Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07J—STEROIDS
  • C07J71/00—Steroids in which the cyclopenta(a)hydrophenanthrene skeleton is condensed with a heterocyclic ring
  • C07J71/0005—Oxygen-containing hetero ring
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
  • A61K31/34—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide
  • A61K31/343—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide condensed with a carbocyclic ring, e.g. coumaran, bufuralol, befunolol, clobenfurol, amiodarone
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
  • A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
  • A01N43/64—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with three nitrogen atoms as the only ring hetero atoms
  • A01N43/647—Triazoles; Hydrogenated triazoles
  • A01N43/653—1,2,4-Triazoles; Hydrogenated 1,2,4-triazoles
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/10—Antimycotics
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D493/00—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system
  • C07D493/02—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system in which the condensed system contains two hetero rings
  • C07D493/08—Bridged systems
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D519/00—Heterocyclic compounds containing more than one system of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring system not provided for in groups C07D453/00 or C07D455/00

Definitions

• the claimed subject matter relates to novel compounds and pharmaceutically acceptable salts, hydrates and prodrugs thereof, compositions containing such compounds, synthesis of such compounds, and use of such compounds as antifungal agents and/or inhibitors of (1,3)- ⁇ -D-glucan synthesis.
• the compounds described herein are derivatives of enfumafungin.
• the novel compounds of this disclosure, their pharmaceutically acceptable salts, hydrates and prodrugs, and compositions comprising such compounds, salts, hydrates and/or prodrugs, are useful for treating and/or preventing antifungal infections and associated diseases and conditions.
• Fungal infection is a major healthcare problem, and the incidence of hospital- acquired fungal diseases continues to rise. Severe systemic fungal infection in hospitals (such as candidiasis, aspergillosis, histoplasmosis, blastomycosis and coccidioidomycosis) is commonly seen in neutropaenic patients following chemotherapy and in other oncology patients with immune suppression, in patients who are immune-compromised due to Acquired Immune Deficiency Syndrome (ADDS) caused by HIV infection, and in patients in intensive care.
• ADDS Acquired Immune Deficiency Syndrome
• Systemic fungal infections cause -25% of infection-related deaths in leukaemics. Infections due to Candida species are the fourth most important cause of nosocomial bloodstream infection.
• Enfumafungin is a hemiacetal triterpene glycoside that is produced in fermentations of a Hormonema spp. associated with living leaves of Juniperus communis (U.S. Pat. No. 5,756,472; Pelaez et al., Systematic and Applied Microbiology, 25:333-343, 2000; Schwartz et al., JACS, 722:4882-4886, 2000; Schwartz, R.E., Expert Opinion on Therapeutic Patents, 11(11): 1761-1772, 2001).
• Enfumafungin is one of the several triterpene glycosides that have in vitro antifungal activities.
• the mode of the antifungal action of enfumafungin and other antifungal triterpenoid glycosides was determined to be the inhibition of fungal cell wall glucan synthesis by their specific action on (1,3)- ⁇ -D-glucan synthase (Onishi et al., Antimicrobial Agents and Chemotherapy, 44:368-377, 2000; Pelaez et al., Systematic and Applied Microbiology, 25:333-343, 2000).
• 1,3- ⁇ -D-Glucan synthase remains an attractive target for antifungal drug action because it is present in many pathogenic fungi which affords broad antifungal spectrum and there is no mammalian counterpart and as such, compounds inhibiting 1,3- ⁇ -D-Glucan synthase have little or no mechanism-based toxicity.
• the present invention relates to enfumafungin derivatives. These compounds, or pharmaceutically acceptable salts thereof, are useful in the inhibition of (1,3)- ⁇ -D-glucan synthase and are useful in the prevention or treatment of mycotic infections caused by one or more of various pathogens including, but are not limited to, Aspergillus, Cryptococcus, Candida, Mucor, Actinomyces, Histoplasma, Dermatophyte, Malassezia, Fusarium, and Pneumocystis carinii.
• the present invention includes a compound of Formula I:
• R 1 is a group of the following structure: wherein W, X', Y, and Z are independently selected from N and CR e provided that two or three of W, X', Y and Z are CR e ;
• R e is independently selected from a) H; b) Halogen; c) NR f R 8 ; d) NHC(O)R 0 ; e) NHC(O)NR f R 8 ; f) NHC(O)OR 0 ; g) NO 2 h) OR 0 ; i) SR 0 ; j) SO 2 R 0 ; k) SO 2 N(R 0 ) 2 ; l) CN; m) C(O)R 0 ; n) C(O)OR 0 ; o) C(O)NR f R g ;
• 3- to 7-membered ring having 0-1 additional heteroatoms independently selected from N, O and S wherein said ring may be optionally substituted on a ring nitrogen atom that is not the point of attachment with C(O)R 0 , CO 2 R 0 , C(O)N(R 0 ) 2 , SO 2 R 0 , or C 1 -C 6 alkyl unsubstituted or substituted with 1 or 2 substituents independently selected from N(R 0 ) 2 , OR 0 , CO 2 R 0 , C(0)N(R 0 ) 2 or halogen; said ring may also be optionally substituted on a sulfur atom with 1 or 2 oxo groups;
• R 2 is a group of the following structure: m is O or 1 ; n is O or 1 ; p is O or 1 ;
• T is NR 6 R 7 or OR 10 ;
• R 5 is H or C 1 -C 6 -alkyl, unsubstituted or substituted with 1 or 2 substituents independently selected from N(R 0 ) 2 and OR 0 ;
• R 6 is H, C 1 -C 6 -alkyl or C 3 -C 6 cycloalkyl;
• R 6 and R 7 are optionally taken together with the attached nitrogen atom to form a 4- to 7-membered saturated, unsaturated or aromatic ring having O or 1 additional heteroatoms independently selected from N, O and S, wherein said ring is optionally substituted on a ring carbon with 1 to 2 substituents independently selected from halogen, CF 3 , N(R 0 K OR 0 , CO 2 R 0 , C(O)N(R 0 K and C 1 -C 6 alkyl unsubstituted or substituted with 1 or 2 substituents independently selected from OR 0 and N(R 0 ) 2 ; said ring may also be optionally substituted on a ring nitrogen atom that is not the point of attachment with C(O)R 0 , CO 2 R 0 , C(O)N(R 0 K SO 2 R 0 or C 1 -C 6 alkyl unsubstituted or substituted with 1 to 3 substituents independently selected from N(R 0 K OR 0
• R 6 and R 8 are optionally taken together to form, with the intervening atoms, a A- to 7-membered saturated ring having O or 1 additional heteroatoms independently selected from N, O and S wherein said ring is optionally substituted as defined above for R 6 and R 7 when joined together to form a ring;
• R 6 and R 5 are optionally taken together to form, with the intervening atoms, a A- to 7-membered saturated ring having O or 1 additional heteroatoms independently selected from N, O and S wherein said ring is optionally substituted as defined above for R 6 and R 7 when joined together to form a ring;
• R 8 is selected from the group consisting of a) hydrogen, b) C 1 -C 6 -alkyl, unsubstituted or substituted with F, OR 0 , N(R 0 ) 2 or SO 2 R 0 , c) C 3 -C 6 -cycloalkyl, d) C 4 -C 7 -cycloalkyl-alkyl, e) aryl, wherein aryl is phenyl or naphthyl and said aryl is unsubstituted or substituted with 1 to 3 substituents selected from C 1 -C 6 -alkyl, halogen, OCF 3 , CF 3 , N(R 0 )2 and OR 0 , and f) heteroaryl, wherein heteroaryl is as defined above in the definition of R e ;
• R 9 is C 1 -C 6 -alkyl, unsubstituted or substituted with OR 0 or SO 2 R 0 ;
• R 8 and R 9 are optionally taken together to form a 3- to 7-membered saturated ring having 0 or 1 additional heteroatoms independently selected from N, O, and S, wherein said ring is optionally substituted as defined above for R 6 and R 7 when joined to form a ring;
• R 10 is selected from the group consisting of a) H, b) C 1 -C 6 -alkyl, unsubstituted or substituted with 1 or 2 substituents selected from N(R 0 ) 2 , OR 0 , CO 2 R 0 , OC(O)R 0 , NHC(O)R 0 , C(O)N(R 0 ) 2 , phenyl, heteroaryl, and heterocyclyl, wherein heteroaryl and heterocyclyl are as defined above in the definition of R e ,
• R 14 is OH, OR 15 or N(R 0 ) 2 ;
• R 15 is C 1 -C 6 -alkyl, unsubstituted or substituted with 1 or 2 substituents independently selected from phenyl and and OC(O)R 0 , wherein said phenyl is optionally substituted with 1 to 3 OR 0 groups;
• compositions comprising the compounds of the invention, optionally in the presence of a second therapeutic agent.
• aspects of the invention relate to methods of preparing a compound of the invention, to methods of preparing compositions of the invention, to methods of treating or preventing fungal infection in patients using a compound of the invention, and to methods of controlling fungal infection in patients using a compound of the invention.
• Other embodiments, aspects and features of the present invention are either further described in or will be apparent from the ensuing description, examples and appended claims.
• the present invention relates to compounds of Formula (I) and pharmaceutically acceptable salts thereof. Different embodiments further describing Formula (I) variables are described below.
• R 3 is C(O)R 14 , wherein R 14 is OH, and the other variables are as provided for in Formula (I) above.
• the compound is of Formula (Ia) wherein all variables are as provided for in Formula (I) above:
• X is H,H, and the other variables are as provided for in Formula (I) above or in the first embodiment.
• X is O, and the other variables are as provided for in Formula (I) above or in the first embodiment.
• R 2 is a group of the following structure: wherein T is OR 10 and the other variables are as provided for in Formula (I) above or in any of the first through third embodiments.
• R 2 is a group of the following structure: wherein T is NR 6 R 7 and the other variables are as provided for in Formula (I) above or in any of the first through third embodiments.
• R 2 is a group of the following structure:
• R 2 is a group of the following structure:
• R and R may be joined together to form a ring providing R of the following structure:
• R is a group of the following structure:
• R is a group of the following structure:
• R and R may be joined together to form a ring providing R of the following structure: and the other variables are as provided for in Formula (I) above, or in any of the first through third embodiments.
• R 2 i •s a group of the following structure:
• R 6 is H or C 1 -C 3 -alkyl
• R 7 is H or methyl
• R 8 is C 1 -C 5 -alkyl, C 3 -C 5 cycloalkyl or C 4 -C 6 cycloalkyl-alkyl;
• R 9 is H or C 1 -C 3 -alkyl; or R 8 and R 9 are optionally taken together to form a 5- to 6-membered saturated ring having 0-1 heteroatom selected from O or S; and the other variables are as provided for in Formula (I) above or in any of the first through third embodiments.
• R 2 is a group of the following structure: wherein
• R is H, methyl, ethyl or n-propyl
• R 8 is ethyl, i-propyl, t-butyl or 1-methylcyclopropyl
• R 9 is methyl or ethyl; or R 8 and R 9 are optionally taken together to form a 6-membered saturated ring containing 0 or 1 oxygen atoms; and the other variables are as provided for in Formula (I) above or in any of the first through third embodiments.
• R 2 is selected from the group consisting of:
• R 1 is a group of the following structure: wherein W, X', Y, and Z are independently selected from N and CR e provided that two of W, X', Y and Z are CR e , and the other variables are as provided for in Formula (I) above or in any of the first through fifth embodiments.
• R 1 is a group of the following structure:
• each R e is independently selected as defined above, and the other variables are as provided for in Formula (I) above or in any of the first through fifth embodiments.
• R 1 is a group of the following structure:
• R e is as defined above, and the other variables are as provided for in Formula (I) above or in any of the first through fifth embodiments.
• R 1 is a group of the following structure: wherein R e is NR f R g , and R f and R 8 and the other variables are as provided for in Formula (I) above or in any of the first through fifth embodiments;
• R 1 is a group of the following structure:
• R e is selected from the following and the other variables are as provided for in Formula (I) above or in any of the first through fifth embodiments: a) H, b) Halogen, c) C(O)NR f R 8 , d) heterocyclyl, wherein the heterocyclyl is a 4- to 6-membered saturated or unsaturated non- aromatic ring having 1 or 2 heteroatoms selected from N, O or S, attached through a carbon or nitrogen on the ring and optionally substituted on a ring carbon with N(R 0 ) 2 , OR 0 , imino or oxo; the heterocyclyl may also be optionally substituted on a ring nitrogen atom that is not the point of attachment with C(O)R 0 , CO 2 R 0 , C(O)N(R 0 ) 2 or C 1 -C 4 alkyl, e) aryl, wherein aryl is phenyl or napthyl and aryl is unsubstitute
• R 1 is a group of the following structure:
• R e is C(O)NR f R 8 and the other variables are as provided for in Formula (I) above or in any of the first through fifth embodiments.
• R 1 groups of this aspect of the invention include but are not limited to the following:
• R 1 is i a group of the following structure:
• R e is heterocyclyl, wherein the heterocyclyl is a 4- to 6-membered saturated or unsaturated non- aromatic ring having 1 or 2 heteroatoms selected from N, O or S, attached through a carbon or nitrogen on the ring and optionally substituted on a ring carbon with N(R ) 2 , OR , imino or oxo; the heterocyclyl may also be optionally substituted on a ring nitrogen atom that is not the point of attachment with C(O)R 0 , CO 2 R 0 , C(O)N(R 0 ) 2 or C 1 -C 4 alkyl; and the other variables are as provided for in Formula (I) above or in any of the first through fifth embodiments.
• R 1 groups of this aspect of the invention include but are not limited to the following:
• R 1 is a group of the following structure:
• R 1 groups of this aspect of the invention include but are not limited to the following:
• R 1 is a group of the following structure:
• R 1 groups of this aspect of the invention include but are not limited to the following:
• R 1 is a group of the following structure: wherein W, X', Y, and Z are independently selected from N and CR e provided that three of W, X', Y and Z are CR e , and the other variables are as provided for in Formula (I) above or in any of the first through fifth embodiments.
• R 1 is a group of the following structure:
• each R e is independently selected as defined above, and the other variables are as provided for in Formula (I) above or in any of the first through fifth embodiments.
• R 1 is a group of the following structure: wherein R e is as defined above, and the other variables are as provided for in Formula (I) above or in any of the first through fifth embodiments.
• R 1 is a group of the following structure:
• X is O or H, H;
• R e is C(O)NR f R g or a 6-membered ring heteroaryl group containing 1 or 2 nitrogen atoms wherein the heteroaryl group is optionally mono-substituted on a ring carbon with fluoro or chloro or on a ring nitrogen with oxygen;
• R f , R 8 , R 6 and R 7 are each independently hydrogen or C 1 -C 3 alkyl
• R 8 is C 1 -C 4 alkyl, C 3 -C 4 cycloalkyl or C 4 -C 5 cycloalkyl-alkyl;
• R 9 is methyl or ethyl
• R 8 and R 9 are optionally taken together to form a 6-membered saturated ring containing 1 oxygen atom.
• R e is either pyridyl or pyrimidinyl optionally mono-substituted on a ring carbon with fluoro or chloro or on a ring nitrogen with oxygen, and the other substituents are as provided in the first aspect or the general formula II.
• R e is 4-pyridyl and the other substituents are as provided in the first aspect or the general formula II.
• R e is C(O)NH 2 or C(O)NH(C 1 -C 3 alkyl) and the other substituents are as provided in the first aspect or the general formula II.
• R is C 1 -C 4 alkyl and R is methyl; and the other substituents are as provided in the first to fourth aspects or the general formula II.
• R is t-butyl, R is methyl; and the other substituents are as provided in the first to fourth aspects or the general formula II.
• R 6 and R 7 are independently selected from hydrogen and methyl and the other substituents are as provided in the first to sixth aspects or the general formula II.
• the compound of the invention is selected from the exemplary species depicted in Examples 1 through 318 shown below (as the free base or a pharmaceutically acceptable salt thereof)-
• compositions comprising a compound of Formula (I) and a carrier, adjuvant, or vehicle;
• composition comprising a compound of Formula (I) and a pharmaceutically acceptable carrier, adjuvant, or vehicle;
• composition of (c), wherein the second therapeutic agent is an azole, a polyene, a purine or pyrimidine nucleotide inhibitor, a pneumocandin or echinocandin derivative, a protein elongation factor inhibitor, a chitin inhibitor, a mannan inhibitor, a bactericidal/permeability-inducing (BPI) protein product, or an immunomodulating agent;
• the second therapeutic agent is an azole, a polyene, a purine or pyrimidine nucleotide inhibitor, a pneumocandin or echinocandin derivative, a protein elongation factor inhibitor, a chitin inhibitor, a mannan inhibitor, a bactericidal/permeability-inducing (BPI) protein product, or an immunomodulating agent;
• a pharmaceutical combination which is (1) a compound of Formula (I) and (2) a second therapeutic agent, wherein the compound of Formula (I) and the second therapeutic agent are each employed in an amount that renders the combination effective for treating or preventing fungal/bacterial infections;
• the second therapeutic agent is an azole, a polyene, a purine or pyrimidine nucleotide inhibitor, a pneumocandin or echinocandin derivative, a protein elongation factor inhibitor, a chitin inhibitor, a mannan inhibitor, a bactericidal/permeability-inducing (BPI) protein product, or an immunomodulating agent;
• a method of treating or preventing mycotic infections in a subject in need thereof comprising administering to the subject a pharmaceutical composition of (b), (c), (d), or (e) or the combination of (f), (g) or (h).
• the present invention also includes a compound of the present invention (i) for use in, (ii) for use as a medicament for, or (iii) for use in the preparation of a medicament for: (a) inhibiting (1,3)- ⁇ -D-glucan synthase in a subject in need thereof, or (b) treating or preventing mycotic infections.
• the compounds of the present invention can optionally be employed in combination, either sequentially or concurrently, with one or more therapeutic agents effective against fungal and/or bacterial infections.
• each embodiment may be combined with one or more other embodiments, to the extent that such a combination provides a stable compound and is consistent with the description of the embodiments.
• the embodiments of compositions and methods provided as (a) through (o) above are understood to include all embodiments of the compounds, including such embodiments as result from combinations of embodiments of the compound.
• the present compounds have antimicrobial (e.g., antifungal) activities against yeasts and fungi, including one or more of the following: Acremonium, Absidia (e.g., Absidia corymbifera), Alternaria, Aspergillus (e.g., Aspergillus clavatus, Aspergillus flavus, Aspergillus fumigatus, Aspergillus nidulans, Aspergillus niger, Aspergillus terreus, and Aspergillus versicolor), Bipolaris, Blastomyces (e.g., Blastomyces dermatitidis), Blastoschizomyces (e.g., Blastoschizomyces capitatus), Candida (e.g., Candida albicans, Candida glabrata (Torulopsis glabrata), Candida guilliermondii, Candida kefyr, Candida krusei, Candida lusit
• capsulatum Malassezia (e.g., Malassezia furfur), Microsporum (e.g., Microsporum canis and Microsporum gypseum), Mucor, Paracoccidioides (e.g., Paracoccidioides brasiliensis), Penicillium (e.g., Penicillium marneffei), Phialophora, Pityrosporum ovale, Pneumocystis (e.g., Pneumocystis carinii), Pseudallescheria (e.g., Pseudallescheria boydii), Rhizopus (e.g., Rhizopus microsporus var.
• Malassezia e.g., Malassezia furfur
• Microsporum e.g., Microsporum canis and Microsporum gypseum
• Mucor Paracoccidioides (e.g., Paracoccidioides brasiliensis)
• Saccharomyces e.g., Saccharomyces cerevisiae
• Scedosporium e.g., Scedosporium apiosperum
• Scopulariopsis e.g., Sporothrix schencku
• Trichoderma Trichoderma, Trichophyton (e.g., Trichophyton mentagrophytes and Trichophyton rubrum), and Trichosporon (e.g., Trichosporon asahii, Trichosporon beigelii and Trichosporon cutaneum).
• the present compounds are not only useful against organisms causing systemic human pathogenic mycotic infections, but also useful against organisms causing superficial fungal infections such as Trichoderma sp. and other Candida spp.
• the compounds of the present invention are particularly effective against Aspergilius flavus, Aspergillus fumigatus, Candida albicans, Candida parapsilosis, Cryptococcus neoformans, Saccharomyces cerevisiae, and Trichophyton mentagrophytes.
• compounds of Formula (I) are useful for the treatment and/or prevention of one or more of a variety of superficial, cutaneous, subcutaneous and systemic mycotic infections in skin, eye, hair, nail, oral mucosa, gastrointestinal tract, bronchus, lung, endocardium, brain, meninges, urinary organ, vaginal portion, oral cavity, ophthalmus, systemic, kidney, bronchus, heart, external auditory canal, bone, nasal cavity, paranasal cavity, spleen, liver, hypodermal tissue, lymph duct, gastrointestine, articulation, muscle, tendon, interstitial plasma cell in lung, blood, and so on.
• compounds of the present invention are useful for preventing and treating one or more of various infectious diseases, such as dermatophytosis (e.g., trichophytosis, ringworm or tinea infections), athletes foot, paronychia, pityriasis versicolor, erythrasma, intertrigo, fungal diaper rash, Candida vulvitis, Candida balanitis, otitis externa, candidiasis (cutaneous and mucocutaneous), chronic mucocandidiasis (e.g.
• dermatophytosis e.g., trichophytosis, ringworm or tinea infections
• athletes foot paronychia
• pityriasis versicolor erythrasma
• intertrigo fungal diaper rash
• Candida vulvitis Candida balanitis
• otitis externa candidiasis (cutaneous and mucocutaneous)
• chronic mucocandidiasis e.g.
• the present compounds may also be used as prophylactic agents to prevent systemic and topical fungal infections.
• prophylactic agents may, for example, be appropriate as part of a selective gut decontamination regimen in the prevention of infection in immuno-compromised patients (e.g. AIDS patients, patients receiving cancer therapy or transplant patients). Prevention of fungal overgrowth during antibiotic treatment may also be desirable in some disease syndromes or iatrogenic states.
• immuno-compromised patients e.g. AIDS patients, patients receiving cancer therapy or transplant patients.
• Prevention of fungal overgrowth during antibiotic treatment may also be desirable in some disease syndromes or iatrogenic states.
• azoles examples include, but are not limited to, fluconazole, voriconazole, itraconazole, ketoconazole, miconazole, ravuconazole, detoconazole, clotrimazole, and posaconazole.
• polyenes examples include, but are not limited to, amphotericin B, nystatin, liposamal and lipid forms thereof such as ABELCET, AMBISOME, and AMPHOCIL.
• Examples of purine or pyrimidine nucleotide inhibitors that may be used in combination with the present compounds include, but are not limited to, flucytosine or polyxins such as nikkomycines, in particular nikkomycine Z or nikkomycine X.
• Another class of therapeutic agents that may be used in combination with the present compounds includes chitin inhibitors.
• Examples of elongation factor inhibitors that may be used in combination with the present compounds include, but are not limited to, sordarin and analogs thereof.
• Examples of pneumocandin or echinocandin derivatives that may be used in combination with the present compounds include, but are not limited to, cilofungin, anidulafungin, micafungin, and caspofungin.
• mannan inhibitors that may be used in combination with the present compounds include but are not limited to predamycin.
• bactericidal/permeability- inducing (BPI) protein products that may be used in combination with the present compounds include but are not limited to XMP.97 and XMP.127.
• immunomodulators that may be used in combination with the present compounds include, but are not limited to, an interferon, (e.g., IL-I, IL-2, IL-3 and IL-8), defensines, tacrolimus and G-CSF (Granulocyte-colony stimulating factor).
• alkyl refers to any linear or branched chain alkyl group having a number of carbon atoms in the specified range.
• C 1-6 alkyl refers to all of the hexyl alkyl and pentyl alkyl isomers as well as n-, iso-, sec- and t-butyl, n- and isopropyl, ethyl and methyl.
• C 1-4 alkyl refers to n-, iso-, sec- and t-butyl, n- and isopropyl, ethyl and methyl.
• cycloalkyl refers to any cyclic ring of an alkane having a number of carbon atoms in the specified range.
• C 3-6 cycloalkyl refers to cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.
• cycloalkyl-alkyl refers to a system that includes an alkyl portion as described above and also includes a cycloalkyl portion as described above. Attachment to a “cycloalkyl-alkyl” (or “alkyl-cycloalkyl”) may be through either the cycloalkyl or the alkyl portion.
• the specified number of carbon atoms in “cycloalkyl-alkyl” systems refers to the total number of carbon atoms in both the alkyl and the cycloalkyl parts.
• C 4 -C 7 cycloalkyl-alkyl examples include but are not limited to methylcyclopropyl, dimethylcyclopropyl, methylcyclobutyl, trimethylcyclobutyl, ethylcyclopentyl, methylcyclohexyl, cyclopropylmethyl, cyclopropylethyl, cyclobutylmethyl, cyclobutylpropyl, cyclopentylethyl and cyclohexylmethyl.
• alkenyl refers to a straight or branched-chain acyclic unsaturated hydrocarbon having a number of carbon atoms in the specified range and containing at least one double bond.
• C 2 -C 3 alkenyl refers to vinyl, (lZ)-1-propenyl, (IE)-I- propenyl, 2-propenyl, or isopropenyl.
• halogen refers to fluorine, chlorine, bromine and iodine (alternatively referred to as fluoro, chloro, bromo, and iodo).
• a heterocyclic ring described as containing from “1 to 4 heteroatoms” means the ring can contain 1, 2, 3 or 4 heteroatoms. It is also to be understood that any range cited herein includes within its scope all of the sub-ranges within that range. Thus, for example, a heterocyclic ring described as containing from “1 to 4 heteroatoms” is intended to include as aspects thereof, heterocyclic rings containing 2 to 4 heteroatoms, 3 or 4 heteroatoms, 1 to 3 heteroatoms, 2 or 3 heteroatoms, 1 or 2 heteroatoms, 1 heteroatom, 2 heteroatoms, and so forth.
• any of the various cycloalkyl and heterocyclic/heteroaryl rings and ring systems defined herein may be attached to the rest of the compound at any ring atom (i.e., any carbon atom or any heteroatom) provided that a stable compound results.
• Suitable 5- or 6-membered heteroaromatic rings include, but are not limited to, pyridyl, pyrrolyl, pyrazinyl, pyrimidinyl, pyridazinyl, triazinyl, thienyl, furanyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isooxazolyl, oxadiazolyl, oxatriazolyl, thiazolyl, isothiazolyl, and thiadiazolyl.
• Suitable 9- or 10- membered heteroaryl rings include, but are not limited to, quinolinyl, isoquinolinyl, indolyl, indazolyl, benzimidazolyl, benztriazoyl, imidazopyridinyl, triazolopyridinyl, and imidazopyrimidinyl.
• Suitable 4- to 6-membered heterocyclyls include, but are not limited to, azetidinyl, piperidinyl, morpholinyl, thiomorpholinyl, thiazolidinyl, isothiazolidinyl, oxazolidinyl, isoxazolidinyl, pyrrolidinyl, imidazolidinyl, piperazinyl, tetrahydrofuranyl, tetrahydrothienyl, pyrazolidinyl, hexahydropyrimidinyl, thiazinanyl, thiadiazinanyl, tetrahydropyranyl, tetrahydrothiopyranyl, and dioxanyl.
• a “stable” compound is a compound which can be prepared and isolated and whose structure and properties remain or can be caused to remain essentially unchanged for a period of time sufficient to allow use of the compound for the purposes described herein (e.g., therapeutic or prophylactic administration to a subject).
• Reference to a compound also includes stable complexes of the compound such as a stable hydrate.
• substituted includes mono- and poly-substitution by a named substituent to the extent such single and multiple substitution (including multiple substitution at the same site) is chemically allowed. Unless expressly stated to the contrary, substitution by a named substituent is permitted on any atom in a ring (e.g., an aryl, a cycloalkyl, a heteroaryl, or a heterocyclyl) provided such ring substitution is chemically allowed and results in a stable compound.
• a ring e.g., an aryl, a cycloalkyl, a heteroaryl, or a heterocyclyl
• a bond terminated by a wavy line is used herein to signify the point of attachment of a substituent group or partial structure. This usage is illustrated by the following example:
• the compounds of this invention are also useful in the preparation and execution of screening assays for antifungal compounds.
• the compounds of this invention are useful for isolating mutants, which are excellent screening tools for more powerful antifungal compounds.
• All compounds of the present invention may be administered in the form of "pharmaceutically acceptable salts" or hydrates as appropriate.
• Other salts may, however, be useful in the preparation of the compounds according to the invention or of their pharmaceutically acceptable salts.
• the compounds of the present invention may be conveniently isolated as trifluoroacetic acid salts (e.g. following HPLC purification). Conversion of the trifluoroacetic acid salts to other salts, including pharmaceutically acceptable salts, may be accomplished by a number of standard methods known in the art. For example, an appropriate ion exchange resin may be employed to generate the desired salt.
• conversion of a trifluoroacetic acid salt to the parent free amine may be accomplished by standard methods known in the art (e.g. neutralization with an appropriate inorganic base such as NaHCO 3 ).
• Other desired amine salts may then be prepared in a conventional manner by reacting the free base with a suitable organic or inorganic acid.
• Representative pharmaceutically acceptable quaternary ammonium salts include the following: hydrochloride, sulfate, phosphate, carbonate, acetate, tartrate, citrate, malate, succinate, lactate, stearate, fumarate, hippurate, maleate, gluconate, ascorbate, adipate, gluceptate, glutamate, glucoronate, propionate, benzoate, mesylate, tosylate, oleate, lactobionate, laurylsulfate, besylate, caprylate, isetionate, gentisate, malonate, napsylate, edisylate, pamoate, xinafoate, napadisylate, hydrobromide, nitrate, oxalate, cinnamate, mandelate, undecylenate, and camsylate.
• suitable pharmaceutically acceptable salts thereof may include alkali metal salts, e.g., sodium or potassium salts; alkaline earth metal salts, e.g., calcium or magnesium salts; and salts formed with suitable organic ligands, e.g., quaternary ammonium salts.
• the present invention includes within its scope prodrugs of the compounds of this invention.
• prodrugs will be functional derivatives of the compounds of this invention which are readily convertible in vivo into the required compound.
• the term “administering” shall encompass the treatment of the various conditions 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 the patient. Conventional procedures for the selection and preparation of suitable prodrug derivatives are described, for example, in "Design of
• administration and variants thereof (e.g., “administering” a compound) in reference to a compound of the invention mean providing the compound or a prodrug of the compound to the subject in need of treatment.
• a compound of the invention or a prodrug thereof is provided in combination with one or more other active agents (e.g., other antifungal/antibacterial agents useful for treating fungal/bacterial infections)
• “administration” and its variants are each understood to include concurrent and sequential provision of the compound or prodrug and other agents.
• composition is intended to encompass a product comprising the specified ingredients, as well as any product which results, directly or indirectly, from combining the specified ingredients.
• pharmaceutically acceptable is meant that the ingredients of the pharmaceutical composition must be compatible with each other and not deleterious to the recipient thereof.
• subject refers to an animal, preferably a mammal, most preferably a human, who has been the object of treatment, observation or experiment.
• effective amount means that amount of active compound or pharmaceutical agent that elicits the biological or medicinal response in a tissue, system, animal or human that is being sought by a researcher, veterinarian, medical doctor or other clinician.
• the effective amount is a "therapeutically effective amount” for the alleviation of the symptoms of the disease or condition being treated.
• the effective amount is a "prophylactically effective amount” for prophylaxis of the symptoms of the disease or condition being prevented or for reducing the likelihood of occurrence.
• the term also includes herein the amount of active compound sufficient to inhibit (1,3)- ⁇ -D-glucan synthase and thereby elicit the response being sought (i.e., an "inhibition effective amount").
• active compound i.e., active ingredient
• references to the amount of active ingredient are to the free acid or free base form of the compound.
• the compounds of the present invention optionally in the form of a salt or a hydrate, can be administered by means that produces contact of the active agent with the agent's site of action.
• the compounds of the invention can, for example, be administered by one or more of the following: orally, parenterally (including subcutaneous injections, intravenous, intramuscular, intrasternal injection or infusion techniques), by inhalation (e.g., nasal or buccal inhalation spray, aerosols from metered dose inhalator, and dry powder inhalator), by nebulizer, ocularly, topically, transdermally, or rectally, in the form of a unit dosage of a pharmaceutical composition containing an effective amount of the compound and conventional non-toxic pharmaceutically-acceptable carriers, adjuvants and vehicles.
• parenterally including subcutaneous injections, intravenous, intramuscular, intrasternal injection or infusion techniques
• inhalation e.g., nasal or buccal inhalation spray, aerosols from metered dose inhalator, and dry powder inhalator
• nebulizer ocularly, topically, transdermally, or rectally, in the form of a unit dosage of a pharmaceutical composition containing
• Liquid preparations suitable for oral administration can be prepared according to techniques known in the art and can employ the usual media such as water, glycols, oils, alcohols and the like.
• Solid preparations suitable for oral administration e.g., powders, pills, capsules and tablets
• Parenteral compositions can be prepared according to techniques known in the art and typically employ sterile water as a carrier and optionally other ingredients, such as a solubility aid.
• Injectable solutions can be prepared according to methods known in the art wherein the carrier comprises a saline solution, a glucose solution or a solution containing a mixture of saline and glucose. Further description of methods suitable for use in preparing pharmaceutical compositions of the present invention and of ingredients suitable for use in said compositions is provided in Remington's Pharmaceutical Sciences. 20 th edition, edited by A. R. Gennaro, Mack Publishing Co., 2000.
• the compounds of this invention can be administered, e.g., orally or intravenously, in a dosage range of, for example, 0.001 to 1000 mg/kg of mammal (e.g., human) body weight per day in a single dose or in divided doses.
• compositions can be provided in the form of tablets or capsules containing, for example, 1.0 to 500 milligrams of the active ingredient, particularly 1, 5, 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 300, 400, and 500 milligrams of the active ingredient for the symptomatic adjustment of the dosage to the patient to be treated.
• the specific dose level and frequency of dosage for any particular patient may be varied and will depend upon a variety of factors including the activity of the specific compound employed, the metabolic stability and length of action of that compound, the age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular condition, and the host undergoing therapy.
• the present invention also includes processes for making compounds of Formula (I).
• the compounds of the present invention may be prepared according to the following reaction schemes and examples, or modifications thereof, from starting material enfumafungin.
• Enfumafungin is a natural product produced from a fungus strain of Hormonema sp. (deposited under the Budapest Treaty in the culture collection of the American Type Culture Collection and assigned accession number ATCC 74360) that was isolated from living leaves of an unidentified shrub collected in Navalquejigo, province of Madrid, Spain, as described in U.S. Pat.
• Scheme A illustrates a method for deglycosylation of the natural product enfumafungin and additional modification to prepare the molecule for further elaboration.
• the lactol group of enfumafungin is reduced by treatment with a suitable reducing agent such as triethylsilane under acidic conditions (e.g. trifluoroacetic acid) to give compound A2.
• a suitable reducing agent such as triethylsilane under acidic conditions (e.g. trifluoroacetic acid) to give compound A2.
• acidic conditions e.g. trifluoroacetic acid
• Removal of the glucose moiety may be accomplished by heating A2 in methanol in the presence of a strong acid such as sulfuric acid. Under these conditions, the acetoxy group at C 14 is also replaced by methoxy to produce the methyl ether compound A3.
• Schemes B to E illustrate methods of introducing the R 2 substituent on the Cl 5 hydroxy group. Additional methods are also described in International Patent Publication No. WO 2007/127012, herein incorporated by reference in its entirety.
• the variables R 6 , R 7 , R 8 and R 9 are as previously defined or are precursor groups thereto. Additional variables are as defined in the individual schemes.
• reaction of A4 with an iV-sulfonyl aziridine (Bl) in the presence of a suitable base such as potassium hydride, sodium hydride or potassium tert- pentylate and optionally in the presence of an appropriate cation complexing agent such as 18- Crown-6 or 15-Crown-5 gives intermediate B2.
• a suitable base such as potassium hydride, sodium hydride or potassium tert- pentylate
• an appropriate cation complexing agent such as 18- Crown-6 or 15-Crown-5
• Scheme C illustrates an alternative method of substituting the amino group by alkylating the iV-sulfonyl intermediate B2 with an appropriate alkylating agent such as methyl iodide, ethyl iodide or allyl bromide in the presence of a suitable base such as sodium hydride to give Cl. Dissolving metal reduction as described previously for B2 then gives C2.
• the synthesis of Scheme C is particularly useful for introducing a single substitution on the aminoether nitrogen.
• introduction of an R 6 group is illustrated, but it will be apparent to one skilled in the art that the synthesis would work equally well for introduction of an R 7 group.
• deprotection Scheme D describes additional methods for introducing the R 2 group.
• Reaction of A4 with the 5-membered cyclic sulfamidate reagent Dl gives intermediate D3. This reaction is carried-out under conditions analogous to those described in Scheme B for coupling with aziridine Bl. An acidic aqueous work-up is carried-out which cleaves the initial N-sulfated product to give the amine D3.
• reaction of A4 with the 6-membered cyclic sulfamidate reagent D2 gives D4 and after removal of the amine protecting group the aminopropyl ether intermediate D5 is obtained.
• Suitable protecting groups for D2 and D4 include t- butyloxycarbonyl (Boc) and benzyloxycarbonyl (Cbz).
• the cyclic sulfamidate reagents Dl and D2 are prepared by methods known in the art (e.g. Tetrahedron 2003, 59, 2581-2616; J. Org. Chem. 2004, 69, 3610-3619; Angew. Chem. Int. Ed. 2005, 44, 3518-3520) and as exemplified further below.
• Scheme E illustrates additional methods for introduction of the R 2 substitution on the C 15 hydroxy group.
• Alkylation of A4 with an allylic halide or other suitably activated allylic species (El) gives the allylic ether E2.
• Suitable bases for this reaction are sodium hydride or potassium hydride and the like.
• Oxidative cleavage of the alkenyl group under standard conditions e.g. OsO 4 ZNaIO 4
• Reductive amination of E3 by standard methods e.g. R 6 R 7 NH, NaBH 3 CN, AcOH, MeOH, THF then gives the aminoether E4.
• E3 can be converted to the sulfinylimine E5 by reaction with an alkyl- or arylsulfinylamide in the presence of a dehydrating agent such as copper sulfate or titanium ethoxide.
• a dehydrating agent such as copper sulfate or titanium ethoxide.
• Reaction of E5 with an alkyllithium reagent e.g. R 9 Li / Me 3 Al
• alkyl Grignard reagent e.g. R 9 MgBr
• R 9 H
• a metal hydride reducing agent e.g. lithium triethylborohydride
• acid treatment e.g. HCl/MeOH
• Schemes G-N illustrate methods for introducing R 1 heterocyclic groups.
• the various methods are exemplified by starting with intermediate B4, but it is understood that the same methods work equally well starting with many other intermediates including but not limited to B3, C2, D3, D5, E4, and E6.
• many of the intermediates and final compounds described in International Patent Publication No. WO 2007/127012 may also serve as starting materials for introduction of R 1 heterocyclic groups as described in Schemes G-N.
• the variables R e , R 6 , R 7 , R 8 and R 9 are as previously defined or are precursor groups thereto.
• Scheme G illustrates the introduction of a 1,2,4-triazole heterocycle at the C 14 position.
• the displacement reaction between B4 and the triazole derivative Gl is promoted by a Lewis acid reagent.
• Suitable Lewis acid reagents include boron trifluoride diethyl etherate, copper trifluoromethansulfonate, zinc trifluoromethanesulfbnate and the like.
• the reaction is conducted in a non-coordinating aprotic solvent such as 1 , 2-dichloroethane at a temperature of between about 20°C and about 100°C. This displacement reaction generally occurs with retention of configuration at C 14, possibly due to participation by the proximal bridging ether oxygen.
• Scheme H illustrates an alternative method for installing a substituted 1,2,4- triazole group.
• Reaction of intermediate B4 with anhydrous hydrazine promoted by a Lewis acid such as boron trifluoride diethyl etherate yields the hydrazine intermediate Hl.
• the reaction is conducted in a non-coordinating aprotic solvent such as 1,2-dichloroethane at a temperature of between about 20°C and 100°C.
• Scheme I describes a variation of the synthesis method of Scheme H which is useful in certain instances.
• the R 1 heterocyclic group is introduced prior to installation of the R 2 ether substituent.
• the starting point for this mode of synthesis is intermediate Il which may be synthesized according to Scheme A.
• Steps 1 and 2 in Scheme I are analogous to Steps 1 and 2 in Scheme H.
• reaction of Il with hydrazine or a protected form of hydrazine promoted by a Lewis acid such as boron trifluoride diethyl etherate gives the hydrazino intermediate 12.
• a Lewis acid such as boron trifluoride diethyl etherate
• a deprotection step may be required at this stage in the synthetic Scheme.
• a bromotriazole compound Jl is employed as a versatile intermediate.
• Compound Jl may be synthesized according to the method of Scheme G. Palladium catalyzed cross-coupling reactions between Jl and a variety of aryl- and heteroaryl- boronic acid and boronate ester derivatives are possible to give product compounds J2. These reactions are generally carried-out in the presence of a base (e.g. cesium carbonate) a palladium catalyst (e.g. palladium (II) acetate) and a phosphine ligand (e.g.
• a base e.g. cesium carbonate
• a palladium catalyst e.g. palladium (II) acetate
• a phosphine ligand e.g.
• 2-dicyclohexylphosphino- 2',4',6'-triisopropylbiphenyl) at an elevated temperature e.g. 50°C to 120°C.
• Such cross- coupling reactions are well-known in the art and are generically referred to as Suzuki couplings.
• Many suitable aryl- and heteroaryl boronic acid and boronate ester derivatives are readily available from commercial sources or are readily prepared by known methods.
• Other related cross-coupling reactions to give product compounds J2 are also possible, such as a Stille cross- coupling reaction between Jl and an aryl or heteroaryl stannane derivative.
• alkenyl groups to give J3 may be accomplished by reaction of Jl with alkenyl trifluoroborate salts (e.g. potassium vinyltrifluoroborate) in the presence of a palladium catalyst. Further reaction of the alkenyl products J3 may also be carried-out (e.g. hydrogenation to give alkyl substitution). Direct reaction of Jl with alkyl trifluoroborate salts is also possible. Other transformations of Jl that may be carried-out include reaction with a thiol derivative in the presence of a base to give products J4 or reaction with an amino derivative under the influence of heat and optionally an additional base to give aminotriazole derivatives J5. Product compounds J4 may also be further derivatized (e.g. oxidation to a sulfone). Scheme K
• Scheme K illustrates the synthesis of another subclass of compounds of the invention.
• the starting cyanotriazole compound Kl may be synthesized by the method of Scheme G. Reaction of Kl with ethylenediamine or a substituted ethylenediamine derivative in the presence of carbon disulfide gives a cyclic amidine derivative K2. This reaction is generally carried-out at a temperature between about 20°C and 100°C. Similarly, reaction of Kl with propylenediamine or a substituted propylenediamine derivative gives a 6-membered ring cyclic amidine derivative K3.
• Scheme L illustrates the synthesis of another subclass of compounds of the invention.
• the starting cyanotriazole compound Kl may be synthesized by the method of Scheme G. Reaction of Kl with ethylenediamine or a substituted ethylenediamine derivative in the presence of carbon disulfide gives a cyclic amidine derivative K2. This reaction is generally carried-out at a temperature between about 20°C and 100°C
• Schemes L to N illustrate the introduction of additional heterocyclic groups.
• Scheme L describes introduction of a mono substituted 1,2,3-triazole heterocycle. This synthesis is analogous to that described in Scheme G for a 1 ,2,4-triazole.
• R e is unsymmetrically substituted
• three regioisomeric products may be formed in this displacement reaction, L2, L3 and L4.
• two or three isomers are formed, it is often possible and desirable to separate them chromatographically or by other means.
• the ratio of the isomers may vary depending on the substituent group R e .
• the starting triazole compounds Ll are generally readily available either from commercial sources or through preparation by known literature methods.
• a mono-substituted triazole is employed.
• a disubstituted triazole may also be introduced in an analogous manner.
• Scheme M illustrates the introduction of a mono-substituted pyrazole heterocycle.
• Scheme N illustrates the introduction of a mono-substituted imidazole heterocycle. This synthesis is analogous to that described in Scheme M for a mono-substituted pyrazole.
• the imidazole starting material is symmetrically substituted as is the case for Nl, then a single product N3 is obtained.
• the imidazole starting material is unsymmetrically substituted (e.g. N2 where R e is not hydrogen) then two regioisomeric products may be formed in this displacement reaction, N4 and N5.
• the ratio of the isomers may vary depending on the substituent group R e .
• the starting imidazole compounds Nl and N2 are generally readily available either from commercial sources or through preparation by known literature methods. While in the examples illustrated in Scheme N, mono-substituted imidazoles are employed, di- and trisubstituted imidazole groups may also be introduced in an analogous manner.
• the antifungal activity of the present compounds can be demonstrated by various assays known in the art, for example, by their glucan synthesis inhibitory activity (IC 50 ), minimum inhibitory concentration (MIC-IOO) or minimum prominent inhibition (MIC-50) against yeasts and minimum effective concentration (MEC) against filamentous moulds and dermatophytes in a broth microdilution assay, or in vivo anti-Candida activity in a mouse (TOKA).
• IC 50 glucan synthesis inhibitory activity
• MIC-IOO minimum inhibitory concentration
• MEC minimum effective concentration
• TOKA in vivo anti-Candida activity in a mouse
• Compounds provided in the Examples were generally found to inhibit the growth of Candida spp. in the range of ⁇ 0.03-32 ⁇ g/mL or to give an MEC against Aspergillus fumigatus in the range of ⁇ 0.03-32 ⁇ g/mL.
• glucan synthase inhibitory activity of compounds was measured in a polymerization assay in 96- well format.
• Each well contained 100 ⁇ L of 3 H- UDPG at 0.5 mM (6000 to 8000 dpm/nmol), 50 mM HEPES pH 7.5 (Sigma), 10% w/v glycerol (Sigma), 1.5 mg/mL bovine serum albumin (Sigma A 9647.
• HARVESTER 40 ⁇ L/well scintillation fluid (Packard ULTIMA GOLD TM-XR) was added and the sealed plates counted in a WALLAC BETA counter in top-counting mode at an efficiency of approximately 40%.
• Graphs of the primary data were created in PRISM software (the average of two determinations) using PRISM' s curve fitting program (sigmoidal dose response non-linear regression). The amount of compound required to inhibit glucan synthase activity by 50% in this assay (IC 50 - ng/mL) was calculated.
• glucan synthase prepared from Candida albicans MY1055 by the following procedure: MY1055 was grown in 10 liters YPD medium (1O g yeast extract, 20 g tryptone, 20 g glucose per liter) with vigorous shaking at 30°C, to early stationary phase. Cells were harvested by centrifugation, the pellet was washed and frozen at -70°C until breakage. Thawed pellets were shaken with an equal volume of breakage buffer (50 mM HEPES pH 7.4, 10% glycerol, 1 mM EDTA, 1 mM PMSF, ImM DTT) and 4 times their weight of 0.5 mm acid washed glass beads for 2 hours at 4°C.
• breakage buffer 50 mM HEPES pH 7.4, 10% glycerol, 1 mM EDTA, 1 mM PMSF, ImM DTT
• Extraction of GS activity from membranes was performed at a protein concentration of 5 mg/mL in extraction buffer (5OmM NaPO 4 pH 7.5, 0.1 M KCl, 0.1M Na citrate, 20% glycerol, 5 ⁇ M GTP- ⁇ -S, ImM DTT, 1 mM PMSF, 3 ⁇ g/mL pepstatin) plus 0.25% Wl by gentle mixing at 4°C for 60 min, followed by centrifugation at 100,000 x g for 60 min. After centrifugation, clear supernatant was removed from a pellet consisting of a hard layer usually with small amounts of gelatinous unextracted membranes above it.
• extraction buffer 5OmM NaPO 4 pH 7.5, 0.1 M KCl, 0.1M Na citrate, 20% glycerol, 5 ⁇ M GTP- ⁇ -S, ImM DTT, 1 mM PMSF, 3 ⁇ g/mL pepstatin
• Trapping was initiated immediately by 5-fold dilution in trapping buffer (50 mM HEPES pH 7.5, 10 mM KF, 1 mM EDTA, 2 mg/mL BSA) plus 2.5 mM UDPG and lO ⁇ M GTP- ⁇ -S. After incubation at 25°C for 60 to 90 minutes, glucan was harvested by low speed centrifugation (3,000 x g, 10 min).
• the soft pellet was washed 3 times with wash buffer (50 mM HEPES, 20% glycerol, 1 mM EDTA) plus 2.5 mM UDPG and 5 ⁇ M GTP- ⁇ -S, once without UDPG, and suspended in about 5 volumes of PE extraction buffer (50 mM HEPES, 30% glycerol, 1 mM EDTA, 20 ⁇ M GTP- ⁇ -S, 0.4% CHAPS, 0.08% cholesterol hemisuccinate) using a DOUNCE homogenizer.
• the suspension was frozen overnight at -70°C, and then centrifuged at 100,000 x g for 10 min. The post-centrifugation supernatant was frozen as aliquots at -70°C for subsequent assays.
• test medium (example: RPMI- 1640 containing 0.165 M MOPS + 3 g/L glutamine w/o sodium bicarbonate or RPMI- 1640 containing 0.165 M MOPS + 3 g/L glutamine w/o sodium bicarbonate with 3.2% DMSO or 2X RPMI- 1640 containing 0.33 M MOPS + 6 g/L glutamine w/o sodium bicarbonate with 6.4% DMSO for the plates with final concentration of 50% serum) was added.
• the test compound was dissolved at concentration of 10 mg/mL in DMSO and diluted 1:78 into appropriate test medium with no DMSO or 1.92% DMSO or 5.12% DMSO.
• the test compound concentration achieved was 128 ⁇ g/ml and DMSO concentration of 3.2%.
• Cryptococcus neoformans MY2062
• Saccharomyces cerevisiae MY2255
• SDA SABOURAUD Dextrose Agar
• From the regrowth 3 to 5 colonies were selected and suspended in 5 mL of sterile normal saline (BBL) and adjusted to match the turbidity of a 0.5 McFarland standard using DADE/BEHRING turbidity meter (preferred OD of 0.06 to 0.12). This resulted in a concentration of approximately 1-5 x 10 6 CFU/mL.
• the inocula were further diluted 1:1000 into RPMI-1640 containing 0.165 M MOPS + 3 g/L glutamine w/o sodium bicarbonate with 3.2% DMSO.
• Assay plates previously titrated with test compound in RPMI- 1640 containing 0.165 M MOPS + 3 g/L glutamine w/o sodium bicarbonate with 3.2% DMSO were then inoculated with 100 ⁇ L/well of this dilution of culture. This resulted in a final organism concentration of 5 x 10 2 to 2.5 x 10 3 CFU/mL and final compound concentrations of 32 to 0.03 ⁇ g/mL.
• albicans (MY1055) was also tested with heat inactivated (1 hour at 55°C) mouse serum which was filtered twice using 0.22 micron GP EXPRESS PLUS MILLIPORE filtration system. This standardized suspension was diluted 1:1000 into mouse serum. Assay plates previously titrated with drug in 2X RPMI-1640 containing 0.33 M MOPS + 6 g/1 glutamine w/o sodium bicarbonate with 6.4% DMSO were then inoculated with 100 ⁇ l/well of this dilution of culture. This resulted in a final organism concentration of 5 x 10 2 to 2.5 x 10 3 CFU/mL and final compound concentration of 32 to 0.03 ⁇ g/ml and 50% mouse serum. Plates were incubated at 35-37°C and MICs were read at 24 hours for Candida and 48 hours for Cryptococcus neoformans. Filamentous fungi
• Each spore suspension was then transferred to another tube and adjusted to match the turbidity of a 0.5 McFarland standard using the DADE/BEHRING turbidity meter (preferred OD of 0.06-0.09) for A. fumigatus and (preferred OD of 0.13-0.17) for dermatophyte T. mentagrophytes. This resulted in a concentration of approximately 1-5 x 10 6 CFU/mL.
• a spore count was performed on each culture suspension with a hemocytometer to insure the correct inoculum. This standardized suspension for A.
• A. fumigatus (MF5668) was also tested with heat inactivated human serum which was filtered once using 0.22 micron GP EXPRESS PLUS MILLIPORE filtration system. This standardized suspension was diluted 1:500 in human serum.
• Assay plates previously titrated with test compound in 2X RPMI-1640 containing 0.33 molar MOPS + 6 g/L glutamine w/o sodium bicarbonate were then inoculated with 100 ⁇ l/well of this dilution of culture. Plates were incubated at 35°C and MICs were read at 48 hours for Aspergillus fumigatus, and plates incubated at 30°C and MICs were read at 96 hours for Dermatophyte T. mentagrophytes.
• the minimum inhibitory concentration (MIC-IOO) for all test compounds is determined to be the lowest concentration of compound at which there was no visible growth as compared to growth control without test compound.
• the minimum prominent inhibition (MIC- 80) in growth is indicated as 80% inhibition in growth compared to growth control without test compound.
• MEC mentagrophytes minimum effective concentration
• mice are treated with test compound, either (1) I.P., b.i.d. for a total of 2 days, or (2) P.O., b.i.d. for a total of 2 days.
• test compound either (1) I.P., b.i.d. for a total of 2 days, or (2) P.O., b.i.d. for a total of 2 days.
• an appropriate sham-treated control group is included.
• Kidneys from euthanized mice are removed four days after challenge using aseptic techniques, weighed and placed in sterile WHIRL PAK bags containing 5 mL sterile saline. Kidneys are homogenized in the bags, serially diluted in saline and aliquots are plated on SD agar plates. Plates are incubated at 35°C and enumerated after 30 to 48 hours for C. albicans colony forming units (CFUs). Means from CFU/g of paired kidneys of treated groups are compared to the means from sham-treated controls.
• CFUs C. albicans colony forming units
• Percent sterilization is indicated by the number of mice with no detectable yeast, where the limit of detection (because of the dilution scheme) is 50 yeast cells per pair of kidneys.
• the limit of detection because of the dilution scheme
• 9.8 is entered into the MICROSOFT EXCEL spread sheets formula [logio (5 x raw count)/paired kidney weight)] so that the counts would be one less than the limit of detection (49 cells per pair of kidneys).
• Compounds provided in the Examples generally have GS IC 50 values less than 500 ng/mL and MIC-100 values against one or more organisms of ⁇ 0.03-32 ⁇ g/mL; however, some compounds may have an IC 50 in the range of from about 500 to more than 10,000 ng/mL.
• Compounds provided in the Examples generally display prominent inhibition of growth in vitro (MIC-50) in the range of ⁇ 0.03-32 ⁇ g/mL and MECs of ⁇ 0.03-32 ⁇ g/mL.
• useful compounds will lower the number of fungal CFU/g kidney by greater than 1 logio unit compared to sham treated controls and compounds that lower the CFU/g by 2 logio units are especially useful.
• Example Numbers correspond to the examples described in the Examples section.
• Step 1 NaBH 4 (94.5 g, 2.498 mol) was charged into a 5 L three-necked flask containing
• the reaction mixture was stirred for 4 h while keeping the temperature below 12°C.
• Water (600 ml) was added to the mixture followed by brine (sat. acqueous NaCl, 350 ml).
• the aqueous layer was extracted with CH 2 Cl 2 (3x500 ml).
• the combined organic layers were dried over Na 2 SO 4 , filtered concentrated.
• the crude product was purified over a pad of silica gel (EtO Ac/Heptanes: 5/95 then 10/90) to afford the title compound as a white solid (36 g).
• Step 2 To a solution of the product from Step 1 (17.0 g, 70 mmol) in MeOH was added
• Step 3 A solution of the amino alcohol from Step 2 (4.98 g, 38.0 mmol) in CH 2 Cl 2 (200 mL) at 0°C was treated with Et 3 N (26 mL, 190 mmol) followed by a solution of p- toluenesulfonyl chloride (8.7 g, 45.6 mmol) in CH 2 Cl 2 (50 mL) over 40 min. The reaction mixture was stirred at room temperature for 4 days.
• Chloramine-T (1Og, 43.9 mmol) was suspended in acetonitrile (146 mL) at room temperature under nitrogen. To this suspension was added 2-ethylbut-1-ene (5.55 g, 65.9 mmol) followed by phenyltrimethylammonium tribromide (1.65 g, 4.39 mmol) in two roughly equal portions. After three days the reaction mixture was concentrated to half volume and then filtered through a sintered glass funnel. The filtrate was concentrated to half volume again which caused further precipitation. This mixture was filtered and the filtrate was partitioned between ethyl acetate and water. The organic phase was dried with MgSO 4 , filtered and concentrated.
• the cooling bath was removed after 30 minutes and the reaction stirred at room temperature and additional reagents were added during the reaction: TsCl (at 16 hours: 5.3 g, at 40 hours: 3 g), triethylamine (at 24 hours: 3 mL) and DMAP (at 20 hours: 200 mg and at 40 hours: 150 mg).
• TsCl at 16 hours: 5.3 g, at 40 hours: 3 g
• triethylamine at 24 hours: 3 mL
• DMAP at 20 hours: 200 mg and at 40 hours: 150 mg.
• After 40 hours at room temperature the reaction was heated to 40°C. After a total of 44 hours the reaction was cooled to room temperature and filtered. The filtrate was concentrated in vacuo then partitioned between ethyl acetate and water. The organic phase was washed with brine, dried over MgSO 4 then filtered and concentrated.
• Phenyltrimethylammonium tribromide (7.80 g, 20.76 mmol) was added in three portions over 10 min. The mixture was stirred at room temperature for 16 hours. The solvent was evaporated and the residue was partitioned between dichloromethane and water. The organic layer was dried over Na 2 SO 4 and the solvent was evaporated. The residue was chromatographed on silica gel with an ISCO Combiflash using EtOAc/hexanes (5-30% gradient) to afford the title compound as a white solid (2.66g).
• Step 1 To a solution of compound (3R,5R)-3-(l,l-dimethylethyl)-3-methyl-5- phenylmorpholin-2-one (100 mg, 0.40 mmol) in MeOH was added formaldehyde (1.40 mL, 37 wt% in water, 16.2 mmol), HOAc (0.14 mL, 2.4 mmol), and sodium cyanoborohydride (100 mg, 1.60 mmol). The reaction mixture was stirred at room temperature overnight, and the solvent was removed under reduced pressure. The residue was partitioned between EtOAc and saturated aqueous NaHCO 3 , and the layers were separated. The organic layer was washed with saturated aqueous NaHCO 3 , saturated aqueous NaCl, dried (Na 2 SO 4 ), and concentrated under reduce pressure to provide the product (99.7 mg, 95%) as a viscous oil.
• Step 3 Following procedures analogous to those described for Steps 3 and 4 of
• reaction solution allowed to cool to room temperature and diluted with dichloromethane (25 mL), washed with water (25 mL), 0.5M hydrochloric acid (25 mL), water (25 mL), dried over MgSO 4 , filtered, and evaporated to solid.
• the solid was flash chromatographed (silica gel, 10-65% ethyl acetate:hexane) to give the product as a white solid (683 mg).
• the vial was cooled to room temperature and unsealed.
• the solid was dissolved in methanol and transferred to a 200 ml flask.
• the solvent was evaporated under reduced pressure and the resulting residue was stripped several times with ethanol to removed excess isopropylamine.
• the solid was suspended in ice cold concentrated ammonium hydroxide (5 ml) and stirred in an ice bath for several minutes. The suspension was filtered, rinsing over with ice cold water (2 x 2 ml). The filtered solid was air dried overnight to give the amide product (313.1 mg) as a white solid.
• the product compound from Step 1 (313.1 mg, 1.612 mmol) and N,N- dimethylformamide diethyl acetal (0.38 ml, 2.217 mmol) were combined and heated to 120°C.
• the reaction mixture was a light purple solution that slowly turned lighter until it had become an orange solution.
• the reaction mixture was slowly placed under vacuum.
• the reaction mixture was cooled to room temperature about five minutes after reaching full vacuum.
• the vacuum was released after the reaction mixture had cooled.
• the reaction mixture was placed under high vacuum and solidified after several hours to give the title compound (390.3 mg) as a tan solid.
• Step 2 N- [( 1 EHdimeth ylamino)meth ylidenel imidazof 1 ,2-al pyridine-7-carboxamide Step 1:
• Step l
• Step 2 To a solution of the solid from above (120 g crude material, -126.9 mmol) in
• the solid was then rinsed with heptane and was allowed to air aspirate to initial dryness.
• the white solid recovered was then transferred to a recrystallizing dish and placed in a vacuum oven at 30°C for four hours to give 52.2 g of white solid.
• a portion of the purified material from Step 1 (800 mg) was dissolved in dimethoxyethane (20 mL) and the solution was chilled to -70°C.
• Ammonia (20 g) was added to the reaction solution and sodium metal (enough to sustain a blue color) was added over the course of 1.5 hours.
• the reaction solution was stirred at -60°C for 2 hours and then warmed to ammonia reflux for 30 minutes.
• the reaction was judged complete and methanol (15 mL) was slowly added.
• the reaction was then warmed to 0°C and water (50 mL) was added.
• the aqueous phase was thrice washed with ethyl acetate (75 mL); the organic phases were combined, dried over magnesium sulfate, and concentrated to give the product as a white solid.
• the reaction was allowed to slowly warm to room temperature and after two hours additional 2-(l,l- dimethylethyl)-2-methyl-1-[(4-methylphenyl)sulfonyl]aziridine (0.43 g, 0,83 mmol) was added. After an additional hour the reaction was quenched by the addition of methanol followed by 1 N aq. HCl. The reaction mixture was partitioned between ethyl acetate and water and the aqueous extracted with ethyl acetate as necessary. The combined organic phase was dried over MgSO 4 , filtered then concentrated.
• reaction was quenched by the addition of water followed by 1 N aq. HCl.
• the reaction mixture was partitioned between ethyl acetate and water and the aqueous extracted with ethyl acetate. The combined organic phase was dried over
• the aqueous layer was extracted with more ethyl acetate (4 x 50 mL) and the combined ethyl acetate layers were dried with sodium sulfate, filtered and evaporated to a solid (6.0 g).
• the solid was purified by reverse phase HPLC using a 19 x 150 mm Sunfire Preparative C18 OBD column. Fractions containing the product were combined, evaporated and freeze-dried from a mixture of ethanol and benzene to give the title compound as a TFA salt (2.0 g).
• the reaction mixture was heated overnight at 50°C and allowed to cool to room temperature.
• the reaction mixture was partitioned between EtOAc and water, and the layers were separated.
• the aqueous layer was extracted with EtOAc.
• the combined organic layers were dried with Na 2 SO 4 and concentrated under reduced pressure.
• the residue was purified by flash chromatography, elution with 0- 50% EtOAc in heptane, to give the product (4.16 g, 66%) as a pale yellow solid.
• Step 2 To a solution of the product from Step 1 (2.71 g, 4.40 mmol) in DMF (30 rnL) was added potassium carbonate (12.0 g, 88.0 mmol) and 4-methoxybenzyl bromide (0.64 mL, 4.40 mmol). The mixture was stirred at room temperature overnight. The reaction mixture was poured into water (ca. 300 mL) and extracted with EtOAc. The combined organic layers were washed with saturated aqueous NaCl, dried with Na 2 SO 4 , and concentrated under reduced pressure. The residue was purified by flash chromatography, elution with 0 - 10% MeOH in CH 2 Cl 2 , to give Intermediate 38 (2.60 g, 80%) as a white solid.
• Methyl 1,2,4-triazole-3-carboxylate (27.1 nig, 0.213 mmol) and BF 3 OEt 2 (54 ⁇ l, 0.426 mmol) were added to a stirred solution of Intermediate 6 (25.9 mg, 0.043 mmol) in 1,2- dichloroethane (0.43 ml).
• the reaction mixture was a light yellow suspension that was heated at 50°C for 7.5 hr and then stirred at room temperature for 64 hr. The solvent was evaporated and the resulting residue was placed under high vacuum.
• WO 2007/127012 herein incorporated by reference in its entirety; 20 mg, 0.03 mmol), 3-chloro-iH-1,2,4-triazole (26.8 mg, 0.259 mmol) and boron trifluoride etherate (75 ⁇ L, 0.592 mmol) in dichloroethane (0.7 mL) was blanketed with nitrogen and placed in a 50°C oil bath for 24 hours. The mixture was cooled to room temperature, evaporated and the residual oil was separated by reverse phase ⁇ PLC using a 19 x 150 mm Sunfire Preparative C18 OBD column.
• EXAMPLE 9A as a white solid (3.4 mg)
• EXAMPLE 9B as a white solid (2.0 mg)
• EXAMPLE 9C as a white solid (2.0 mg).
• EXAMPLE 29B 1H NMR (CD 3 OD, 600MHz, ppm) 0.76 (s, 3H, Me), 0.77 (d, 3H, Me), 0.81 (s,
• EXAMPLE 32B (7.8 mg) as a white solid.
• the regiochemistry of the two isomers was assigned based on an 1 H NMR NOE from H 14 to the methine proton of the cyclopropyl which was observed for EXAMPLE 32A, but not for EXAMPLE 32B.
• EXAMPLE 33 (1S,4a ⁇ ,6aS,7R,8R,10aR,10bR,12aR,14R,15R)-15-[[(2R)-2-amino-2,3- dimethylbutyl]oxy]-14-(3,5-dibromo-4H-1,2,4-triazol-4-yl)-8-[(lR)-1,2-dimethylpropyl]- 1 ,6,6a,7,8,9, 10, 10a, 10b, 11 , 12, 12a-dodecahydro- 1 ,6a,8, 10a-tetramethyl-4H- 1 ,4a-propano-2H- phenanthro[1,2-c]pyran-7-carboxylic acid (EXAMPLE 33A) and (1S,4aR,6aS,7R,8R,10aR,10bR,12aR,14R,15R)-15-[[(2R)-2-amino-2,3- dimethylbuty
• N-(l-methylethyl)-1H-1,2,4-triazole-3-carboxamide (42.0 mg, 0.272 mmol) and BF 3 OEt 2 (68 ⁇ l, 0.537 mmol) were added to a stirred solution of Intermediate 6 (32.6 mg, 0.054 mmol) in 1,2-dichloroethane (1.0 ml).
• the reaction mixture was a pale yellow solution that was heated to 50°C. After 4.5 hr, LCMS and 1 H NMR showed about 75% conversion of Intermediate 6 to a mixture of the three triazole regioisomers at C 14. After 4.75 hours, additional BF 3 OEt 2 (30 ⁇ l, 0.237 mmol) was added to the reaction mixture.
• the reaction mixture was cooled to room temperature, the solvent was evaporated, and the resulting residue was placed under high vacuum.
• the residue was dissolved in methanol and separated using a single HPLC run on a 19 x 150 mm Sunfire Prep C18 OBD 10 ⁇ m column by eluting with acetonitrile/water + 0.1% TFA.
• the total flow rate was 20 ml/min and the HPLC method employed a 12 minute 20%- 100% acetonitrile/water gradient followed by a 6 minute acetonitrile flush.
• N-(l-methylethyl)-1H-1,2,4-triazole-3-carboxamide 753.1 mg, 4.88 mmol
• BF 3 OEt 2 1.3 ml, 10.26 mmol
• the reaction mixture was a white suspension that was heated to 50°C. After 6 hr, LCMS and 1 H NMR showed complete consumption of.
• the total flow rate was 15 ml/min and the HPLC method employed a 12 minute 20%- 100% acetonitrile/water gradient followed by a 7 minute acetonitrile flush.
• the HPLC fractions containing the desired product were combined and the solvent was evaporated under reduced pressure to give the title compound (450 mg) as a colorless residue.
• N-(l-methylethyl)-7H-1,2,4-triazole-3-carboxamide (82.3 mg, 0.534 mmol) and BF 3 OEt 2 (200 ⁇ l, 1.578 mmol) were added to a stirred solution of
• the reaction mixture was a light amber solution that was heated to 50°C. After 50 hours, the reaction mixture was cooled to room temperature, the solvent was evaporated under reduced pressure, and the resulting residue was placed under high vacuum. The residue was dissolved in methanol and purified using two HPLC runs (-35 mg / run) on a 19 x 150 mm Sunfire Prep C18 OBD 10 ⁇ m column by eluting with acetonitrile/water + 0.1% TFA. The total flow rate was 15 ml/min and the HPLC method employed a 12 minute 20%- 100% acetonitrile/water gradient followed by a 7 minute acetonitrile flush. The product HPLC fractions were combined, the solvent was evaporated under reduced pressure, and the residue was lyophilized from ethanol and benzene to give the title compound (24.2 mg, 0.028 mmol) as a white solid.
• N-(triethylsilyl)-iH-1,2,4-triazole-3-carboxamide (78.6 mg, 0.347 mmol) and BF 3 OEt 2 (130 ⁇ l, 1.026 mmol) were added to a stirred solution of Intermediate 6 (41.6 mg, 0.069 mmol) in 1,2-dichloroethane (1.15 ml).
• the reaction mixture was a tan suspension that was heated to 50°C. After 2 hr, LCMS and 1 H NMR showed complete consumption of Intermediate 6.
• the reaction mixture was cooled to room temperature, the solvent was evaporated, and the resulting residue was placed under high vacuum.
• the residue was dissolved in methanol and purified using a single HPLC run on a 19 x 150 mm Sunfire Prep C18 OBD 10 ⁇ m column by eluting with acetonitrile/water + 0.1% TFA.
• the total flow rate was 15 ml/min and the HPLC method employed a 12 minute 20%- 100% acetonitrile/water gradient followed by a 7 minute acetonitrile flush.
• the HPLC fractions containing the desired product were combined, the solvent was evaporated under reduced pressure, and the residue was lyophilized from ethanol and benzene to give the title compound (9.0 mg) as a white solid.

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