WO2000076316A1

WO2000076316A1 - Methods related to metabolism of parasites and mycobacteria

Info

Publication number: WO2000076316A1
Application number: PCT/US2000/016709
Authority: WO
Inventors: Glenn D. Prestwich; Frederick S. Buckner; Jerald C. Hinshaw
Original assignee: University of Utah Research Foundation Inc
Current assignee: University of Utah Research Foundation Inc
Priority date: 1999-06-16
Filing date: 2000-06-16
Publication date: 2000-12-21
Anticipated expiration: 2001-12-16
Also published as: AU5876800A

Abstract

The present invention relates to the field of microorganism metabolism. In one aspect, the present invention relates to parasite and mycobacterial steroid compound biosynthesis, including methods to inhibit the steroid compound biosynthesis. The present invention therefore relates broadly to microbiology, pharmaceutical chemistery, and disease treatments.

Description

Methods Related to Metabolism of Parasites and Mycobacteria

The present invention was supported in part by a grant from the National Institutes of Health, Grant Number GM48632; the U.S. Government may have certain rights in this invention. This application claims priority to US provisional Patent Applications 60/139,418, filed June 16, 1999, 60/140,071, filed June 21, 1999 and 60/140,578, filed June 23, 1999.

FIELD OF THE INVENTION

The present invention relates to the field of microorganismmetabolism. In one aspect, the present invention relates to parasite and mycobacterial steroid compound biosynthesis, including methods to inhibit the steroid compound biosynthesis. The present invention therefore relates broadly to microbiology, pharmaceutical chemistry, and disease treatments.

BACKGROUND OF THE INVENTION

Although sterol biosynthesis has been studied extensively in mammals, eubacteria and yeast, relatively little is known about sterol biosynthesis in parasites and mycobacteria. Speculation regarding the reasons for so little research runs the gamut from lack of financial backing to fight organisms primarily present in third world countries, to difficulty in culturing these wiley types.

Research in the field of parasite sterol biosynthesis has revealed more clues than that of mycobacterial research. For instance, it is known that most parasites synthesize steroids, and that inhibition of certain enzymes has been successful in lowering the numbers of parasites in culture. Researchers have not fully characterized the parasitic sterol biosynthesis pathway in such a way as to identify a parasite-equivalent to the oxidosqualene cyclase found in humans, eubacteria and yeast. Until the present disclosure, it was uncertain whether parasites borrowed the precursor or precursor-converting enzyme from host tissues.

Likewise, oxidosqualene cyclase activity had not been identified in mycobacteria. In the case of mycobacteria, however, sterol biosynthesis itself had not been confirmed. Conflicting reports had fueled the debate over the existence of mycobacterial sterol biosynthesis, but those in the art continued to question whether mycobacteria borrowed steroid compounds from their host or made them. Citation of the above documents is not intended as an admission that any of the foregoing is prior art. All statements as to the date or representation as to the contents of these documents is based on subjective characterization of information available to the applicant, and does not constitute any admission as to the accuracy of the dates or contents of the documents.

SUMMARY OF THE INVENTION

The present invention provides methods to inhibit physical development of at least one parasite, comprising affecting parasitic oxidosqualene cyclase activity of said parasite.

Also provided are methods to alter sterol biosynthesis in a parasite, comprising inhibiting parasitic oxidosqualene cyclase activity of said parasite.

Also provided are methods to treat parasite infection in a mammal in need of such treatment, comprising administering to said mammal a pharmaceutically-acceptable parasitic oxidosqualene cyclase inhibitor.

In particular, there are provided such methods wherein said oxidosqualene cyclase inhibitor is a compound selected from the group consisting of: [4'-(6-allyl-methyl-amino-hexyloxy)- 2-fluoro-phenyl]-(4-bromophenyl)-methanone; trans-N-(4-chlorobenzoyl)-N-methyl-(4- dimethylaminomethylphenyl)-cyclohexylamine; 2-Nitro-N-(phenylmethyl)- 1 H-imidazole- 1 - acetamide; N-benyl-2-nitroimidazole-l-acetamide; 3-[(4-chlorobenzoyl)-4- phenoxy quinuclidine; (Z)-3-[4-(4-Bromobenzoyl)pheacylidene]quinuclidine. Those methods wherein said oxidosqualene cyclase inhibitor is [4'-(6-allyl-methyl-amino- hexyloxy)-2-fluoro-phenyl]-(4-bromophenyl)-methanone are preferred. However, particularly provided are those methods wherein the parasite is selected from the group consisting of: nematodes; trematodes; and parasitic protozoa. Those methods wherein said parasite is selected from the group consisting of: Ascaris lumbricoides; Ancylostoma duodenale; Necator americanus; Trichuria trichuria; Enterobius vermicularis; Strongyloides stercoralis; Wuchereria Bancrofti; Brugia malayi; Brugia timori; onchocerciasis; Onchocerca; Trichinella spiralis; Schistosoma spp; Trypanosoma cruzi; Leishmania spp; Trypanosoma brucei; Naegleria; and Acanthamoeba are preferred. More preferred are those wherein said parasite is selected from the group consisting of: T. cruzi; and L. mexicana. Preferred methods are those wherein said treatment further comprises and adjuvant, particularly those wherein said adjuvant is selected from the group consisting of: an antimonial; amphotericin B; allopurinol; pentamidine; an azole anti-fungal; an aromatic antifungal; ketoconazole; itraconazole; and sodium stibogluconate.

The present invention additionally provides methods to affect physical development of at least one mycobacterium, comprising affecting mycobacterial sterol biosynthesis of said mycobacterium.

Also provided are methods to treat mycobacterial infection in a mammal in need of such treatment, comprising administering a pharmaceutically acceptable oxidosqualene cyclase inhibitor.

Also provided are methods to treat tuberculosis in a mammal in need of such treatment, comprising administering a pharmaceutically acceptable oxidosqualene cyclase inhibitor.

In particular, there are provided such methods wherein said oxidosqualene cyclase inhibitor is a compound selected from the group consisting of: [4'-(6-allyl-methyl-amino-hexyloxy)- 2-fluoro-phenyl]-(4-bromophenyl)-methanone; trans-N-(4-chlorobenzoyl)-N-methyl-(4- dimethylaminomethylphenyl)-cyclohexylamine; 2-Nitro-N-(phenylmethyl)-lH-imidazole-l- acetamide; N-benyl-2-nitroimidazole-l-acetamide; 3-[(4-chlorobenzoyl)-4- phenoxy]quinuclidine; (Z)-3-[4-(4-Bromobenzoyl)pheacylidene]quinuclidine. Those methods wherein said oxidosqualene cyclase inhibitor is [4'-(6-allyl-methyl-amino- hexyloxy)-2-fluoro-phenyl]-(4-bromophenyl)-methanone are preferred. However, particularly provided are those methods wherein the mycobacterium is selected from the group consisting of: Mycobacterium tuberculosis; Mycobacterium avium complex; and Mycobacterium leprae are provided, with Mycobacterium tuberculosis being preferred.

Preferred methods are those wherein said treatment further comprises and adjuvant, particularly those wherein said adjuvant is selected from the group consisting of: an antimonial; amphotericin B; allopurinol; pentamidine; an azole anti-fungal; an aromatic antifungal; ketoconazole; itraconazole; and sodium stibogluconate.

Moreover, for the purposes of the present invention, the term "a" or "an" entity refers to one or more of that entity; for example, "a protein" or "a nucleic acid molecule" refers to one or more of those compounds or at least one compound. As such, the terms

"a" (or "an"), "one or more" and "at least one" can be used interchangeably herein. It is also to be noted that the terms "comprising", "including", and "having" can be used interchangeably. Furthermore, a compound "selected from the group consisting of refers to one or more of the compounds in the list that follows, including mixtures (i.e., combinations) of two or more of the compounds. According to the present invention, an isolated, or biologically pure, protein or nucleic acid molecule is a compound that has been removed from its natural milieu. As such, "isolated" and "biologically pure" do not necessarily reflect the extent to which the compound has been purified. An isolated compound of the present invention can be obtained from its natural source, can be produced using molecular biology techniques or can be produced by chemical synthesis.

Definitions:

"Affecting" means any change, including increase, decrease, change in structure, change in the ability to react with other compounds, change in potentcy, etc.

"Inhibiting" means negatively impacting in the form of reduction of numbers, activity, ability to interact, etc.

"Mycobacteria" includes all species of the genus Mycobacterium, as that term is generally accepted by those in the relevant art.

"Oxidosqualene cyclase" means either a) and enzyme with oxidosqualene cyclase activity or b) an enzyme that is inhibited by known oxidosqualene cyclase inhibitors, such as Ro 48- 8071.

"Parasite" means an organism which requires, for at least a portion of its lifecycle, a host to survive, not including viruses or bacteria. "Parasitic" does not include mammalian, amphibian or reptilian carnivorous or herbivorous behaviour.

Brief Description of the Drawings

Figure 1. Inhibition of Mycobacterium tuberculosis H37Ra by OSC inhibitors DETAILED DESCRIPTION OF THE INVENTION

Also provided are methods to alter sterol biosynthesis in a parasite, comprising inhibiting parasitic oxidosqualene cyclase activy of said parasite.

In particular, there are provided such methods wherein said oxidosqualene cyclase inhibitor is a compound selected from the group consisting of: [4'-(6-allyl-methyl-amino-hexyloxy)- 2-fluoro-phenyl]-(4-bromophenyl)-mefhanone; trans-N-(4-chlorobenzoyl)-N-methyl-(4- dimethylaminomethylphenyl)-cyclohexylamine; 2-Nitro-N-(phenylmethyl)-lH-imidazole-l- acetamide; N-benyl-2-nitroimidazole-l-acetamide; 3-[(4-chlorobenzoyl)-4- phenoxy]quinuclidine; (Z)-3-[4-(4-Bromobenzoyl)pheacylidene]quinuclidine. Those methods wherein said oxidosqualene cyclase inhibitor is [4'-(6-allyl-methyl-amino- hexyloxy)-2-fluoro-phenyl]-(4-bromophenyl)-methanone are preferred. However, particularly provided are those methods wherein the parasite is selected from the group consisting of: nematodes; trematodes; and parasitic protozoa. Those methods wherein said parasite is selected from the group consisting of: Ascaris lumbricoides; Ancylostoma duodenale; Necator americanus; Trichuria trichuria; Enterobius vermicularis; Strongyloides stercoralis; Wuchereria Bancrofti; Brugia malayi; Brugia timori; onchocerciasis; Onchocerca; Trichinella spiralis; Schistosoma spp; Trypanosoma cruzi; Leishmania spp; Trypanosoma brucei; Naegleria; and Acanthamoeba are preferred. More preferred are those wherein said parasite is selected from the group consisting of: : T. cruzi; and L. mexicana.

Preferred methods are those wherein said treatment further comprises and adjuvant, particularly those wherein said adjuvant is selected from the group consisting of: an antimonial; amphotericin B; allopurinol; pentamidine; an azole anti-fungal; an aromatic antifungal; ketoconazole; itraconazole; and sodium stibogluconate. The present invention additionally provides methods to affect physical development of at least one mycobacterium, comprising affecting mycobacterial sterol biosynthesis of said mycobacterium.

In particular, there are provided such methods wherein said oxidosqualene cyclase inhibitor is a compound selected from the group consisting of: [4'-(6-allyl-methyl-amino-hexyloxy)- 2-fluoro-phenyl]-(4-bromophenyl)-methanone; trans-N-(4-chlorobenzoyl)-N-methyl-(4- dimethylaminomethylphenyl)-cyclohexylamine; 2-Nitro-N-(phenylmethyl)- 1 H-imidazole- 1 - acetamide; N-benyl-2-nitroimidazole-l-acetamide; 3-[(4-chlorobenzoyl)-4- phenoxyjquinuclidine; (Z)-3-[4-(4-Bromobenzoyl)pheacylidene]quinuclidine. Those methods wherein said oxidosqualene cyclase inhibitor is [4'-(6-allyl-methyl-amino- hexyloxy)-2-fluoro-phenyl]-(4-bromophenyl)-methanone are preferred. However, particularly provided are those methods wherein wherein the mycobacterium is selected from the group consisting of: Mycobacterium tuberculosis; Mycobacterium avium complex; and Mycobacterium leprae are provided, with Mycobacterium tuberculosis being preferred.

Virtually any cholesterol-lowering agent is a candidate in the present invention. In that regard, issued US patents which are hereby incorporated by reference in their entirety into this patent application are: US Patent Serial Number 5574071, 5637771, 5495048, 5856503, 5731,323, and 5714496.

In the present methods, therapeutic compositions can be formulated in an excipient that the animal to be treated can tolerate. Examples of such excipients include water, saline, Ringer's solution, dextrose solution, Hank's solution, and other aqueous physiologically balanced salt solutions. Nonaqueous vehicles, such as fixed oils, sesame oil, ethyl oleate, or triglycerides may also be used. Other useful formulations include suspensions containing viscosity enhancing agents, such as sodium carboxymethylcellulose, sorbitol, or dextran. Excipients can also contain minor amounts of additives, such as substances that enhance isotonicity and chemical stability. Examples of buffers include phosphate buffer, bicarbonate buffer and Tris buffer, while examples of preservatives include thimerosal, cresols, formalin and benzyl alcohol. Standard formulations can either be liquid injectables or solids which can be taken up in a suitable liquid as a suspension or solution for injection. Thus, in a non-liquid formulation, the excipient can comprise dextrose, human serum albumin, preservatives, etc., to which sterile water or saline can be added prior to administration.

Administration of the compounds can be by a variety of routes known to those skilled in the art including, but not limited to, subcutaneous, intradermal, intravenous, intranasal, oral, transdermal, intramuscular routes and other parenteral routes.

In one embodiment of the present methods, a therapeutic composition can include an adjuvant. Adjuvants are agents that are capable of increasing the immune response of an animal to a specific antigen. Protein adjuvants of the present invention can be delivered in the form of the protein themselves or of nucleic acid molecules encoding such proteins using the methods described herein.

In another embodiment of the present methods, a therapeutic composition can include a carrier. Carriers include compounds that increase the half-life of a therapeutic composition in the treated animal. Suitable carriers include, but are not limited to, polymeric controlled release vehicles, biodegradable implants, liposomes, bacteria, viruses, other cells, oils, esters, and glycols.

Another embodiment of the present methods is a controlled release formulation that is capable of slowly releasing a composition of the present invention into an animal. As used herein, a controlled release formulation comprises a composition of the present invention in a controlled release vehicle. Suitable controlled release vehicles include, but are not limited to, biocompatible polymers, other polymeric matrices, capsules, microcapsules, microparticles, bolus preparations, osmotic pumps, diffusion devices, liposomes, lipospheres, and transdermal delivery systems. Other controlled release formulations of the present invention include liquids that, upon administration to an animal, form a solid or a gel in situ. Preferred controlled release formulations are biodegradable (i.e., bioerodible).

A preferred controlled release formulation of the present invention is capable of releasing a composition of the present invention into the blood of an animal at a constant rate sufficient to attain therapeutic dose levels in the animal. The therapeutic composition is preferably released over a period of time ranging from about 1 day to about 12 months, and include release over a 2, 3, 4 , 5, 6, 7 day through a 30 day time period.

Acceptable protocols to administer therapeutic compositions of the present invention in an effective manner include individual dose size, number of doses, frequency of dose administration, and mode of administration. Determination of such protocols can be accomplished by those skilled in the art. A suitable single dose is a dose that is capable of protecting (i.e., preventing or treating) an animal from disease when administered one or more times over a suitable time period. The need for additional administrations of a therapeutic composition can be determined by one of skill in the art in accordance with the given condition of a patient.

Pharmaceutically useful compositions comprising herein proteins, may be formulated according to known methods such as by the admixture of a pharmaceutically acceptable carrier, or by modification with additional chemical moieties so as to form a chemical derivative. Examples of such carriers, modifications and methods of formulation may be found in Remington's Pharmaceutical Sciences. To form a pharmaceutically acceptable composition suitable for effective administration, such compositions will contain an effective amount of the protein or DNA.

The present invention also has the objective of providing suitable topical, oral, systemic and parenteral formulations of the pharmaceutical compounds herein provided. The formulations can be administered in a wide variety of therapeutic dosage forms in conventional vehicles for administration. For example, the compounds can be formulated for oral administration in the form of tablets, capsules (each including timed release and sustained release formulations), pills, powders, granules, elixirs, tinctures, solutions, suspensions, syrups and emulsions, or by injection. Likewise, they may also be administered intravenously (both bolus and infusion), during angioplasty/catheterization, intraperitoneally, subcutaneously, topically with or without occlusion, or intramuscularly, all using forms well known to those of ordinary skill in the pharmaceutical arts.

A molecule can be combined with a buffer in which the molecule is solubilized, and/or with a carrier. Suitable buffers and carriers are known to those skilled in the art. Examples of suitable buffers include any buffer in which a molecule can function to inhibit its target enzyme(s), such as, but not Hmited to, phosphate buffered saline, water, saline, phosphate buffer, bicarbonate buffer, HEPES buffer (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid buffered saline), TES buffer (Tris-EDTA buffered saline), Tris buffer and TAE buffer (Tris-acetate-EDTA). Examples of carriers include, but are not limited to, polymeric matrices, toxoids, and serum albumins, such as bovine serum albumin.

In the methods of the present invention, the compounds herein described in detail can form the active ingredient, and are typically administered in admixture with suitable pharmaceutical diluents, excipients or carriers (collectively referred to herein as "carrier" materials) suitably selected with respect to the intended form of administration, that is, oral tablets, capsules, elixirs, syrups and the like, and consistent with conventional pharmaceutical practices.

For instance, for oral administration in the form of a tablet or capsule, the active drug component can be combined with an oral, non-toxic pharmaceutically acceptable inert carrier such as ethanol, glycerol, water, and the like. Moreover, when desired or necessary, suitable binders, lubricants, disintegrating agents and coloring agents can also be incorporated into the mixture. Suitable binders include, without limitation, starch, gelatin, natural sugars such as glucose or beta lactose, corn sweeteners, natural and synthetic gums such as acacia, tragacanth or sodium alginate, carboxymethylcellulose, polyethylene glycol, waxes and the like. Lubricants used in these dosage forms include, without limitation, sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, and the like. Disintegrators include, without limitation, starch, methyl cellulose, agar, bentonite, xanthan gum and the like.

For liquid forms the active drug component can be combined in suitably flavored suspending or dispersing agents such as the synthetic and natural gums, for example, tragacanth, acacia, methylcellulose and the like. Other dispersing agents which may be employed include glycerin and the like. For parenteral administration, sterile suspensions and solutions are desired. Isotonic preparations which generally contain suitable preservatives are employed when intravenous administration is desired.

Topical preparations containing the active drug component can be admixed with a variety of carrier materials well known in the art, such as, e.g., alcohols, aloe vera gel, allantoin, glycerine, vitamin A and E oils, mineral oil, PPG2 myristyl propionate, and the like, to form, e.g., alcoholic solutions, topical cleansers, cleansing creams, skin gels, skin lotions formulations. The compounds of the present invention can also be administered in the form of liposome delivery systems, such as small unilamellar vesicles, large unilamellar vesicles and multilamellar vesicles. Liposomes can be formed from a variety of phospholipids, such as cholesterol, stearylamine or phosphatidylcholines. The compounds of the present invention may also be coupled with soluble polymers as targetable drug carriers. Such polymers can include polyvinyl-pyrrolidone, pyran copolymer, polyhydroxypropylmethacryl-amidephenol, polyhydroxy- ethylaspartamidephenol, or polyethyleneoxidepolylysine substituted with palmitoyl residues. Furthermore, the compounds of the present invention may be coupled to a class of biodegradable polymers useful in achieving controlled release of a drug, for example, polylactic acid, polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydro-pyrans, polycyanoacrylates and cross-linked or amphipathic block copolymers of hydrogels.

The following examples illustrate the present invention without, however, limiting it.

It is to be noted that the Examples include a number of molecular biology, microbiology, immunology and biochemistry techniques considered to be known to those skilled in the art.

Disclosure of such techniques can be found, for example, in Sambrook et al., ibid., and related references .

EXAMPLES

Example 1. Synthesis of inhibitors

Synthesis of Ro 48-8071

The full chemical name of the Ro-48-8071 (Ro-48 on Fig 1.) is [4'-(6-allyl- methylaminohexyloxy)-2-fluorophenyl](4-bromophenyl)methanone. The compound was prepared for these studies as reported in: Morand, O. H., Aebi, J. D., Dehmlow, H., Ji, Y. H., Gains, N., Lengsfeld, H., and Himber, J. (1997) Ro 48-8071, a new 2,3- oxidosqualene:lanosterol cyclase inhibitor lowering plasma cholesterol in hamsters, squirrel monkeys, and minipigs: Comparison to simvastatin. J Lipid Res 38, 373-390.

The following steps indicate the intermediates made during synthesis of Ro 48-8071. All intermediates and Ro 48-8071 were charactereized by 250 MHz Η-NMR, IR, MS, and microanalyses. Melting points (uncorrected) were determined using a Biichi 510 apparatus. Proton NMR spectra were recorded on a Bruker AC250 spectometer, and delta values are given in ppm relative to tetramethylsilane. IR spectra of KBr pellets were recorded using a Nicolet 7199-FT IR spectrometer. Mass spectra (MS) were obtained using the pneumatically assisted electrospray technique (Perkin-Elmer Sciex. Type API-Ill). Results of elemental analyses were within 0.3% fo theoretical values. (4-Bromo-phenyl)-(2'-fluoro-4'-methoxy-phenyl)-methanone [1]

Aluminum chloride (144 g. 1.08mol) was added to 450 ml precooled nitrobenzene keeping the temperature <8°C. Then, a suspension of 219.5 g (1 mol) 4-bromobenzoyl chloride in 200 ml nitrobenzene was added over 20 min. Followed 10 min later by 108.5 ml (0.95 mol) 3 fluoroanisole. The reaction mixture was warmed to room temperature overnight, mixed into iced water (1.51), and extracted with 3 x 11 dichloromefhane. The three organic phases were washed sequentially with 2 x 11 water, pooled, and dried (Na₂SO₄). Evaporation (85°C, 1 Torr) provided a mixture of (4-bromo-phenyl)-(2-fluoro- 4-methoxy-phenyl)-methanone and (4-bromo-phenyl)-(4' fluoro-2'-methoxy-phenyl)- methanone which was immediately dissolved in 300 ml ethyl acetate, and crystallized at room temperature. The crystals were filtered off and washed with 100 ml ethyl acetate and 3 x 100 ml cyclohexane to give pure (4-bromo-phenyl)-(2'-fluoro-4 -methoxy-phenyl) methanone [1] (122.4 g, 41.6%): mp 125-126°C; IR 1643 cm-1; 'H-NMR (CDCl₃),delta 3.87 (s, OCH₃), 6.66 (dd, J = 12.1, 2.4 Hz IH, 3'-H), 6.80 (dd, J = 8.7, 2.4 Hz, IH, 5XH), 7.54-7.68 (m, 5H, arom H): EIMS m/z=308 (M, 1 Br). Calculated analysis for Cl₄H₁₀BrFO₂C, 54.40; H, 3.26; F, 6.15; Br, 25.85. Found: C, 54.57; H, 3.35; F, 6.21; Br, 26.07.

(4-Bromo-phenyl)-(2'-fluoro-4'-hydroxy-phenyl)-methanone [2]

A suspension of 61.8 g (200 mmol) of [I] in 400 ml acetic acid was treated with 230 ml 62%-aqueous hydrobromic acid, and stirred at 125°C for 8 h prior to evaporation. The residue was dissolved in 500 ml ethyl acetate and washed with 300 ml saturated sodium bicarbonate and 300 ml 10%-sodium chloride solution. The aqueous phases were extracted with 2 x 500 ml ethyl acetate. The organic phase was dried (Na_jSO and evaporated to give orange crystals of (4-bromo phenyl)-(2'-fluoro-4'-hydroxy-phenyl)-methanone [2] (57.2 g, 96.9%): mp 62-63°C: IR 1652 cm 1; 1H-NMR (DMSO-d6) 3 6.68 (dd, J = 12.6, 2.2Hz, IH, 3'-H), 6.77 (dd, J = 8.5, 2.2 Hz, IH, 5' H), 7.49 (dd, j = 8.5, 85.Hz, IH, 6'-H), 7.64 and 7.75 (AATB', 4H, 2,3,5,6-H), 10.85 (br s, IH, OH): EMS m/z= 294 (Ml, IBr), Calculated analysis for Cl₃H₈BrFO₂: C, 52.91; H, 2.73; F,6.44; Br, 27.08. Found: C, 52.94; H, 2.74; F, 6.42; Br, 26.84.

[4'-(6-Allyl-methyl-amino-hexyloxy)-2'-fluoro-phenyl]-(4-bromophenyl)-methanone fumarate [3]

A mixture of 35.4 g (120 mmol) [2], 54.9 ml (360 mmol) 1,6-dibromohexane and 49.8 g (360 mmol) potassium carbonate in 1100 ml acetone was vigorously stirred at 75°C for 5 h. After filtration and evaporation, the residue was dissolved in dichloromethane treated with sodium sultfate, filtered again, and evaporated. Crystallization with 400 ml cyclohexane-hexane 1:3 (v/v) first at 0°C and then at -78°C gave 53.2 g (116 mmol) crude [4'-(6-bromo-hexyloxy)-2'-fluoro-phenyl]-(4 bromophenyl)-methanone. This product was dissolved in 390 ml N.N-dimethylacctamide, cooled to 0°C, and 22.5 ml (232 mmol) N- allylmethylamine was added dropwise. After 22 h at room temperature the reaction was cooled to 0°C, and treated again with 22.5 ml (232 mmol) N allylmethylamine. After 5 h the solution was evaporated (70°C, 1 Torr), neutralized with 300 ml saturated sodium bicarbonate, and extracted with 3 x 400 ml dichloromethane. The organic phase was dried (NaoSO₄), evaporated to dryness, and purified by flash column chromatography (silica gel 0.04-0.063 mm, dicchloromethane-methanol 95:5 (v/v), producing 37.7 g (84.1 mmol) of [4 ' (6-allyl-methyl-amino-hexyloxy)-2 '-fluoro-phenyl]-(4-bromo-phenyl)-methanone. The free amine and 8,8 g (75.7 mmol) of fumaric acid were dissolved in 200 ml ethanol, evaporated, and crystallized from acetone-ethylacetate-ether to give [4'-(6-allyl-methyl- amino-hexyloxy)-2'-fluoro phenyl]-(4-bromo-phenyl)-methanone fumarate [3] (36.2 g, 53.4%): mp 86-88°C; IR 1653 cm -1; 'H-NMR (DMSO-d₆) θ 1.25-1.60 (m, 6H, OCH₂CH₂CH₂CH₂CH₂CH₂N), 1.70-1.80 (m, 2H, OCH₂CH₂CH₂CH₂CH₂CH₂N), 2.24 (S, 3H, NCH₃), 2.40-2.50 (m, 2H, OCH₂CH₂CH₂CH₂CH₂CH₂N), 3.11 (d, J = 6.5 Ηz, 2Η, NCH₂CHCH₂), 4.08 ( t, J = 6.4 Hz, 2H, OCH₂CH₂CH₂CH₂CH₂CH₂N), 5.17-5.27 (m, 2H, NCH₂CHCH₂, 5.75-5.90 (m, 1Η, NCΗ₂CΗCΗ₂), 6.67 (s, ²H, fumarate), 6.91-7.00 (m, 2H, 3',5'-H), 7.56 (dd, J = 8.6.8.6 Hz, IH, 6'-H), 7.65 and 7.76 (AAΕB', 4H, 2,3,5,6-H): EIMS m/z 448 (M+, 1 Br). Calculated analysis for C₂₃H₂₇NBRFO₂ ^' C₄H₄O₄; C, 57.45; H, 5.54; N, 2.48; F, 3.37; Br, 14.16. Found: C, 57.39; H, 5.57; N, 2.50; F, 3.38; Br, 14.15.

Synthesis of BIBX 79 (trans-N-(4-chlorobenzoyl)-N- methyl(4- dimethylaminomethylphenyl)-cyclohexylamine)

(1) 4-(4-Dimethylaminomethylphenyl) cyclohexanone. To a solution of n-butly lithium (1.21 1, 1.6 mol in hexane) in THF (1.5 1) a solution of 4-bromo-N, N- dimethylbenzylamine (364 g, 1.7 mol, from 4-bromobenzylbromide and dimethylamine in toluene, b.p.l l 105°C) in THF (500 ml) was added (-65°C, 1.4 cyclohexandionmonoethylendetal (276.4 g, 1.72 nol) in THF (1.1 1) was added, the mixture was stirred for 30 min at -65°C, then stirred overnight at room temperature (N2) and poured into a mixture of ice water (6 1) and ethyl acetate (1.8 1). After stirring for 10 min, the aqueous phase was extracted with ethyl acetate (4 x 800 ml) and the combined extracts were worked up (brine, Na₂SO₄) to give a residue that was crystallized from diisopropyl ether (1.2 1) to give 1 (4-dimethylaminomethylphenyl)-4-ethylendioxycyclohexanol (419 g, 84.6%): mp 84-86°C. This product (224.4 g, 0.77 mol) was dehydrated by refluxing in toluene (2.4 1, 3.5 h) in the presence of p-toluenesulfonic acid monohydrate (150.3 g, 0.79 mol) and ethylene glycol (390 ml) with continuous removal of the formed water. The mixture was poured into ice water (1 1), the pH was raised to 12-13 (2 N NaOH) and the aqueous phase was extracted with toluene (2 x 500 ml). Workup (brine, Na_jSO of the combined extracts gave l-(4-dimethylaminomethylphenyl)-4 ethylendioxy-cyclohex-1-ene (211 g, 100%) as a yellow oil.

The crude material (325 g, 1.19 mol) dissolved in a mixture of ethyl acetate (3240 ml) and methanol (1380 ml) was hydrogenated over Pd/BaSO₄ (78 g) at room temperature (1.5 h, 5 bar). Removal of catalyst and solvents gave a yellow brownish oil (305 g) consisting of a mixture of 1 (4-dimethylaminomethylphenyl)-4-ethyl-endioxycyclohexane as the main product and l-(4 methylphenyl)-4-ethylendioxycyclohexane as the by-product. This oil (132 g) was stirred in 2 N HCl (730 ml) for 3.5 h at room temperature. To remove the by-product, the acid solution was extracted with ethyl acetate (3 x 300 ml), then the pH was raised to 13-14 under cooling (50% NaOH, N₂) and the solution was extracted with ethyl acetate (3 x 400 ml). Workup (brine, Na₂SO₄) of the combined extracts gave 4-(4- dimethylamino-methyl-phenyl) cyclohexanone (87.3 g): mp 64-67°C.

(II) trans 4-(4-Dimethylyaminomethylphenyl)-N-methylcyclohexylamine. 4-(4 Dimethylyaminomethylphenyl)-cyclohexanone (118, 0.51 mol), molecular sieve A 3 (190 g, Fluka) and a solution of methylamine in toluene (640 ml, 2.04 mol) were vigorously stirred for 15 h at room temperature and then molecular sieve and solvent were removed. The residue was dissolved in methanol (650 ml): sodium borohydride (14 g, 0.37 mol) was added during 1 h with cooling (-8 to -4°C): the mixture was stirred for 3 h at room temperature, and then the solvent was removed. The residue was suspended in water (600 ml) and concentrated hydrochloric acid was added to pH 1-2 with cooling. After 1 h at room temperature the pH was brought to 12-13 (50% NaOH) with cooling; the solution was extraced with ethyl acetate under pH control and the extract was concentrated (Na₂SO₄). To separate the isomers, the residue was dissolved in ethyl acetate (50 ml). A solution of benzoic acid (62 g, 0.51 mol) in hot ethyl acetate (220 ml) was added, followed by hot diisopropyl ether (200 ml). After 2 h at -15°C, the solid was filtered off, dissolved in a mixture of ice water (500 ml) and methylene chloride (200 ml), the pH was adjusted to 14 (50% NaOH), and the aqueous phase was extracted with methylene chloride (3 x 150 ml). Workup (Na₂SO₄) of the combined extracts gave trans 4-(4-dimethylaminomethylphenyl- N-methylcyclohexylamine) (55.3 g, 44%): mp 56-58°C. (III) trans-N-(4-Chlorobenzoyl)-N-methyl-4-(4-dimethylaminomethylphenyl) cyclohexylamine. A solution of 4-chlorobenzoyl chloride (7.1 g, 40.6 mmol) in methylene chloride (20 ml) was added to a solution of trans 4-(4-dimethylaminomethylphenyl)-N methylcyclohexylamine (10 g, 40.5 mmol) in methylene chloride (100 ml) at room temperature. After 2 h the mixture was poured into water, the aqueous phase was made alkaline (NaOH) and extracted three times with methylene chloride. Workup (Na₂SO₄) of the combined extracts and purification by chromatography (alumina N, act. πi, ICN, ethyl acetate) gave trans-N-(4 chlorobenzoly)-N-methyl-4-(4-dimethylaminomethylphenyl) cyclohexylamine (12.5 g, 80%): mp 154-156°C.

Typical Synthesis of a 3-Oxy Linked Quinuclidine (Multiple-Parallel Synthesis), 3-[(4-Chlorobenzoyl)-4-phenoxylquinuclidine (13a).

Compound 11a, (0.64 g, 5 mmol) in THF (4.5 mL) and DMF (2.5 mLO was added to a stirred solution of 4-chloro-4'-hydroxybenzophenone (1.29 g, 5.5 mmol), PPh3 (1.70 g, 6.5 mmol) and DEAD (0.94 mL, 6.0 mmol) in THF (4 mL) at 5-10°C, and the mixture was stirred for 18 h. The solvent was evaporated and the residue dissolved in MTBE (8mL); the solution was extracted with 2 M HCl, and the extracts were washed with MTBE. The extract was made basic with 4 M NaOH to pH = 11, and the mixture was stirred for 1 h and filtered to give a solid, which was triturated in EtOH to give, on crystallization from, EtOH 13A (0.37 g, 22%): mp 147 - 149°C; IH NMR (CDC1₃) delta 1.4 (m, IH), 1.67 (m, 2H), 2.0 (m, IH), 2.20 (m, IH), 2.95 (m, 5H), 3.32 (m, IH), 4.47 (m, IH), 6.90 (d, 2H), 7.34 (d, 2H), 7.75 (q, 4H); EI-MS m/z 342 (M + H). Anal. (C₂₀OH₂₀OC1NO₂) C, H, N.

Meyer-Schuster Rearrangement Reaction. (Z)-3-[4-(4-Bromobenzoyl) phenacylidene] quinuclidine (16).

3-[2-(4-Bromobenzophenone) ethynyl] quinuclidin-3-ol (0.5 g, 1.2 mmol) was added to 98% H₂SO₄ (5 mL) with stirring. The reaction mixture was stirred at ambient temperature for 18 h, and H₂0 (10 mL) was added cautiously. The diluted solution was poured onto ice/10 M NaOH to precipitate a solid, and the solid was extracted into CH₂C1₂ (2 x 50 mL). The extracts were combined, washed with brine, dried, and evaporated. The residue was purified by flash chromatography on silica gel, eluting with EtHO/EtOAc/Et₃N (80;20;3) to give 16 as a colorless solid (0.25 g 50%); mp 182 - 183°C; 'HNMR (DMSO-d6) delta 1.6 - 1.7 (m, 2H), 1.8-1.95 (m, 2H) 2.7 (m, IH), 2.7 - 2.95 (m, 4H), 3.95 (s, 2H), 7.1 (m, IH), 7.6 - 7.9 (m, 6H), 8.1 ( , 2H); EI-MS m/z 410 (M + H). Anal. (C₂₂H₂₀0BrNO₂ • 0.25 H₂O) C, H, N. Synthesis of JCH-1

The full chemical name of JCH-1 is: [4-(l-azabicyclo[2.2.2]oct-3-yloxy)phenyl](4- chlorophenyl)methanone or 3-[4-chlorobenzoyl)-4-phenoxy]quinuclidine. This compound was prepared for these studies as reported in :

Brown, G. R., Hollinshead, D. M., Stokes, E. S. E., Clarke, D. S., Eakin, M. A., Foubister, A. J., Glossop, S. G., Griffiths, D., Johnson, M. C, McTaggart, F., Mirrlees, D. J., Smith, G. J., and Wood, R. (1999) Quinuclidine inhibitors of 2,3-oxidosqualene cyclase-lanosterol synthase: Optimization from lipid profiles. J Med Chem 42, 1306-1311.

Example 2. Oxidoqualene cyclase inhibitors inhibit parasite development

In vitro activity of OSC inhibitors against parasites: The indicated values are the concentrations that resulted in 50% inhibition of growth (IC₅₀). Note the selective inhibition of parasites over mammalian cells.

^Trypanosoma cruzi were grown in co-culture with mouse fibroblasts **Leishmania mexicana promastigotes ***Mouse 3T3 fibroblasts

Example 2 continued

In vivo activity of OSC inhibitors against parasites. The following figure shows the inhibitory effect of Ro48-8071 against Trypanosoma cruzi in a mouse model of Chagas disease.

0

5 Mouse Study with Ro 48-8071

Days post-infection

Figure legend. Parasitemia in mice treated with Ro 48-8071. Mice were administered drug (100 μmol/kg/dose) or placebo by oral gavage at the times indicated (■I). Parasites were quantified in tail blood by light microscopy in 50 high power fields (hpf). Differences in parasitemia were significant at the indicated points (* P<0.01).

Example 3. Oxidoqualene cyclase inhibitors inhibit mycobacterial development

Two known OSC/SHC inhibitors for activity in M. tuberculosis cell assays were evaluated. The two compounds designated Ro-48-8071 and JCH-1 are among the most potent OSC/SHC inhibitors reported. Both compounds showed significant inhibition of M. tuberculosis HR37Ra cell growth. See Figure 1.

JHC1 Ro-48-8071

IC₅₀ = 120 nM A. acidocaldarius SHC ICj₀ = 9 nM A. acidocaldarius SHC

IC₅₀ = 96 nM Rat OSC IC₅₀ = 40 nM Rat OSC

Furthermore, since Ro-48-8071 covalently modifies the recombinant putative cyclase enzyme in cell lysates upon photactivation, we will synthesize and examine other reported potent OSC and SHC for inhibition of the cloned M. tuberculosis cyclase enzyme. Important candidates include BIBB 515, BIBX 79, and Ro-44-2103.

BIBX 79 Ro-44-2103 BIBB 515

IC₅₀ = 70 nM SHC IC₅₀ = ~100nM C. albicans OSC IC₅ 9nM Human OSC

IC₅₀ = lOOnM Rat OSC

These compounds (as well as Ro-48-8071 and JCH-1) have a general structure with an amine portion linked to a relatively rigid hydrophopic portion. Example 4. Oxidoqualene cyclase inhibitors bind to a mycobacterial enzyme

Encouraged by these results, the M. tuberculosis gene was PCR amplified, ligated into pBAD TOPO vector (Invitrogen) and transformed into TOPIO E. coli cells (Invitrogen) induced with L-arabinose. SDS-PAGE of the lysed cells showed a predicted protein band of 55 kDa which was not present in control E. coli cell lysate. The cloned cell lysate was incubated with with [³H]- Ro-48-8071 and irradiated with 360 nm UV light. A fluorogram of the SDS-PAGE gel protein separation revealed that the 55 kDa protein had been specifically labeled with the photoactivatableinhibitor.

A: 1 2 3 4 B: 1 2 3 4

(A) SDS PAGE and Fluorogram (B) of E. coli cell lysates photolabeled with [³H]- Ro-48-8071. Lane 1: Lysis by lysozyme in phosphate buffer (pH 7.4); Lane 2: lysis by lysozyme in Tris buffer (pH 7.4); Lane 3: lysis by French press in phosphate buffer; Lane 4: lysis by French press in Tris buffer.

Claims

We claim:

1. A method to inhibit physical development of at least one parasite, comprising affecting parasitic oxidosqualene cyclase activity of said parasite.

2. A method to alter sterol biosynthesis in a parasite, comprising inhibiting parasitic oxidosqualene cyclase activy of said parasite.

3. A method to treat parasite infection in a mammal in need of such treatment, comprising administering to said mammal a pharmaceutically-acceptable parasitic oxidosqualene cyclase inhibitor.

4. A method of claim 3, wherein said oxidosqualene cyclase inhibitor is a compound selected from the group consisting of: [4'-(6-allyl-methyl-amino-hexyloxy)-2- fluoro-phenyl]-(4-bromophenyl)-methanone; trans-N-(4-chlorobenzoyl)-N-methyl-

(4-dimethylaminomethylphenyl)-cyclohexylamine; 2-Nitro-N-(phenylmethyl)-lH- imidazole-1-acetamide; N-benyl-2-nitroimidazole-l-acetamide; 3-[(4-chlorobenzoyl)-4-phenoxy]quinuclidine; (Z)-3-[4-(4-Bromobenzoyl)pheacylidene]quinuclidine.

5. A method of claim 3, wherein said oxidosqualene cyclase inhibitor is [4'-(6-allyl- methyl-amino-hexyloxy)-2-fluoro-phenyl]-(4-bromophenyl)-methanone.

6. A method of claim 3, wherein the parasite is selected from the group consisting of: nematodes; trematodes; and parasitic protozoa.

7. A method of claim 3, wherein said parasite is selected from the group consisting of: Ascaris lumbricoides; Ancylostoma duodenale; Necator americanus; Trichuria trichuria; Enterobius vermicularis; Strongyloides stercoralis; Wuchereria Bancrofti; Brugia malayi; Brugia timori; onchocerciasis; Onchocerca; Trichinella spiralis; Schistosoma spp; Trypanosoma cruzi; Leishmania spp; Trypanosoma brucei; Naegleria; and Acanthamoeba.

8. A method of claim 3, wherein said parasite is selected from the group consisting of: T. cruzi; and L. mexicana.

9. A method of claim 5, wherein said treatment further comprises and adjuvant.

0. A method of claim 7, wherein said adjuvant is selected from the group consisting of: an antimonial; amphotericin B; allopurinol; pentamidine; an azole anti-fungal; an aromatic antifungal; ketoconazole; itraconazole; and sodium stibogluconate.

11. A method to affect physical development of at least one mycobacterium, comprising affecting mycobacterial sterol biosynthesis of said mycobacterium.

12. A method to treat tuberculosis in a mammal in need of such treatment, comprising administering a pharmaceutically acceptable oxidosqualene cyclase inhibitor.

13. A method to treat mycobacterial infection in a mammal in need of such treatment, comprising administering a pharmaceutically acceptable oxidosqualene cyclase inhibitor.

14. A method of claim 13, wherein said oxidosqualene cyclase inhibitor is a compound selected from the group consisting of: : [4'-(6-allyl-methyl-amino-hexyloxy)-2- fluoro-phenyl]-(4-bromophenyl)-methanone; trans-N-(4-chlorobenzoyl)-N-methyl- (4-dimethylaminomethylphenyl)-cyclohexylamine; 2-Nitro-N-(phenylmethyl)-lH- imidazole- 1 -acetamide; N-benyl-2-nitroimidazole- 1 -acetamide; 3-[(4-chlorobenzoyl)-4-phenoxy]quinuclidine;

(Z)-3-[4-(4-Bromobenzoyl)pheacylidene]quinuclidine.

15. A method of claim 13, wherein said oxidosqualene cyclase inhibitor is [4'-(6-allyl- methyl-amino-hexyloxy)-2-fluoro-phenyl]-(4-bromophenyl)-methanone.

16. A method of claim 13, wherein the mycobacterium is selected from the group consisting of: Mycobacterium tuberculosis; Mycobacterium avium; and Mycobacterium leprae.

17. A method of claim 13, wherein said mycobacterium is Mycobacterium tuberculosis.

18. A method of claim 15, wherein said treatment further comprises and adjuvant.

19. A method of claim 18, wherein said adjuvant is selected from the group consisting of: an antimonial; amphotericin B; allopurinol; pentamidine; an azole anti-fungal; an aromatic antifungal; ketoconazole; itraconazole; and sodium stibogluconate.