyl), cycloalkyl(C₂-C₅ alkenyl), or cycloalkyl(C₂-C₅ alkynyl), provided that R⁹ is not H when Z is O;

R¹² and R¹³ together are O, or R¹² is H and R¹³ is R⁵;

Y is O or N-OR⁵; and

Z is, independently for each occurrence thereof, O or NH.

2. The method of claim 1, wherein said compound represented by formula I has a structure represented by formula II:

3. The method of claim 1, wherein said compound represented by formula I has a structure represented by formula I-A:

4. The method of claim 1 , wherein said compound represented by formula I has a structure represented by formula H-A:

5. The method of claim 1 , wherein the compound represented by formula I has a structure represented by formula H-D

6. The method of claim 1 , wherein the compound represented by formula I is ambruticin VS-3, ambruticin VS-4, or ambruticin VS-5.

7. The method of claim 1, wherein R² is H, CH₃, aryl(CH₂), cycloalkyl(CH₂), or cycloalkyl; and R³ is C₂-C₅ alkyl, aryl(CH₂), cycloalkyl(CH₂), or cycloalkyl.

8. The method of claim 1, wherein R³ is CH₃CH₂, CH₂CH₂OH, (CHj)₂CH, CH₃CH₂CH₂, CH₃CH₂CH₂CH₂, COCF₃, CH₂CH₂F₅CH₂CHF₂, CH₂CF₃,

or

9. The method of claim 3, wherein R⁴ is H, and R¹, R² and R³ are according to the combinations set forth in the following table:

10. The method of claim 1, wherein the compound represented by formula I is 20,21- dihydroambruticin VS-3.

11. The method of claim 1 , wherein the Coccidioides infection is coccidioidomycosis.

12. The method of claim 1 , wherein the subj ect is immunocompromised.

13. The method of claim 1 , wherein said administering results in the inhibition of the growth of the Coccidioides spp. in the subject.

14. The method of claim 13 , wherein said administering results in the subj ect becoming cleared of the Coccidioides infection.

15. The use of a compound according to claim 1 for the preparation of a medicament for treating or reducing the probability of a Coccidioides infection.

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