EP4598906A2 - Sterylglucosidase inhibiting compositions and method of using - Google Patents

Sterylglucosidase inhibiting compositions and method of using

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Publication number
EP4598906A2
EP4598906A2 EP23875757.9A EP23875757A EP4598906A2 EP 4598906 A2 EP4598906 A2 EP 4598906A2 EP 23875757 A EP23875757 A EP 23875757A EP 4598906 A2 EP4598906 A2 EP 4598906A2
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EP
European Patent Office
Prior art keywords
alkyl
aryl
heteroaryl
heterocycloalkyl
compound
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
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EP23875757.9A
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German (de)
French (fr)
Inventor
Iwao Ojima
Maurizio Del Poeta
Michael AIROLA
Nivea PEREIRA DE SA
Seung Youn SHIN
Kalani JAYANETTI
Dominick RENDINA
Ananya SHIBANA THENNARASU
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Research Foundation of the State University of New York
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Research Foundation of the State University of New York
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Publication of EP4598906A2 publication Critical patent/EP4598906A2/en
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
    • C07D403/06—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings linked by a carbon chain containing only aliphatic carbon atoms
    • 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
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/4025—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil not condensed and containing further heterocyclic rings, e.g. cromakalim
    • 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
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/506—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim not condensed and containing further heterocyclic rings
    • 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
    • A61K31/53—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with three nitrogens as the only ring hetero atoms, e.g. chlorazanil, melamine
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing three or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
    • C07D403/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings linked by a chain containing hetero atoms as chain links
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing three or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
    • C07D405/14—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing three or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D413/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms
    • C07D413/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings
    • C07D413/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings linked by a chain containing hetero atoms as chain links
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D413/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms
    • C07D413/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing three or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
    • C07D417/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing three or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
    • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
    • C07D487/04—Ortho-condensed 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

  • Invasive fungal infections are a leading cause of death in immunocompromised patients. While much is known about the cellular processes required for the pathogenesis of these infections, translating understanding into tangible clinical benefit has been difficult because these fungal pathogens and their hosts have similar physiology. As a result, current antifungal agents have limited clinical efficacy, are poorly fungicidal in the host, are occasionally toxic, and are increasingly ineffective due to emerging resistance. Thus, innovative antifungal agents are needed.
  • the present invention provides a method of inhibiting growth of a fungus in a subject, comprising reducing the activity of sterylglucosidase 1 (Sigil) and/or sterylglucosidase A (SglA) in the fungus.
  • Sigil sterylglucosidase 1
  • SglA sterylglucosidase A
  • the present invention provides a compound having the structure: wherein R 1 and R 2 are independently H, alkyl, alkenyl, alkynyl carbonyl, amine; -CH-, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, C(O)-alkyl, C(O)-haloalkyl, C(O)-cycloalkyl, C(O)- heterocycloalkyl, C(O)-aryl, C(O)-heteroaryl, CO 2 -alkyl, CO 2 -aryl, CO 2 -heteroaryl, or CO 2 - heterocycloalkyl; preferably, R 1 and R 2 are independently H, alkyl, alkenyl, alkynyl carbonyl, or amine; or a pharmaceutically acceptable salt thereof.
  • the present invention provides a compound having the following structure: wherein R 17 , R 18 , R 19 are each independently CH, N. or S: wherein R 20 is NH 2 .
  • R 17 , R 18 , R 19 are each independently CH, N. or S: wherein R 20 is NH 2 .
  • NH-NHC(S)- alkyl alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, C(O)-alkyl, C(O)-haloalkyl, C(O)- cycloalkyl, C(O)-heterocycloalkyl, C(O)-aryl, C(O)-heteroaryl.
  • CO 2 -alkyl CO 2 -aryl.
  • R 21 is aryl, heteroaryl, cycloalkyl, heterocycloalkyl, C(O)-heterocycloalkyl, C(O)-aryl, C(O)-heteroaryl, CO 2 -aryl, CO 2 -heteroaryl, CO 2 -heterocycloalkyl, SO 2 -cycloalkyl, SO 2 - heterocycloalkyl, SO 2 -aryl, or SO 2 -heteroaiyl; preferably, R 21 is aryl, heteroaryl, C(O)-aryL C(O)- heteroaryl, CO 2 -aryl, CO 2
  • FIG. 1 Deletion of SGL1 in Cn or SGLA gene in Af abolishes virulence.
  • mice infected with 5x10 4 conidia of Af 293 wild-type (A/WT) or with the reconstituted (Af ⁇ sglA+SGLA) strained died within 18 days.
  • n 10 mice/group.
  • IS immunosuppression: all mice in B received a subcutaneous administration of triamcinolone (400 mg/kg) one day prior to the injection of Af conidia. *P ⁇ 0.001, ⁇ sgll or ⁇ sgla vs WT by Kruskal-Wallis test.
  • FIG. 1 Effect of Sgl 1 deletion/inhibition of growth of C. Neqformans (Cn). Deletion of Sgl l gene in Cn ( ⁇ sgll) affect growth in DMEM agar at low oxygen (B vs. A). Treatment on Cn WT with 100 pM Hit 1(c) inhibits growth similarly to ⁇ sgll (B).
  • FIG. 3 Effect of SglA deletion/inhibition on hyphal formation in A.fumigatus (Af). Deletion of SglA gene in Af ( ⁇ sglAI) dramatically effects hyphal elongation.
  • B compared to WT
  • A Treatment of AfWT with Hit b for 12 hours.
  • C recapitulates tire phenotype of ⁇ sglA.
  • D Quantitative analysis of hyphal length of AfWT untreated (Un). Af ⁇ sglA-Un. and AfWT treated with different concentrations of SglA inhibitors Hit b (10 and 100 pM) or Hit c (10, 100 and 500 pM). Both compounds significantly inhibit hyphal elongation in Aj 'WT similarly to ⁇ sglA. Black bar in A, B and C, 20 pm. *** in D, P ⁇ 0.01 , by ANOVA.
  • Figure 4 Percentage of SglA inhibition of Hit b (B) and its 7 derivatives (B1-B7) from ChemBridge. Dose-response curves using the native substrate ergosterol 30-D-glucoside. Reactions were performed using 0.5 mol% ergosterol glucoside in Triton-X-100 mixed micelles with 20 min reaction time at 37 °C. **Ergosterol was detected at 282 nm by UV absorption after HPLC separation. *, P ⁇ 0.001, B7 or Bl versus Hit B. Statistic by one-way ANOVA. Tukey’s multiple Comparation Test.
  • Hit 1 prevents dissemination of C. neoformans to the brain.
  • CBA/J mice were inoculated with 1x10 6 Cn WT H99. After 6 hours, treatment started intraperitoneally with 10 mg/kg/day of Hit l or fluconazole (F).
  • F fluconazole
  • No Cn cells were found in the brain when mice were treated with Hit 1 or Hit 1 + F.
  • the Hit 1 + F combination also showed a significant reduction in lung CFU.
  • FIG. 7 B7 Prolongs survival of mice infected with Af.
  • A) Mice were infected with 5x10 4 Af conidia. After 24 hours they received an intraperitoneal injection of B7 5mg/Kg/day or twice a day, which they continued daily. n 10 mice in each group. * P ⁇ 0.01, B7 5 mg/Kg/twice day versus untreated by Kruskal-Wallis test.
  • FIG. 8 Structural analysis of Cn Sgll with its substrate and with its inhibitor.
  • FIG. 9 Docking analysis of Hit 9 with Sgll (A) and Hit b with SglA (B).
  • Hit 9 (purple) binds to Glu 587, Lys 47 and Glu 270 of Sgll in the active site.
  • Hit b (pink) binds to Asp 127 and Glu 247 of SglA in the active site. Others amino acids in the active site are shown.
  • FIG. 10 2D structures, 3 D poses, DOCK 6 fitness scores, and ligand descriptors for Hit 9, (green in C) and 182 analogs (orange in D). complexed with Sgll. Key protein residues involved with H- bonding (magenta) are also shown. Analogs were constructed using an isosteric swapping protocol in which the cognate ligand sidechain (shaded oval in A) was employed to identify 500 isosteres which were then sampled at position R (shaded oval in B). Out of 500 sidechains sample, 182 analogs had a new bond connection previously seen in a large drug-like library and the pose was geometrically and energetically compatible with the binding site.
  • FIG. 11 A) Binding pocket of Hit 9 in Sgll; B) SS-103 (and SS-104) in the binding pocket; C) Enzyme Inhibitory assay of Hit 9 and SS-103 w hich ergosterol 3-[3-glucoside; D, E) SG accumulation in C. neoformans H99 with SS-103 (D) and Hit 9 (E). [0019] Figure 12. Selected branched Hit 9 analogs with high docking scores.
  • Figure 14 Overlay of SglA with Hit b, Hit c and Hit 9.
  • Figure 15 Selected branched Hit b analogs with high docking scores.
  • FIG. 16 A) Deletion of Sgll ( ⁇ sgll) accumulates ergosterol-3-[3-glucoside in C. albicans cells. B) IC 50 (50% inhibition) of C. neoformans (Cn) Sgll, A. fumigalus (Af) SglA or Ca Sgll by Hit b, Hit 8 and Hit 11 in vitro. *, p ⁇ 0.001 by ANOVA.
  • Figure 17 SglA enzyme inhibitory assay of Hit 9, B7 and DR-SglA-l ⁇ 3 with natural substrate, ergosteryl 3-b-D-glucoside.
  • FIG. Biological Potency Evaluations for SS-103.
  • an in vitro enzyme inhibitory assay of Hit 9 purple line, circles
  • SS-103 black line, squares
  • erg-glc natural substrate
  • an erg-glc accumulation study in C. neoformans strain H99 with Hit 9 (left) and SS-103 (right) is shown.
  • the present invention provides a method of inhibiting growth of a fungus in a subject, comprising reducing the activity of stcrylghicosidasc 1 (Sigil) and/or sterylglucosidase A (SglA) in the fungus.
  • Sigil stcrylghicosidasc 1
  • SglA sterylglucosidase A
  • the present invention provides a method of reducing the activity of Sigil and/or SglA comprises:
  • the present invention provides a method of inhibiting growth of a fungus comprising contacting the fungus with an effective amount of a compound having the structure: wherein R 1 and R2 are independently H, alkyl, alkenyl, alkynyl carbonyl, amine; -CH-, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, C(O)-alkyl, C(O)-haloalkyl, C(O)-cycloalkyl, C(O)-heterocycloalkyl, C(O)- aryl.
  • R 1 and R2 are independently H, alkyl, alkenyl, alkynyl carbonyl, amine; -CH-, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, C(O)-alkyl, C(O)-haloalkyl, C(O)-cycloalkyl, C(O)-heterocycloalkyl,
  • R, and R 2 are independently H, alkyl, alkenyl, alkynyl carbonyl, or amine.
  • Hie present invention provides a method of inhibiting growth of a fungus comprising contacting the fungus with an effective amount of a compound having the structure: wherein R17, R 1 g, R 19 are each independently CH, N, or S; wherein R 20 is NH 2 , NH-alkyl, NH-C(O)-alkyl, NH-C(S)-alkyl, NH-NHC(O)-alkyl. NH-NHC(S)- alkyl, alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, C(O)-alkyl.
  • R 20 is NH-alkyl, NH-C(O)-alkyl, NH-C(S)-alkyl, NH-NHC(O)-alkyl, NH- NHC(S)-alkyl, alkyl, alkenyl, alkynyl, C(O)-alkyl, C(O)-haloalkyl, CO 2 -alkyl, alkyl-NH 2 , SO 2 -alkyl, or SO 2 -haloalkyl.
  • R 20 is NH-alkyl, NH-C(O)-alkyl, NH-C(S)-alkyl, NH-NHC(O)-alkyl, NH- NHC(S)-alkyl, alkyl, alkenyl, alkynyl, C(O)-alkyl, or C(O)-haloalkyl.
  • R 20 is NH-alkyl, NH-C(O)-alkyl, NH-C(S)-alkyl, NH-NHC(O)-alkyl, NH- NHC(S)-alkyl, alkyl, C(O)-alkyl, or C(O)-haloalkyl.
  • R 20 is NH-alkyl or alkyl.
  • R 21 is aryl, heteroaryl, C(O)-aryl, C(O)-heteroaryl, CO 2 -aryl. CO 2 - heteroaryl, SO 2 -aryl, or SO?-hctcroaryl.
  • R 21 is heteroaryl
  • R 1 and R 2 are independently H, alkyl, alkenyl, alkynyl carbonyl, or amine.
  • the present invention provides a compound having the following structure:
  • the present invention provides a compound having the following structure: wherein R, is 0, NH, CH 2 , or S; wherein R 4 is -CH-, -N-, -NH-N-, -NH-C(O)-, -NH-C(S)-, -NH-NHC(O)-, -NH-NHC(S)-, alkyl, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, C(O)-alkyl, C(O)-haloalkyl, C(O)-cycloalkyl, C(O)- heterocycloalkyl, C(O)-arvl, C(O)-heteroaryl, CO 2 -alkyl, CO 2 -aryl, CO 2 -heteroaryl, CO 2 -heterocycloalkyl, alkyl-N-, SO 2 -alkyL SO 2 -haloalkyl.
  • R 5 and Rg are each independently H, ary l, heteroaryl, cycloalkyl, heterocycloalkyl, CO 2 - aryl, CO 2 -heteroaryl, CO 2 -cycloalkyl, or CO 2 -heterocycloalkyl.
  • R3 is 0, NH. or S.
  • R3 is O, or NH.
  • R3 is O.
  • R4 is -CH-, -N-, -NH-N-, -NH-C(O)-, -NH-C(S)-, -NH-NHC(O)-, -NH- NHC(S)-, or heteroaryl.
  • R4 is -CH-.
  • R4 is heteroaryl
  • the present invention provides a compound having the following structure:
  • R4 is heteroaryl, C(O)-heteroaryl, CO 2 -heteroaryl, or SO 2 -heteroaryl.
  • the heteroaryl is pyran, pyridine, piperidine, pyrimidine, isoxazole, oxazole, silole, 6H- 1,2, 5 -thiadiazine, 2H,6H-l,5,2-dithiazine, 1,4-thiazepine, triazine, oxirane, thiirane or azirine.
  • R4 is pyridine, pyrimidine or triazine.
  • R4 is triazine
  • the present invention provides a compound having the following structure: wherein R 5 and Re are each independently H, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, CO 2 - aryl, CO 2 -heteroaryl, CO 2 -cycloalkyl, or CO 2 -heterocycloalkyl.
  • R 5 and Re are each independently aryl, heteroaryl, cycloalkyl, or heterocycloalkyl.
  • R 5 and Re are each independently aryl or cycloalkyl.
  • R 5 and Re are each independently aryl.
  • R 5 and Re are each independently cycloalkyl.
  • R 5 and Re are each independently substituted.
  • R 5 and Re are each independently substituted aryl or substituted cycloalkyl.
  • R 5 and Re are each independently substituted with alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, alkoxy, carbonyl, carboxyl, amino, or amide.
  • R 5 and Re are each independently substituted with aryl, heterocycloalkyl, carbonyl or carboxyl.
  • R5 and Re are each independently substituted with aryl or heterocycloalkyl.
  • R 5 and Re are each independently:
  • U is N or CH; wherein V, X and Y are each independently C, NH, 0, S, -N-, -N-NH 2 , -N-C(O)-, -N-C(S)-, -N- NHC(O)-, -N-NHC(S), -N-alkyl, -N-alkyne, -N-alkynl, -N-aryl, -N-heteroaryl, -N-cycloalkyl, -N- hctcrocycloalkyl, or -N-haloalkyl; wherein W and Z are each independently H, NH 2 .
  • each occurrence of R 14 is independently alkyl, alkenyl, alkynyl, aryl, or heteroaryl, wherein each occurrence of R 15 is independently -H, alkyl, alkenyl, alkynyl, aryl, or heteroaryl, wherein each occurrence of R 16 is independently -H, alkyl, alkenyl, alkynyl, aryl, or hcteroaryl.
  • R 5 and Re arc each independently:
  • R 6 and R 6 are the same.
  • R 5 and R 6 are different.
  • the present invention provides a compound having the following structure:
  • the present invention provides a compound having the following structure: wherein R 17 , R 18 , R 19 are each independently CH, N, or S; wherein R 20 is NH 2 , NH-alkyl, NH-C(O)-alkyl, NH-C(S)-alkyl, NH-NHC(O)-alkyl, NH-NHC(S)- alkyl, alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, C(O)-alkyl. C(O)-haloalkyl, C(O)- cycloalkyl, C(O)-heterocycloalkyl.
  • R 22 when R 22 is H, R 20 is NH 2 .
  • R 21 is aryl, heteroaryl, C(O)-aryl, C(O)-heteroaryl, CO 2 -aryl, CO 2 - heteroaryl, SO 2 -aryl, or SO 2 -heteroaryL
  • R 21 is heteroaryl
  • R 20 is NH-alkyl, NH-C(O)-alkyl, NH-C(S)-alkyl, NH-NHC(O)-alkyl, NH- NHC(S)-alkyl, alkyl, alkenyl, alkynyl, C(O)-alkyl, C(O)-haloalkyl, CO 2 -alkyl, alkyl-NH 2 , SO 2 -alkyl, or SO 2 -haloalkyl.
  • R 20 is NH-alkyl, NH-C(O)-alkyl, NH-C(S) -alkyl, NH-NHC(O)-alkyl, NH- NHC(S)-alkyl, alkyl, alkenyl, alkynyl, C(O)-alkyl, or C(O)-haloalkyl.
  • R 20 is NH-alkyl, NH-C(O)-alkyl, NH-C(S)-alkyl, NH-NHC(O)-alkyl, NH- NHC(S)-alkyl, alkyl, C(O)-alkyl, or C(O)-haloalkyl.
  • R 20 is NH-alkyl or alkyl.
  • R 20 is NH-alkyl
  • R 20 is alkyl
  • the present invention provides a compound having the following structure:
  • R 17 , R 18 , R 19 are CH, N, N.
  • R 17 , R 18 , R 19 are N, CH, N.
  • R 17 , R 18 , R 19 are CH. CH, N.
  • R17, R 18 , R 19 are N, CH, CH.
  • R 17 , R 18 , R 19 are CH, CH, CH.
  • R 22 is NH- C 1-6 alkyl or C 1-6 alkyl.
  • R 21 is an aryl or hctcroaryl.
  • R 21 is an aryl
  • R 21 is a heteroaryl
  • aryl is phenyl, p-toluenyl (4-methylphenyl), naphthyl, tetrahydro-naphthyl, indanyl, biphenyl, phenanthryl, anthryl or acenaphthyl.
  • aryl is phenyl, p-toluenyl (4-methylphenyl).
  • aryl is phenyl
  • heteroaryl is pyridine, pyridazine. pyrimidine, pyrazine, 1,2,4-triazine, 1,3,5-triazine, 1,4,5, 6-tetrahydrocyclopenta[b]pyrrole, l,3a,4,61-tetrahydropyrrolo[3,2-b]pyrrole, 1,4- dihydropyrrole[3,2-b]pyrrole, l,6-dihydropyrrolo[2,3-b]pyrrole, indoline, 3/7-indolc. 1/7-indole, 2H- isoindole, indolizine, IH-indazolc. benzimidazole, 7-azaindole, 4-azaindole, 5-azaindole, or 97/-carbazolc.
  • heteroaryl is 3H-indole, 1/7-indole, 2H-isoindole, indolizine, l/7-indazolc. benzimidazole, 7-azaindole, 4-azaindole, 5-azaindole, or 9H-carbazole.
  • heteroaryl is 1H-indazolc or 9/7-carbazolc.
  • the aryl or heteroaryl is substituted.
  • aryl or heteroaryl is substituted with alkyl, alkenyl, alkynyl. aryl, heteroaryl, cycloalkyl, heterocycloalkyl, alkoxy, carbonyl, carboxyl, amino, or amide.
  • aryl or heteroaryl is substituted with alkyl, alkenyl, alkynyl, aryl, or heteroaryl.
  • alkyl is C 1-6 alkyl.
  • alkyl is C 1-3 alkyl. [0112] In some embodiments, alkyl is methyl or ethyl.
  • R 21 has the following structure:
  • U is N or CH; wherein V, X and Y are each independently C, NH, 0, S, -N-, -N-NH 2 , -N-C(O)-, -N-C(S)-, -N- NHC(O)-, -N-NHC(S), -N-alkyl, -N-alkyne, -N-alkynl, -N-aryl, -N-heteroaryl, -N-cycloalkyl, -N- heterocycloalkyl, or -N-haloalkyl; wherein W and Z are each independently H, NH 2 .
  • R 21 has the following structure: ,
  • R 21 has the following structure:
  • R 21 has the following structure:
  • the present invention provides a compound having the following structure:
  • the present invention provides a pharmaceutical composition comprising compounds described in the current invention and a pharmaceutically acceptable carrier.
  • the present invention provides a method of treating a subject with fungal infection comprising administering an effective amount of the compound described in any one of paragraphs [0039]-[0117],
  • the present invention provides a method of treating a subject with fungal infection comprising administering an effective amount of the compounds having the structure: wherein R 1 and R 2 are independently H, alkyl, alkenyl, alkynyl carbonyl, amine; -CH-, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, C(O)-alkyl, C(O)-haloalkyl, C(O)-cycloalkyl, C(O)-heterocycloalkyl, C(O)- aryl, C(O)-heteroaryl, CO 2 -alkyl, CO 2 -aryl, CO 2 -heteroaryl, or CO 2 -heterocycloalkyl.
  • R 1 and R 2 are independently H, alkyl, alkenyl, alkynyl carbonyl, amine; -CH-, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, C(
  • R 1 and R 2 are independently H, alkyl, alkenyl, alkynyl carbonyl, or amine.
  • the present invention provides a method of treating a subject with fungal infection comprising administering an effective amount of the compounds having the structure: wherein R 17 , R 18 , R 19 are each independently CH, N, or S; wherein R 20 is NH 2 , NH-alkyl, NH-C(O)-alkyl, NH-C(S)-alkyL NH-NHC(O)-alkyl, NH-NHC(S)- alkyl, alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, C(O)-alkyl, C(O)-haloalkyl, C(O)- cycloalkyl, C(O)-heterocycloalkyl, C(O)-aryl.
  • the method further comprises administering to the subject an effective amount of an anti-fungal agent.
  • the anti-fungal agent is fluconazole, amphotericin B, caspofungin, tunicamycin or aureobasidin A or a combination thereof.
  • the anti-fungal agent is azole, flucytosine, amphotericin B or echinocandins, or a combination thereof.
  • the fungus is a saprotrophic fungus.
  • the fungus is Cryptococcus Neoformans, Cryptococcus gattii, Candida albicans, Candida krusei, Candida glabrata, Candida parapsilosis, Candida guilliermondii, Aspergillus fumigatus, Rhizopus oryzae, Rhizopus spp. , Blastomyces dermatitis, Histoplasma capsulatum, Coccidioides spp., Paecilomyces variotii, Pneumocystis murina, Pneumocystis proved, Histoplasma capsulatum, Aspergillus spp., or Sporothrix brasiliensis.
  • the fungus is Cryptococcus Neoformans or Aspergillus fumigatus.
  • the fungal infection is caused by Candida, Aspergillus, Cryptococcus , Histoplasma, Pneumocystis, Stachybotrys or Mycrorales fungus.
  • the fungal infection is caused by Cryptococcus Neoformans.
  • the fungal infection is Cryptococcus neoformans cryptococcosis.
  • the fungal infection is caused by Sporothrix.
  • the fungal infection is caused by 5. brasiliensis, S. schenckii, S. globosa, S. mexicana, S. chilensis. S. luriei, and S. pallida.
  • the fungal infection is caused by 5. brasiliensis.
  • the fungal infection is caused by a fungus other than Cryptococcus Neoformans .
  • the fungal infection is a fungal infection other than Cryptococcus neoformans cryptococcosis.
  • the fungal infection is Aspergillosis. Blastomycosis, Candidiasis, Coccidioidomycosis, Cryptococcus gattii cryptococcosis. Fungal Keratitis, Dermatophytes, Histoplasmosis, Mucormycosis, Pneumocystis pneumonia (PCP), or Sporotrichosis.
  • the fungal infection is Sporotrichosis.
  • the fungal infection is caused by Cryptococcus gattii, Candida albicans, Candida krusei, Candida glabrata, Candida parapsilosis, Candida guilliermondii, Aspergillus fumigatus, Rhizopus oryzae, Rhizopus spp. , Blastomyces dermatitis, Histoplasma capsulatum, Coccidioides spp., Paecilomyces variotii, Pneumocystis murina, Pneumocystis jiroveci, Histoplasma capsulatum, Aspergillus spp., or dimorphic fungi.
  • the anti-fungal agent is fluconazole, amphotericin B, caspofungin, tunicamycin or aureobasidin A.
  • the fungal infection is a fungal infection on a plant.
  • the fungal infection is a fungal infection in human.
  • the fungal infection is an internal fungal infection.
  • the fungal infection is an invasive fungal infection.
  • the fungal infection is a fungal infection of the skin or lung.
  • the compound has a fungistatic effect on the fungus.
  • the compound is administered orally to the subject.
  • the compound is administered topically to the subject.
  • the subject is also afflicted with an immunodeficiency disorder.
  • the subject is also afflicted with human immunodeficiency vims (HIV).
  • HAV human immunodeficiency vims
  • the subject is also afflicted with pulmonary aspergillosis.
  • the subject is also afflicted with cryptococcal meningoencephalitis.
  • the antifungal agent is Amphotericin B, Candicidin, Filipin, Hamycin, Natamycin, Nystatin, Rimocidin, Clotrimazole, Bifonazole, Butoconazole, Clotrimazole, Econazole, Fenticonazole, Isoconazole, Ketoconazole, Luliconazole, Miconazole, Omoconazole, Oxiconazole, Sertaconazole, Sulconazole, Tioconazole, Albaconazole, Fluconazole, Isavuconazole, Itraconazole, Posaconazole, Ravuconazole, Terconazole, Voriconazole, Abafungin, Amorolfin, Butenafine, Naftifine, Terbinafine, Anidulafungin, Caspofungin, Micafungin, Ciclopirox, Flucytosine, Griseofulvin, Haloprogin, Toln
  • the present invention provides a pharmaceutical composition comprising a compound of the present invention and an antifungal agent, and at least one pharmaceutically acceptable carrier for use in treating a fungal infection.
  • a pharmaceutical composition comprising an amount of the compound of the present invention for use in treating a subject afflicted with a fungal infection as an add-on therapy or in combination with, or simultaneously, contemporaneously or concomitantly with an anti-fungal agent.
  • the subject is a human.
  • the compound and/or anti-fungal agent is orally administered to the subject.
  • the present invention provides identifying a compound that inhibits the activity of sterylglucosidase using structure-based computer-aided drug design (CADD) software; or determining the crystal structure of Sigil and/or SglA.
  • CADD computer-aided drug design
  • the compound and/or anti-fungal agent is topically administered to the subject.
  • the fungus or fungal infection has developed resistance to one or more drugs.
  • a drug resistant fungal infection may have developed drug-resistance to an azole antifungal drug, a polyene antifungal drug and/or an echinocandin antifungal drug.
  • the compound targets APL5, COS111,MKK1, and STE2 in the fungus. [0163] In some embodiments of any of the above methods or uses, the compound targets at least one of APL5, COS111,MKK1, or STE2 in the fungus.
  • the compound disrupts vesicular transport mediate by APL5.
  • the fungus carries non-mutated APL5, COS111,MKK1, and STE2.
  • the fungus carries at least one of non- mutated APL5, COS111. MKK1, and STE2.
  • a “symptom” associated with a fungal infection includes any clinical or laboratory manifestation associated with the fungal infection and is not limited to what the subject can feel or observe.
  • treating e.g. of a fungal infection, encompasses inducing prevention, inhibition, regression, or stasis of the disease or a symptom or condition associated with the infection.
  • modifying means a change in a subject, which may be an increase or decrease in amount, activity, rate of production, rate of inactivation, rate of breakdown, delay of onset, earlier onset, addition or removal of material, mutation, or any combination of these, so long as there is a reduced level or activity of starch synthase II.
  • the compounds of the present invention include all hydrates, solvates, and complexes of the compounds used by this invention. If a chiral center or another form of an isomeric center is present in a compound of the present invention, all forms of such isomer or isomers, including enantiomers and diastereomers, are intended to be covered herein.
  • Compounds containing a chiral center may be used as a racemic mixture, an enantiomerically enriched mixture, or the racemic mixture may be separated using well-known techniques and an individual enantiomer may be used alone.
  • Tire compounds described in the present invention are in racemic form or as individual enantiomers.
  • Tire enantiomers can be separated using known techniques, such as those described in Pure and Applied Chemistry 69. 1469-1474, (1997) IUPAC. In cases in which compounds have unsaturated carbon-carbon double bonds, both the cis (Z) and trans (E) isomers are within the scope of this invention.
  • the compounds of the subject invention may have spontaneous tautomeric forms.
  • compounds may exist in tautomeric forms, such as keto-enol tautomers, each tautomeric form is contemplated as being included within this invention whether existing in equilibrium or predominantly in one form.
  • This invention also provides isotopic variants of the compounds disclosed herein, including wherein the isotopic atom is 2 H and/or wherein the isotopic atom 13 C. Accordingly, in the compounds provided herein hydrogen can be enriched in the deuterium isotope. It is to be understood that the invention encompasses all such isotopic fomrs.
  • each stereogenic carbon may be of the R or S configuration.
  • isomers arising from such asymmetry' e.g., all enantiomers and diastereomers
  • Such isomers can be obtained in substantially pure fonn by classical separation techniques and by stereochemically controlled synthesis, such as those described in "Enantiomers, Racemates and Resolutions" by J. Jacques, A. Collet and S. Wilen, Pub. John Wiley & Sons, NY, 1981.
  • the resolution may be carried out by preparative chromatography on a chiral column.
  • the subject invention is also intended to include all isotopes of atoms occurring on the compounds disclosed herein.
  • Isotopes include those atoms having the same atomic number but different mass numbers.
  • isotopes of hydrogen include tritium and deuterium.
  • isotopes of carbon include C-13 and C-14.
  • any notation of a carbon in structures throughout this application when used without further notation, are intended to represent all isotopes of carbon, such as 12 C, 13 C, or 14 C.
  • any compounds containing 13 C or 14 C may specifically have the structure of any of the compounds disclosed herein.
  • any notation of a hydrogen in structures throughout this application when used without further notation, are intended to represent all isotopes of hydrogen, such as 1 H, 2 H, or ? H.
  • any compounds containing 2 H or 3 H may specifically have the structure of any of the compounds disclosed herein.
  • Isotopically-labeled compounds can generally be prepared by conventional techniques known to those skilled in the art using appropriate isotopically-labeled reagents in place of the non-labeled reagents employed.
  • the substituents may be substituted or unsubstituted, unless specifically defined otherwise.
  • alkyl, heteroalkyl, monocycle, bicycle, aryl, hctcroaryl and heterocycle groups can be further substituted by replacing one or more hydrogen atoms with alternative non-hydrogen groups.
  • non-hydrogen groups include, but are not limited to, halo, hydroxy, mercapto, amino, carboxy, cyano, carbamoyl and aminocarbonyl and aminothiocarbonyl.
  • substituents and substitution patterns on the compounds used in the method of the present invention can be selected by one of ordinary skill in the art to provide compounds that are chemically stable and that can be readily synthesized by techniques known in the art from readily available starting materials. If a substituent is itself substituted with more than one group, it is understood that these multiple groups may be on the same carbon or on different carbons, so long as a stable structure result.
  • alkyl is intended to include both branched and straight-chain saturated aliphatic hydrocarbon groups having the specified number of carbon atoms.
  • Ci-Cn as in “Ci-C n alkyl” is defined to include groups having 1, , n-1 or n carbons in a linear or branched arrangement, and specifically includes methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, isopropyl, isobutyl, sec-butyl and so on.
  • alkenyl refers to a non-aromatic hydrocarbon radical, straight or branched, containing at least 1 carbon to carbon double bond, and up to the maximum possible number of non-aromatic carbon- carbon double bonds may be present.
  • Tirus, C 2 -C11 alkenyl is defined to include groups having 1, 2...., n-1 or n carbons.
  • C 2 -C6 alkenyl means an alkenyl radical having 2, 3, 4, 5, or 6 carbon atoms, and at least 1 carbon-carbon double bond, and up to, for example, 3 carbon-carbon double bonds in the case of a C 6 alkenyl, respectively.
  • Alkenyl groups include ethenyl, propenyl, butenyl and cyclohexenyl. As described above with respect to alkyl, the straight, branched or cyclic portion of the alkenyl group may contain double bonds and may be substituted if a substituted alkenyl group is indicated.
  • An embodiment can be C 2 -C 12 alkenyl, C 3 -C 12 alkenyl, C 4 -C 12 alkenyl and so on.
  • alkynyl refers to a hydrocarbon radical straight or branched, containing at least 1 carbon to carbon triple bond, and up to the maximum possible number of non-aromatic carbon-carbon triple bonds may be present.
  • C 2 -C n alkynyl is defined to include groups having 1, 2...., n-1 or n carbons.
  • C 2 -C6 alkynyl means an alkynyl radical having 2 or 3 carbon atoms, and 1 carbon-carbon triple bond, or having 4 or 5 carbon atoms, and up to 2 carbon-carbon triple bonds, or having 6 carbon atoms, and up to 3 carbon-carbon triple bonds.
  • Alkynyl groups include ethynyl, propynyl and butynyl. As described above with respect to alkyl, the straight or branched portion of the alkynyl group may contain triple bonds and may be substituted if a substituted alkynyl group is indicated.
  • An embodiment can be a C 2 -C n alkynyl.
  • An embodiment can be C 2 -C 12 alkynyl, C 3 -C 12 alkynyl, C 4 -C 12 alkynyl and so on
  • alkylene alkenylene and alkynylene shall mean, respectively, a divalent alkane, alkene and alkyne radical, respectively. It is understood that an alkylene, alkenylene, and alkynylene may be straight or branched. An alkylene, alkenylene, and alkynylene may be unsubstituted or substituted.
  • heteroalkyl includes both branched and straight-chain saturated aliphatic hydrocarbon groups having the specified number of carbon atoms and at least 1 heteroatom within the chain or branch.
  • cycloalkyl shall mean cyclic rings of alkanes of three to eight total carbon atoms, or any number within this range (i.e., cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl or cyclooctyl).
  • monocycle includes any stable polyatomic carbon ring of up to 10 atoms and may be unsubstituted or substituted.
  • non-aromatic monocycle elements include but are not limited to: cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl.
  • aromatic monocycle elements include but are not limited to: phenyl.
  • bicycle includes any stable polyatomic carbon ring of up to 10 atoms that is fused to a polyatomic carbon ring of up to 10 atoms with each ring being independently unsubstituted or substituted.
  • non-aromatic bicycle elements include but are not limited to: decahydronaphthalene.
  • aromatic bicycle elements include but are not limited to: naphthalene.
  • aryl is intended to mean any stable monocyclic, bicyclic or polycyclic carbon ring of up to 10 atoms in each ring, wherein at least one ring is aromatic, and may be unsubstituted or substituted.
  • aryl elements examples include phenyl, p-toluenyl (4-methylphenyl), naphthyl, tetrahydro-naphthyl, indanyl, biphenyl, phenanthryl, anthryl or acenaphthyl.
  • aryl substituent is bicyclic and one ring is non-aromatic, it is understood that attachment is via the aromatic ring.
  • polycyclic refers to unsaturated or partially unsaturated multiple fused ring structures, which may be unsubstituted or substituted.
  • arylalkyl refers to alkyl groups as described above wherein one or more bonds to hydrogen contained therein are replaced by a bond to an aryl group as described above. It is understood that an '‘arylalkyl” group is connected to a core molecule through a bond from the alkyl group and that the aryl group acts as a substituent on the alkyl group.
  • arylalkyl moieties include, but are not limited to, benzyl (phenylmethyl), p-trifluoromethylbenzyl (4-trifluoromethylphenylmethyl), 1 -phenylethyl, 2- phenylethyl, 3-phenylpropyl, 2-phenylpropyl and the like.
  • heteroaryl represents a stable monocyclic, bicyclic or polycyclic ring of up to 10 atoms in each ring, wherein at least one ring is aromatic and contains from 1 to 4 hctcroatoms selected from the group consisting of O, N and S.
  • Heteroaryl groups within the scope of this definition include but are not limited to: benzimidazolyl, benzofuranyl, benzofurazanyl. benzopyrazolyl, benzotriazolyl, benzothiophenyl, benzoxazolyl, carbazolyl, carbolinyl, quinolyl, fiiranyl, indolinyl, indolyl, indolazinyl, indazolyl, isobenzofiiranyl, isoindolyl, isoquinolyl, isothiazolyl, isoxazolyl, naphthpyridinyl, oxadiazolyl, oxazolyl, isoxazoline, pyranyl, pyrazinyl, pyrazolyl, pyridazinyl, pyridopyridinyl, pyridazinyl, pyridyl, pyrimidyl, pyrrolyl, quin
  • heteroarylalkyl refers to alkyl groups as described above wherein one or more bonds to hydrogen contained therein are replaced by a bond to an heteroaryl group as described above. It is understood that an “heteroarylalkyl” group is connected to a core molecule through a bond from the alkyl group and that the heteroaryl group acts as a substituent on the alkyl group. Examples of heteroarylalkylmoieties include, but are not limited to, -CEE-CCsFEN). -CH2-CH2-(C5H4N) and tire like.
  • heterocycle refers to a mono- or poly-cyclic ring system which can be saturated or contains one or more degrees of unsaturation and contains one or more heteroatoms.
  • Preferred heteroatoms include N, 0. and/or S, including N-oxides, sulfur oxides, and dioxides.
  • the ring is three to ten-membered and is either saturated or has one or more degrees of unsaturation. More preferably the ring is three to four-membered and has one or more degrees of unsaturation.
  • the heterocycle may be unsubstituted or substituted, with multiple degrees of substitution being allowed.
  • alkyl, alkenyl, alkynyl, aryl, hetcroaryl and heterocyclyl substituents may be substituted or unsubstituted, unless specifically defined otherwise.
  • alkyl, alkenyl, alkynyl, and, heterocyclyl and heteroary l groups can be further substituted by replacing one or more hydrogen atoms with alternative non-hydrogen groups. These include, but are not limited to, halo, hydroxy, mercapto, amino, carboxy, cyano and carbamoyl.
  • halogen refers to F, Cl, Br, and I.
  • Tire tenns “substitution”, “substituted” and “substituent” refer to a functional group as described above in which one or more bonds to a hydrogen atom contained therein are replaced by a bond to non- hydrogen or non-carbon atoms, provided that normal valencies are maintained and that the substitution results in a stable compound.
  • Substituted groups also include groups in which one or more bonds to a carbon(s) or hydrogen(s) atom are replaced by one or more bonds, including double or triple bonds, to a heteroatom.
  • substituent groups include the functional groups described above, and halogens (i.e., F, Cl, Br, and I); alkyl groups, such as methyl, ethyl, n-propyl, isopropryl, n-butyl, tert-butyl, and trifluoromethyl; hydroxyl: alkoxy groups, such as methoxy, ethoxy, n-propoxy, and isopropoxy: aryloxy groups, such as phenoxy: arylalkyloxy, such as benzyloxy (phenylmethoxy) and p- trifluoromethylbenzyloxy (4-trifluoromethylphenylmethoxy); heteroaryloxy groups; sulfonyl groups, such as trifluoromethanesulfonyl, methanesulfonyl, and p-toluenesulfonyl; nitro, nitrosyl; mercapto; sulfanyl groups, such
  • substituted compound can be independently substituted by one or more of the disclosed or claimed substituent moieties, singly or pluraly.
  • independently substituted it is meant that the (two or more) substituents can be the same or different.
  • Tire various R groups attached to the aromatic rings of the compounds disclosed herein may be added to the rings by standard procedures, for example those set forth in Advanced Organic Chemistry: Part B: Reaction and Synthesis, Francis Carey and Richard Sundberg, (Springer) 5th ed. Edition. (2007), the content of which is hereby incorporated by reference.
  • the compounds used in the method of the present invention may be prepared by techniques described in Vogel’s Textbook of Practical Organic Chemistry, A.I. Vogel, A.R. Tatchell, B.S. Fumis, A.J. Hannaford, P.W.G. Smith, (Prentice Hall) 5 th Edition (1996), March's Advanced Organic Chemistry : Reactions, Mechanisms, and Structure, Michael B. Smith, Jerry March, (Wiley-Interscience) 5 th Edition (2007), and references therein, which are incorporated by reference herein. However, these may not be the only means by which to synthesize or obtain the desired compounds.
  • Another aspect of the invention comprises a compound used in the method of the present invention as a pharmaceutical composition.
  • compositions which have pendant carboxylic acid groups may be modified in accordance with the present invention using standard esterification reactions and methods readily available and known to those having ordinary skill in the art of chemical synthesis. Where a phannaceutically active agent does not possess a carboxylic acid group, the ordinarily skilled artisan will be able to design and incorporate a carboxylic acid group into the pharmaceutically active agent where esterification may subsequently be carried out so long as the modification does not interfere with the pharmaceutically active agent’s biological activity or effect.
  • the compounds used in the method of the present invention may be in a salt form.
  • a “salt” is a salt of the instant compounds which has been modified by making acid or base salts of the compounds.
  • the salt is pharmaceutically acceptable.
  • pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as phenols.
  • Hie salts can be made using an organic or inorganic acid.
  • Such acid salts are chlorides, bromides, sulfates, nitrates, phosphates, sulfonates, formates, tartrates, maleates, malates, citrates, benzoates, salicylates, ascorbates, and the like.
  • Phenolate salts are the alkaline earth metal salts, sodium, potassium or lithium.
  • pharmaceutically acceptable salt in this respect, refers to the relatively non-toxic, inorganic and organic acid or base addition salts of compounds of the present invention.
  • salts can be prepared in situ during the final isolation and purification of the compounds of the invention, or by separately reacting a purified compound of tire invention in its free base or free acid form with a suitable organic or inorganic acid or base, and isolating the salt thus formed.
  • Representative salts include the hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, napthylatc, mesylate, glucoheptonate, lactobionatc, and lauryl sulphonate salts and the like. (See, e.g., Berge el al. (1977) "Phannaceutical Salts", J Pharm. Sci. 66: 1-19).
  • Tire compounds of the present invention may also form salts with basic amino acids such a lysine, arginine, etc. and with basic sugars such as N-methylglucamine, 2-amino-2-deoxyglucose, etc. and any other physiologically non-toxic basic substance.
  • “administering” an agent may be performed using any of the various methods or delivery systems well known to those skilled in the art.
  • Tire administering can be performed, for example, orally, parenterally, intraperitoneally, intravenously, intraarterially, transdermally, sublingually, intramuscularly, rectally, transbuccally, intranasally, liposomally, via inhalation, vaginally, intraoccularly, via local delivery, subcutaneously, intraadiposally, intraarticularly, intrathecally, into a cerebral ventricle, intraventicularly, intratumorally, into cerebral parenchyma or intraparenchchymally.
  • the compounds used in the method of the present invention may be administered in various fomrs, including those detailed herein.
  • Tire treatment with the compound may be a component of a combination therapy or an adjunct therapy, i.e. the subject or patient in need of the drug is treated or given another dmg for the disease in conjunction with one or more of the instant compounds.
  • This combination therapy can be sequential therapy where the patient is treated first with one drug and then the other or the two drugs are given simultaneously.
  • These can be administered independently by the same route or by two or more different routes of administration depending on the dosage fonns employed.
  • a "pharmaceutically acceptable carrier” is a pharmaceutically acceptable solvent, suspending agent or vehicle, for delivering the instant compounds to the animal or human.
  • the carrier may be liquid or solid and is selected with the planned manner of administration in mind.
  • Liposomes are also a pharmaceutically acceptable carrier as are slow-release vehicles.
  • a dosage unit of the compounds used in the method of the present invention may comprise a single compound or mixtures thereof with additional antitumor agents.
  • Hie compounds can be administered in oral dosage forms as tablets, capsules, pills, powders, granules, elixirs, tinctures, suspensions, syrups, and emulsions.
  • the compounds may also be administered in intravenous (bolus or infusion), intraperitoneal, subcutaneous, or intramuscular form, or introduced directly, e.g. by injection, topical application, or other methods, into or topically onto a site of disease or lesion, all using dosage fonns well known to those of ordinary skill in the pharmaceutical arts.
  • Tire compounds used in the method of the present invention can be administered in admixture with suitable phannaceutical diluents, extenders, excipients, or in carriers such as the novel programmable sustained-release multi-compartmental nanospheres (collectively referred to herein as a phannaceutically acceptable carrier) suitably selected with respect to the intended form of administration and as consistent with conventional pharmaceutical practices.
  • a phannaceutically acceptable carrier suitably selected with respect to the intended form of administration and as consistent with conventional pharmaceutical practices.
  • the unit will be in a form suitable for oral, nasal, rectal, topical, intravenous or direct injection or parenteral administration.
  • the compounds can be administered alone or mixed with a phannaceutically acceptable carrier.
  • This carrier can be a solid or liquid, and the type of carrier is generally chosen based on the type of administration being used.
  • Tire active agent can be co-administered in the fomr of a tablet or capsule, liposome, as an agglomerated powder or in a liquid form.
  • suitable solid carriers include lactose, sucrose, gelatin and agar.
  • Capsule or tablets can be easily formulated and can be made easy to swallow or chew; other solid forms include granules, and bulk powders. Tablets may contain suitable binders, lubricants, diluents, disintegrating agents, coloring agents, flavoring agents, flow-inducing agents, and melting agents.
  • liquid dosage forms examples include solutions or suspensions in water, phannaceutically acceptable fats and oils, alcohols or other organic solvents, including esters, emulsions, symps or elixirs, suspensions, solutions and/or suspensions reconstituted from non-effervescent granules and effervescent preparations reconstituted from effervescent granules.
  • Such liquid dosage forms may contain, for example, suitable solvents, preservatives, emulsifying agents, suspending agents, diluents, sweeteners, thickeners, and melting agents.
  • Oral dosage forms optionally contain flavorants and coloring agents.
  • Parenteral and intravenous forms may also include minerals and other materials to make them compatible with the type of injection or delivery system chosen.
  • Tablets may contain suitable binders, lubricants, disintegrating agents, coloring agents, flavoring agents, flow-inducing agents, and melting agents.
  • the active drug component can be combined with an oral, non-toxic, pharmaceutically acceptable, inert carrier such as lactose, gelatin, agar, starch, sucrose, glucose, methyl cellulose, magnesium stearate, dicalcium phosphate, calcium sulfate, mannitol, sorbitol and the like.
  • Suitable binders include starch, gelatin, natural sugars such as glucose or beta-lactose, com 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 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.
  • the compounds used in the method of the present invention may 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 lecithin, sphingomyelin, proteolipids, protein-encapsulated vesicles or from cholesterol, stearylamine, or phosphatidylcholines.
  • Hie compounds may be administered as components of tissue-targeted emulsions.
  • the compounds used in the method of the present invention may also be coupled to soluble polymers as targetable drug carriers or as a prodrug.
  • soluble polymers include polyvinylpyrrolidone, pyran copolymer, polyhydroxylpropylmethacrylamide-phenol, polyhydroxyethylasparta-midephenol, or polyethyleneoxide-polylysine substituted with palmitoyl residues.
  • the compounds may be coupled to a class of biodegradable polymers useful in achieving controlled release of a drug, for example, polylactic acid, polyglycolic acid, copolymers of polylactic and polyglycolic acid, polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydropyrans, polycyanoacylates, and crosslinked or amphipathic block copolymers of hydrogels.
  • a class of biodegradable polymers useful in achieving controlled release of a drug
  • a drug for example, polylactic acid, polyglycolic acid, copolymers of polylactic and polyglycolic acid, polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydropyrans, polycyanoacylates, and crosslinked or amphipathic block copolymers of hydrogels.
  • Gelatin capsules may contain the active ingredient compounds and powdered carriers, such as lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, and the like. Similar diluents can be used to make compressed tablets. Both tablets and capsules can be manufactured as immediate release products or as sustained release products to provide for continuous release of medication over a period of hours. Compressed tablets can be sugar-coated or fdm-coated to mask any unpleasant taste and protect the tablet from the atmosphere, or enteric coated for selective disintegration in the gastrointestinal tract.
  • powdered carriers such as lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, and the like. Similar diluents can be used to make compressed tablets. Both tablets and capsules can be manufactured as immediate release products or as sustained release products to provide for continuous release of medication over a period of hours. Compressed tablets can be sugar-coated or fdm-coated to mask any unpleasant taste and protect the tablet from the atmosphere, or enteric
  • liquid dosage form For oral administration in liquid dosage form, the oral drug components are combined with any oral, non-toxic, pharmaceutically acceptable inert carrier such as ethanol, glycerol, water, and tire like.
  • suitable liquid dosage forms include solutions or suspensions in water, phannaceutically acceptable fats and oils, alcohols or other organic solvents, including esters, emulsions, syrups or elixirs, suspensions, solutions and/or suspensions reconstituted from non-effervescent granules and effervescent preparations reconstituted from effervescent granules.
  • Such liquid dosage forms may contain, for example, suitable solvents, preservatives, emulsifying agents, suspending agents, diluents, sweeteners, thickeners, and melting agents.
  • Liquid dosage forms for oral administration can contain coloring and flavoring to increase patient acceptance.
  • water, asuitable oil, saline, aqueous dextrose (glucose), and related sugar solutions and glycols such as propylene glycol or polyethylene glycols are suitable carriers for parenteral solutions.
  • Solutions for parenteral administration preferably contain a water-soluble salt of the active ingredient, suitable stabilizing agents, and if necessary, buffer substances.
  • Antioxidizing agents such as sodium bisulfite, sodium sulfite, or ascorbic acid, either alone or combined, are suitable stabilizing agents.
  • citric acid and its salts and sodium EDTA are also used.
  • Tire compounds used in the method of the present invention may also be administered in intranasal form via use of suitable intranasal vehicles, or via transdermal routes, using those forms of transdermal skin patches well known to those of ordinary skill in that art.
  • the dosage administration will generally be continuous rather than intermittent throughout the dosage regimen.
  • Parenteral and intravenous forms may also include minerals and other materials such as solutol and/or ethanol to make them compatible with the type of injection or delivery system chosen.
  • the active ingredient can be administered orally in solid dosage forms, such as capsules, tablets, powders, and chewing gum; or in liquid dosage forms, such as elixirs, syrups, and suspensions, including, but not limited to, mouthwash and toothpaste. It can also be administered parentally, in sterile liquid dosage fonns.
  • Solid dosage forms such as capsules and tablets, may be enteric-coated to prevent release of the active ingredient compounds before they reach the small intestine.
  • Materials that may be used as enteric coatings include, but are not limited to, sugars, fatty acids, proteinaceous substances such as gelatin, waxes, shellac, cellulose acetate phthalate (CAP), methyl acrylate-methacrylic acid copolymers, cellulose acetate succinate, hydroxy propyl methyl cellulose phthalate, hydroxy propyl methyl cellulose acetate succinate (hypromellose acetate succinate), polyvinyl acetate phthalate (PVAP), and methyl methacrylate - methacrylic acid copolymers.
  • CAP cellulose acetate phthalate
  • PVAP polyvinyl acetate phthalate
  • Tire compounds and compositions of the invention can be coated onto stents for temporary or permanent implantation into the cardiovascular system of a subject.
  • CNAG 05607 While working on the characterization of the gene involved in the catabolism of the sphingolipids, CNAG 05607 was discovered as the gene homolog to Cn EGCrPl, which is the glucosylceramidase active at neutral and alkaline pH (shibashi Y et al. 2012). CNAG 05607 was initially thought to be a second glucosylceramidase; but the biochemical analysis suggested that CNAG 05607 was a sterylglucosidase. In fact. CNAG_05607 from either Cryptococcus neoformans (Cn) (Rella A, et al., 2015).
  • Cn Cryptococcus neoformans
  • CNAG_05607 was renamed sterylglucosidase 1 (Cn Sgll), the first sterylglucosidase ever isolated from any living organism. (Rella A, et al., 2015).
  • Virulence studies showed that upon inhalation of Cn ⁇ sgll, 100% of mice were still alive after 3 months of observation (Fig. 1A) (Rella A, et al., 2015). Tissue burden studies showed that the Cn ⁇ sgll is promptly eliminated from the lung between 9 and 14 days after inhalation (Rella A, et al., 2015). More importantly. ⁇ sgll never gains access to the brain, where the WT causes a lethal meningo-encephalitis instead. Similarly, virulence studies performed with the Af ⁇ sglA showed that the mutant is not virulent, and 100% mice were alive and healthy after 30 days of observation (Fig. IB) (Rella A, et al., 2015).
  • Cn ⁇ sgll and Af ⁇ sglA mutants have growth defect in host physiological conditions.
  • a DiverSet-CL ChemBridge library comprising 50,000 synthetic compounds in 96-well format was screened (Pereira de Sa N, et al. 2021). three compounds to inhibit specifically Sgll enzyme with a IC50 of IpM or less were found: Hit 1, Hit 9 and Hit 15.
  • Hit 1 4-(hydroxymethyl)-l-[2-(3-methoxy- phenyl)-l,3-thiazol-5-yl]methylpiperidin-4-ol (ChemBridge ID&59928901); Hit 9, N-[(3R,5S)-5- (hydroxym ethyl)- 1 -mcthylpyrrolidin-3 -yl] -2-(3 -oxo-3,4-dihydro-2H- 1 ,4-bcnzoxazin-6-yl)acctamidc (Chembridge ID#23645796); and Hit 15 (2S,4R)-4-[(6-isopropyl-l-methyl-lH-pyrazolo[3,4-d]pyrimidin- 4-yl)amino]-l -methyl -2 -pyrrolidinyl methanol (ChemBridge ID#86711567).
  • Hit b and Hit c two compounds to inhibit specifically this enzyme with a IC50 of IpM or less were also found: Hit b and Hit c: Hit b, N-[(2,3-dimethyl-lH-indol-5-yl)methyl]pyrimidine-4,6-diamine (ChemBridge ID&88182154) and Hit c, 2-5 -[2-( 1 ,3-benzodioxol-5-yl)- l-methylethyl]-4-phenyl- IH-imidazol- 1 -ylethanol (ChemBridge ID#44349962) (Pereira de Sa N, et al. 2022).
  • Treating Cn WT or Aj WT cells with Hit 1 or Hit b inhibitors recapitulate the in vitro phenotypes observed with the respective mutants under physiological growing conditions (Fig. 2C and Fig. 3C), indicating that these compounds have potent antifungal activity in vitro when fungal cells are exposed to host environments (e.g. low oxygen or/and low glucose).
  • Tire ChemBridge database for compounds having similar scaffold w as analyzed and 10 compounds similar to Hit 1, 9 or 15, and 7 compounds similar to Hit b were found. Tire ten compounds similar to Hit 1, 9 or 15 were tested but none inhibited Sgll (or SglA) at a concentration of 100 pM or less (data not shown). Tirus, DOCK6 was used to initiate a computer-aided drug design to predict derivatives for Hit 1, 9 and 15 (inhibitors of Sgll) (see below). The 7 compounds similar to Hit b were also tested and B7 was 10-fold more potent than Hit b, whereas Bl was almost ineffective in inhibiting SglA activity (Fig. 4). Structural-activity relationship (SAR) studies were initiated based on these results.
  • Sgll or SglA
  • Hit 1 was studied to see whether it would have antifungal activity in the animal model. This was possible even without comprehensive pharmacokinetics (PK) data because these compounds have favorable “druggable” properties. For instance, based on its biophysical properties and on pkCSM software (Pires DE, Blundell TL, et al. 2015), predicted PK and toxicity properties of Hit 1 were highly favorable. Hit 1 was tested in the cryptococcal animal model, whereas Hit b in the Aspergillus animal model. For the cryptococcal model, mice were infected intranasally and 6 hours, later Hit 1 was administered intraperitoneally every day, alone or in combination with fluconazole for 14 days, and lung and brain tissue burden was examined.
  • PK pharmacokinetics
  • FIG. 6 A 50% survival in the group receiving 20 mg/kg/day (Fig. 6) was observed. Importantly, the 5 mice treated with Hit b that survived for 30 days did not show any lung fungal burden, suggesting that in these mice Hit b treatment eradicated the lung infection (Fig. 6). The lung fungal burden was assessed by qPCR determination of the concentration of Af 18 S rDNA compared to the concentration of mouse lung GAPDH at the time of death for 5 mice untreated, and at day 30 for 5 mice treated with 20 mg/Kg/day of Hit b.
  • B7 which is more potent than Hit b in inhibiting SglA (Fig. 4) is also more efficacious in the animal model and treatment with only 5 mg/Kg/day initiated 1 hour after the infection is sufficient to obtain a 50% survival (data not shown). B7 is also efficacious in improving mice survival and lung infectivity when treatment is started 24 hours after the infection, when all conidia already germinated in hyphae. As illustrated in Fig. 7, treatment with 5 mg/kg/twice day of B7 produced a 50% survival (Fig. 7A), and the surviving mice at day 30 do not have any fungal burden in their lung (Fig. 7B).
  • Hit 9 was selected as the parent structure for generating Hit 9 derivatives against Cn Sgll, such as SS-103 (see below).
  • Hit b (Fig. 9) or Hit c binds in the active site of SglA, thus preventing the binding of SGs to SglA.
  • Hit 9 is the best Sgl 1 inhibitor among tire three Hit compounds in hand for further computer- aided drug design (CADD).
  • Hit 9 caused a higher accumulation of SGs in Cn cells compared to Hit 1 or Hit 15 (Pereira de Sa N, et al. 2021).
  • Fig. 10 shows proof-of-concept refinement examples, starting from Hit 9 (pdb code 7LPQ), generated using a powerful new isosteric swapping protocol.48 Briefly, the ability to use a reference ligand fragment and generate an aligned group of "related fragments" (isosteres) which can then be "swapped” during computational refinement has implemented into DOCK6 (Pereira de Sa N, et al. 2021). In the present example, the benzoxazinone sidechain on Hit 9 (Fig.
  • a CADD strategy was used, similar to the one for the hit-to-lead optimization of Sgll inhibitors, described above.
  • An excellent binding site model through the overlay of SglA with Sgll -Hit 9 high resolution crystal structures was produced.
  • Computational structure analysis of SglA and Sgll revealed that the surface area and volume of the binding site of SglA is substantially smaller than those of Sgll, z.e., surface area: SglA 693 A2 vs. Sgl l 1,392 A2; volume: SglA 555 A3 vs. Sgl 1 967 A3.
  • Mutated clones are directly ordered from Bio Basic Inc., sequenced to make sure each clone displays only the desired mutation, expressed and used in the biochemical assay and for crystallography studies comparing the in vitro activity and the 3D structure of the active site of Sgll/SglA mutated forms with the WT Sgll/SglA.
  • the conidial suspension in water was obtained, and a pellet with 1 x 10 7 conidia was treated in minimal medium broth pH 6.5 (MM or MM+UU) ( 1 % [wt/vol] glucose, 1 x high-nitrate salts, 1 x trace elements) for 48 h at 37°C under agitation, with the hits selected, according to the toxicity criteria, at various concentrations.
  • a previously described MIC assay was performed in accordance with the guidelines in the CLSI document M38-A2, to determine the MIC and select a concentration range that does not affect mold growth. After that, the resultant hyphal pellet was used for lipid extraction.
  • (2S,4A)-4-amino-l-methylpyrrolidin-2-ylmethanol (44 mg, 0.332 mmol, 1 equiv.) in N,N- dimethylformamide (1.0 mL) was added to the solution and stirred overnight at room temperature. The progress of the reaction was monitored by TLC. Tire reaction mixture was diluted with dichloromethane and a saturated Nal ICO; solution was added. Tire organic layer was separated, and the aqueous layer was extracted with dichloromethane (5 mL x 3).
  • Step 1 Preparation of 2.2-bis(dibcnzo
  • Total erg-glc and ergosterol was detected using absorbance at 282 nm on a C-8 column with a flow rate of 0.5 ml/min in methanol/water 90: 10 ratio buffered with 1 mM ammonium formate and 0.2% formic acid.
  • the total area of product was normalized by the total area of its respective substrate.
  • C. neoformans H99 was cultivated in yeast nitrogen base (YNB) broth for 24 h at 37 °C with shaking. A pellet with 5 x 10 8 cells was treated during 24 h with all the hits selected, according to the toxicity criteria, in various concentrations. A previous minimal inhibitory concentration assay was performed in accordance with the guidelines in the CLSI document M27-A3, to determine the minimal inhibitory concentration and selecting a concentration range that did not affect the yeast growth. After that, the resultant pellets were re-counted and used for lipid extraction. Thenceforth the total lipid was extracted and dried samples were resuspended in chloroform/methanol 2: 1 ratio for LC-MS analysis. A standard erg- glc from Avanti Polar Lipids was used as a control for the calibration curve. Data were normalized to the total inorganic phosphate content in the sample.
  • Cryptococcus neoformans is an environmental fungal pathogen that, upon entering the lung and disseminating through the bloodstream, causes a life-threatening meningo-encephalitis in susceptible patients, particularly HIV+ subjects, leading to high morbidity and mortality.
  • Current antifungals such as azoles, flucytosine, amphotericin B and echinocandins have limitations: amphotericin B and flucytosine are toxic; flucytosine is not available everywhere; echinocandins have a narrow spectrum of activity and not active against cryptococcosis; and azoles have limited use due to drug-interaction and resistance (Farowski F et al. 2012 and 2013, Odabasi Z et al. 2007, Saribas Z et al. 2012, Yanni SB et al. 2012 and Mukheijee PK et al. 2011) .
  • this invention proposes a new class of antifungals targeting the sterylglucosidase 1, Sgll, (and its homolog sterylglucosidase A, SglA), an enzyme present in fungi but not in human cells.
  • This invention describes the promising Hits and the crystal structures of both Sgll and SglA alone and with its specific inhibitor (Pereira de Sa N, et al. 2021 and 2022).
  • Tire process described in tire present invention matches a rational drug design focused on the discovery of the Hit compound(s) and created a second-generation library for the identification of a lead compound(s) more potent than the Hit compound(s). This process will be iterative, requiring information on activity and mechanism of action to maximize efficacy, fungal target specificity, and lack of mammalian toxicity.
  • the compounds described in the present invention target the fungal sterylglucosidase s and will provide new in vitro and in vivo insights regarding the therapeutic efficacy of such compounds against invasive fungal infections.
  • the compound described in the present invention are also believed to have broad antifungal activity because Sgll is present in many yeasts, molds and dimorphic fungi (Grille S, Zaslawski A et al. 2010 and Normile TG et al. 2020).
  • the present invention targeted pathways present in fungal and not mammalian cells (Sgll and its homologs are not present in mammalian cells).
  • the present invention discovered the interesting association of SGs with the host immune response.
  • the Cn ⁇ sgll and Af ⁇ sgla are potent stimulators of the host immunity through the adjuvant action of SGs on y/ 5 T cells (Normile TG ct al. 2022).
  • both Cn ⁇ sgll and Af ⁇ sgla arc exciting vaccine candidates because they are highly effective in preventing a secondary infection either as live-attenuated or as heat-killed (Normile TG et al. 2020 and 2022 and Rella A, et al. 2015).
  • tire present invention envisions that a drug targeting Sgll/SglA, and thus increasing SGs, would stimulate a protective immunity which will help in the clearance of the primary infection and potentially in preventing the recurrence of a secondary infection.
  • This type of treatment could be ideal in patients waiting for transplants (susceptible to aspergillosis) and in patients affected with HIV (susceptible to cryptococcosis), because these fungal vaccines are effective in condition of neutropenia (a condition that favors aspergillosis) or in condition of CD4+ T cell deficiency (a condition that favors cryptococcosis).
  • neutropenia a condition that favors aspergillosis
  • CD4+ T cell deficiency a condition that favors cryptococcosis

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Abstract

The present invention provides a method of inhibiting growth of a fungus in a subject, comprising reducing the activity of sterylglucosidase I (SigIl) and/or sterylglucosidase A (SglA) in the fungus.

Description

STERYLGLUCOSIDASE INHIBITING COMPOSITIONS AND METHOD OF USING
[0001] This application claims priority of U.S. Provisional Application No. 63/378.403, filed October 5, 2022, the contents of which are hereby incorporated by reference.
[0002] Throughout this application, various publications are referenced, including referenced in parenthesis. The disclosures of all publications mentioned in this application in their entireties are hereby incorporated by reference into this application in order to provide additional description of the art to which this invention pertains and of the features in the art which can be employed with this invention.
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0001] This invention was made with government support under All 16420 awarded by National Institutes of Health. The government has certain rights in the invention.
BACKGROUND OF THE INVENTION
[0002] Invasive fungal infections are a leading cause of death in immunocompromised patients. While much is known about the cellular processes required for the pathogenesis of these infections, translating understanding into tangible clinical benefit has been difficult because these fungal pathogens and their hosts have similar physiology. As a result, current antifungal agents have limited clinical efficacy, are poorly fungicidal in the host, are occasionally toxic, and are increasingly ineffective due to emerging resistance. Thus, innovative antifungal agents are needed.
[0003] When the steryl-glucosidase 1 (SGL1) gene was deleted in Cryptococcus neoformans (Cn) and its homolog (SGLA) gene was deleted in Aspergillus fumigatus ( Af), it was found that the resulting mutants (Cn Δsgll or Af Δsgla) accumulates steryl glucosides (SGs) and are not pathogenic in a mouse model of infection. Sterylglucosidases are enzymes found in plants and fungi but not in humans. Amazingly, mice receiving an intranasal administration of Cn Δsgll or Af Δsgla rapidly eliminate the respective mutant because these mutants are not viable at 5% CO2 and low O2 (5-10%). Uris indicated that the fungal sterylglucosidase is necessary for fungal pathogenicity and it is a promising novel drug target.
[0004] Therefore, a high throughput screening (HTS) assay was developed and used to screen a ChemBridge DiverSet library and inhibitors to Sgll/SglA enzymes were identified in this invention. The crystal structures of both Sgl 1 and SglA alone were obtained and specific inhibitors of Sgl 1/SglA were identified. In addition to inhibiting sterylglucosidase activity in vitro, these compounds also decrease Sgll/SglA activity in living fungal cells, accumulating SGs. Treatment with Sgll inhibitor protects mice from developing cryptococcal meningoencephalitis and treatment with SglA inhibitor significantly improved mice survival upon pulmonary aspergillosis. BRIEF SUMMARY OF THE INVENTION
[0005] The present invention provides a method of inhibiting growth of a fungus in a subject, comprising reducing the activity of sterylglucosidase 1 (Sigil) and/or sterylglucosidase A (SglA) in the fungus.
[0006] The present invention provides a compound having the structure: wherein R1 and R2 are independently H, alkyl, alkenyl, alkynyl carbonyl, amine; -CH-, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, C(O)-alkyl, C(O)-haloalkyl, C(O)-cycloalkyl, C(O)- heterocycloalkyl, C(O)-aryl, C(O)-heteroaryl, CO2-alkyl, CO2-aryl, CO2-heteroaryl, or CO2- heterocycloalkyl; preferably, R1 and R2 are independently H, alkyl, alkenyl, alkynyl carbonyl, or amine; or a pharmaceutically acceptable salt thereof.
[0007] The present invention provides a compound having the following structure: wherein R17, R18, R19 are each independently CH, N. or S: wherein R20 is NH2. NH-alkyl, NH-C(O)-alkyl, NH-C(S)-alkyl. NH-NHC(O)-alkyl. NH-NHC(S)- alkyl, alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, C(O)-alkyl, C(O)-haloalkyl, C(O)- cycloalkyl, C(O)-heterocycloalkyl, C(O)-aryl, C(O)-heteroaryl. CO2-alkyl, CO2-aryl. CO2- heteroaryl, CO2-heterocycloalkyl, alkyl-N-, SO2-alkyl, SO2-haloalkyl, SO2-cycloalkyl, SO2- heterocycloalkyl, SO2-aryl, or SO2-heteroaryl; wherein R21 is aryl, heteroaryl, cycloalkyl, heterocycloalkyl, C(O)-heterocycloalkyl, C(O)-aryl, C(O)-heteroaryl, CO2-aryl, CO2-heteroaryl, CO2-heterocycloalkyl, SO2-cycloalkyl, SO2- heterocycloalkyl, SO2-aryl, or SO2-heteroaiyl; preferably, R21 is aryl, heteroaryl, C(O)-aryL C(O)- heteroaryl, CO2-aryl, CO2-heteroaryl, SO2-aryl, or SO2-heteroaryl; more preferably, R21 is heteroaryl; and wherein R22 is alkyl or -NH-alkyl; and wherein when R22 is H, R20 is NH2; or a pharmaceutically acceptable salt thereof.
BRIEF DESCRIPTION OF THE DRAWINGS
[0008] Figure 1. Deletion of SGL1 in Cn or SGLA gene in Af abolishes virulence. (A) Virulence studies showing that 100% of mice infected with Cn Δsgll survived the infection whereas mice infected with Cn wild-type (TW) H99 or with Δsgll +SGL1 reconstituted strain died within 24±6 and 21±7 days, respectively. n=10 mice in each group. (B) Virulence studies showing that 100% of mice receiving an intranasal administration of 5x104 conidia of AfΔsglA survived the infection whereas mice infected with 5x104 conidia of Af 293 wild-type (A/WT) or with the reconstituted (Af ΔsglA+SGLA) strained died within 18 days. n=10 mice/group. IS, immunosuppression: all mice in B received a subcutaneous administration of triamcinolone (400 mg/kg) one day prior to the injection of Af conidia. *P <0.001, Δsgll or Δsgla vs WT by Kruskal-Wallis test.
[0009] Figure 2. Effect of Sgl 1 deletion/inhibition of growth of C. Neqformans (Cn). Deletion of Sgl l gene in Cn (Δsgll) affect growth in DMEM agar at low oxygen (B vs. A). Treatment on Cn WT with 100 pM Hit 1(c) inhibits growth similarly to Δsgll (B).
[0010] Figure 3. Effect of SglA deletion/inhibition on hyphal formation in A.fumigatus (Af). Deletion of SglA gene in Af (ΔsglAI) dramatically effects hyphal elongation. (B) compared to WT (A). Treatment of AfWT with Hit b for 12 hours. (C) recapitulates tire phenotype of ΔsglA. (D) Quantitative analysis of hyphal length of AfWT untreated (Un). Af ΔsglA-Un. and AfWT treated with different concentrations of SglA inhibitors Hit b (10 and 100 pM) or Hit c (10, 100 and 500 pM). Both compounds significantly inhibit hyphal elongation in Aj 'WT similarly to ΔsglA. Black bar in A, B and C, 20 pm. *** in D, P<0.01 , by ANOVA.
[0011] Figure 4. Percentage of SglA inhibition of Hit b (B) and its 7 derivatives (B1-B7) from ChemBridge. Dose-response curves using the native substrate ergosterol 30-D-glucoside. Reactions were performed using 0.5 mol% ergosterol glucoside in Triton-X-100 mixed micelles with 20 min reaction time at 37 °C. **Ergosterol was detected at 282 nm by UV absorption after HPLC separation. *, P <0.001, B7 or Bl versus Hit B. Statistic by one-way ANOVA. Tukey’s multiple Comparation Test.
[0012] Figure 5. Hit 1 prevents dissemination of C. neoformans to the brain. CBA/J mice were inoculated with 1x106 Cn WT H99. After 6 hours, treatment started intraperitoneally with 10 mg/kg/day of Hit l or fluconazole (F). Five mice in each group where euthanized and lung CFUs were analyzed at days 3, 6, 9, and 14 after infcction/trcatment (A) whereas brain CFU only at day 14 (B). No Cn cells were found in the brain when mice were treated with Hit 1 or Hit 1 + F. The Hit 1 + F combination also showed a significant reduction in lung CFU. One-way ANOVA, Tukey’s Multiple Comparison Test. Ns, not significant; *, p<0.05; **<p<0.001, treated versus untreated. [0013] Figure 6. Hit b prolongs survival of mice infected with Af A) Mice were infected with 5x104 Af conidia. After ~lh they received an intraperitoneal injection of Hit b 5mg/Kg or 20 mg/Kg, which they continued daily. n=10 mice in each group. * P<0.01, Hit b 20 mg/Kg versus untreated by Kruskal-Wallis test. B) No fungal DNA was found in lungs treated with Hit b. Graphed data represent the means ± SD of the fold changes in 18S fungal rDNA/mouse lung GAPDH. Statistic was determined via two-way ANOVA using Tukey’s multiple Comparation Test. *, p<0.001 for Hit b-treated versus untreated. IS, Immunosuppression.
[0014] Figure 7. B7 Prolongs survival of mice infected with Af. A) Mice were infected with 5x104 Af conidia. After 24 hours they received an intraperitoneal injection of B7 5mg/Kg/day or twice a day, which they continued daily. n=10 mice in each group. * P<0.01, B7 5 mg/Kg/twice day versus untreated by Kruskal-Wallis test. B) No fungal DNA was found in lungs of 5 surviving mice treated with B7 (twice a day for 30 days). Graphed data represent the means ± SD of the fold changes in 18S fungal DNA/mouse lung GAPDH. Statistic was determined via two-way ANOVA using Tukey’s multiple Comparation Test. *, p<0.001 for B7-treated versus untreated. IS, Immunosuppression.
[0015] Figure 8. Structural analysis of Cn Sgll with its substrate and with its inhibitor. A) Sgll structure revealed a two domains architecture: a catalytic domain and a [3-sandwich domain. The active site is enclosed in a pocket. B) Interactions of ergosterol-3-|3-glucoside with the Sgl 1 active site. Glucose interacts with Lys 47 and Trp 570. The sterol component makes hydrophobic interaction with Glu 270 and Leu 431. C) Hit 1 (green) binds to Glu 270 and Trp 570. All other amino acids in the active site are shown.
[0016] Figure 9. Docking analysis of Hit 9 with Sgll (A) and Hit b with SglA (B). Hit 9 (purple) binds to Glu 587, Lys 47 and Glu 270 of Sgll in the active site. Hit b (pink) binds to Asp 127 and Glu 247 of SglA in the active site. Others amino acids in the active site are shown.
[0017] Figure 10. 2D structures, 3 D poses, DOCK 6 fitness scores, and ligand descriptors for Hit 9, (green in C) and 182 analogs (orange in D). complexed with Sgll. Key protein residues involved with H- bonding (magenta) are also shown. Analogs were constructed using an isosteric swapping protocol in which the cognate ligand sidechain (shaded oval in A) was employed to identify 500 isosteres which were then sampled at position R (shaded oval in B). Out of 500 sidechains sample, 182 analogs had a new bond connection previously seen in a large drug-like library and the pose was geometrically and energetically compatible with the binding site.
[0018] Figure 11. A) Binding pocket of Hit 9 in Sgll; B) SS-103 (and SS-104) in the binding pocket; C) Enzyme Inhibitory assay of Hit 9 and SS-103 w hich ergosterol 3-[3-glucoside; D, E) SG accumulation in C. neoformans H99 with SS-103 (D) and Hit 9 (E). [0019] Figure 12. Selected branched Hit 9 analogs with high docking scores.
[0020] Figure 13. Fragment-based design of Sgll inhibitors.
[0021] Figure 14. Overlay of SglA with Hit b, Hit c and Hit 9.
[0022] Figure 15. Selected branched Hit b analogs with high docking scores.
[0023] Figure 16. A) Deletion of Sgll (Δsgll) accumulates ergosterol-3-[3-glucoside in C. albicans cells. B) IC50 (50% inhibition) of C. neoformans (Cn) Sgll, A. fumigalus (Af) SglA or Ca Sgll by Hit b, Hit 8 and Hit 11 in vitro. *, p<0.001 by ANOVA.
[0024] Figure 17. SglA enzyme inhibitory assay of Hit 9, B7 and DR-SglA-l~3 with natural substrate, ergosteryl 3-b-D-glucoside.
[0025] Figure 18. Biological Potency Evaluations for SS-103. In the top panels, an in vitro enzyme inhibitory assay of Hit 9 (purple line, circles) and SS-103 (black line, squares) with erg-glc (natural substrate) is shown. In the bottom panels, an erg-glc accumulation study in C. neoformans strain H99 with Hit 9 (left) and SS-103 (right) is shown.
DETAILED DESCRIPTION OF THE INVENTION
[0026] The present invention provides a method of inhibiting growth of a fungus in a subject, comprising reducing the activity of stcrylghicosidasc 1 (Sigil) and/or sterylglucosidase A (SglA) in the fungus.
[0027] The present invention provides a method of reducing the activity of Sigil and/or SglA comprises:
(a) inhibiting the synthesis of Sigil and/or SglA;
(b) reducing the level of Slgl 1 and/or SglA; and/or
(c) blocking the active site of Slgl 1 and/or SglA.
[0028] The present invention provides a method of inhibiting growth of a fungus comprising contacting the fungus with an effective amount of a compound having the structure: wherein R1 and R2 are independently H, alkyl, alkenyl, alkynyl carbonyl, amine; -CH-, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, C(O)-alkyl, C(O)-haloalkyl, C(O)-cycloalkyl, C(O)-heterocycloalkyl, C(O)- aryl. C(O)-heteroaryl, CCE-alkyl, CO2-aryl, CO2-heteroaryl, or CO2-heterocycloalkyl. [0029] In some embodiments, R, and R2 are independently H, alkyl, alkenyl, alkynyl carbonyl, or amine.
[0030] Hie present invention provides a method of inhibiting growth of a fungus comprising contacting the fungus with an effective amount of a compound having the structure: wherein R17, R1g, R19 are each independently CH, N, or S; wherein R20is NH2, NH-alkyl, NH-C(O)-alkyl, NH-C(S)-alkyl, NH-NHC(O)-alkyl. NH-NHC(S)- alkyl, alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, C(O)-alkyl. C(O)-haloalkyl, C(O)- cycloalkyl, C(O)-heterocycloalkyl, C(O)-aryL C(O)-heteroaryl, CO2-alkyl, CO2-aryl, CO2- heteroaryl, CO2-heterocycloalkyl, alkyl-N-, SO2-alkyl, SO2-haloalkyl, SO2-cycloalkyl, S02- heterocycloalkyl, SO2-aryl, or SO2-heteroaryl; wherein R21 is aryl, hctcroaryl, cycloalkyl, hctcrocycloalkyl, C(O)-hctcrocycloalkyl, C(O)-aryl, C(O)-heteroaryl, CO2-aryl, CO2-heteroaryl, CO2-heterocycloalkyl, SO2-cycloalkyl, S02- heterocycloalkyl, SO2-aryl, or SO2-heteroaryl; wherein R22 is H, alkyl or -NH-alkyl; and wherein when R22 is H, R20 is NH2; or a pharmaceutically acceptable salt thereof.
[0031] In some embodiments, R20 is NH-alkyl, NH-C(O)-alkyl, NH-C(S)-alkyl, NH-NHC(O)-alkyl, NH- NHC(S)-alkyl, alkyl, alkenyl, alkynyl, C(O)-alkyl, C(O)-haloalkyl, CO2-alkyl, alkyl-NH2, SO2-alkyl, or SO2-haloalkyl.
[0032] In some embodiments, R20 is NH-alkyl, NH-C(O)-alkyl, NH-C(S)-alkyl, NH-NHC(O)-alkyl, NH- NHC(S)-alkyl, alkyl, alkenyl, alkynyl, C(O)-alkyl, or C(O)-haloalkyl.
[0033] In some embodiments, R20 is NH-alkyl, NH-C(O)-alkyl, NH-C(S)-alkyl, NH-NHC(O)-alkyl, NH- NHC(S)-alkyl, alkyl, C(O)-alkyl, or C(O)-haloalkyl.
[0034] In some embodiments, R20 is NH-alkyl or alkyl.
[0035] In some embodiments, R20 is NH-alkyl. [0036] In some embodiments, R20 is alkyl.
[0037] In some embodiments, R21 is aryl, heteroaryl, C(O)-aryl, C(O)-heteroaryl, CO2-aryl. CO2- heteroaryl, SO2-aryl, or SO?-hctcroaryl.
[0038] In some embodiments, R21 is heteroaryl.
[0039] The present invention provides a compound having the structure: wherein R1 and R2 are independently H, alkyl, alkenyl, alkynyl carbonyl, amine: -CH-, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, C(O)-alkyl, C(O)-haloalkyl, C(O)-cycloalkyl, C(O)- heterocycloalkyl, C(O)-aryl, C(O)-hcteroaryl. CO2-alkyl, CO2-aryl, CO2-heteroaryl, or CO2- heterocycloalkyl; or a pharmaceutically acceptable salt thereof.
[0040] In some embodiments, R1 and R2 are independently H, alkyl, alkenyl, alkynyl carbonyl, or amine.
[0041] In some embodiments, the present invention provides a compound having the following structure:
[0042] In some embodiments, the present invention provides a compound having the following structure: wherein R, is 0, NH, CH2, or S; wherein R4 is -CH-, -N-, -NH-N-, -NH-C(O)-, -NH-C(S)-, -NH-NHC(O)-, -NH-NHC(S)-, alkyl, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, C(O)-alkyl, C(O)-haloalkyl, C(O)-cycloalkyl, C(O)- heterocycloalkyl, C(O)-arvl, C(O)-heteroaryl, CO2-alkyl, CO2-aryl, CO2-heteroaryl, CO2-heterocycloalkyl, alkyl-N-, SO2-alkyL SO2-haloalkyl. SO2-cycloalkyl, SO2-hctcrocycloalkyl. SO2-aryl, or SO?-hctcroaryl; and wherein R5 and Rg are each independently H, ary l, heteroaryl, cycloalkyl, heterocycloalkyl, CO2- aryl, CO2-heteroaryl, CO2-cycloalkyl, or CO2-heterocycloalkyl.
[0043] In some embodiments, R3 is 0, NH. or S.
[0044] In some embodiments, R3 is O, or NH.
[0045] In some embodiments, R3 is O.
[0046] In some embodiments, R3 is NH.
[0047] In some embodiments, R4 is -CH-, -N-, -NH-N-, -NH-C(O)-, -NH-C(S)-, -NH-NHC(O)-, -NH- NHC(S)-, or heteroaryl.
[0048] In some embodiments, R4 is -CH- or heteroaryl.
[0049] In some embodiments, R4 is -CH-.
[0050] In some embodiments, R4 is heteroaryl.
[0051] In some embodiments, the present invention provides a compound having the following structure:
[0053] In some embodiments, R4 is heteroaryl, C(O)-heteroaryl, CO2-heteroaryl, or SO2-heteroaryl.
[0054] In some embodiments, the heteroaryl is pyran, pyridine, piperidine, pyrimidine, isoxazole, oxazole, silole, 6H- 1,2, 5 -thiadiazine, 2H,6H-l,5,2-dithiazine, 1,4-thiazepine, triazine, oxirane, thiirane or azirine.
[0055] In some embodiments, R4 is heteroaryl.
[0056] In some embodiments, R4 is pyridine, pyrimidine or triazine.
[0057] In some embodiments, R4 is triazine.
[0058] In some embodiments, the present invention provides a compound having the following structure: wherein R5 and Re are each independently H, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, CO2- aryl, CO2-heteroaryl, CO2-cycloalkyl, or CO2-heterocycloalkyl.
[0059] In some embodiments, R5 and Re are each independently aryl, heteroaryl, cycloalkyl, or heterocycloalkyl.
[0060] In some embodiments, R5 and Re are each independently aryl or cycloalkyl.
[0061] In some embodiments, R5 and Re are each independently aryl.
[0062] In some embodiments, R5 and Re are each independently cycloalkyl.
[0063] In some embodiments, R5 and Re are each independently substituted.
[0064] In some embodiments, R5 and Re are each independently substituted aryl or substituted cycloalkyl.
[0065] In some embodiments, R5 and Re are each independently substituted with alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, alkoxy, carbonyl, carboxyl, amino, or amide.
[0066] In some embodiments, R5 and Re are each independently substituted with aryl, heterocycloalkyl, carbonyl or carboxyl.
[0067] In some embodiments, R5 and Re are each independently substituted with aryl or heterocycloalkyl.
[0068] In some embodiments, R5 and Re are each independently:
wherein U is N or CH; wherein V, X and Y are each independently C, NH, 0, S, -N-, -N-NH2, -N-C(O)-, -N-C(S)-, -N- NHC(O)-, -N-NHC(S), -N-alkyl, -N-alkyne, -N-alkynl, -N-aryl, -N-heteroaryl, -N-cycloalkyl, -N- hctcrocycloalkyl, or -N-haloalkyl; wherein W and Z are each independently H, NH2. alkyl, alkyne, alkynl, -NH-alkyl, -O-alkyl, -O- haloalkyl, -O-cycloalkyl, O-heterocycloalkyL O-aryl; O-heteroaryl; carbonyl or alkoxy; and wherein R9, R1o, R11, R12, RB are each independently -H, -OH, -COOH, -P(=O)(OH)2, halogen, CN, -CF3, -CHF2, -OCF3, -NO2, alkyl, alkenyl, alkynyl, and, heteroaryl, -OAc, -ORu. -COR14, - SH, -SR14, -SO2RW, -NH2, -NHR14, -NR15R16, -NHCOR15, or -CONR15R16; wherein each occurrence of R 14 is independently alkyl, alkenyl, alkynyl, aryl, or heteroaryl, wherein each occurrence of R 15 is independently -H, alkyl, alkenyl, alkynyl, aryl, or heteroaryl, wherein each occurrence of R16 is independently -H, alkyl, alkenyl, alkynyl, aryl, or hcteroaryl.
[0069] In some embodiments, R5 and Re arc each independently:
[0070] In some embodiments, R6 and R6 are the same.
[0071] In some embodiments, R5 and R6 are different.
[0072] In some embodiments, the present invention provides a compound having the following structure:
[0073] The present invention provides a compound having the following structure: wherein R17, R18, R19 are each independently CH, N, or S; wherein R20 is NH2, NH-alkyl, NH-C(O)-alkyl, NH-C(S)-alkyl, NH-NHC(O)-alkyl, NH-NHC(S)- alkyl, alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, C(O)-alkyl. C(O)-haloalkyl, C(O)- cycloalkyl, C(O)-heterocycloalkyl. C(O)-aryl, C(O)-heteroaryl, CO2-alkyl, CO2-aryl, CO2- heteroaryL CO2-heterocycloalkyL alkyl-N-, SO2-alkyl, SO2-haloalkyl. SCf-cycloalkyl. SO2- heterocycloalkyl, SO2-aryl, or SO2-heteroaryl; wherein R21 is aryl, heteroaryl, cycloalkyl, heterocycloalkyl, C(O)-heterocycloalkyl, C(O)-aryl, C(O)-hctcroaryl. CO2-aryl, CO2 hctcroaryl. CO2-hctcrocycloalkyl, SO2-cycloalkyl, SO2- heterocycloalkyl, SO2-aryl, or SO2-heteroaryl; wherein R22 is alkyl or -NH-alkyk and wherein when R22 is H, R20 is NH2; or a pharmaceutically acceptable salt thereof.
[0074] In some embodiments, when R22 is H, R20 is NH2. [0075] In some embodiments, R21 is aryl, heteroaryl, C(O)-aryl, C(O)-heteroaryl, CO2-aryl, CO2- heteroaryl, SO2-aryl, or SO2-heteroaryL
[0076] In some embodiments, R21 is heteroaryl.
[0077] In some embodiments, R20 is NH-alkyl, NH-C(O)-alkyl, NH-C(S)-alkyl, NH-NHC(O)-alkyl, NH- NHC(S)-alkyl, alkyl, alkenyl, alkynyl, C(O)-alkyl, C(O)-haloalkyl, CO2-alkyl, alkyl-NH2, SO2-alkyl, or SO2-haloalkyl.
[0078] In some embodiments, R20 is NH-alkyl, NH-C(O)-alkyl, NH-C(S) -alkyl, NH-NHC(O)-alkyl, NH- NHC(S)-alkyl, alkyl, alkenyl, alkynyl, C(O)-alkyl, or C(O)-haloalkyl.
[0079] In some embodiments, R20 is NH-alkyl, NH-C(O)-alkyl, NH-C(S)-alkyl, NH-NHC(O)-alkyl, NH- NHC(S)-alkyl, alkyl, C(O)-alkyl, or C(O)-haloalkyl.
[0080] In some embodiments, R20 is NH-alkyl or alkyl.
[0081] In some embodiments, R20 is NH-alkyl.
[0082] In some embodiments, R20 is alkyl.
[0083] In some embodiments, the present invention provides a compound having the following structure:
[0084] In some embodiments, R17, R18, R19 are CH, N, N.
[0085] In some embodiments, R17, R18, R19 are N, CH, N.
[0086] In some embodiments, R17, R18, R19 are N, N.CH.
[0087] In some embodiments, R17, R18, R19 are CH. CH, N.
[0088] In some embodiments, R17, R18, R19 are CH. N, CH.
[0089] In some embodiments, R17, R18, R19 are N, CH, CH.
[0090] In some embodiments, R17, R18, R19 are CH, CH, CH.
[0091] In some embodiments, R17, R18, R19 are N, N, N. [0092] In some embodiments, R22 is NH-alkyl, NH-C(O)-alkyl, NH-C(S)-alkyl, NH-NHC(O)-alkyL NH- NHC(S)-alkyl, alkyl, C(O)-alkyl, or C(O)-haloalkyl.
[0093] In some embodiments, R.22 is NH-alkyl or alkyl.
[0094] In some embodiments, R22 is NH- C1-6 alkyl or C1-6 alkyl.
[0095] In some embodiments, R22 is NH-C1-3 alkyl or C1-3 alkyl.
[0096] In some embodiments, R22 is NH-CH3 or CH3.
[0097] In some embodiments, R21 is an aryl or hctcroaryl.
[0098] In some embodiments, R21 is an aryl.
[0099] In some embodiments, R21 is a heteroaryl.
[0100] In some embodiments, aryl is phenyl, p-toluenyl (4-methylphenyl), naphthyl, tetrahydro-naphthyl, indanyl, biphenyl, phenanthryl, anthryl or acenaphthyl.
[0101] In some embodiments, aryl is phenyl, p-toluenyl (4-methylphenyl).
[0102] In some embodiments, aryl is phenyl.
[0103] In some embodiments, heteroaryl is pyridine, pyridazine. pyrimidine, pyrazine, 1,2,4-triazine, 1,3,5-triazine, 1,4,5, 6-tetrahydrocyclopenta[b]pyrrole, l,3a,4,61-tetrahydropyrrolo[3,2-b]pyrrole, 1,4- dihydropyrrole[3,2-b]pyrrole, l,6-dihydropyrrolo[2,3-b]pyrrole, indoline, 3/7-indolc. 1/7-indole, 2H- isoindole, indolizine, IH-indazolc. benzimidazole, 7-azaindole, 4-azaindole, 5-azaindole, or 97/-carbazolc.
[0104] In some embodiments, heteroaryl is 3H-indole, 1/7-indole, 2H-isoindole, indolizine, l/7-indazolc. benzimidazole, 7-azaindole, 4-azaindole, 5-azaindole, or 9H-carbazole.
[0105] In some embodiments, heteroaryl is 1H-indazolc or 9/7-carbazolc.
[0106] In some embodiments, the aryl or heteroaryl is substituted.
[0107] In some embodiments, aryl or heteroaryl is substituted with alkyl, alkenyl, alkynyl. aryl, heteroaryl, cycloalkyl, heterocycloalkyl, alkoxy, carbonyl, carboxyl, amino, or amide.
[0108] In some embodiments, aryl or heteroaryl is substituted with alkyl, alkenyl, alkynyl, aryl, or heteroaryl.
[0109] In some embodiments, and or heteroaryl is substituted with alkyl.
[0110] In some embodiments, alkyl is C1-6 alkyl.
[0111] In some embodiments, alkyl is C1-3 alkyl. [0112] In some embodiments, alkyl is methyl or ethyl.
[0113] In some embodiments, R21 has the following structure:
wherein U is N or CH; wherein V, X and Y are each independently C, NH, 0, S, -N-, -N-NH2, -N-C(O)-, -N-C(S)-, -N- NHC(O)-, -N-NHC(S), -N-alkyl, -N-alkyne, -N-alkynl, -N-aryl, -N-heteroaryl, -N-cycloalkyl, -N- heterocycloalkyl, or -N-haloalkyl; wherein W and Z are each independently H, NH2. alkyl, alkyne, alkynl, -NH-alkyl, -O-alkyl, -O- haloalkyl, -O-cycloalkyl, O-heterocycloalkyl, O-aryl; O-heteroaryl; carbonyl or alkoxy: and wherein R9, R1o, Rn, R12, R13 are each independently -H, -OH, -C00H, -P(=O)(OH)2, halogen, CN, -CF3, -CHF2, -OCF3, -NO2, alkyl, alkenyl, alkynyl, aryl, heteroaryl, -OAc, -OR14, -CORu, -SH, - SR14, -SO2R14, -NH2, -NHR14, -NR15R16, -NHCOR15, or -CONR15R16; wherein each occurrence of R14 is independently alkyl, alkenyl, alkynyl, aryl, or heteroaryl, wherein each occurrence of R15 is independently -H, alkyl, alkenyl, alkynyl, aryl, or heteroaryl, wherein each occurrence of R16 is independently -H, alkyl, alkenyl, alkynyl, aryl, or heteroaryl.
[0114] In some embodiments, R21 has the following structure: ,
[0115] In some embodiments, R21 has the following structure:
[0116] In some embodiments, R21 has the following structure:
[0117] In some embodiments, the present invention provides a compound having the following structure:
[0118] In some embodiments, the present invention provides a pharmaceutical composition comprising compounds described in the current invention and a pharmaceutically acceptable carrier.
[0119] In some embodiments, the pharmaceutical composition further comprises an effective amount of an anti-fungal agent.
[0120] In some embodiments, the present invention provides a method of treating a subject with fungal infection comprising administering an effective amount of the compound described in any one of paragraphs [0039]-[0117],
[0121] In some embodiments, the present invention provides a method of treating a subject with fungal infection comprising administering an effective amount of the compounds having the structure: wherein R1 and R2 are independently H, alkyl, alkenyl, alkynyl carbonyl, amine; -CH-, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, C(O)-alkyl, C(O)-haloalkyl, C(O)-cycloalkyl, C(O)-heterocycloalkyl, C(O)- aryl, C(O)-heteroaryl, CO2-alkyl, CO2-aryl, CO2-heteroaryl, or CO2-heterocycloalkyl.
[0122] In some embodiments, R1 and R2 are independently H, alkyl, alkenyl, alkynyl carbonyl, or amine.
[0123] In some embodiments, the present invention provides a method of treating a subject with fungal infection comprising administering an effective amount of the compounds having the structure: wherein R17, R18, R19 are each independently CH, N, or S; wherein R20 is NH2, NH-alkyl, NH-C(O)-alkyl, NH-C(S)-alkyL NH-NHC(O)-alkyl, NH-NHC(S)- alkyl, alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, C(O)-alkyl, C(O)-haloalkyl, C(O)- cycloalkyl, C(O)-heterocycloalkyl, C(O)-aryl. C(O)-heteroaryl. CO2-alkyl, CO2-aryl, CO2- heteroaryl, CO2-heterocycloalkyl, alkyl-N-, SO2-alkyl, SO2-haloalkyl, SO2-cycloalkyl, SO2- heterocycloalkyl, SO2-aryl, or SO2-heteroaryl; wherein R21 is aryl, heteroaryl, cycloalkyl, heterocycloalkyl, C(O)-heterocycloalkyl, C(O)-aryl, C(O)-heteroaryl, CO2-aryl, CO2-heteroaryl, CO2-heterocycloalkyl, SO2-cycloalkyl, SO2- heterocycloalkyl, SO2-aryl, or SO2-heteroaryl; wherein R22 is H, alkyl or -NH-alkyl; and wherein when R22 is H, R20 is NH2; or a pharmaceutically acceptable salt thereof.
[0124] In some embodiments, the method further comprises administering to the subject an effective amount of an anti-fungal agent.
[0125] In some embodiments of the method, the anti-fungal agent is fluconazole, amphotericin B, caspofungin, tunicamycin or aureobasidin A or a combination thereof.
[0126] In some embodiments of the method, the anti-fungal agent is azole, flucytosine, amphotericin B or echinocandins, or a combination thereof.
[0127] In some embodiments of the method, the fungus is a saprotrophic fungus.
[0128] In some embodiments, the fungus is Cryptococcus Neoformans, Cryptococcus gattii, Candida albicans, Candida krusei, Candida glabrata, Candida parapsilosis, Candida guilliermondii, Aspergillus fumigatus, Rhizopus oryzae, Rhizopus spp. , Blastomyces dermatitis, Histoplasma capsulatum, Coccidioides spp., Paecilomyces variotii, Pneumocystis murina, Pneumocystis proved, Histoplasma capsulatum, Aspergillus spp., or Sporothrix brasiliensis.
[0129] In some embodiments of the method, the fungus is Cryptococcus Neoformans or Aspergillus fumigatus.
[0130] In some embodiments of tire method, the fungal infection is caused by Candida, Aspergillus, Cryptococcus , Histoplasma, Pneumocystis, Stachybotrys or Mycrorales fungus.
[0131] In some embodiments of the method, the fungal infection is caused by Cryptococcus Neoformans. [0132] In some embodiments of the method, the fungal infection is Cryptococcus neoformans cryptococcosis.
[0133] In some embodiments of the method, the fungal infection is caused by Sporothrix.
[0134] In some embodiments of the method, the fungal infection is caused by 5. brasiliensis, S. schenckii, S. globosa, S. mexicana, S. chilensis. S. luriei, and S. pallida.
[0135] In some embodiments of the method, the fungal infection is caused by 5. brasiliensis.
[0136] In some embodiments of the method, the fungal infection is caused by a fungus other than Cryptococcus Neoformans .
[0137] In some embodiments of the method, the fungal infection is a fungal infection other than Cryptococcus neoformans cryptococcosis.
[0138] In some embodiments of the method, the fungal infection is Aspergillosis. Blastomycosis, Candidiasis, Coccidioidomycosis, Cryptococcus gattii cryptococcosis. Fungal Keratitis, Dermatophytes, Histoplasmosis, Mucormycosis, Pneumocystis pneumonia (PCP), or Sporotrichosis.
[0139] In some embodiments of the method, the fungal infection is Sporotrichosis.
[0140] In some embodiments of the method, the fungal infection is caused by Cryptococcus gattii, Candida albicans, Candida krusei, Candida glabrata, Candida parapsilosis, Candida guilliermondii, Aspergillus fumigatus, Rhizopus oryzae, Rhizopus spp. , Blastomyces dermatitis, Histoplasma capsulatum, Coccidioides spp., Paecilomyces variotii, Pneumocystis murina, Pneumocystis jiroveci, Histoplasma capsulatum, Aspergillus spp., or dimorphic fungi.
[0141] In some embodiments of the method, the anti-fungal agent is fluconazole, amphotericin B, caspofungin, tunicamycin or aureobasidin A.
[0142] In some embodiments, the fungal infection is a fungal infection on a plant.
[0143] In some embodiments, the fungal infection is a fungal infection in human.
[0144] In some embodiments, the fungal infection is an internal fungal infection.
[0145] In some embodiments, the fungal infection is an invasive fungal infection.
[Of 46] In some embodiments, the fungal infection is a fungal infection of the skin or lung.
[0147] In some embodiments, the compound has a fungistatic effect on the fungus.
[0148] In some embodiments, the compound is administered orally to the subject.
[0149] In some embodiments, the compound is administered topically to the subject. [0150] In some embodiments, the subject is also afflicted with an immunodeficiency disorder.
[0151] In some embodiments, the subject is also afflicted with human immunodeficiency vims (HIV).
[0152] In some embodiments, the subject is also afflicted with pulmonary aspergillosis.
[0153] In some embodiments, the subject is also afflicted with cryptococcal meningoencephalitis.
[0154] In some embodiments, the antifungal agent is Amphotericin B, Candicidin, Filipin, Hamycin, Natamycin, Nystatin, Rimocidin, Clotrimazole, Bifonazole, Butoconazole, Clotrimazole, Econazole, Fenticonazole, Isoconazole, Ketoconazole, Luliconazole, Miconazole, Omoconazole, Oxiconazole, Sertaconazole, Sulconazole, Tioconazole, Albaconazole, Fluconazole, Isavuconazole, Itraconazole, Posaconazole, Ravuconazole, Terconazole, Voriconazole, Abafungin, Amorolfin, Butenafine, Naftifine, Terbinafine, Anidulafungin, Caspofungin, Micafungin, Ciclopirox, Flucytosine, Griseofulvin, Haloprogin, Tolnaftatc, or Undccylcnic acid.
[0155] In some embodiments, the present invention provides a pharmaceutical composition comprising a compound of the present invention and an antifungal agent, and at least one pharmaceutically acceptable carrier for use in treating a fungal infection.
[0156] In some embodiments, a pharmaceutical composition comprising an amount of the compound of the present invention for use in treating a subject afflicted with a fungal infection as an add-on therapy or in combination with, or simultaneously, contemporaneously or concomitantly with an anti-fungal agent.
[0157] In some embodiments of any of the above methods or uses, the subject is a human.
[0158] In some embodiments of any of the above methods or uses, the compound and/or anti-fungal agent is orally administered to the subject.
[0159] In some embodiments, the present invention provides identifying a compound that inhibits the activity of sterylglucosidase using structure-based computer-aided drug design (CADD) software; or determining the crystal structure of Sigil and/or SglA.
[0160] In some embodiments of any of the above methods or uses, the compound and/or anti-fungal agent is topically administered to the subject.
[0161] In some embodiments, the fungus or fungal infection has developed resistance to one or more drugs. For example, a drug resistant fungal infection may have developed drug-resistance to an azole antifungal drug, a polyene antifungal drug and/or an echinocandin antifungal drug.
[0162] In some embodiments of any of the above methods or uses, the compound targets APL5, COS111,MKK1, and STE2 in the fungus. [0163] In some embodiments of any of the above methods or uses, the compound targets at least one of APL5, COS111,MKK1, or STE2 in the fungus.
[0164] In some embodiments of any of the above methods or uses, the compound disrupts vesicular transport mediate by APL5.
[0165] In some embodiments of any of the above methods or uses, the fungus carries non-mutated APL5, COS111,MKK1, and STE2.
[0166] In some embodiments of any of the above methods or uses, the fungus carries at least one of non- mutated APL5, COS111. MKK1, and STE2.
[0167] As used herein, a “symptom” associated with a fungal infection includes any clinical or laboratory manifestation associated with the fungal infection and is not limited to what the subject can feel or observe.
[0168] As used herein, “treating”, e.g. of a fungal infection, encompasses inducing prevention, inhibition, regression, or stasis of the disease or a symptom or condition associated with the infection.
[0169] As used herein, "modifying" or "modified" means a change in a subject, which may be an increase or decrease in amount, activity, rate of production, rate of inactivation, rate of breakdown, delay of onset, earlier onset, addition or removal of material, mutation, or any combination of these, so long as there is a reduced level or activity of starch synthase II.
[0170] Tire contents of U.S. Application No. 16/097,558, now patented as U.S. Patent No. 11,072,633, are hereby incorporated by reference.
[0171] The compounds of the present invention include all hydrates, solvates, and complexes of the compounds used by this invention. If a chiral center or another form of an isomeric center is present in a compound of the present invention, all forms of such isomer or isomers, including enantiomers and diastereomers, are intended to be covered herein. Compounds containing a chiral center may be used as a racemic mixture, an enantiomerically enriched mixture, or the racemic mixture may be separated using well-known techniques and an individual enantiomer may be used alone. Tire compounds described in the present invention are in racemic form or as individual enantiomers. Tire enantiomers can be separated using known techniques, such as those described in Pure and Applied Chemistry 69. 1469-1474, (1997) IUPAC. In cases in which compounds have unsaturated carbon-carbon double bonds, both the cis (Z) and trans (E) isomers are within the scope of this invention.
[0172] The compounds of the subject invention may have spontaneous tautomeric forms. In cases wherein compounds may exist in tautomeric forms, such as keto-enol tautomers, each tautomeric form is contemplated as being included within this invention whether existing in equilibrium or predominantly in one form.
[0173] In the compound structures depicted herein, hydrogen atoms are not shown for carbon atoms having less than four bonds to non-hydrogen atoms. However, it is understood that enough hydrogen atoms exist on said carbon atoms to satisfy the octet rule.
[0174] This invention also provides isotopic variants of the compounds disclosed herein, including wherein the isotopic atom is 2H and/or wherein the isotopic atom 13C. Accordingly, in the compounds provided herein hydrogen can be enriched in the deuterium isotope. It is to be understood that the invention encompasses all such isotopic fomrs.
[0175] It is understood that the structures described in the embodiments of the methods hereinabove can be the same as the structures of the compounds described hereinabove.
[0176] It is understood that where a numerical range is recited herein, the present invention contemplates each integer between, and including, the upper and lower limits, unless otherwise stated.
[0177] Except where otherwise specified, if the structure of a compound of this invention includes an asymmetric carbon atom, it is understood that the compound occurs as a racemate, racemic mixture, and isolated single enantiomer. All such isomeric forms of these compounds are expressly included in this invention. Except where otherwise specified, each stereogenic carbon may be of the R or S configuration. It is to be understood accordingly that the isomers arising from such asymmetry' (e.g., all enantiomers and diastereomers) are included within the scope of this invention, unless indicated otherwise. Such isomers can be obtained in substantially pure fonn by classical separation techniques and by stereochemically controlled synthesis, such as those described in "Enantiomers, Racemates and Resolutions" by J. Jacques, A. Collet and S. Wilen, Pub. John Wiley & Sons, NY, 1981. For example, the resolution may be carried out by preparative chromatography on a chiral column.
[0178] The subject invention is also intended to include all isotopes of atoms occurring on the compounds disclosed herein. Isotopes include those atoms having the same atomic number but different mass numbers. By way of general example and without limitation, isotopes of hydrogen include tritium and deuterium. Isotopes of carbon include C-13 and C-14.
[0179] It will be noted that any notation of a carbon in structures throughout this application, when used without further notation, are intended to represent all isotopes of carbon, such as 12C, 13C, or 14C. Furthermore, any compounds containing 13C or 14C may specifically have the structure of any of the compounds disclosed herein. [0180] It will also be noted that any notation of a hydrogen in structures throughout this application, when used without further notation, are intended to represent all isotopes of hydrogen, such as 1 H, 2H, or ?H.
[0181] Furthermore, any compounds containing 2H or 3H may specifically have the structure of any of the compounds disclosed herein.
[0182] Isotopically-labeled compounds can generally be prepared by conventional techniques known to those skilled in the art using appropriate isotopically-labeled reagents in place of the non-labeled reagents employed.
[0183] In the compounds used in the method of the present invention, the substituents may be substituted or unsubstituted, unless specifically defined otherwise.
[0184] In the compounds used in the method of the present invention, alkyl, heteroalkyl, monocycle, bicycle, aryl, hctcroaryl and heterocycle groups can be further substituted by replacing one or more hydrogen atoms with alternative non-hydrogen groups. These include, but are not limited to, halo, hydroxy, mercapto, amino, carboxy, cyano, carbamoyl and aminocarbonyl and aminothiocarbonyl.
[0185] It is understood that substituents and substitution patterns on the compounds used in the method of the present invention can be selected by one of ordinary skill in the art to provide compounds that are chemically stable and that can be readily synthesized by techniques known in the art from readily available starting materials. If a substituent is itself substituted with more than one group, it is understood that these multiple groups may be on the same carbon or on different carbons, so long as a stable structure result.
[0186] In choosing the compounds used in the method of the present invention, one of ordinary skill in the art will recognize that the various substituents, i.e. R1, R2, etc. are to be chosen in conformity with well- known principles of chemical structure connectivity.
[0187] As used herein, "alkyl" is intended to include both branched and straight-chain saturated aliphatic hydrocarbon groups having the specified number of carbon atoms. Thus, Ci-Cn as in “Ci-Cn alkyl" is defined to include groups having 1, , n-1 or n carbons in a linear or branched arrangement, and specifically includes methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, isopropyl, isobutyl, sec-butyl and so on. An embodiment can be C1-C12 alkyl, C2-C12 alkyl, C3-C12 alkyl, C4-C12 alkyl and so on. “Alkoxy" represents an alkyl group as described above attached through an oxygen bridge.
[0188] The term "alkenyl" refers to a non-aromatic hydrocarbon radical, straight or branched, containing at least 1 carbon to carbon double bond, and up to the maximum possible number of non-aromatic carbon- carbon double bonds may be present. Tirus, C2-C11 alkenyl is defined to include groups having 1, 2...., n-1 or n carbons. For example, "C2-C6 alkenyl" means an alkenyl radical having 2, 3, 4, 5, or 6 carbon atoms, and at least 1 carbon-carbon double bond, and up to, for example, 3 carbon-carbon double bonds in the case of a C6 alkenyl, respectively. Alkenyl groups include ethenyl, propenyl, butenyl and cyclohexenyl. As described above with respect to alkyl, the straight, branched or cyclic portion of the alkenyl group may contain double bonds and may be substituted if a substituted alkenyl group is indicated. An embodiment can be C2-C12 alkenyl, C3-C12 alkenyl, C4-C12 alkenyl and so on.
[0189] The term "alkynyl" refers to a hydrocarbon radical straight or branched, containing at least 1 carbon to carbon triple bond, and up to the maximum possible number of non-aromatic carbon-carbon triple bonds may be present. Thus, C2-Cn alkynyl is defined to include groups having 1, 2...., n-1 or n carbons. For example, "C2-C6 alkynyl" means an alkynyl radical having 2 or 3 carbon atoms, and 1 carbon-carbon triple bond, or having 4 or 5 carbon atoms, and up to 2 carbon-carbon triple bonds, or having 6 carbon atoms, and up to 3 carbon-carbon triple bonds. Alkynyl groups include ethynyl, propynyl and butynyl. As described above with respect to alkyl, the straight or branched portion of the alkynyl group may contain triple bonds and may be substituted if a substituted alkynyl group is indicated. An embodiment can be a C2-Cn alkynyl. An embodiment can be C2-C12 alkynyl, C3-C12 alkynyl, C4-C12 alkynyl and so on
[0190] "‘Alkylene”, “alkenylene” and “alkynylene” shall mean, respectively, a divalent alkane, alkene and alkyne radical, respectively. It is understood that an alkylene, alkenylene, and alkynylene may be straight or branched. An alkylene, alkenylene, and alkynylene may be unsubstituted or substituted.
[0191] As used herein, "heteroalkyl" includes both branched and straight-chain saturated aliphatic hydrocarbon groups having the specified number of carbon atoms and at least 1 heteroatom within the chain or branch.
[0192] As herein, "cycloalkyl" shall mean cyclic rings of alkanes of three to eight total carbon atoms, or any number within this range (i.e., cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl or cyclooctyl).
[0193] As used herein, "monocycle" includes any stable polyatomic carbon ring of up to 10 atoms and may be unsubstituted or substituted. Examples of such non-aromatic monocycle elements include but are not limited to: cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl. Examples of such aromatic monocycle elements include but are not limited to: phenyl.
[0194] As used herein, "bicycle" includes any stable polyatomic carbon ring of up to 10 atoms that is fused to a polyatomic carbon ring of up to 10 atoms with each ring being independently unsubstituted or substituted. Examples of such non-aromatic bicycle elements include but are not limited to: decahydronaphthalene. Examples of such aromatic bicycle elements include but are not limited to: naphthalene. [0195] As used herein, "aryl" is intended to mean any stable monocyclic, bicyclic or polycyclic carbon ring of up to 10 atoms in each ring, wherein at least one ring is aromatic, and may be unsubstituted or substituted. Examples of such aryl elements include phenyl, p-toluenyl (4-methylphenyl), naphthyl, tetrahydro-naphthyl, indanyl, biphenyl, phenanthryl, anthryl or acenaphthyl. In cases where the aryl substituent is bicyclic and one ring is non-aromatic, it is understood that attachment is via the aromatic ring.
[0196] As used herein, the term “polycyclic” refers to unsaturated or partially unsaturated multiple fused ring structures, which may be unsubstituted or substituted.
[0197] The term “arylalkyl” refers to alkyl groups as described above wherein one or more bonds to hydrogen contained therein are replaced by a bond to an aryl group as described above. It is understood that an '‘arylalkyl” group is connected to a core molecule through a bond from the alkyl group and that the aryl group acts as a substituent on the alkyl group. Examples of arylalkyl moieties include, but are not limited to, benzyl (phenylmethyl), p-trifluoromethylbenzyl (4-trifluoromethylphenylmethyl), 1 -phenylethyl, 2- phenylethyl, 3-phenylpropyl, 2-phenylpropyl and the like.
[0198] The term "heteroaryl", as used herein, represents a stable monocyclic, bicyclic or polycyclic ring of up to 10 atoms in each ring, wherein at least one ring is aromatic and contains from 1 to 4 hctcroatoms selected from the group consisting of O, N and S. Bicyclic aromatic heteroaryl groups include phenyl, pyridine, pyrimidine or pyridizine rings that are (a) fused to a 6-membered aromatic (unsaturated) heterocyclic ring having one nitrogen atom; (b) fused to a 5- or 6-membered aromatic (unsaturated) heterocyclic ring having two nitrogen atoms; (c) fused to a 5-membered aromatic (unsaturated) heterocyclic ring having one nitrogen atom together with either one oxygen or one sulfur atom; or (d) fused to a 5- membered aromatic (unsaturated) heterocyclic ring having one hctcroatom selected from O, N or S. Heteroaryl groups within the scope of this definition include but are not limited to: benzimidazolyl, benzofuranyl, benzofurazanyl. benzopyrazolyl, benzotriazolyl, benzothiophenyl, benzoxazolyl, carbazolyl, carbolinyl, quinolyl, fiiranyl, indolinyl, indolyl, indolazinyl, indazolyl, isobenzofiiranyl, isoindolyl, isoquinolyl, isothiazolyl, isoxazolyl, naphthpyridinyl, oxadiazolyl, oxazolyl, isoxazoline, pyranyl, pyrazinyl, pyrazolyl, pyridazinyl, pyridopyridinyl, pyridazinyl, pyridyl, pyrimidyl, pyrrolyl, quinazolinyl, quinolyl, quinoxalinyl, tetrazolyl, tetrazolopyridyl, thiadiazolyl, thiazolyl, thienyl, triazolyl, acridinyl, carbazolyl, quinoxalinyl, pyrrazolyl, indolyl, benzotriazolyl, benzothiazolyl, benzoxazolyl, isoxazolyl, isothiazolyl, furanyl, thienyl, benzothienyl, benzofuranyl. quinolinyl. isoquinolinyl, oxazolyl, isoxazolyl, indolyl, pyrazinyl, pyridazinyl, pyridinyl, pyrimidinyl, or pyrrolyl. In cases where the heteroaryl substituent is bicyclic and one ring is non-aromatic or contains no heteroatoms, it is understood that attachment is via the aromatic ring or via the heteroatom containing ring, respectively. If the heteroaryl contains nitrogen atoms, it is understood that the corresponding N-oxides thereof are also encompassed by this definition. [0199] The term “heteroarylalkyl” refers to alkyl groups as described above wherein one or more bonds to hydrogen contained therein are replaced by a bond to an heteroaryl group as described above. It is understood that an “heteroarylalkyl” group is connected to a core molecule through a bond from the alkyl group and that the heteroaryl group acts as a substituent on the alkyl group. Examples of heteroarylalkylmoieties include, but are not limited to, -CEE-CCsFEN). -CH2-CH2-(C5H4N) and tire like.
[0200] The temi "heterocycle" or “heterocyclyl” refers to a mono- or poly-cyclic ring system which can be saturated or contains one or more degrees of unsaturation and contains one or more heteroatoms. Preferred heteroatoms include N, 0. and/or S, including N-oxides, sulfur oxides, and dioxides. Preferably the ring is three to ten-membered and is either saturated or has one or more degrees of unsaturation. More preferably the ring is three to four-membered and has one or more degrees of unsaturation. The heterocycle may be unsubstituted or substituted, with multiple degrees of substitution being allowed. Such rings may be optionally fused to one or more of another "heterocyclic" ring(s), heteroaryl ring(s), aryl ring(s), or cycloalkyl ring(s). Examples of heterocycles include, but are not limited to, aziridine, azirine, diazirine, oxirane, thiirane, azetidine, oxetane, thetane, tetrahydrofuran, pyran, 1,4-dioxane, 1,3-dioxane, piperidine, piperazine, pyrrolidine, morpholine, thiomorpholine, tetrahydrothiopyran, tetrahydrothiophene, 1,3- oxathiolane, and the like.
[0201] The alkyl, alkenyl, alkynyl, aryl, hetcroaryl and heterocyclyl substituents may be substituted or unsubstituted, unless specifically defined otherwise. In the compounds of the present invention, alkyl, alkenyl, alkynyl, and, heterocyclyl and heteroary l groups can be further substituted by replacing one or more hydrogen atoms with alternative non-hydrogen groups. These include, but are not limited to, halo, hydroxy, mercapto, amino, carboxy, cyano and carbamoyl.
[0202] As used herein, the term “halogen” refers to F, Cl, Br, and I.
[0203] Tire tenns “substitution”, “substituted” and “substituent” refer to a functional group as described above in which one or more bonds to a hydrogen atom contained therein are replaced by a bond to non- hydrogen or non-carbon atoms, provided that normal valencies are maintained and that the substitution results in a stable compound. Substituted groups also include groups in which one or more bonds to a carbon(s) or hydrogen(s) atom are replaced by one or more bonds, including double or triple bonds, to a heteroatom. Examples of substituent groups include the functional groups described above, and halogens (i.e., F, Cl, Br, and I); alkyl groups, such as methyl, ethyl, n-propyl, isopropryl, n-butyl, tert-butyl, and trifluoromethyl; hydroxyl: alkoxy groups, such as methoxy, ethoxy, n-propoxy, and isopropoxy: aryloxy groups, such as phenoxy: arylalkyloxy, such as benzyloxy (phenylmethoxy) and p- trifluoromethylbenzyloxy (4-trifluoromethylphenylmethoxy); heteroaryloxy groups; sulfonyl groups, such as trifluoromethanesulfonyl, methanesulfonyl, and p-toluenesulfonyl; nitro, nitrosyl; mercapto; sulfanyl groups, such as methylsulfanyl, ethylsulfanyl and propylsulfanyl; cyano; amino groups, such as amino, methylamino, dimethylamino, ethylamino, and diethylamino; and carboxyl. Where multiple substituent moieties are disclosed or claimed, the substituted compound can be independently substituted by one or more of the disclosed or claimed substituent moieties, singly or pluraly. By independently substituted, it is meant that the (two or more) substituents can be the same or different.
[0204] It is understood that substituents and substitution patterns on the compounds of the instant invention can be selected by one of ordinary skill in the art to provide compounds that are chemically stable and that can be readily synthesized by techniques known in the art, as well as those methods set forth below, from readily available starting materials. If a substituent is itself substituted with more than one group, it is understood that these multiple groups may be on the same carbon or on different carbons, so long as a stable structure result.
[0205] In choosing the compounds of the present invention, one of ordinary skill in the art will recognize that the various substituents, i.e. R1, R2, etc. are to be chosen in conformity with well-known principles of chemical structure connectivity.
[0206] Tire various R groups attached to the aromatic rings of the compounds disclosed herein may be added to the rings by standard procedures, for example those set forth in Advanced Organic Chemistry: Part B: Reaction and Synthesis, Francis Carey and Richard Sundberg, (Springer) 5th ed. Edition. (2007), the content of which is hereby incorporated by reference.
[0207] The compounds used in the method of the present invention may be prepared by techniques well known in organic synthesis and familiar to a practitioner ordinarily skilled in the art. However, these may not be the only means by which to synthesize or obtain the desired compounds.
[0208] The compounds used in the method of the present invention may be prepared by techniques described in Vogel’s Textbook of Practical Organic Chemistry, A.I. Vogel, A.R. Tatchell, B.S. Fumis, A.J. Hannaford, P.W.G. Smith, (Prentice Hall) 5th Edition (1996), March's Advanced Organic Chemistry : Reactions, Mechanisms, and Structure, Michael B. Smith, Jerry March, (Wiley-Interscience) 5th Edition (2007), and references therein, which are incorporated by reference herein. However, these may not be the only means by which to synthesize or obtain the desired compounds.
[0209] Another aspect of the invention comprises a compound used in the method of the present invention as a pharmaceutical composition.
[0210] In some embodiments, a pharmaceutical composition comprising the compound of the present invention and a pharmaceutically acceptable carrier. [0211] As used herein, the term “pharmaceutically active agent” means any substance or compound suitable for administration to a subject and furnishes biological activity or other direct effect in the treatment, cure, mitigation, diagnosis, or prevention of disease, or affects the structure or any function of the subject. Phannaceutically active agents include, but are not limited to, substances and compounds described in the Physicians' Desk Reference (PDR Network, LLC; 64th edition; November 15, 2009) and “Approved Drug Products with Therapeutic Equivalence Evaluations” (U.S. Department Of Health And Human Services, 30th edition, 2010), which are hereby incorporated by reference. Pharmaceutically active agents which have pendant carboxylic acid groups may be modified in accordance with the present invention using standard esterification reactions and methods readily available and known to those having ordinary skill in the art of chemical synthesis. Where a phannaceutically active agent does not possess a carboxylic acid group, the ordinarily skilled artisan will be able to design and incorporate a carboxylic acid group into the pharmaceutically active agent where esterification may subsequently be carried out so long as the modification does not interfere with the pharmaceutically active agent’s biological activity or effect.
[0212] The compounds used in the method of the present invention may be in a salt form. As used herein, a “salt” is a salt of the instant compounds which has been modified by making acid or base salts of the compounds. In the case of compounds used to treat an infection or disease caused by a pathogen, the salt is pharmaceutically acceptable. Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as phenols. Hie salts can be made using an organic or inorganic acid. Such acid salts are chlorides, bromides, sulfates, nitrates, phosphates, sulfonates, formates, tartrates, maleates, malates, citrates, benzoates, salicylates, ascorbates, and the like. Phenolate salts are the alkaline earth metal salts, sodium, potassium or lithium. The term "pharmaceutically acceptable salt" in this respect, refers to the relatively non-toxic, inorganic and organic acid or base addition salts of compounds of the present invention. These salts can be prepared in situ during the final isolation and purification of the compounds of the invention, or by separately reacting a purified compound of tire invention in its free base or free acid form with a suitable organic or inorganic acid or base, and isolating the salt thus formed. Representative salts include the hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, napthylatc, mesylate, glucoheptonate, lactobionatc, and lauryl sulphonate salts and the like. (See, e.g., Berge el al. (1977) "Phannaceutical Salts", J Pharm. Sci. 66: 1-19).
[0213] Tire compounds of the present invention may also form salts with basic amino acids such a lysine, arginine, etc. and with basic sugars such as N-methylglucamine, 2-amino-2-deoxyglucose, etc. and any other physiologically non-toxic basic substance. [0214] As used herein, “administering” an agent may be performed using any of the various methods or delivery systems well known to those skilled in the art. Tire administering can be performed, for example, orally, parenterally, intraperitoneally, intravenously, intraarterially, transdermally, sublingually, intramuscularly, rectally, transbuccally, intranasally, liposomally, via inhalation, vaginally, intraoccularly, via local delivery, subcutaneously, intraadiposally, intraarticularly, intrathecally, into a cerebral ventricle, intraventicularly, intratumorally, into cerebral parenchyma or intraparenchchymally.
[0215] The compounds used in the method of the present invention may be administered in various fomrs, including those detailed herein. Tire treatment with the compound may be a component of a combination therapy or an adjunct therapy, i.e. the subject or patient in need of the drug is treated or given another dmg for the disease in conjunction with one or more of the instant compounds. This combination therapy can be sequential therapy where the patient is treated first with one drug and then the other or the two drugs are given simultaneously. These can be administered independently by the same route or by two or more different routes of administration depending on the dosage fonns employed.
[0216] As used herein, a "pharmaceutically acceptable carrier" is a pharmaceutically acceptable solvent, suspending agent or vehicle, for delivering the instant compounds to the animal or human. The carrier may be liquid or solid and is selected with the planned manner of administration in mind. Liposomes are also a pharmaceutically acceptable carrier as are slow-release vehicles.
[0217] The dosage of the compounds administered in treatment will vary depending upon factors such as the pharmacodynamic characteristics of a specific chemotherapeutic agent and its mode and route of administration; the age, sex, metabolic rate, absorptive efficiency, health and weight of the recipient; the nature and extent of the symptoms; the kind of concurrent treatment being administered; the frequency of treatment with; and the desired therapeutic effect.
[0218] A dosage unit of the compounds used in the method of the present invention may comprise a single compound or mixtures thereof with additional antitumor agents. Hie compounds can be administered in oral dosage forms as tablets, capsules, pills, powders, granules, elixirs, tinctures, suspensions, syrups, and emulsions. The compounds may also be administered in intravenous (bolus or infusion), intraperitoneal, subcutaneous, or intramuscular form, or introduced directly, e.g. by injection, topical application, or other methods, into or topically onto a site of disease or lesion, all using dosage fonns well known to those of ordinary skill in the pharmaceutical arts.
[0219] Tire compounds used in the method of the present invention can be administered in admixture with suitable phannaceutical diluents, extenders, excipients, or in carriers such as the novel programmable sustained-release multi-compartmental nanospheres (collectively referred to herein as a phannaceutically acceptable carrier) suitably selected with respect to the intended form of administration and as consistent with conventional pharmaceutical practices. The unit will be in a form suitable for oral, nasal, rectal, topical, intravenous or direct injection or parenteral administration. The compounds can be administered alone or mixed with a phannaceutically acceptable carrier. This carrier can be a solid or liquid, and the type of carrier is generally chosen based on the type of administration being used. Tire active agent can be co-administered in the fomr of a tablet or capsule, liposome, as an agglomerated powder or in a liquid form. Examples of suitable solid carriers include lactose, sucrose, gelatin and agar. Capsule or tablets can be easily formulated and can be made easy to swallow or chew; other solid forms include granules, and bulk powders. Tablets may contain suitable binders, lubricants, diluents, disintegrating agents, coloring agents, flavoring agents, flow-inducing agents, and melting agents. Examples of suitable liquid dosage forms include solutions or suspensions in water, phannaceutically acceptable fats and oils, alcohols or other organic solvents, including esters, emulsions, symps or elixirs, suspensions, solutions and/or suspensions reconstituted from non-effervescent granules and effervescent preparations reconstituted from effervescent granules. Such liquid dosage forms may contain, for example, suitable solvents, preservatives, emulsifying agents, suspending agents, diluents, sweeteners, thickeners, and melting agents. Oral dosage forms optionally contain flavorants and coloring agents. Parenteral and intravenous forms may also include minerals and other materials to make them compatible with the type of injection or delivery system chosen.
[0220] Techniques and compositions for making dosage forms useful in the present invention are described in the following references: 7 Modem Pharmaceutics, Chapters 9 and 10 (Banker & Rhodes, Editors, 1979); Pharmaceutical Dosage Forms: Tablets (Lieberman et al., 1981); Ansel, Introduction to Pharmaceutical Dosage Forms 2nd Edition ( 1976); Remington's Pharmaceutical Sciences, 17th ed. (Mack Publishing Company, Easton, Pa., 1985); Advances in Pharmaceutical Sciences (David Ganderton, Trevor Jones, Eds., 1992); Advances in Pharmaceutical Sciences Vol. 7. (David Ganderton, Trevor Jones, James McGinity, Eds., 1995); Aqueous Polymeric Coatings for Phannaceutical Dosage Forms (Drugs and the Pharmaceutical Sciences, Series 36 (James McGinity, Ed., 1989); Phannaceutical Particulate Carriers: Therapeutic Applications: Drugs and the Pharmaceutical Sciences, Vol 61 (Alain Rolland, Ed., 1993); Drug Delivery to the Gastrointestinal Tract (Ellis Horwood Books in the Biological Sciences. Series in Pharmaceutical Technology; J. G. Hardy, S. S. Davis, Clive G. Wilson, Eds.); Modem Pharmaceutics Drugs and the Pharmaceutical Sciences, Vol 40 (Gilbert S. Banker, Christopher T. Rhodes, Eds.). All of the aforementioned publications are incorporated by reference herein.
[0221] Tablets may contain suitable binders, lubricants, disintegrating agents, coloring agents, flavoring agents, flow-inducing agents, and melting agents. For instance, for oral administration in the dosage unit form of a tablet or capsule, the active drug component can be combined with an oral, non-toxic, pharmaceutically acceptable, inert carrier such as lactose, gelatin, agar, starch, sucrose, glucose, methyl cellulose, magnesium stearate, dicalcium phosphate, calcium sulfate, mannitol, sorbitol and the like. Suitable binders include starch, gelatin, natural sugars such as glucose or beta-lactose, com 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 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.
[0222] The compounds used in the method of the present invention may 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 lecithin, sphingomyelin, proteolipids, protein-encapsulated vesicles or from cholesterol, stearylamine, or phosphatidylcholines. Hie compounds may be administered as components of tissue-targeted emulsions.
[0223] The compounds used in the method of the present invention may also be coupled to soluble polymers as targetable drug carriers or as a prodrug. Such polymers include polyvinylpyrrolidone, pyran copolymer, polyhydroxylpropylmethacrylamide-phenol, polyhydroxyethylasparta-midephenol, or polyethyleneoxide-polylysine substituted with palmitoyl residues. Furthermore, the compounds may be coupled to a class of biodegradable polymers useful in achieving controlled release of a drug, for example, polylactic acid, polyglycolic acid, copolymers of polylactic and polyglycolic acid, polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydropyrans, polycyanoacylates, and crosslinked or amphipathic block copolymers of hydrogels.
[0224] Gelatin capsules may contain the active ingredient compounds and powdered carriers, such as lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, and the like. Similar diluents can be used to make compressed tablets. Both tablets and capsules can be manufactured as immediate release products or as sustained release products to provide for continuous release of medication over a period of hours. Compressed tablets can be sugar-coated or fdm-coated to mask any unpleasant taste and protect the tablet from the atmosphere, or enteric coated for selective disintegration in the gastrointestinal tract.
[0225] For oral administration in liquid dosage form, the oral drug components are combined with any oral, non-toxic, pharmaceutically acceptable inert carrier such as ethanol, glycerol, water, and tire like. Examples of suitable liquid dosage forms include solutions or suspensions in water, phannaceutically acceptable fats and oils, alcohols or other organic solvents, including esters, emulsions, syrups or elixirs, suspensions, solutions and/or suspensions reconstituted from non-effervescent granules and effervescent preparations reconstituted from effervescent granules. Such liquid dosage forms may contain, for example, suitable solvents, preservatives, emulsifying agents, suspending agents, diluents, sweeteners, thickeners, and melting agents.
[0226] Liquid dosage forms for oral administration can contain coloring and flavoring to increase patient acceptance. In general, water, asuitable oil, saline, aqueous dextrose (glucose), and related sugar solutions and glycols such as propylene glycol or polyethylene glycols are suitable carriers for parenteral solutions. Solutions for parenteral administration preferably contain a water-soluble salt of the active ingredient, suitable stabilizing agents, and if necessary, buffer substances. Antioxidizing agents such as sodium bisulfite, sodium sulfite, or ascorbic acid, either alone or combined, are suitable stabilizing agents. Also used are citric acid and its salts and sodium EDTA. In addition, parenteral solutions can contain preservatives, such as benzalkonium chloride, methyl- or propyl-paraben, and chlorobactene. Suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences, Mack Publishing Company, a standard reference text in this field.
[0227] Tire compounds used in the method of the present invention may also be administered in intranasal form via use of suitable intranasal vehicles, or via transdermal routes, using those forms of transdermal skin patches well known to those of ordinary skill in that art. To be administered in the form of a transdermal delivery system, the dosage administration will generally be continuous rather than intermittent throughout the dosage regimen.
[0228] Parenteral and intravenous forms may also include minerals and other materials such as solutol and/or ethanol to make them compatible with the type of injection or delivery system chosen.
[0229] The compounds and compositions of the present invention can be administered in oral dosage forms as tablets, capsules, pills, powders, granules, elixirs, tinctures, suspensions, syrups, and emulsions. The compounds may also be administered in intravenous (bolus or infusion), intraperitoneal, subcutaneous, or intramuscular form, or introduced directly, e.g. by topical administration, injection or other methods, to the afflicted area, such as a wound, including ulcers of the skin, all using dosage fonns well known to those of ordinary skill in the pharmaceutical arts.
[0230] Specific examples of pharmaceutically acceptable carriers and excipients that may be used to formulate oral dosage forms of the present invention are described in U.S. Pat. No. 3,903.297 to Robert, issued Sept. 2, 1975. Techniques and compositions for making dosage forms useful in the present invention are described-in the following references: 7 Modem Pharmaceutics, Chapters 9 and 10 (Banker & Rhodes, Editors, 1979); Pharmaceutical Dosage Forms: Tablets (Lieberman et al., 1981); Ansel, Introduction to Pharmaceutical Dosage Forms 2nd Edition (1976); Remington's Pharmaceutical Sciences, 17th ed. (Mack Publishing Company, Easton, Pa., 1985); Advances in Pharmaceutical Sciences (David Ganderton, Trevor Jones, Eds., 1992); Advances in Pharmaceutical Sciences Vol 7. (David Ganderton, Trevor Jones, James McGinity, Eds., 1995); Aqueous Polymeric Coatings for Pharmaceutical Dosage Forms (Drugs and the Pharmaceutical Sciences, Series 36 (James McGinity, Ed., 1989); Pharmaceutical Particulate Carriers: Therapeutic Applications: Drugs and the Pharmaceutical Sciences, Vol 61 (Alain Rolland, Ed., 1993); Drug Delivery to the Gastrointestinal Tract (Ellis Horwood Books in the Biological Sciences. Series in Pharmaceutical Technology; J. G. Hardy, S. S. Davis, Clive G. Wilson. Eds.); Modem Pharmaceutics Drugs and the Pharmaceutical Sciences, Vol 40 (Gilbert S. Banker, Christopher T. Rhodes, Eds.). All of the aforementioned publications are incorporated by reference herein.
[0231] The active ingredient can be administered orally in solid dosage forms, such as capsules, tablets, powders, and chewing gum; or in liquid dosage forms, such as elixirs, syrups, and suspensions, including, but not limited to, mouthwash and toothpaste. It can also be administered parentally, in sterile liquid dosage fonns.
[0232] Solid dosage forms, such as capsules and tablets, may be enteric-coated to prevent release of the active ingredient compounds before they reach the small intestine. Materials that may be used as enteric coatings include, but are not limited to, sugars, fatty acids, proteinaceous substances such as gelatin, waxes, shellac, cellulose acetate phthalate (CAP), methyl acrylate-methacrylic acid copolymers, cellulose acetate succinate, hydroxy propyl methyl cellulose phthalate, hydroxy propyl methyl cellulose acetate succinate (hypromellose acetate succinate), polyvinyl acetate phthalate (PVAP), and methyl methacrylate - methacrylic acid copolymers.
[0233] Tire compounds and compositions of the invention can be coated onto stents for temporary or permanent implantation into the cardiovascular system of a subject.
[0234] Variations on those general synthetic methods will be readily apparent to those of ordinary skill in the art and are deemed to be within the scope of the present invention.
[0235] Each embodiment disclosed herein is contemplated as being applicable to each of the other disclosed embodiments. Thus, all combinations of the various elements described herein are within the scope of the invention.
[0236] This invention will be better understood by reference to the Experimental Details which follow, but those skilled in the art will readily appreciate that the specific experiments detailed are only illustrative of the invention as described more fully in the claims which follow thereafter. EXAMPLES
[0237] The Identification of The First Fungal Sterylglucosidase.
[0238] While working on the characterization of the gene involved in the catabolism of the sphingolipids, CNAG 05607 was discovered as the gene homolog to Cn EGCrPl, which is the glucosylceramidase active at neutral and alkaline pH (shibashi Y et al. 2012). CNAG 05607 was initially thought to be a second glucosylceramidase; but the biochemical analysis suggested that CNAG 05607 was a sterylglucosidase. In fact. CNAG_05607 from either Cryptococcus neoformans (Cn) (Rella A, et al., 2015). and its homologs from Aspergillus fumigatus (Af) (Pereira de Sa N, et al. 2020), metabolize sterylglucosides (SGs) and not glucosylceramide (GlcCer), as claimed improperly by an early publication which used as a substrate a different, short chain, non-physiological GlcCer (Watanabe T, et al. 2015). The biochemically studies were confirmed genetically because deletion of Sgl 1 in Cn does not cause any change in the level of endogenous GlcCer but rather a dramatic accumulation of SGs, as measured by thin layer chromatography (TLC) (Rella A. et al.. 2015). and confirmed by both gas chromatography mass spectrometry and by liquid chromatography mass spectrometry (Rella A, et al., 2015). This was confirmed recently using the crystal structures, showing that GlcCer does not fit the active site of Sgll/SglA (Pereira de Sa N, et al., 2021 and 2022). Thus, CNAG_05607 was renamed sterylglucosidase 1 (Cn Sgll), the first sterylglucosidase ever isolated from any living organism. (Rella A, et al., 2015).
[0239] Cn Sgll andA/Sgla are Required for Pathogenicity.
[0240] Virulence studies showed that upon inhalation of Cn Δsgll, 100% of mice were still alive after 3 months of observation (Fig. 1A) (Rella A, et al., 2015). Tissue burden studies showed that the Cn Δsgll is promptly eliminated from the lung between 9 and 14 days after inhalation (Rella A, et al., 2015). More importantly. Δsgll never gains access to the brain, where the WT causes a lethal meningo-encephalitis instead. Similarly, virulence studies performed with the Af ΔsglA showed that the mutant is not virulent, and 100% mice were alive and healthy after 30 days of observation (Fig. IB) (Rella A, et al., 2015). At 30 days post-infection, tissue burden analysis showed that the Af ΔsglA mutant is not found in the lung or in any other organs (data not shown), suggesting that clearance of Af ' ΔsglA also occurred early after the inhalation of conidia (Fernandes CM, et al. 2022). During the study, the animals receiving Δsgll or Δsgla appeared healthy, showed normal physical activity with no weight loss.
[0241] Cn Δsgll and Af ΔsglA mutants have growth defect in host physiological conditions.
[0242] The initial characterization of Cn Δsgll revealed that the mutant does not have an altered growth at alkaline or acidic pH, or a defect in melanin production or in capsular size (Rella A, et al., 2015). Also, there were no difference among wild-type (WT), Δsgll or Δsgll + SGL1 reconstituted strain when cells were grown intracellularly (within macrophages) or when they were exposed to hydrogen peroxide or nitrosative stress (data not shown) (Rella A, et al . , 2015) . No difference in urease activity or in the secreted phospholipase Bl activity (data not shown) was found. However, when the Cn Δsgll mutant is incubated in physiological media, such as Minimal Media (MM), Dulbecco’s Modified Eagle Medium (DMEM) or Yeast Nitrogen Base (YNB) containing physiological low concentration of glucose (0.1%) and low oxygen (—5-10%) it cannot grow, and, eventually, it dies (Fig. 2). For ΔsglA, the growing defect is more pronounced, and this mutant cannot elongate its hyphae when exposed to MM, containing 0.5% (or less) glucose (Fig. 3). These results suggest that accumulation of SGs is not tolerated by either Cn or Af cells when exposed to in vitro conditions mimicking physiological host environments. Thus, it was reasoned to obtain the same benefits by targeting Sgll or SglA pharmacologically.
[0243] Screening of a DiverSet-CL ChemBridge library and identification of specific Sgll/SglA inhibitors and solving the crystal structure of both Cn Sgll and Af SglA.
[0244] The Cn Sgll and 4/' SglA proteins in E. coli were cloned and overexpressed for two purposes: a) to produce large quantity of recombinant protein for a high-throughput screening (HTS) assay and b) to produce crystal structure of the enzymes.
[0245] A) To set up the HTS assay, the Anorogenic glucosidase substrate resorufin p-D-glucopyranoside was used. This substrate is ideal because it has been successfiilly implemented in HTS assays for other glucosidases (Urban DJ, et al. 2008). Initial parameters were established to ensure linearity vs. time and [enzyme] . KM and Vinax were determined to ensure we screen below the KM for competitive inhibitors. Other critical factors were assessed, including enzyme stability upon long-tenn storage (Pereira de Sa N, et al. 2021). Lastly, the Z-score was calculated to assess the robustness of our assay and found to be 0.94, which is an outstanding score.
[0246] A DiverSet-CL ChemBridge library comprising 50,000 synthetic compounds in 96-well format was screened (Pereira de Sa N, et al. 2021). three compounds to inhibit specifically Sgll enzyme with a IC50 of IpM or less were found: Hit 1, Hit 9 and Hit 15. Hit 1, 4-(hydroxymethyl)-l-[2-(3-methoxy- phenyl)-l,3-thiazol-5-yl]methylpiperidin-4-ol (ChemBridge ID&59928901); Hit 9, N-[(3R,5S)-5- (hydroxym ethyl)- 1 -mcthylpyrrolidin-3 -yl] -2-(3 -oxo-3,4-dihydro-2H- 1 ,4-bcnzoxazin-6-yl)acctamidc (Chembridge ID#23645796); and Hit 15 (2S,4R)-4-[(6-isopropyl-l-methyl-lH-pyrazolo[3,4-d]pyrimidin- 4-yl)amino]-l -methyl -2 -pyrrolidinyl methanol (ChemBridge ID#86711567). Against SglA, two compounds to inhibit specifically this enzyme with a IC50 of IpM or less were also found: Hit b and Hit c: Hit b, N-[(2,3-dimethyl-lH-indol-5-yl)methyl]pyrimidine-4,6-diamine (ChemBridge ID&88182154) and Hit c, 2-5 -[2-( 1 ,3-benzodioxol-5-yl)- l-methylethyl]-4-phenyl- IH-imidazol- 1 -ylethanol (ChemBridge ID#44349962) (Pereira de Sa N, et al. 2022). [0247] Treating Cn WT or Aj WT cells with Hit 1 or Hit b inhibitors recapitulate the in vitro phenotypes observed with the respective mutants under physiological growing conditions (Fig. 2C and Fig. 3C), indicating that these compounds have potent antifungal activity in vitro when fungal cells are exposed to host environments (e.g. low oxygen or/and low glucose).
[0248] Tire ChemBridge database for compounds having similar scaffold w as analyzed and 10 compounds similar to Hit 1, 9 or 15, and 7 compounds similar to Hit b were found. Tire ten compounds similar to Hit 1, 9 or 15 were tested but none inhibited Sgll (or SglA) at a concentration of 100 pM or less (data not shown). Tirus, DOCK6 was used to initiate a computer-aided drug design to predict derivatives for Hit 1, 9 and 15 (inhibitors of Sgll) (see below). The 7 compounds similar to Hit b were also tested and B7 was 10-fold more potent than Hit b, whereas Bl was almost ineffective in inhibiting SglA activity (Fig. 4). Structural-activity relationship (SAR) studies were initiated based on these results.
[0249] Hit 1 was studied to see whether it would have antifungal activity in the animal model. This was possible even without comprehensive pharmacokinetics (PK) data because these compounds have favorable “druggable” properties. For instance, based on its biophysical properties and on pkCSM software (Pires DE, Blundell TL, et al. 2015), predicted PK and toxicity properties of Hit 1 were highly favorable. Hit 1 was tested in the cryptococcal animal model, whereas Hit b in the Aspergillus animal model. For the cryptococcal model, mice were infected intranasally and 6 hours, later Hit 1 was administered intraperitoneally every day, alone or in combination with fluconazole for 14 days, and lung and brain tissue burden was examined. Tire results were preliminary but Hit 1 totally blocked the dissemination of Cn WT cells to the brain (Fig. 5). a phenotype also observed with the Δsgll mutant (Rella A, et al. 2015). Hit 1 also synergized with fluconazole in decreasing the lung fungal burden (Fig. 5). It is a promising result considering that Hit 1 increases SGs in Cn WT cells by only 15-20% compared to the increase generated by the deletion of Sgl 1 (Cn Δsgll) (Pereira de Sa N et al. 2021).
[0250] Treatment of WT Af cells with Hit b increases SGs by ~50% compared to the increase generated by the deletion of SglA (AfΔsgla) (data not shown) (Pereira de Sa N, et al. 2023). Thus, for Hit b, both survival and tissue burden studies were performed using a mouse model of aspergillosis . Mice were first immunosuppressed with a subcutaneous administration of triamcinolone (40 mg/kg) one day prior to the injection. Next day, mice received an intranasal administration of 5x104 conidia ofd/'WT 293 strain. One hour later, they received an intraperitoneal injection of Hit b at 5 mg/kg/day or 20 mg/kg/day and mice survival was monitored. A 50% survival in the group receiving 20 mg/kg/day (Fig. 6) was observed. Importantly, the 5 mice treated with Hit b that survived for 30 days did not show any lung fungal burden, suggesting that in these mice Hit b treatment eradicated the lung infection (Fig. 6). The lung fungal burden was assessed by qPCR determination of the concentration of Af 18 S rDNA compared to the concentration of mouse lung GAPDH at the time of death for 5 mice untreated, and at day 30 for 5 mice treated with 20 mg/Kg/day of Hit b.
[0251] B7, which is more potent than Hit b in inhibiting SglA (Fig. 4) is also more efficacious in the animal model and treatment with only 5 mg/Kg/day initiated 1 hour after the infection is sufficient to obtain a 50% survival (data not shown). B7 is also efficacious in improving mice survival and lung infectivity when treatment is started 24 hours after the infection, when all conidia already germinated in hyphae. As illustrated in Fig. 7, treatment with 5 mg/kg/twice day of B7 produced a 50% survival (Fig. 7A), and the surviving mice at day 30 do not have any fungal burden in their lung (Fig. 7B). This suggests that inhibitors of SglA are effective not only in preventing conidia from germinating in vitro (Fig 6) but are also effective in blocking hyphal growth in the lung (Fig. 7). These results are very promising because this animal model of aspergillosis is very susceptible to /(/'(all untreated animals are dead by 8 days) and a lower dose of B7 is not efficacious. This suggests there is a dose dependent effect of B7.
[0252] Cn Sgll structure at 1.9 A resolution (Fig. 8) and d/SglA structure at 2.0 A resolution were solved (Pereira de Sa N, et al. 2022). A co-crystal structure analysis revealed that Hit 1 binds in the active site of Sgll (Fig. 8) thus preventing the binding of SGs to Sgll, and structural analysis showed that Hit 9 binds even more strongly to Sgll than Hit 1 or Hit 15 (Fig. 9) (Pereira de Sa N, et al. 2021). In fact, Cn WT cells treated with Hit 9 accumulate more SGs than those treated with Hit 1 or Hit 15 (Pereira de Sa N, et al. 2021). Thus, Hit 9 was selected as the parent structure for generating Hit 9 derivatives against Cn Sgll, such as SS-103 (see below). Similarly, Hit b (Fig. 9) or Hit c binds in the active site of SglA, thus preventing the binding of SGs to SglA. These structural studies have been published (Pereira de Sa N, et al. 202 l and 2022).
[0253] Noteworthy, the Cn Sgll and the Af SglA crystal structures show that the active pocket of the enzyme interacts with SGs but not with the natural/physiological fungal GlcCer, because the long hydrophobic tail of fungal GlcCer (C18-C9methyl GlcCer) does not fit in the active site. The short chain non-physiological GlcCer (C6-GlcCer), used in the studies by Watanabe et al. does fit the active site of Sgll(Pereira de Sa N, et al. 2021). Thus, the biochemical, genetic and now7 structural data conclusively demonstrated that Sgll/SglA is a sterylglucosidase only, explain the controversial data published by other authors, and emphasize that biochemical enzymatic characteristics about substrate specificity should be attributed only upon testing natural/physiological and not artificial/non-physiological substrates (Watanabe T ef al., 2015). [0254] Summary of the preliminary data
[0255] A new class of antifiingals targeting a fungal specific enzyme(s) was discovered that is required for pathogenicity (Table 1) (Pereira de Sa N, et al. 2021 and 2022). The crystal structures ofboth Cn Sgll and A/SglA were solved, conclusively clarified their substrate specificity and initiated a computer-based drug design coupled with a medicinal chemistry effort. Importantly, the Hit compounds have antifungal activity in vitro and in vivo and, in this invention, many compounds were identified.
[0256] Structure-based computer-aided drug design (CADD), synthesis and biochemical studies for hit-to-lead and lead optimization of novel Sgll/SglA inhibitors.
[0257] In the preliminary studies, Cn Sgl 1 and d/'Sgl A were biochemically characterized, and their crystal structure were solved (Fig. 8 and 9), and a high throughput screening (HTS) assay for assessing fungal Sgl activity was developed, the screening of a ChemBridge DiverSet library was completed and putative inhibitor(s) of Sgll and SglA (Table 1) were identified and Hit compounds having antifungal activity in vitro and in vivo were found.
[0258] Table 1. Top Hit Compounds against Sgll and SglA
Table 1. Top Hit compounds Sgll SglA LD50 cLogP against Sgll and SglA ic50 IC50 (pM) (pM) (pM) 1.0 8.0 655 1.49
0.5 8.0 585 0.58 1.0 8.0 512 0.91 4.0 1.0 350 1.90
8.0 2.0 1,461 3.14 [0259] Molecular docking analysis of Hit compounds for Sgll (Hit 1, 9 and 15), as well as ca. 200 newly designed analogs using AutoDock and DOCK6 programs was performed. These structural studies also confirmed that Hit 9 is the best Sgl 1 inhibitor among tire three Hit compounds in hand for further computer- aided drug design (CADD). In fact, Hit 9 caused a higher accumulation of SGs in Cn cells compared to Hit 1 or Hit 15 (Pereira de Sa N, et al. 2021).
[0260] Fig. 10 shows proof-of-concept refinement examples, starting from Hit 9 (pdb code 7LPQ), generated using a powerful new isosteric swapping protocol.48 Briefly, the ability to use a reference ligand fragment and generate an aligned group of "related fragments" (isosteres) which can then be "swapped" during computational refinement has implemented into DOCK6 (Pereira de Sa N, et al. 2021). In the present example, the benzoxazinone sidechain on Hit 9 (Fig. 10) was used and searched, 124 geometrically aligned sidechains culled from 13M drug-like compounds for which the top 500 fragments based on Hungarian overlap + Volume overlap + Tanimoto overlap were retained (Brzankalski GE, et al. and Allen WJ, Balius TE, et al. 2015). As shown in Fig. 10, these 500 sidechains were sampled using de novo DOCK50 which yielded 182 compatible analogs. Importantly, the designed analogs in a number of cases had better scores than the cognate ligand. Further, they showed a wide range of logP and logS scores which provide flexibility when prioritizing analogs for potential synthesis. From a structural perspective, a number of analogs showed the potential for additional H-bonding at position Gly 455, and in one case Glu 499 (Fig. 10), in addition to binding at Glu 587, Lys 47 and Glu 270 (Fig. 10).
[0261] Furthennore, the surface binding analysis of Hit 9 in the binding pocket revealed that there are two rather spacy regions to explore (Fig 11 A, red circles). This strategy led to the design of novel branched Hit 9 analogs, many of which gave substantially enhanced docking scores (Fig. 11B). Among ca. 80 Y-shaped branched analogs designed and docked, SS-103 was selected for synthesis and biochemical evaluations (Fig. 11). SS-103 was synthesized from commercially available starting materials in 5 steps uneventfully and fully characterized, which showed slightly lower enzyme inhibitory activity than Hit 9 using ergosterol 3[3-D-glucoside as the substrate (Fig. 11C). However, SS-103 exhibited much better efficacy than Hit 9 in the SG accumulation assay in Cn WT cells (compare Fig. 11D and HE), i.e.. Maximal accumulation occurred at the highest concentration for both compounds, but SS-103 led to a significantly higher accumulation of ergosterol 3p-D-glucoside than Hit 9, which is closer to the mutant lacking Sgll (Cn dsgiiy
[0262] Although preliminary, these results provided a highly encouraging proof for the validity of CADD, featuring branched Hit 9 analogs for hit-to-lcad and lead optimization. SS-103 was selected first, because of its high docking score, as well as its symmetrical and rather simple structure for chemical synthesis. SS- 103 congeners and other Hit 9 analogs with excellent docking scores are in the process of being synthesized (Fig. 12). It was found that the replacement of the amide linkage to the corresponding aminomethyl linkage gave even higher docking scores in many cases. For the construction of branched Hit 9 analogs, the 1,3,5- triazine framework provides a versatile platform for CADD and some analogs scored very high. Several representative Hit 9 analogs in this series out of ca. 500 compounds, designed and docked, are shown in Fig. 12. It is likely that several highly efficacious Sgll inhibitor lead compounds will be identified through this library of compounds, which have already been designed.
[0263] Since Hit 9 analogs were the analogs of focus, the (3/?.5.S)-l -mcthyl-3-aminoprolinol moiety was kept as a critical fragment, which secures putative canonical interactions with Lys 47, Glu 270 and Glu 587 through H-bonding. However, this moiety would be replaceable with other groups bearing H-bonding donor and acceptor, such as 4-dihydroxymethyl-piperidinyl group in Hit 1 (and diaminopyrimidinyl group in Hit b) (Table 1). The LD5o for Hit compounds against A549 cell line are very favorable (see Table 1), predicting low toxicity to mammalian cells.
[0264] A CADD strategy was used, similar to the one for the hit-to-lead optimization of Sgll inhibitors, described above. An excellent binding site model through the overlay of SglA with Sgll -Hit 9 high resolution crystal structures was produced. Computational structure analysis of SglA and Sgll revealed that the surface area and volume of the binding site of SglA is substantially smaller than those of Sgll, z.e., surface area: SglA 693 A2 vs. Sgl l 1,392 A2; volume: SglA 555 A3 vs. Sgl 1 967 A3. These differences in the size of the binding site and mutations in amino acid residues are likely to be reflected in the preferred structures of SglA-selective inhibitors.
[0265] Tire overlay of the inhibitor-bound structures of SglA with Hit b, Hit c (7? and S isomers) and Hit 9 is shown in Fig. 14. As it is very clear from the overlay, Hit b shares the “canonical'’ binding site with Hit 9, while Hit c (R/S) occupies a binding site in a deep pocket. Tirus, Hit b is one of the good hits. An analog of Hit b. B7. was found to be a more potent SglA inhibitor than Hit b, which was predicted by the molecular docking analysis, as shown in Fig. 15. The docking analysis also predicted that the introduction of triazine (B7-001) instead of pyrimidine (B7) substantially increases the binding score. The replacement of indole with carbazole increased the docking score (B7-013), and that of pyrimidine/triazine with 3- amino-N-methylprolinol was predicted to be we 11 -tolerated (KJ-046). Then, the introduction of Y-shaped skeleton with indol-5-yl groups scored high (KJ-072).
[0266] Structure of Sgll and SglA and how they bind to SGs and their inhibitors has been recently proposed and (Fig. 8) (Pereira de Sa N, et al. 2021 and 2022). The structure revealed a two domains architecture: a catalytic domain and a [3-sandwich domain. The active site is enclosed in a pocket. The glucose of the substrate ergosterol-3-0-D-glucoside (SGs) interacts with Lys 47 and Trp 570 whereas the sterol component makes hydrophobic interaction with Glu 270 and Leu 431 (Fig 8). Mutations of key residues involved in the organization of the active site of Sgll/SglA will be essential to conclusively demonstrate the direct involvement of these residues in binding to SGs or/and to the inhibitor(s). Trp 570, Lys 47, Glu 270 and Leu 431 will be focused on, which interact with SGs. Glu 270 and Trp 570 residues in Sgl 1 also interact with Hit 1, whereas Glu 587, Lys 47 and Glu 270 interact with Hit 9. Asp 127 and Glu 247 of SglA interact with Hit b. Mutated clones are directly ordered from Bio Basic Inc., sequenced to make sure each clone displays only the desired mutation, expressed and used in the biochemical assay and for crystallography studies comparing the in vitro activity and the 3D structure of the active site of Sgll/SglA mutated forms with the WT Sgll/SglA.
[0267] In the preliminary' studies, the treatment of WT cells with Sgll and SglA inhibitors was found to recapitulate the respective mutant phenotypes of Cn Δsgll and Af Δsgla, respectively (Fig. 2 and Fig. 3) (Pereira de Sa N, et al. 2021). Hit 1 exerts an antifungal activity in the animal model of cryptococcosis (Fig. 5), and Hit b exerts antifungal activity against aspergillosis in cell culture (Fig. 3) and in the animal model (Fig. 6 and 7) (Pereira de Sa N. et al. 2021).
[0268] As Fig. 16 shows, the compounds which are efficacious against Cn Sgll and Af SglA are also efficacious against Candida albicans (Ca) Sgll (Fig. 16). This was expected because the catalytic domain of Ca Sgll is highly similar to Cn Sgll and Af SglA. It was also expected that these compounds will be active against other fungi, as Sgll is highly conserved in the fungal kingdom. The lack of Sgll in mammalian cells should minimize toxicity. A blast analysis identified a Sgll homolog in Ca with an e- value of 4.5xlO 150. The deletion mutant (Ca Δsgll) does indeed accumulate SGs in Ca cells (Fig. 16A), and some inhibitors of Cn Sgll or 4/ Sgl A also inhibit Ca Sgll (Fig. 16B).
[0269] Synthesis of SglA inhibitors
[0270] Experimental procedure for the synthesis of A2-(2-ethyl-3-methyl-lL7-indol-5-ylmethyl)-Ad,A6- dimethyl-l,3,5-triazine-2,4,6-triamine (DR-SglA-1)
[0272] To a screw cap vial, 2, 4-diaminomethyl-6-chloro- 1,3, 5 -triazine (50 mg, 0.288 mmol, 1.0 eqv.), 3- ethyl-2-methyl-5-methylaminoindole (67 mg, 0.356 mmol, 1 .2 equiv.), 0.58 M A''. A'-dimcthylacctamidc (0.5 mL), and diisopropylethylamine (0.432mmol, 1.5eqv.) were added. The reaction mixture was heated at 100 °C and stirred for 24 hours. Tire reaction mixture was taken up with ethyl acetate and water. Tire aqueous layer was extracted with ethyl acetate (30 mL x 3), and then the organic layer was washed with brine (30 mL x 3), dried over magnesium sulfate, filtered, and concentrated in vacuo. The residue was purified by flash column chromatography on silica gel with 100% ethyl acetate as eluent to afford the title compound (58.6 mg, 0.180 mmol, 63% yield) as an off-white solid: mp. 169-170°C; 1H NMR (700 MHz, CD3OD) 5 7.38 (s, 1H). 7.19 (d, J= 8.2 Hz, 1H), 7.03 (s, 1H), 4.61 (s, 2H). 3.45 - 3.27 (m, 3H), 2.85 (d, J= 33.4 Hz, 5H). 2.75 (q, J= 7.6 Hz. 2H), 2.20 (s, 3H). 1.28 (t. J= 7.6 Hz, 3H); 13C NMR (176 MHz, CDCI3) 5 166.83, 166.01, 137.03, 134.45, 129.73, 129.57, 121.22, 117.40, 110.17, 106.22, 45.51, 27.58, 19.43, 13.99, 8.38; HRMS (ESI-TOF) m/z calcd. for C17H24N7 + [M+H]+ 326.2088, found 326.2081 (A = 2.03 ppm). HPLC purity: 98.5% at 254 nm.
[0273] In a similar manner, DR-SglA-2 and DR-SglA-3 were synthesized and characterized.
[0274] N2-(3-Ethyl-2-methyl-lH-indol-5-ylmethyl)-N4,N6-dimethyl-1,3,5-triazine-2,4,6-triamine (DR- SglA-2):
[0276] White solid; 72.6% yield; mp. 189-190 °C; 1H NMR (700 MHz, CD3OD) 5 7.42 (s, 1H), 7.18 (d, J = 8.2 Hz, 1H), 7.02 (s, 1H), 4.61 (s, 2H), 2.88 (s, 6H), 2.70 (q, J = 7.6 Hz, 2H), 2.35 (s, 3H), E20 (t, J = 7.6 Hz, 3H); 13C NMR (176 MHz, CDCI3) 5 166.69, 165.82, 134.62, 130.68, 129.58, 128.60. 121.12, 117.44, 113.97, 110.17, 45.59, 27.57. 17.32, 15.46. 11.57; HRMS (ESI-TOF) m z calcd. for CI7H24N7 + [M+H]+ 326.2088, found 326.2083 (A = 1.43 ppm). HPLC purity: 99.9% at 254 nm.
[0277] A2-(2-Ethyl-3-methyl-lH-indol-5-ylmethyl)-6-methyl-l,3,5-triazine-2,4-diamine (DR-SglA-3):
[0279] White solid; 68% yield; mp. 108-109 °C. 1H NMR (700 MHz, CD3OD) 5 7.35 (s, 1H), 7.20 (d, J= 8.2 Hz, 1H), 7.00 (d, J= 8.0 Hz, 1H), 4.62 (s, 2H), 2.75 (q, J= 7.6 Hz, 2H), 2.20 (s, 5H), 1.27 (t, J= 7.6 Hz, 3H); 13C NMR (176 MHz, CD3OD) 5 174.59, 166.70, 165.21, 137.40, 135.02. 129.35, 128.23, 119.80, 116.16, 109.85, 104.40, 44.49, 23.08, 18.83, 13.15, 7.01; HRMS (ESI-TOF) m/z calcd. for CRH^ INL [M+H]+ 297.1822, found 297.1816 (A = 2.2 ppm). HPLC purity: 95.5% at 254 nm.
[0280] Enzymatic assay
[0281] A 50-pL volume of wild-type Af SglA containing 10 ng of protein (0. 11 pmol) and 50 pL of mixed micelles of lipid and Triton X-100 in 50 mM sodium acetate buffer (pH 5.5) with 150 mM NaCl, 5 mM |3ME, and 5 mM DTT were mixed and incubated at 37°C for 20 min (ErgGlc; Avanti Polar Lipids). After the incubation period, each reaction was quenched with a 2: 1 chloroform -methanol solution, and the organic phase was collected and dried. Then, the lipid content was resuspended in 50 mL methanol and analyzed by HPLC using Agilent 1260 Infinity II (Agilent Technologies). Total ErgGlc and ergosterol were detected at 282 nm on a Cx column with a flow rate of 0.5 mL/min in methanol-water (90: 10) buffered with 1 mM ammonium formate and 0.2% formic acid.
[0282] Ergosterol 3-P-D-glucoside accumulation in Aspergillus fumigatus
[0283] Wild-type Af AKu80pyrGla and the mutant Ssgla strain were cultivated in yeast extract-glucose medium (YAG) (2% [wt/vol] glucose, 0.5% [wt/vol] yeast extract, l x trace elements, l x amino acid solution, 2% [wt/vol] bacteriological agar) for 48 h at 37°C. High-nitrate salts, trace elements, and amino acid solutions were prepared. The medium was supplemented with 1.2 g/L of uracil and uridine (UU), generating YAG+UU medium when tire strain AKu80pyrGla was used. After that, the conidial suspension in water was obtained, and a pellet with 1 x 107 conidia was treated in minimal medium broth pH 6.5 (MM or MM+UU) ( 1 % [wt/vol] glucose, 1 x high-nitrate salts, 1 x trace elements) for 48 h at 37°C under agitation, with the hits selected, according to the toxicity criteria, at various concentrations. A previously described MIC assay was performed in accordance with the guidelines in the CLSI document M38-A2, to determine the MIC and select a concentration range that does not affect mold growth. After that, the resultant hyphal pellet was used for lipid extraction. Then, the total lipid was extracted, and dried samples were resuspended in 2: 1 chloroform-methanol for liquid chromatography-mass spectrometry (LC-MS) analysis. A standard ErgGlc from Avanti Polar Lipids was used as a control for the calibration curve. Data were normalized to the total inorganic phosphate content in the sample.
[0284] Computer-aided drug design (CADD) was used to explore novel structures with better SglA enzyme inhibitory activities than Hit 9, B7 and three B7 analogs bearing the triazine moiety. The CADD using the Dock 6.9 program correctly predicted the enzyme inhibitory activities of these three compounds as shown in Table 2 (entries 1-3). Table 1 summarizes novel compounds bearing l-methyl-4-aminoprolinol moiety, which exhibit substantially better docking scores than so far, the best compound, DR-SglA-2, indicating superior enzyme inhibitory activities. [0285] Table 2. Docking scores for DR-SglA-l~3 and novel compounds designed by Dock 6.9 program.
[0286] Examples for Sgll inhibitors
[0287] Synthesis of 2,2-Bis(dibenzo[b,d]furan-2-yl)-2V-(37?,55)-5-(hydroxylmethyl)-l- methylpyrrolidin-3-yl)acetamide (SS-103)
[0288] The title compound (SS-103) was synthesized from commercially available 2 -bromodibenzofuran and dibenzofuran-2-carboxaldehyde in 4 steps as shown below.
[0289] Step 1. Preparation of bis(dibenzo[b,d]furan-2-yl)methanol.
[0291] To a 50 mL oven dried three-necked flask with a reflux condenser, charged with magnesium turnings (1.4 g, 10 equiv.), was added THF (1.0 mL) and iodine (1 scoop of spatula). To this suspension 2- bromodibenzofuran (2.0 g, 7.8 mmol, 1.36 equiv.) in THF (3 mL) was added dropwise at reflux with stirring and reacted for 40 min to generate the corresponding Grignard reagent. Then, the solution was cooled to 0 °C and dibenzofuran-2-carboxaldehyde (1 g. 5.7 mmol, 1 equiv.) was added dropwise. The reaction mixture was allowed to warm to room temperature and left for 3 hrs with stirring. Then, methanol (3.0 mL) was added to quench the reaction. Saturated aqueous NH4C1 solution was added slowly, followed by ethyl acetate (25 mL), and the layers were separated. The aqueous layer was extracted with ethyl acetate and the combined organic extracts were washed with water and brine. The resulting solution was dried over MgSO4. filtered and concentrated to give a crude product, which was purified by flash column chromatography on silica gel using hexanes/ethyl acetate as eluent to give the title compound as a white solid (1.82 g, 4.99 mmol, 88 % yield): H NMR (500 MHz, DMSO-d6) 5 8.24 (s, 1 H), 8.15 (d, J = 7.65 Hz, 2 H), 7.68 (d, J= 8.2 Hz, 2 H), 7.64 (d, J= 8.55 Hz, 2 H), 7.57 (dd, J=1.4 Hz, J =8.55 Hz, 2 H), 7.51 (t, J= 7.3 Hz, 2 H), 7.39 (t, J =1.5 Hz, 2 H), 6.20 (d, J= 1.88 Hz, 1 H), 6.09 (d, J= 1.83 Hz, 1H); 13C NMR (500 MHz, DMSO-iL) 8 156.28, 154.94, 141.62, 121.98, 126.69, 124.09, 123.79, 123.52, 121.61, 119.10, 112.12, 111.69, 74.67: FIA-MS (ESI) m/z: ealed. for C25H16O3 364.11, found 364.9.
[0292] Step 2. Preparation of 2,2-bis(dibenzo[b,d]furan-2-yl)acetonitrile [0293]
[0294] A 100 mL round-bottomed flask was charged with bis(dibenzo[b,d]furan-2-yl)methanol (1.465 g, 4.01 mmol, 1 equiv.) in dichloromethane (30 mL), LizCCL (59 mg, 0.804 mmol, 0.2 equiv.), trimethylsilyl cyanide (2.26 ml, 18.086 mmol, 4.5 equiv.), and L (2.04 g, 8.038 mmol, 2 equiv.) successively. Then, the reaction mixture was stirred at 35 °C overnight. The reaction was quenched with saturated NazSzCL solution. The organic layer was separated, and the aqueous layer was extracted with dichloromethane. Tire combined organic layers were dried over MgSOr and concentrated under reduced pressure. The resulting residue was purified by recrystallization to give the title compound as a pale-yellow solid (1.458 g, 97 %): 1H NMR (400 MHz, DMSO-ds) 5 8.30 (s, 2H), 8.20 (d, J= 3.82 Hz, 2H), 7.77 (d, J= 4.28 Hz, 2H), 7.71 (d, J= 4.12 Hz, 2H); l3C NMR (400 MHz, DMSO-d6) 5 156.46, 155.33, 132.55, 128.59, 127.63, 124.76, 123.79, 123.55, 121.97, 121.29, 120.73, 112.89, 112.27; FIA-MS (ESI) m/z calcd for C26H15O2 373.11, found 373.1.
[0295] Step 3. Preparation of 2.2-bis(dibcnzo|6.r/|furan-2-yl)acctic acid
[0297] To 2,2-bis(dibenzo[b,d]furan-2-yl)acetonitrile (1.26 g, 3.36 mmol, 1 equiv.), a solution of 1,4- dioxane, concentrated acetic acid , concentrated sulfuric acid and water (4.5: 4.5: 1: 1) was added and refluxed at 130 °C for 2 days. After cooling the reaction mixture to room temperature, solid NaOH was added in small portions to adjust the pH of the reaction mixture to ~4. The organic layer was separated and the aqueous layer was extracted with dichloromethane. Tire combined organic layers were dried over MgSCL, and concentrated under reduced pressure to give the title compound as a pale brown solid (1.16 g, 88% yield): 'HNMR (400 MHz, DMSO-r/fi 5 12.92 (s, 1H), 8.19 (s, 2H), 8.14 (d, J= 3.8 Hz, 2H), 7.70 (s, 1H), 7.68 (d, J= 1.38 Hz, 2H), 7.67 (s, 1H), 7.56 (d, J= 4.1 Hz, 2H), 7.52 (t, J= 7.68 Hz, 2H), 7.39 (t, J= 7.56 Hz, 2H); 1?C NMR (400 MHz, DMSO<4) 5 174.34, 156.29, 154.89, 135.55, 128.73, 128.14, 124.11, 123.90, 123.59, 121.68, 121.47, 112.15, 112.01, 56.53; FIA-MS (ESI) m'z calcd. for C26H16O4 392.10, found 392.4. [0298] Step 4. Synthesis of 2.2-Bis(dibcnzo|/rc/|furan-2-yl)-A'-((3/?.5.S)-5-(hydroxylmcthyl)- l - methylpyrrolidin-3-yl)acetamide (SS-103)
[0300] 2,2-Bis(dibenzo[Z?,J|furan-2-yl)acetic acid (100 mg, 0.26 mmol) in A'A'-dimethylformamidc (2.0 mL) was stirred for 10 min. N, JV-diisopropylethylamine (0.132 mL, 0.76 mmol, 3 equiv.) was added dropwise with stirring, followed by the addition of l-[bis(dimethylamino)methylene]-lH-l,2,3- triazolo[4,5-6]pyridinium 3-oxidehexafluorophosphate, hexafluorophosphate azabenzotriazole tetramethyl uronium (144 mg, 0.38 mmol, 1.5 equiv.) in N. A'-dimcthylformamidc (2.0 mL) dropwise for 15 min. Lastly, (2S,4A)-4-amino-l-methylpyrrolidin-2-ylmethanol (44 mg, 0.332 mmol, 1 equiv.) in N,N- dimethylformamide (1.0 mL) was added to the solution and stirred overnight at room temperature. The progress of the reaction was monitored by TLC. Tire reaction mixture was diluted with dichloromethane and a saturated Nal ICO; solution was added. Tire organic layer was separated, and the aqueous layer was extracted with dichloromethane (5 mL x 3). Tire combined organic layers were dried over MgSCL and concentrated under reduced pressure to give a crude product, which was purified by flash column chromatography on silica gel (10 % CFLCL/McOH) to give the title compound as a white solid (41 mg, 32 % yield): mp. 195-198 °C; ’H NMR (700 MHz, DMSO-ds ) 5 1.76 - 1.66 (m, 1H), 1.97 - 1.87 (m, 1H), 2.09 (t, 1H), 2.28 (s, 3H), 2.42 (s, 1H), 3.30 - 3.22 (m, 2H), 3.44 - 3.39 (m, 1H), 4.24 - 4.12 (m, 1H), 4.47 (s, 1H), 5.31 (s, 1H), 7.38 (t. J = 7.5 Hz, 2H), 7.56 - 7.49 (m, 4H), 7.68 (t, J = 8.3 Hz, 4H), 8.15 - 8.08 (m, 4H). 8.58 (d, J = 7.0 Hz. 1H); 13C NMR (700 MHz, DMSO-de) 5 14.09. 20.77, 35.23, 40.80, 45.68, 46.97, 55.92. 59.76, 62.49. 63.09, 65.69, 111.41. 111.67. 120.82. 121.17. 123.10. 123.47. 123.49, 127.62, 128.18, 135.79, 135.81, 154.37, 155.80, 170.35, 171.1 1; HRMS (TOF) m/z calcd for C32H28N2O4+: 505.21218, found: 505.21427 (A = -4.13 ppm). HPLC Purity: 99% at 254 nm, 99% at 210 nm.
[0301] Synthesis of (2A,47?)-4-((2,2-bis(dibenzo[Z>,J]furan-2-ylethyl)amino)-l-methyl-2- hydroxymethylpyrrolidine (SS-103A)
[0302] The title compound (SS-103 A) was synthesized from 2,2-bis(dibenzo[A<7|furan-2-yl)acetic acid (see Step 3 shown above) in 3 steps as shown below.
[0303] Step 1. Preparation of 2.2-bis(dibcnzo|A.r/|furan-2-yl)cthan-l -ol from 2,2-bis(dibenzo[Z7,</]furan-2- yl)acetic acid
[0305] To a solution of 2,2-bis(dibenzo[b,d]furan-2-yl)acetic acid (1.06 g. 2.7 mmol, 1 equiv.) in tetrahydrofuran (10 mL), was added borane-tetrahydrofuran (15.9 mL, 16.2 mmol, 6 equiv.) at 0 °C and the resulting mixture was allowed to warm to room temperature with stirring for 6 hrs. The completion of the reaction was monitored by TLC and a saturated NaCl solution and ethyl acetate (30 mL) were added to the reaction mixture. The organic layer was separated, and the aqueous layer was extracted with ethyl acetate. The combined organic layers were dried over MgSCL. fdtered and concentrated under reduced pressure. The resulting residue was purified by column chromatography on silica gel (hexane s/ethyl acetate) to give 2,2-bis(dibenzo[b,d]furan-2-yl)ethan-l-ol as a white solid (811 mg, 2.14, 79% yield): NMR (400 MHz, DMSO-d6) δ 12.87 (s, 1H), 8.16 (s, 2H), 8.13 (d, J = 3.72 Hz, 2H), 7.66 (d, J= 4.1 Hz, 2H), 7.61 (d, J = 4.26, 2H), 7.49 (m, 4H), 7.38 (t, 7.52 Hz, 2H), 4.95 (t, J= 5.16 Hz, 1H), 4.48 (t, J= 136 Hz, 1H), 4.198
(dd, J = 1.76 Hz, J = 2.68 Hz, 2H); 13C NMR (400 MHz, DMSO-d6) δ 156.22, 154.58, 138.82, 128.49, 127.91, 124.10, 123.99, 123.45, 121.57, 120.89. 112.09. 111.81.
[0306] Step 2. Preparation of 2,2-bis(dibenzo[b,d]furan-2-yl)acetaldehyde using Dess-Martin periodinane
[0308] To a solution of 2,2-bis(diben l (800 mg, 2.133 mmol, 1 equiv.) in dichloromethane (10 mL), 1,1,1-tris(acetoxy)-l,l-dihydro-l,2-benziodoxol-3-(177)-one (1.08 g, 2.56 mmol, 1.2 equiv.) was added at 0 °C and the resulting mixture was allowed to warm to room temperature and stirred overnight. The completion of the reaction was monitored by TLC. To the reaction mixture, saturated of NaHCO3 solution was added and the organic layer was separated. The aqueous layer was extracted with dichloromethane and the combined organic layers were dried over magnesium sulfate, filtered and concentrated under reduced pressure. The resulting crude product was purified by flask column chromatography on silica gel (hexane s/ethyl acetate) to give the title compound (212 mg, 0.56 mmol, 27% yield) as a pale white solid: Tl NMR (400 MHz, DMSO-d6) δ 10.186 (s, 1H), 8.24 (d, 2H), 8.17 (d, 2H), 7.72 (t, 4H), 7.55 (t, 2H), 7.39 (t, 2H), 7.11 (s, 1H); 13C NMR (400 MHz, DMSO-d6) δ 200.61. 156.36, 155.32, 136.98, 128.29, 127.47, 124.01. 123.89. 123.71. 121.81, 120.32, 112.19, 112.02, 83.12. [0309] Step 3. Synthesis of (2S,47?)-4-((2,2-bis(dibenzo[b,d]furan-2-yl)ethylainino)-l-methyl-2- hydroxymethylpyrrolidinc (SS-103A)
[0311] To a solution of 2.2-bis(dibcnzo|Az/|furan-2-yl [acetaldehyde (110 mg, 0.29 mmol, lequiv.) in N,N- dimethylacetamide (5.0 mL), (2.S'.4/?)-4-amino- l-methyl-2-hydroxymethylpyrrolidine (48 mg, 0.35 mmol, 1.2 equiv.) and acetic acid (0.03 mL, 0.35 mmol, 1.2 equiv) were added at room temperature and the mixture was allowed to stir for 3 hrs. To the reaction mixture, sodium cyanoborohydride (22 mg, 0.35 mmol, 1.2 equiv.) was added and the reaction mixture was stirred at room temperature overnight. The completion of the reaction was monitored by TLC. The reaction mixture was distributed in saturated NaCl solution and ethyl acetate. The organic layer was separated, and the aqueous layer was extracted with ethyl acetate. The combined organic layers were dried over magnesium sulfate, fdtered and concentrated under reduced pressure. Tire resulting crude product was purified by flask column chromatography on silica gel to give the title compound (47mg. 0.089 mmol, 31% yield) as an ivory colored solid: 1H NMR (700 MHz, DMSO- de) 5 8.34 (t. J= 2.03 Hz, 2H). 8.15 (t. J= 6.7 Hz, 2H), 7.67 (d, J= 4.1 Hz, 2H), 7.63 (d. J= 2.66 Hz , 2H), 7.60 (m, 2H), 7.50 (t, J = 7.49 Hz, 2H), 7.39 (t, J= 7.49 Hz, 2H), 5.88 (s, 1H), 4.92 (s, 1H), 3.76 (t, J = 5.18 Hz, 1H), 3.47 (m, 3H), 2.95 (s, 1H), 2.89 (t, J= 3.71 Hz, 1H), 2.78 (s, 1H), 2.75 (d, J= 6.72 Hz, 3H), 1.95 (s, 1H); 13C NMR (700 MHz, DMSO-tL) 8 156.52, 154.61, 128.18, 127.92, 126.65, 124.25, 123.51, 121.61, 121.57, 118.91, 112.11, 111.34, 79.63, 77.26, 72.41, 69.42. 60.24, 58.13, 57.96, 56.40, 54.91, 51.48, 40.45, 37.91, 34.91, 21.87. HRMS (ESI-TOF) wz calcd. for C32H30N2O3 + 491.2329, found 491.2347 (A = -3.56 ppm). HPLC Purity: 97 % at 254 nm, 96 % at 210 nm.
[0312] Enzymatic assay
[0313] A 50 pL volume of Cn Sgll wild type and 50 pL of mixed micelles of lipid and Triton X-100 in 50 mM Bis-Tris buffer with 150 mM NaCl, 5 mM [3ME, and 5 mM DTT were mixed and incubated at 37 °C for 20 min (erg-glc, Avanti Polar Lipids). The amount of protein was 5 ng. After the incubation period, each reaction was quenched with a 2: 1 chlorofomrmethanol solution and the organic phase was collected and dried. Then, the lipid content was resuspended in 50 pL methanol and applied to HPLC (Agilent Technologies). Total erg-glc and ergosterol was detected using absorbance at 282 nm on a C-8 column with a flow rate of 0.5 ml/min in methanol/water 90: 10 ratio buffered with 1 mM ammonium formate and 0.2% formic acid. The total area of product was normalized by the total area of its respective substrate.
[0314] Ergosteryl 3 P-D -glucoside accumulation in Cryptococcus neoformcins
[0315] C. neoformans H99 was cultivated in yeast nitrogen base (YNB) broth for 24 h at 37 °C with shaking. A pellet with 5 x 108 cells was treated during 24 h with all the hits selected, according to the toxicity criteria, in various concentrations. A previous minimal inhibitory concentration assay was performed in accordance with the guidelines in the CLSI document M27-A3, to determine the minimal inhibitory concentration and selecting a concentration range that did not affect the yeast growth. After that, the resultant pellets were re-counted and used for lipid extraction. Thenceforth the total lipid was extracted and dried samples were resuspended in chloroform/methanol 2: 1 ratio for LC-MS analysis. A standard erg- glc from Avanti Polar Lipids was used as a control for the calibration curve. Data were normalized to the total inorganic phosphate content in the sample.
[0316] Table 3. Docking scores for SS-103, SS-103A and novel compounds designed by Dock 6.9 program.
DISCUSSION
[0317] Cryptococcus neoformans (Cn) is an environmental fungal pathogen that, upon entering the lung and disseminating through the bloodstream, causes a life-threatening meningo-encephalitis in susceptible patients, particularly HIV+ subjects, leading to high morbidity and mortality. Current antifungals such as azoles, flucytosine, amphotericin B and echinocandins have limitations: amphotericin B and flucytosine are toxic; flucytosine is not available everywhere; echinocandins have a narrow spectrum of activity and not active against cryptococcosis; and azoles have limited use due to drug-interaction and resistance (Farowski F et al. 2012 and 2013, Odabasi Z et al. 2007, Saribas Z et al. 2012, Yanni SB et al. 2012 and Mukheijee PK et al. 2011) .
[0318] Aspergillus fumigatus (Af) is also a saprotrophic fungus ubiquitously found in the environment that, upon inhalation, causes both acute and chronic illnesses in at-risk individuals Tekaia F, Latge J-P. 2005). It is estimated that humans inhale hundreds of Af conidia every day that readily reach tire alveolar spaces due to their relatively small size (2-3 pm), indicating a constant daily battle at the host-pathogen interface in the upper respiratory tract and lower airways (Wassano NS et al. 2020 and Latge J-P. 1999). Healthy individuals exposed to Af mount an appropriate immune response resulting in tire pulmonary clearance of the fungus (Becker KL, et al. 2015). However, individuals with compromised immunity fail to control the inhaled conidia, which germinate to hyphae upon entering lung parenchyma, causing invasive disease. In addition, injury to the respiratory tract, for example caused by the SARS-CoV-2 virus (Livermore JM. 2021). does stimulate fungal invasion and the emergence of COVID-19-associated pulmonary aspergillosis (Chong WH et al. 2021 and Egger M et al. 2022). The incidence of invasive aspergillosis, due to Af has increased three-fold in the last decade (Marr Ka et al. 2022), and its mortality has risen by over 300% (McNeil MM et al. 2021). The Infectious Diseases Society of America listed d / as one of six pathogens for which a substantive treatment breakthrough is urgently needed (Talbot GH, et al. 2006).
[0319] Tire classic polyene antifungal, amphotericin B dcoxycholatc, was discovered over 50 years ago, and the triazole antifungals were approved in the early 1990s. Second-generation triazoles, voriconazole and posaconazole. expanded the antifungal spectrum but were only pharmacologic moiety modifications and suffer many of the same drawbacks and resistance pitfalls as their predecessors. The echinocandin antifungals, which act on the fungal cell wall, were developed in the mid-1990s and approved for use in the early 2000s (Steinbach WJ et al. 2003). Thus, the growing numbers of patients with invasive fungal infections has greatly outpaced antifungal development, at least in part due to pharmaceutical companies shifting away from this field. Additionally, antifungal resistance is increasing and hampering effective treatment (Cowen LE, Steinbach WJ. 2008). As a result, there is a greater need for bold and innovative approaches to discovering broad new molecular antifungal targets and their inhibitors. [0320] To meet this need, this invention proposes a new class of antifungals targeting the sterylglucosidase 1, Sgll, (and its homolog sterylglucosidase A, SglA), an enzyme present in fungi but not in human cells. This invention describes the promising Hits and the crystal structures of both Sgll and SglA alone and with its specific inhibitor (Pereira de Sa N, et al. 2021 and 2022).
[0321] Tire process described in tire present invention matches a rational drug design focused on the discovery of the Hit compound(s) and created a second-generation library for the identification of a lead compound(s) more potent than the Hit compound(s). This process will be iterative, requiring information on activity and mechanism of action to maximize efficacy, fungal target specificity, and lack of mammalian toxicity.
[0322] The methods used in this invention to test the efficacy of candidate drugs against fungal infections are well established and are used extensively (Rhome R, et al. 201 1, Shea JM et al. 2006 and Kechichian TB, et al. 2007). The present invention is innovative conceptually and technologically.
[0323] The compounds described in the present invention target the fungal sterylglucosidase s and will provide new in vitro and in vivo insights regarding the therapeutic efficacy of such compounds against invasive fungal infections.
[0324] The compound described in the present invention are also believed to have broad antifungal activity because Sgll is present in many yeasts, molds and dimorphic fungi (Grille S, Zaslawski A et al. 2010 and Normile TG et al. 2020).
[0325] By introducing a totally new class of antifungals, these antifungals can be combined with existing compounds as a combination therapy, with the goal of decreasing the development of resistance while maintaining efficacy. In fact, the compounds described in the present invention are synergistic when combined with fluconazole or voriconazole (Pereira de Sa N, et al. 2021 and 2022).
[0326] To generate effective and safe antifungals, the present invention targeted pathways present in fungal and not mammalian cells (Sgll and its homologs are not present in mammalian cells).
[0327] The present invention discovered the intriguing association of SGs with the host immune response. In fact, the Cn Δsgll and AfΔsgla are potent stimulators of the host immunity through the adjuvant action of SGs on y/ 5 T cells (Normile TG ct al. 2022). Thus, both Cn Δsgll and Af Δsgla arc exciting vaccine candidates because they are highly effective in preventing a secondary infection either as live-attenuated or as heat-killed (Normile TG et al. 2020 and 2022 and Rella A, et al. 2015). Hence, tire present invention envisions that a drug targeting Sgll/SglA, and thus increasing SGs, would stimulate a protective immunity which will help in the clearance of the primary infection and potentially in preventing the recurrence of a secondary infection. This type of treatment could be ideal in patients waiting for transplants (susceptible to aspergillosis) and in patients affected with HIV (susceptible to cryptococcosis), because these fungal vaccines are effective in condition of neutropenia (a condition that favors aspergillosis) or in condition of CD4+ T cell deficiency (a condition that favors cryptococcosis). This invention will open the avenue to a totally new field of basic research and clinical investigation.
[0328] Structure-based computer-aided drug design (CADD) software was used in the present invention to provide synthesis and biochemical studies for hit-to-lead and lead optimization of novel Sgll/SglA inhibitors. Employ CADD design of ca. 2,000 compounds, followed by in silico screening of pharmacological properties and synthetic feasibility assessment to generate ca. 100-compound library for chemical synthesis, based on the co-crystal structures of Sgll/SglA inhibitors determined in these laboratories. Thus, the following were performed: A) Identification of lead Sgll-specific inhibitors; B) Identification of lead SglA inhibitors; and C) Structural and in vitro biochemical inhibition studies (MOA) of the new derivatives. This initial screening will also confirm the mechanism of action (MOA). study resistance, and assess solubility to identify lead compounds.
[0329] PK, toxicology and antifungal activity will be studied. With tire compounds synthesized in the present invention, the present invention will: A) Perform preliminary pharmacokinetics (PK). preliminary toxicology studies; B) Test the derivatives for antifungal activity' in vitro and in vivo (animals); and C) Assess the host immune response during anti-Sgl 1 /SglA treatment. The present invention will iterate library based on observed Structure-Activity Relationship (SAR) in vitro and in the animals.
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Claims

1. A method of inhibiting growth of a fungus in a subject, comprising reducing the activity of sterylghicosidase 1 (Sigil) and/or sterylglucosidase A (SglA) in tire fungus.
2. The method of claim 1, wherein reducing the activity of Sigil and/or SglA comprises:
(a) inhibiting the synthesis of Sigil and/or SglA:
(b) reducing the level of Sigil and/or SglA; and/or
(c) blocking the active site of Sigil and/or SglA.
3. The method of any one of claims 1-2, comprises contacting the fungus with an effective amount of a compound having the structure: wherein R1 and R? are independently H, alkyl, alkenyl, alkynyl carbonyl, amine; -CH-, aryl, hctcroaryl, cycloalkyl, hctcrocycloalkyl, C(O)-alkyl, C(O)-haloalkyl, C(O)-cycloalkyl, C(O)- heterocycloalkyl, C(O)-aryl, C(O)-heteroaryl, CO2-alkyl, CO2-aryl, CO2-heteroaryl, or CO2- heterocycloalkyl; preferably, R1 and R2 are independently H. alkyl, alkenyl, alkynyl carbonyl, or amine; and/or a compound having the structure: wherein R17, R18, R19 are each independently CH, N, or S; wherein R20is NH2, NH-alkyl, NH-C(O)-alkyl, NH-C(S)-alkyl, NH-NHC(O)-alkyl, NH-NHC(S)- alkyl, alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, C(O)-alkyl, C(O)-haloalkyl, C(O)- cycloalkyl, C(O)-heterocycloalkyl, C(O)-aryl, C(O)-heteroaryl, CO2-alkyl, CO2-aryl, CO2- heteroaryl, CO2-heterocycloalkyl, alkyl-N-, SCL-alkyl, SCL-haloalkyl, SCf-cycloalkyl. SO2- heterocycloalkyl, SCL-aryl, or SO2-heteroaryl; preferably, R20 is NH-alkyl. NH-C(O)-alkyl, NH- C(S)-alkyl, NH-NHC(O)-alkyl, NH-NHC(S)-alkyl, alkyl, alkenyl, alkynyl, C(O)-alkyL C(O)- haloalkyl, CO2-alkyl, alkyl-NH2, SO2-alkyl, or SO2-haloalkyl; more preferably, R20 is NH-alkyl, NH-C(O)-alkyl, NH-C(S)-alkyl, NH-NHC(O)-alkyl, NH-NHC(S)-alkyl, alkyl, alkenyl, alkynyl, C(O)-alkyl, or C(O)-haloalkyl; more preferably, R20 is NH-alkyl, NH-C(O)-alkyl, NH-C(S)-alkyl, NH-NHC(O)-alkyl, NH-NHC(S)-alkyl, alkyl, C(O)-alkyl, or C(O)-haloalkyl; more preferably, R20 is NH-alkyl or alkyl; wherein R21 is aryl, heteroaryl, cycloalkyL heterocycloalkyl, C(O)-heterocycloalkyl, C(O)-aryl, C(O)-heteroaryl, CO2-aryl, CO2-hcteroaryl. CO2-heterocycloalkyl, SO2-cycloalkyl, SO2- heterocycloalkyl, SO2-aryl, or SO2-heteroaryl; preferably, R21 is aryl, heteroaryl, C(O)-aryl, C(O)- hctcroaryl, CO2-aryl, CO2-hctcroaryl, SO2-aryl. or SO2-hctcroaryl; more preferably, R21 is heteroaryl; wherein R22 is H, alkyl or -NH-alkyl; and wherein when R22 is H, R20 is NH2; or a pharmaceutically acceptable salt thereof. A compound having the structure: wherein R1 and R2 are independently H, alkyl, alkenyl, alkynyl carbonyl, amine; -CH-, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, C(O)-alkyl, C(O)-haloalkyl, C(O)-cycloalkyl, C(O)- heterocycloalkyl, C(O)-aryl, C(O)-heteroaryl, CO2-alkyl, CO2-aryl, CO2-heteroaryl, or CO2- heterocycloalkyl; preferably, R1 and R2 are independently H. alkyl, alkenyl, alkynyl carbonyl, or amine; or a pharmaceutically acceptable salt thereof. The compound of claim 4 having the following structure: The compound of any one of claims 4-5 having the following structure: wherein R; is 0, NH, CH2, or S; p more preferably, R3 is 0, or NH; wherein R4 is -CH-, -N-, -NH-N-, -NH-C(O)-, -NH-C(S)-, -NH-NHC(O)-. -NH-NHC(S)-, alkyl, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, C(O)-alkyl, C(O)-haloalkyl, C(O)-cycloalkyl. C(O)- heterocycloalkyl, C(O)-aryl, C(O)-heteroaryl, CO2-alkyl, CO2-aryl, CCb-hctcroaryl. CO2- heterocycloalkyl, alkyl-N-, SO2-alkyl. SO2-haloalkyl, SO2-cycloalkyl. SO2-heterocycloalkyl, SO2- aryl, or SO2-heteroaryl; and wherein R5 and R, are each independently H, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, CO2- aryl, CO2-hcteroaryl. CO2-cycloalkyl, or CO2-hctcrocycloalkyl. The compound of any one of claims 4-6 having the structure: The compound of any one of claims 4-7 having the structure: The compound of claim 8, wherein R4 is heteroaryl, C(O)-heteroaryl, CO-hctcroaryl. or SO2- heteroaryl; preferably, the hetcroaryl is pyran, pyridine, piperidine, pyrimidine, isoxazole, oxazole, silole, 6H-l,2,5-thiadiazine, 2H,6H-l,5,2-dithiazine, 1,4-thiazepine, triazine, oxirane, thiirane or azirine; preferably, heteroaryl is pyridine, pyrimidine or triazine; more preferably, heteroaryl is triazine. The compound of claim 9 having the structure: wherein R5 and Rs are each independently H, ary l, heteroaryl, cycloalkyl, heterocycloalkyl, CO2- aryl, CO2-heteroaryl, CO2-cycloalkyl, or CO2-heterocycloalkyl. The compound of any one of claims 4-10, wherein R5 and Re are each independently aryl, heteroaryl, cycloalkyl, or heterocycloalkyl; preferably, R5 and Rs are each independently aryl or cycloalkyl. The compound of any one of claims 4-11, wherein R5 and Rs are each independently substituted aryl or substituted cycloalkyl: preferably, R5 and Rs are each independently substituted with alkyl, alkenyl, alkynyl, and, heteroaryl, cycloalkyl, heterocycloalkyl, alkoxy, carbonyl, carboxyl, amino, or amide; preferably, R5 and R5 are each independently substituted with aryl, heterocycloalkyl, carbonyl or carboxyl; more preferably, R5 and Rs are each independently substituted with aryl or heterocycloalkyl. The compound of claim 12. wherein R5 and Rs are each independently:
wherein U is N or CH; wherein V, X and Y are each independently C, NH, 0, S, -N-, -N-NH2, -N-C(O)-, -N-C(S)-, -N- NHC(O)-, -N-NHC(S), -N-alkyl, -N-alkyne, -N-alkynl, -N-aryl, -N-heteroaryl, -N-cycloalkyl, -N- heterocycloalkyl, or -N-haloalkyl; wherein W and Z are each independently H, NH2. alkyl, alkyne, alkynl, -NH-alkyl, -O-alkyl, -O- haloalkyl, -O-cycloalkyl, O-heterocycloalkyL O-aryl; O-heteroaryl; carbonyl or alkoxy; and wherein R9, R1o, Rn, R12, R13 are each independently -H, -OH, -C00H, -P(=O)(OH)2, halogen, CN, -CF3, -CHF2, -OCF3, -NO2, alkyl, alkenyl, alkynyl, aryl, heteroaryl, -OAc, -ORu. -COR14, - SH, -SR14, -SO2R14, -NH2, -NHR14, -NR15R16, -NHCOR15, or -CONR15R16; preferably, R9, R1o, Rn, R12, R13 are each independently -H, -OH, -C00H. -P(=0)(0H)2, wherein each occurrence of RH is independently alkyl, alkenyl, alkynyl, and, or heteroaryl, wherein each occurrence of R15 is independently -H, alkyl, alkenyl, alkynyl, aryl, or hctcroaryl, wherein each occurrence of R1e is independently -H, alkyl, alkenyl, alkynyl, aryl, or heteroaryl.
14. The compound of claim 13, wherein R5 and Re are each independently
15. The compound of claim 14, wherein R5 and Re are the same: or wherein R5 and Re are different.
16. Tire compound of claim 4, wherein the compound having the structure:
A compound having the structure: wherein R17, R18, R19 are each independently CH, N, or S: wherein R20 IS NH2. NH-alkyl, NH-C(O)-alkyl, NH-C(S)-alkyL NH-NHC(O)-alkyl, NH-NHC(S)- alkyl, alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, C(O)-alkyl, C(O)-haloalkyl, C(O)- cycloalkyl, C(O)-heterocycloalkyl, C(O)-aryl, C(O)-heteroaryl. CO2-alkyl, CO2-aryl. CO2- heteroaryl. CO2-heterocycloalkyl, alkyl-N-, SO2-alkyl, SO2-haloalkyl, SO2-cycloalkyl, SO2- heterocycloalkyl, SO2-aryl, or SO2-heteroaryl; preferably, R20 is NH-alkyl. NH-C(O)-alkyl, NH- C(S)-alkyl, NH-NHC(O)-alkyl. NH-NHC(S)-alkyl, alkyl, alkenyl, alkynyl, C(O)-alkyl, C(O)- haloalkyl, CO2-alkyl, alkyl-NH2, SO2-alkyl, or SO2-haloalkyl; more preferably, R20 is NH-alkyl, NH-C(O)-alkyl, NH-C(S)-alkyl, NH-NHC(O)-alkyl, NH-NHC(S)-alkyl, alkyl, alkenyl, alkynyl, C(O)-alkyl, or C(O) -haloalky 1; more preferably, R20 is NH-alkyl, NH-C(O)-alkyl, NH-C(S)-alkyl, NH-NHC(O)-alkyl, NH-NHC(S)-alkyl, alkyl, C(O)-alkyl, or C(O)-haloalkyl; more preferably, R20 is NH-alkyl or alkyl; wherein R21 is aryl, heteroaryl, cycloalkyl, heterocycloalkyl, C(O)-heterocycloalkyl, C(O)-aryl, C(O)-heteroaryl, CO2-aryl, CO2-heteroaryl, CO2-heterocycloalkyl, SO2-cycloalkyl, SO2- heterocycloalkyl, SO2-aryl, or SO2-heteroaiyl; preferably, R21 is aryl, heteroaryl, C(O)-aryL C(O)- heteroaryl, CO2-aryl. CO2-heteroaryl, SO2-aryl, or SO2-heteroaryl; more preferably, R21 is heteroaryl; and wherein R22 is alkyl or -NH-alkyl; and wherein when R22 is H, R20 is NH2; or a pharmaceutically acceptable salt thereof. The compound of claim 17, wherein the compound having the following structure: The compound of claim 18, wherein
(a) R17. RIS, R19 are CH, N. N;
(b) R17. RIS, R19 are N, CH. N;
(c) R17, RIS, R19 are N, N,CH;
(d) R17, RIS, R19 are CH, CH, N;
(e) R17, RIS, R19 are CH, N, CH;
(f) R17, RIS, R19 are N, CH, CH;
(g) R17. RIS, R19 are CH, CH, CH, or
(h) R17, RIS, R19 are N, N, N. The compound of any one of claims 17-19, wherein R21 is an aryl or heteroaryl. The compound of claim 20, wherein aryl is phenyl, p-toluenyl (4-methylphenyl), naphthyl, tetrahydro-naphthyl, indanyl, biphenyl, phenanthryl, anthryl or acenaphthyl; preferably aryl is phenyl, p-toluenyl (4-methylphenyl); more preferably, aryl is phenyl. Tire compound of claim 21, wherein heteroaryl is pyridine, pyridazine, pyrimidine, pyrazine, 1,2,4- triazinc, 1,3, 5 -triazinc, l,4,5,6-tctrahydrocyclopcnta[b]pyrrolc, 1,3a,4,61-tctrahydropyrrolo[3,2- b]pyrrole, 1,4-dihydropyrrole[3,2-b]pyrrole, 1,6-dihydropyrrolo[2,3-b]pyrrole, indoline, 3H- indole, 1H-indole. 2/7-isoindole, indolizine, 1H-indazole. benzimidazole, 7-azaindole, 4-azaindole, 5-azaindole, or 9H-carbazole; preferably, heteroaryl is 3/7-indole, 1H-indole. 2H-isoindole, indolizine, 1H-indazolc. benzimidazole, 7-azaindole, 4-azaindole, 5-azaindole, or 9H-carbazolc: more preferably, heteroaryl is 1H-indazole or 9H-carbazolc. The compound of any one of claims 21-22, wherein the aryl or heteroaryl is substituted; preferably, aryl or hctcroaryl is substituted with alkyl, alkenyl, alkynyl, aryl, hctcroaryl. cycloalkyl, heterocycloalkyl, alkoxy, carbonyl, carboxyl, amino, or amide; more preferably, aryl or hctcroaryl is substituted with alkyl, alkenyl, alkynyl, aryl, or heteroaryl; more preferably, aryl or heteroaryl is substituted with alkyl. The compound of claim 23, wherein alkyl is C1-6 alkyl; preferably, alkyl is C1-3 alkyl; more preferably, alkyl is methyl or ethyl. The compound of any one of claims 17-24, wherein R21 has the following structure:
wherein U is N or CH; wherein V, X and Y are each independently C, NH, 0, S, -N-, -N-NH2, -N-C(O)-, -N-C(S)-, -N- NHC(O)-, -N-NHC(S), -N-alkyl, -N-alkyne, -N-alkynl, -N-aryl, -N-heteroaryl, -N-cycloalkyl, -N- heterocycloalkyl, or -N-haloalkyl; wherein W and Z are each independently H, NH2. alkyl, alkyne, alkynl, -NH-alkyl, -O-alkyl, -O- haloalkyl, -O-cycloalkyl, O-heterocycloalkyl, O-aryl; O-heteroaryl; carbonyl or alkoxy: and wherein R9, R1o, Rn, R12, R13 are each independently -H, -OH, -COOH, -P(=O)(OH)2, halogen, CN, -CF3, -CHF2, -OCF3, -NO2, alkyl, alkenyl, alkynyl, aryl, heteroaryl, -OAc, -OR14, -COR14, -SH, - SR14, -SO2R14, -NH2, -NHR14, -NR15R16, -NHCOR15, or -CONR15R16; preferably, R9, R1o, R11, R12, RB are each independently -H, -OH, -COOH, -P(=O)(OH)2. wherein each occurrence of RH is independently alkyl, alkenyl, alkynyl, ary l, or heteroaryl, wherein each occurrence of R15 is independently -H, alkyl, alkenyl, alkynyl, aryl, or hctcroaryl, wherein each occurrence of R16 is independently -H, alkyl, alkenyl, alkynyl, aryl, or heteroaryl. The compound of claim 25, wherein R21 has the following structure: 26, wherein R21 has the following structure: Tire compound of claim 27, wherein R21 has the following structure: Tire compound of any one of claims 17-28, wherein R20 is NH-alkyl, NH-C(O)-alkyl, NH-C(S)- alkyl, NH-NHC(O)-alkyl, NH-NHC(S) -alkyl, alkyl, C(O)-alkyl, or C(O)-haloalkyl; more preferably, R20 is NH-alkyl or alkyl. The compound of claim 29, wherein R20 is NH-C1-6 alkyl or C1-6 alkyl; preferably, R20 is NH-C1-3 alkyl or C1-3 alkyl; more preferably, R20 is NH-CH3 or CH3. The compound of claim 17, wherein the compound having the structure:
A pharmaceutical composition comprising compound of any one of claims 4-31 and a pharmaceutically acceptable carrier. The pharmaceutical composition of claim 32, further comprising an effective amount of an anti- fungal agent. A method of inhibiting growth of a fungus comprising contacting the fungus with an effective amount of the compound of any one of claims 4-31 to the subject. A method of treating a subject with fungal infection comprising administering an effective amount of the compounds of any one of claims 4-31 to the subject.
36. The method of claim 35, further comprising administering to the subject an effective amount of an anti-fungal agent.
37. Tire method of claim 36, wherein the anti-fungal agent is azole, flucytosine, amphotericin B or echinocandins, or a combination thereof.
38. The method of any one of claims 1-3 and 34-37, wherein the fungus is a saprotrophic fungus.
39. The method of claim 38, wherein the fungus is Cryptococcus Neoformans, Cryptococcus gattii, Candida albicans, Candida krusei, Candida glabrata, Candida parapsilosis, Candida guilliermondii, Aspergillus fumigatus, Rhizopus oryzae, Rhizopus spp. , Blastomyces dermatitis, Histoplasma capsulatum, Coccidioides spp., Paecilomyces variotii, Pneumocystis murina, Pneumocystis jiroveci, Histoplasma capsulatum, Aspergillus spp., or Sporothrix brasiliensis.
40. The method of claim 39, wherein the fungus is Cryptococcus Neoformans or Aspergillus fumigatus.
41. A compound of formula (III) or a pharmaceutically acceptable salt thereof: wherein:
X1 is C0-6 alkyl, NR2, or carbonyl;
R1 is
X2, X3, X4, and X5 are each independently selected from 0, NR7, or CH2;
R2, R3, R4, R5, R6, and R7 are each independently selected from one or more of C0-ealkyl. -OCo ealkyl halogen, carbonyl, -CN, -CF3, -OCF3, -NO2, C0-ealkenyl, Co ealkynyl, C0-earyl, C0-eheteroaryl, C0- heterocycle. -OAc, -COR8, -CH2OR8, -SH, -SR8, -SO2R8, -NH2, -NHR8, -NR9R10, -NHCOR9, -CONR’R10, wherein each occurrence of R8, R9, and R10 are independently one or more of C0-6alkyl, C0-6alkenyl, C0- 6alkynyl, C0-6aryl. or C0-6 heteroaryl.
42. A pharmaceutical composition comprising the compound of claim 41 and a pharmaceutically acceptable carrier.
43. A method of inhibiting the growth of a fungus comprising contacting the fungus with an effective amount of a compound of claim 41 or a pharmaceutically acceptable salt or ester thereof, so as to thereby inhibit the growth of the fungus.
44. A method of inhibiting fungal sterylglucosidase synthesis in a fungus comprising contacting the fungus with an effective amount of claim 41 or a pharmaceutically acceptable salt or ester thereof, so as to thereby inhibit sterylglucosidase synthesis in the fungus.
45. The method of any one of claims 41-44, further comprising contacting the fungus with an amount of an anti-fungal agent.
46. Tire method of any one of claims 41-45, wherein the method further comprises inhibiting the growth of a fungus in a plant.
47. The method of any one of claims 41-46, wherein the fungus is Cryptococcus Neoformans, Cryptococcus gailii. Candida albicans, Candida krusei, Candida glabrata, Candida parapsilosis, Candida guilliermondii, Aspergillus fumigatus, Rhizopus oryzae, Rhizopus spp., Blastomyces dermatitis, Histoplasma capsulatum, Coccidioides spp., Paecilomyces variotii, Pneumocystis murina, Pneumocystis jiroveci, Histoplasma capsulatum, Aspergillus spp., S. brasiliensis, S. schenckii, S. globosa, S. mexicana, S. chilensis, S. luriei, or S. pallida.
48. The method of claim 47, wherein the fungus is Cryptococcus Neoformans.
49. The method of claim 47, wherein the fungus is Aspergillus fumigatus.
EP23875757.9A 2022-10-05 2023-10-04 Sterylglucosidase inhibiting compositions and method of using Pending EP4598906A2 (en)

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