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WO2024137780A1 - Pank modulators and methods of treatment using same - Google Patents

Pank modulators and methods of treatment using same Download PDF

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Publication number
WO2024137780A1
WO2024137780A1 PCT/US2023/085075 US2023085075W WO2024137780A1 WO 2024137780 A1 WO2024137780 A1 WO 2024137780A1 US 2023085075 W US2023085075 W US 2023085075W WO 2024137780 A1 WO2024137780 A1 WO 2024137780A1
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compound
triazolo
methyl
pyrimidin
amino
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French (fr)
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Choukri BEN MAMOUN
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Yale University
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Yale University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/10—Antimycotics
    • 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
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
    • A61K31/197—Carboxylic acids, e.g. valproic acid having an amino group the amino and the carboxyl groups being attached to the same acyclic carbon chain, e.g. gamma-aminobutyric acid [GABA], beta-alanine, epsilon-aminocaproic acid or pantothenic acid
    • 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
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with 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
    • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
    • A61K31/5375—1,4-Oxazines, e.g. morpholine
    • A61K31/5377—1,4-Oxazines, e.g. morpholine not condensed and containing further heterocyclic rings, e.g. timolol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca

Definitions

  • the disclosure provides a method of reducing, minimizing, and/or preventing drug resistance against an antifungal agent used to treat, ameliorate, and/or prevent a fungal infection in a subject. In one aspect, the disclosure provides a method of sensitizing a fungus to an antifungal agent administered to a subject to treat, ameliorate, and/or prevent the fungal infection in the subject.
  • the method comprises administering a therapeutically effective amount of a compound which is a pantothenate kinase (PanK) inhibitor and/or modulator to a subject being administered the antifungal agent.
  • a compound which is a pantothenate kinase (PanK) inhibitor and/or modulator to a subject being administered the antifungal agent.
  • FIGs. 1 A-1H illustrate relationship between pantothenate utilization and yeast susceptibility to amphotericin B.
  • FIG. 1G depicts relative times for treated cells to reach mid-log phase compared to untreated cells, calculated from FIG. 1A. Time for untreated cells to grow to mid-log was normalized to 1, and is represented by the dashed line.
  • FIG. 1A Time for untreated cells to grow to mid-log was normalized to 1, and is represented by the dashed line.
  • FIG. 2 illustrates a non-limiting model of the mechanism by which inhibition of Cab 1 activity and CoA biosynthesis leads to enhanced susceptibility to antifungal drugs.
  • inhibition or reduction of Cab Ip activity through genetic mutation (use of Cabl mutants) or enzyme inhibition (using Cabl inhibitors or analogs of pantothenic acid such as a-PanAm) results in altered vacuolar function (wherein the vacuole plays a key role in drug detoxification in fungi), which increases susceptibility of fungi to antifungals.
  • FIG. 4 illustrates the finding that reduced fungal PanK activity in 6 S. cerevisiae mutants carrying different Cabl alleles leads to enhanced susceptibility to a large panel of antifungal drugs.
  • Addition of the wild type CAB1 gene restores drug susceptibility to wild ty pe levels.
  • Drug susceptibility was assessed using a limiting dilution assay on YPD-based agar plates lacking or supplemented with each of the 8 drugs at the indicated concentrations.
  • FIG. 5 shows the MIC50 values of wild type and cabl G351s strains for 7 antifungal drugs and the ratio of WT MICso/ cabl G351s MICso to represent the fold increase in susceptibility to these antifungal drugs caused by the G351S mutation.
  • FIG. 6 is a graphical representation of the data in FIG. 5.
  • FIG. 7 illustrates the finding that susceptibility’ of the S. cerevisiae wild type strain to terbinafine (5 and 10 pg/ml) is significantly enhanced by the commercially available drug PZ-2891 at 2 different concentrations (50 pM and 100 pM). Addition of PZ -2891 further increased the susceptibility' of this strain to terbinafine.
  • the compound (PZ-2891) itself had no effect on fungal growth at concentrations up to 200 pM.
  • FIG. 9 illustrates the finding that susceptibility’ of a Candida parapsilosis clinical isolate to terbinafine (5 pg/ml) is significantly enhanced by the commercially available drug PZ-2891 at 2 different concentrations (50 pM and 100 pM).
  • the compound (PZ-2891) itself had no effect on fungal growth at concentrations up to 200 pM.
  • FIG. 10 illustrates the finding that susceptibility of a Candida albicans clinical isolate to terbinafine (20 pg/ml) is significantly enhanced by the commercially available drug PZ- 2891 at 2 different concentrations (50 pM and 100 pM).
  • the compound (PZ-2891) itself had no effect on fungal growth at concentrations up to 200 pM.
  • FIG. 11 illustrates how Cabl mutants display growth defects and increased susceptibility toward ergosterol targeting drugs.
  • A Schematic diagram of Coenzyme A synthetic pathway and biological roles of the Acetyl-CoA.
  • Pantothenate kinase PanK
  • PanK Pantothenate kinase
  • Phosphopantothenoylcysteine synthase converts 4’ -phosphopantothenic acid to phosphopantothenoylcysteine.
  • Phosphopantothenoylcysteine decarboxylase catalyzes the decarboxylation of phosphopantothenoylcysteine to form pantetheine.
  • Pantetheine kinase PANK
  • PANK pantothenoylcysteine
  • DPCK Dephospho-CoA kinase
  • FIGs. 12A-12B shows the cabl mutants display susceptibility toward drugs targeting ergosterol pathway and non-ergosterol targeting pathway.
  • Yeast liquid growth curve assay with known AFDs targeting the ergosterol biosynthesis pathway was done using yeast cells harboring different CAB1 mutants. Cells were inoculated into 100 pL of YPD liquid media containing the antifungals in serial dilutions at 10 cells per pL ratio and incubated at 30°C while cell grow th was monitored by ODeoo. % of cell growth of individual mutant strain in presence of AFDs was obtained compared to the cell growth in the vehicle control.
  • FIG. 13A illustrates normalized relative MICso of yeast strains producing Cabl variants against AFDs.
  • MICso values w ere determined on MICso data obtained from the liquid growth assays presented in FIG. 12A and 12B. These values were calculated by dividing the MICso value of the cabl mutant or the wild type strain for the specified AFD by the MICso value of the wild type strain.
  • FIG. 13B depicts complementation of the w ild type CAB1 gene restores AFD resistance in the cabl mutants to the similar level as that observed in wild-type cells.
  • the cab 1 ⁇ w303-l strain harboring 1) wild type CABL 2) cabl mutar,t , or 3) cabl mutant + wild t pe CAB were inoculated into synthetic glucose medium and grown overnight. Cells were harvested and re-suspended in 0.9% NaCl.
  • FIG. 14 illustrates how yeast strains expressing various CAB1 mutations have defects in detoxification and vacuolar function and structure.
  • the cabl mutations altered yeast capacity to overcome metal toxicity. Solid growth assays were performed with the yeast strains described above in glucose media supplemented with 7 mM FeSC or with 10 mM CuSO 4 .
  • FIG. 15A shows morphological analysis of vacuolar defects shows that cabl mutant strains have unusually enlarged vacuoles.
  • Immuno-fluorescence microscopy images of cablX + cad7 mutant strains described above show enlarged and/or fragmented vacuoles, while the cablX + CAB!'''' 2 and the cablX + cabl mutant + CABl ⁇ 1 strains have typical vacuolar morphology.
  • FIG. 15B shows quantitative analysis of vacuolar area size (as a proportion of total cell area) as a function of CAB1 status.
  • FIG. 16 illustrates Electron microscopy images of cab ⁇ + CABl lVT and cab 1 X+cab 7 G351S single cells confirm enlargement of vacuole induced by G351S mutation.
  • FIG. 17 illustrates how yeast cablX strains harboring various CAB1 mutations display defects in mitochondrial function. Solid growth assays reveal that mutations in CAB1 alter strains’ ability to utilize non-fermentable carbon sources. Solid growth assays using ten-fold serial dilutions plated onto YPD (glucose), YPL (lactate), YPG (glycerol), or YPE (ethanol) media and observed over 3-4 days.
  • YPD glucose
  • YPL lactate
  • YPG glycerol
  • YPE ethanol
  • FIGs. 18A-18B show oxygen consumption rate (OCR) of yeast cells harboring CAB1 variants. OCR profile of cablX strains using Seahorse 96X and Mito Stress kit. Dashed lines represent the injections of mitochondrial uncoupling drugs; oligomycin, FCCP, antimycin A, and rotenone.
  • FIG. 19 illustrates cablX strains exhibit mitochondrial structural defects. Immunofluorescence microscopy images of cablX strains reveal aberrant mitochondria structures.
  • FIG. 20 shows cablX strains have increased levels of reactive oxygen species (ROS). ROS analysis was performed using dihydrorodamine 123.
  • FIG. 21 A illustrates metabolic defects in yeast strains harboring cabl mutations.
  • PA utilization in the cablX strains harboring various CABl mutations Cell free extracts of yeast cabl mutants were used to measure the endogenous PA utilization of cablX + CAB1, cablX + cabl G25IS , cablX + cabl G25IS + CABL cablX + cabl SI58A , cablX + cabl SI5SA + CABL and cablX + cabl 812901 .
  • FIG. 21B depicts cellular levels of CoA in cablS strains. CoA levels were measured using metabolite extracts from the yeast strains mentioned above grown in the presence of 0.2 pM PA.
  • FIG. 22 depicts cysteine cellular levels of cahl ⁇ strains harboring various CABl mutations.
  • FIG. 23 A illustrates a non-limiting schematic of the connection between the PCA pathway and the SUL/MET pathways.
  • Transcription of the SUIAMET genes responsible for synthesizing the crucial sulfur-containing amino acids methionine and cysteine, is mediated by the transcription factor Met4.
  • Met4 This regulatory’ process is sensitive to changes in cellular cysteine levels.
  • Met4 undergoes ubiquitination by the ubiquitin ligase Met30, leading to Met4’s inactivation and subsequent repression of the SUL/MET genes.
  • Table 3 The gene list with annotations shown in Table 3.
  • FIG. 24A illustrates chemical inhibition of Acs2 and V-type ATPase increases susceptibility to a variety' of AFDs via a schematic of the relevant portions of the PCA pathway.
  • FIG. 24B shows the growth of acs2 tet-off strain is inhibited by increasing concentrations of doxycycline.
  • FIG. 25 A illustrates AR- 12 potentiates caspofungin. fluconazole, and terbinafine by factors of ⁇ 100x against S. cerevisiae WT.
  • FIG. 25B illustrates Concanamycin A increases yeast susceptibility 7 to antifungal drugs.
  • FIG. 26A illustrates PZ-2891 increases AFD susceptibility in A cerevisiae. C. albicans and A. fumigatus . Solid growth assays showing increased S', cerevisiae and C. albicans susceptibility to amphotericin B when potentiated by PZ-2891.
  • FIG. 26B shows liquid growth assays showing increased C. albicans susceptibility to amphotericin B (125 ng/mL) and caspofungin (3.9 ng/mL) is potentiated by PZ-2891.
  • FIGs. 27A-27B illustrates average colony diameter rate and captures of solid media assay (up to 72h of growth) with A. fumigatus cells under caspofungin treatment (20 pg/ml) in the presence or absence of PZ-2891 (50 pM).
  • FIG. 28A illustrates cellular CoA levels in S cerevisiae following treatment with PZ- 2891 .
  • CoA levels were measured using the metabolite extracts from the S cerevisiae cells grown in minimal glucose medium supplemented with 1 pM PA in the presence or absence of 50 pM PZ-2891.
  • FIG. 28B shows yeast acetyl CoA synthetase activity.
  • the in vitro activity of purified enzyme from S cerevisiae was measured using a standard hydroxylamine-coupled assay, at 37 °C for 30 minutes, in the presence of absence of 18 pM of AR- 12 or PZ-2891
  • FIG. 29 illustrates a model for PCA pathway-mediated regulation of vacuolar detoxification and susceptibility to antifungal drugs in fungi.
  • FIGs. 30A-30B illustrate cellular CoA (FIG. 30A) and cysteine (FIG. 30B) levels in cab IX strains harboring various CAB1 mutations. CoA and cysteine levels were measured using the metabolite extracts from the yeast strains grown in the presence of 1 pM PA
  • FIG. 31 illustrates RNA-Seq analysis for cysteine and sulfur homeostasis genes expressed in cab IX strains. The results are based on normalized TMM compared with the expression profile of the WT parent strain. The gene list with annotations shown in Table 3.
  • FIG. 33A illustrates the effect of modulation of downstream steps from the PCA pathway on the growth of S. cerevisiae.
  • S. cerevisiae WT or acs2-tetoff mutant, as mentioned
  • FIG. 33B shows AR-12
  • FIG. 33C shows concanamycin A, at 30°C for 24- 48 h.
  • FIG. 34 illustrates PA utilization in the cabl ' strains harboring various CAB1 mutations.
  • Cell free extracts of yeast expressing cabl mutants w ere used to measure the endogenous PA utilization of cabl variants using D-[1- 14 C] pantothenate as a substrate for 10 min at 30°C.
  • the extracts PA utilization was measured in the absence or presence of 20 pM PZ-2891 (hPanK3 activator), a-PanAm (known Cabl inhibitor), and YU385599 (reported Cabl inhibitor).
  • FIG. 35A illustrates potentiation of PZ-2891 on antifungal susceptibility' in different yeast species.
  • S', cerevisiae cells were inoculated in the presence or absence PZ-2891, in combination with caspofungin treatment at 30°C for 24-48 h.
  • FIG. 35B shows potentiation of terbinafine efficacy in C. albicans with PZ-2891.
  • C. albicans spotting growth assays w ere performed when cells were inoculated into YPD overnight, harvested, w ashed, and re-suspended in 0.9% NaCl. Serial dilutions of cells were spotted onto YPD plates containing terbinafine (20 pg/mL) in the presence or absence of PZ- 2891 (50 pM) at 30°C for 4 days.
  • FIG. 36 show s the average growth rate (based on colony diameter) of A. fumigatus colonies in the presence or absence of PZ-2891 (50 pM) in combination with caspofungin treatment (20 pg/ml). The results were calculated after 72 h of growth.
  • FIG. 37A illustrates PZ-2891 does not have inhibitory effect on either . cerevisiae Cabl enzymatic activity or S. cerevisiae cell growth as shown in the dose-response curve for AcCoA effect on recombinant Cabl enzyme activity.
  • FIG. 37B shows the dose-response curve for PZ-2891 effect on recombinant Cabl enzyme activity.
  • FIG. 38 shows a dose-response curve for AcCoA effect on recombinant Cabl enzyme activity in the absence of presence of PZ-2891 or YU385599 (inhibitor control).
  • FIG. 39 illustrates liquid growth assay for cables strains harboring various CAB1 mutations in the presence or absence of 1-100 pM PZ-2891.
  • the disclosure relates, in certain aspects, to certain compounds that are useful to treat, ameliorate, and/or prevent fungal infections in a subject.
  • the fungus causing the fungal infection comprises C. albicans, C. parapsilosis, and/or A. fumigatus.
  • the compounds of the disclosure comprise any pantothenate kinase (PanK) inhibitor and/or modulator known in the art.
  • PanK pantothenate kinase
  • the compounds of the disclosure comprise any pantazine know n in the art.
  • pantazines include PZ-2891, any PanK modulator disclosed in WO2017/223474, WO2019/133635, US20190300499 Al, US20210246113 Al, and US20210061788 Al (each of which is incorporated herein in its entirety by reference), any PanK modulator disclosed in Sharma, el al.. 2018, "A therapeutic approach to pantothenate kinase associated neurodegeneration ” , Nature Comm., 9(1):4399, doi: 10.
  • the compound is PZ-2891. In certain embodiments, the compound is not PZ-2891.
  • the compounds of the disclosure comprise any pantothenic acid analogue and/or derivative.
  • pantothenic acid analogues and/or derivatives include a-PanAm, any pantothenamide analogue disclosed in WO2016/072854 and US20180282279 Al (all of which are incorporated herein in their entireties by reference), any pantothenamide analogue disclosed in de Vries, et al., 2022, “Preclinical characterization and target validation of the antimalarial pantothenamide MMV693183”, ⁇ aX Covarr.
  • the compound is a-PanAm. In certain embodiments, the compound is not a-PanAm.
  • the compounds of the disclosure comprise certain compounds disclosed herein, such as but not limited to a compound of formula (la) or (lb).
  • the compounds of the disclosure potentiate the biological activity of known antifungal drugs.
  • the compounds of the disclosure have synergistic inhibitory effects against fungi when used in combination with other antifungal agents.
  • the contemplated antifungal agents include but are not limited to agents that disrupt synthesis and/or activity of ergosterol, such as but not limited to azoles, morpholines, allylamines, and polyenes, as well as inhibitors of other metabolic pathways such as cell wall metabolism, as well as nucleic acid and protein synthesis.
  • Non-limiting examples of azole antifungal agents contemplated herein include:
  • imidazoles bifonazole, butoconazole. clotrimazole, econazole, fenticonazole, isoconazole, ketoconazole, luliconazole, miconazole, omoconazole, oxiconazole, sertaconazole, sulconazole, tioconazole.
  • Non-limiting examples of allylamine antifungal agents contemplated herein include butenafme, naftifme, and terbinafme.
  • Non-limiting examples of polyene antifungal agents contemplated herein include amphotericin B, candicidin, filipin, hamycin, natamycin, nystatin, and rimocidin.
  • Non-limiting examples of morpholine antifungal agents contemplated herein include amorolfme and fenpropimorph.
  • Non-limiting examples of inhibitors of other metabolic pathways include cycloheximide (a protein synthesis inhibitor), echinocandins (inhibitors of fungal cell wall synthesis; non-limiting examples include anidulafungin, caspofungin, and/or micafungin), cytosine analogues (such as but not limited to 5-flucytosine), aminoglycosides (such as but not limited to hygromy cin B).
  • the compounds of the disclosure increase potency of an antifungal agent against a fungal infection in a subject.
  • the antifungal agent comprises for example agents that disrupt synthesis and/or activity of ergosterol, such as but not limited to azoles, morpholines, allylamines, and polyenes, as well as inhibitors of other metabolic pathways.
  • the compounds of the disclosure reduce at least one toxicity effect of an antifungal agent against a fungal infection in a subject when a compound of the disclosure is co-administered with the antifungal agent.
  • administration of a compound of the disclosure and the antifungal agent allows for a lower dose of the antifungal agent as compared to the dose of the antifungal agent alone required to achieve equivalent antifungal effect.
  • the antifungal agent comprises for example agents that disrupt synthesis and/or activity of ergosterol, such as but not limited to azoles, morpholines, allylamines, and polyenes, as well as inhibitors of other metabolic pathways.
  • the compounds of the disclosure reduce drug resistance against an antifungal agent administered to a subject to treat, ameliorate, and/or prevent a fungal infection in a subject when a compound of the disclosure is co-administered with the antifungal agent.
  • administration of a compound of the disclosure and the antifungal agent allows for a lower dose/lower dosing frequency /lower overall dosing period of the antifungal agent as compared to the dose of the antifungal agent alone required to achieve equivalent antifungal effect.
  • the antifungal agent comprises for example agents that disrupt synthesis and/or activity of ergosterol, such as but not limited to azoles, morpholines, allylamines, and polyenes, as well as inhibitors of other metabolic pathways.
  • the compounds of the disclosure sensitize fungal pathogens to an antifungal agent administered to a subject to treat, ameliorate, and/or prevent a fungal infection in a subject when a compound of the disclosure is co-administered with the antifungal agent.
  • the antifungal agent comprises for example agents that disrupt synthesis and/or activity of ergosterol, such as but not limited to azoles, morpholines, allylamines, and polyenes, as well as inhibitors of other metabolic pathways.
  • an element or component is said to be included in and/or selected from a list of recited elements or components, it should be understood that the element or component can be any one of the recited elements or components and can be selected from a group consisting of two or more of the recited elements or components.
  • the acts can be carried out in any order, except when a temporal or operational sequence is explicitly recited. Furthermore, specified acts can be carried out concurrently unless explicit claim language recites that they be carried out separately. For example, a claimed act of doing X and a claimed act of doing Y can be conducted simultaneously within a single operation, and the resulting process will fall within the literal scope of the claimed process.
  • the term “about” will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which it is used. As used herein, “about” when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ⁇ 20%, ⁇ 10%, ⁇ 5%, ⁇ 1%, or ⁇ 0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.
  • alkenyl means, unless otherwise stated, a stable monounsaturated or diunsaturated straight chain or branched chain hydrocarbon group having the stated number of carbon atoms. Examples include vinyl, propenyl (or allyl), crotyl, isopentenyl, butadienyl, 1,3-pentadienyl, 1,4-pentadienyl, and the higher homologs and isomers.
  • alkoxy employed alone or in combination with other terms means, unless otherwise stated, an alkyl group having the designated number of carbon atoms, as defined elsewhere herein, connected to the rest of the molecule via an oxygen atom, such as, for example, methoxy, ethoxy, 1 -propoxy, 2-propoxy (or isopropoxy) and the higher homologs and isomers.
  • oxygen atom such as, for example, methoxy, ethoxy, 1 -propoxy, 2-propoxy (or isopropoxy) and the higher homologs and isomers.
  • Si-Cs alkoxy
  • alkyl by itself or as part of another substituent means, unless otherwise stated, a straight or branched chain hydrocarbon having the number of carbon atoms designated (i.e., C1-C10 means one to ten carbon atoms) and includes straight, branched chain, or cyclic substituent groups. Examples include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, neopentyl, hexyl, and cyclopropylmethyl.
  • a specific embodiment is (Ci-Ce)alkyl, such as, but not limited to, ethyl, methyl, isopropyl, isobutyl, /7-pentyl, n-hexyl, and cyclopropylmethyl.
  • alkynyl employed alone or in combination with other terms means, unless otherwise stated, a stable straight chain or branched chain hydrocarbon group with a triple carbon-carbon bond, having the stated number of carbon atoms. Nonlimiting examples include ethynyl and propynyl, and the higher homologs and isomers.
  • ethynyl and propynyl and the higher homologs and isomers.
  • ethynyl and propynyl and the higher homologs and isomers.
  • aromatic refers to a carbocycle or heterocycle with one or more polyunsaturated rings and having aromatic character, z.e., having (4n+2) delocalized n (pi) electrons, where ‘n’ is an integer.
  • aryl employed alone or in combination with other terms means, unless otherwise stated, a carbocyclic aromatic system containing one or more rings (typically one, two or three rings) wherein such rings may be attached together in a pendent manner, such as a biphenyl, or may be fused, such as naphthalene. Examples include phenyl, anthracyl and naphthyl.
  • Aryl groups also include, for example, phenyl or naphthyl rings fused with one or more saturated or partially saturated carbon rings (e.g, bicyclo [4.2.0] octa-1, 3,5- trienyl, or indanyl), which can be substituted at one or more carbon atoms of the aromatic and/or saturated or partially saturated rings.
  • saturated or partially saturated carbon rings e.g, bicyclo [4.2.0] octa-1, 3,5- trienyl, or indanyl
  • aryl-(Ci-C6)alkyl refers to a functional group wherein a one-to-six carbon alky lene chain is attached to an aryl group, e.g., -CEECEb-phenyl or -CH2- phenyl (or benzyl). Specific examples are aryl-CEE- and ary 1-CH(CH3)-.
  • substituted aryl-(Ci-C6)alkyl refers to an aryl-(Ci-C6)alkyl functional group in which the aryl group is substituted. A specific example is substituted aryl(CH2)-.
  • Non-limiting examples of cycloalkyl groups include: cyclopropyl, 2-methyl-cyclopropyl, cyclopropenyl, cyclobutyl, 2,3-dihydroxycyclobutyl, cyclobutenyl, cyclopentyl, cyclopentenyl, cyclopentadienyl, cyclohexyl, cyclohexenyl, cycloheptyl, cyclooctanyl, decalinyl, 2.5-dimethylcyclopentyl. 3,5- dichlorocyclohexyl, 4-hydroxy cyclohexyl, 3,3,5-trimethylcyclohex-l-yl, octahydropental enyl.
  • octahydro- I/7-indenyl 3a.4.5.6.7.7a-hexahydro-3//-inden-4-yl.
  • decahydroazulenyk bicyclo[6.2.0]decanyl, decahydronaphthalenyl, and dodecahydro- 1/7- fluorenyl.
  • cycloalky l also includes bicyclic hydrocarbon rings, non-limiting examples of which include, bicyclo[2.1.1]hexanyl, bicyclo[2.2.1]heptanyl, bicyclo[3.1.1]heptanyl, l,3-dimethyl[2.2.1]heptan-2-yl, bicyclo[2.2.2]octanyl, and bicyclo[3.3.3]undecanyl.
  • a “disease” is a state of health of a subject wherein the subject cannot maintain homeostasis, and wherein if the disease is not ameliorated then the subject’s health continues to deteriorate.
  • a “disorder” in a subject is a state of health in which the subject is able to maintain homeostasis, but in which the subject’s state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the subject's state of health.
  • halide refers to a halogen atom bearing a negative charge.
  • the halide anions are fluoride (F-), chloride (CL), bromide (Br ), and iodide (I ).
  • halo or “halogen” alone or as part of another substituent refers to, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom.
  • heteroalkenyl by itself or in combination with another term refers to, unless otherwise stated, a stable straight or branched chain monounsaturated or diunsaturated hydrocarbon group consisting of the stated number of carbon atoms and one or two heteroatoms selected from the group consisting of O, N, and S, and wherein the nitrogen and sulfur atoms may optionally be oxidized and the nitrogen heteroatom may optionally be quatemized. Up to two heteroatoms may be placed consecutively.
  • heteroalkyl by itself or in combination with another term refers to, unless otherwise stated, a stable straight or branched chain alkyl group consisting of the stated number of carbon atoms and one or two heteroatoms selected from the group consisting of O, N, and S, and wherein the nitrogen and sulfur atoms may be optionally oxidized and the nitrogen heteroatom may be optionally quatemized.
  • the heteroatom(s) maybe placed at any position of the heteroalkyl group, including between the rest of the heteroalkyl group and the fragment to which it is attached, as well as attached to the most distal carbon atom in the heteroalkyl group. Examples include: -OCH 2 CH 2 CH 3 , - CH 2 CH 2 CH 2 OH.
  • Up to two heteroatoms may be consecutive, such as, for example, -CH 2 NH-OCH 3 , or -CH 2 CH 2 SSCH 3 .
  • heteroaryl or “heteroaromatic” refers to a heterocycle having aromatic character.
  • a polycyclic heteroaryl may- include one or more rings that are partially saturated. Examples include tetrahydroquinoline and 2,3-dihydrobenzofuryl.
  • heterocycle or “heterocyclyl” or “heterocyclic” by itself or as part of another substituent refers to. unless otherwise stated, an unsubstituted or substituted, stable, mono- or multi-cyclic heterocyclic ring system that comprises carbon atoms and at least one heteroatom selected from the group consisting of N, O, and S, and wherein the nitrogen and sulfur heteroatoms may be optionally oxidized, and the nitrogen atom may be optionally quatemized.
  • the heterocyclic system may be attached, unless otherwise stated, at any heteroatom or carbon atom that affords a stable structure.
  • a heterocycle may be aromatic or non-aromatic in nature. In certain embodiments, the heterocycle is a heteroaryl.
  • non-aromatic heterocycles include monocyclic groups such as aziridine, oxirane, thiirane, azetidine, oxetane, thietane, pyrrolidine, pyrroline, imidazoline, pyrazolidine, dioxolane, sulfolane, 2,3-dihydrofuran, 2,5-dihydrofuran, tetrahydrofuran, thiophane, piperidine, 1,2,3,6-tetrahydropyridine, 1.4-dihydropyridine, piperazine, morpholine, thiomorpholine, pyran, 2.3-dihydropyran, tetrahydropyran, 1,4-di oxane. 1,3- dioxane, homopiperazine, homopiperidine, 1,3-dioxepane, 4,7-dihydro-l,3-dioxepin, and hexamethyleneoxid
  • heteroaryl groups include pyridyl, pyrazinyl, pyrimidinyl (such as, but not limited to, 2- and 4-pyrimidinyl), pyridazinyl. thienyl, furyl, pyrrolyl. imidazolyl, thiazolyl, oxazolyl, pyrazolyl, isothiazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, 1,3,4-triazolyl, tetrazolyl, 1,2,3-thiadiazolyl, 1,2,3-oxadiazolyl, 1,3,4-thiadiazolyl, and 1,3,4-oxadiazolyl.
  • polycyclic heterocycles include indolyl (such as, but not limited to, 3-, 4- , 5-, 6- and 7-indolyl), indolinyl.
  • 4-, 5-, 6-, and 7-benzothienyl benzoxazolyl, benzothiazolyl (such as, but not limited to, 2-benzothiazolyl and 5-benzothiazolyl), purinyl, benzimidazolyl, benztriazolyl, thioxanthinyl, carbazolyl, carbolinyl, acridinyl, pyrrolizidinyl, and quinolizidinyl.
  • heterocyclyl and heteroaryl moi eties are intended to be representative and not limiting.
  • composition refers to a mixture of at least one compound useful within the disclosure with a pharmaceutically acceptable carrier.
  • the pharmaceutical composition facilitates administration of the compound to a subject.
  • the term “pharmaceutically acceptable” refers to a material, such as a carrier or diluent, which does not abrogate the biological activity or properties of the compound useful within the disclosure, and is relatively non-toxic, z.e., the material may be administered to a subject without causing undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained.
  • the term “pharmaceutically acceptable carrier” means a pharmaceutically acceptable material, composition or earner, such as a liquid or solid filler, stabilizer, dispersing agent, suspending agent, diluent, excipient, thickening agent, solvent or encapsulating material, involved in carrying or transporting a compound useful within the disclosure within or to the subject such that it may perform its intended function.
  • a pharmaceutically acceptable material such as a liquid or solid filler, stabilizer, dispersing agent, suspending agent, diluent, excipient, thickening agent, solvent or encapsulating material, involved in carrying or transporting a compound useful within the disclosure within or to the subject such that it may perform its intended function.
  • Such constructs are carried or transported from one organ, or portion of the body, to another organ, or portion of the body.
  • Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation, including the compound useful within the disclosure, and not injurious to the subject.
  • materials that may serve as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as com starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, com oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; surface active agents; alginic acid; pyrogen-free water; isotonic s
  • “pharmaceutically acceptable carrier” also includes any and all coatings, antibacterial and antifungal agents, and absorption delaying agents, and the like that are compatible with the activity of the compound useful within the disclosure, and are physiologically acceptable to the subject. Supplementary active compounds may also be incorporated into the compositions.
  • the “pharmaceutically acceptable carrier” may further include a pharmaceutically acceptable salt of the compound useful within the disclosure.
  • Other additional ingredients that may be included in the pharmaceutical compositions used in the practice of the disclosure are known in the art and described, for example in Remington’s Pharmaceutical Sciences (Genaro, Ed., Mack Publishing Co., 1985. Easton, PA), which is incorporated herein by reference.
  • pharmaceutically acceptable salt refers to a salt of the administered compound prepared from pharmaceutically acceptable non-toxic acids and/or bases, including inorganic acids, inorganic bases, organic acids, inorganic bases, solvates (including hydrates) and clathrates thereof.
  • a “pharmaceutically effective amount,” “therapeutically effective amount,’' or “effective amount” of a compound is that amount of compound that is sufficient to provide a beneficial effect to the subject to which the compound is administered.
  • prevent means avoiding or delaying the onset of symptoms associated with a disease or condition in a subject that has not developed such symptoms at the time the administering of an agent or compound commences.
  • Disease, condition and disorder are used interchangeably herein.
  • PZ2891 corresponds to the compound 6-(4-(2-(4- isopropylphenyl)acetyl)piperazin-l-yl)pyridazine-3-carbonitrile or salt or solvate thereof.
  • PZ-2891 is a pantothenate kinase (PANK) modulator with ICso values of 40.2nM, 0.7nM and 1.3nM for human pantothenate kinases PANK10, PANK2, and PANK3, respectively.
  • PANK pantothenate kinase
  • the terms “subj ect” and “individual” and “patient” can be used interchangeably and may refer to a human or non-human mammal or a bird.
  • Non-human mammals include, for example, livestock and pets, such as ovine, bovine, porcine, canine, feline and murine mammals.
  • the subject is human.
  • substituted refers to that an atom or group of atoms has replaced hydrogen as the substituent attached to another group.
  • substituted alkyl refers to alkyl, cycloalkyl, alkenyl, or alkynyl, as defined elsewhere herein, substituted by one, two or three substituents independently selected from the group consisting of halogen, -OH, alkoxy, tetrahydro-2 -H-pyranyl, -NH2. -NH(Ci-Ce alkyl), -N(Ci-Ce alkyl)2.
  • substituted alkyls include, but are not limited to, 2,2-difluoropropyl, 2- carboxy cyclopentyl and 3-chloropropyl.
  • the term ‘'substituted’’ as applied to the rings of these groups refers to any level of substitution, namely mono-, di-, tri-, tetra-, or penta-substitution, where such substitution is permitted.
  • the substituents are independently selected, and substitution may be at any chemically accessible position. In certain embodiments, the substituents vary in number between one and four. In other embodiments, the substituents vary in number between one and three. In yet other embodiments, the substituents vary 7 in number between one and two.
  • the substituents are independently selected from the group consisting of Ci-Ce alkyl, -OH, Ci-Ce alkoxy, halo, amino, acetamido and nitro.
  • the carbon chain may be branched, straight or cyclic.
  • each occurrence of alkyl, alkenyl, alkynyl, or cycloalkyl is independently optionally substituted with at least one substituent selected from the group consisting of Ci-Ce alkyl, C3-C8 cycloalkyl, halo, cyano (-CN), -OR a .
  • Ci-Ce alky l optionally substituted Ci-Ce alky l, optionally substituted C3-C8 cycloalkyl, optionally substituted aryl, or optionally substituted heteroaryl, or two R a groups combine with the N to which they are bound to form a heterocycle.
  • R b the alkyl or cycloalkyl is optionally substituted with at least one selected from the group consisting of halogen, -OH, Ci-Ce alkoxy, and heteroaryd; or substituents on two adjacent carbon atoms combine to form -O(CH 2 )i-3O-.
  • R b is independently H, Ci-Ce alkyl, or Cs-Cs cycloalkyl, wherein in R b the alkyd or cycloalkyl is optionally substituted with at least one selected from the group consisting of halogen, -OH, Ci-Ce alkoxy, and heteroaryl; or substituents on two adjacent carbon atoms combine to form - O(CH 2 )I-3O-.
  • the alkyl, alkenyl, alkynyl, cycloalkyl, heteroaryl, heterocyclyl, aryl, or benzyl group is optionally independently substituted with at least one group selected from the group consisting of Ci-Ce alkyd; Ci-Ce alkoxy; Ci-Ce haloalky l; Ci- C6 haloalkoxy; -NH2, -NH(CI-C6 alkyl), -N(Ci-C& alkyl)(Ci-C6 alk d), halogen, -OH; -CN; phenoxy.
  • each occurrence of the heteroary 1 is independently selected from the group consisting of quinolinyl, imidazo[l,2-a]pyridyl, pyridyl, pyrimidyl, pyrazinyl, imidazolyl, thiazolyl, pyrazolyl. isoxazolyl. oxadiazolyl (including 1.2.3-, 1,2,4-. 1,2,5-. and 1,3,4-oxadiazole), and triazolyl (such as 1 ,2,3-triazolyl and 1,2,4-triazolyl).
  • the ring can have carbon atoms and optionally 7 one or more (e.g, 1 to 3) additional heteroatoms independently 7 selected from nitrogen, oxygen, or sulfur.
  • the ring can be saturated or partially saturated, and can be optionally substituted.
  • substituents of compounds are disclosed in groups or in ranges. It is specifically intended that the description include each and every individual subcombination of the members of such groups and ranges.
  • the term “Ci-6 alkyl” is specifically intended to individually disclose Ci, C2, C3, C4, Cs, Ce, Ci-Ce, C1-C5, C1-C4, C1-C3, C1-C2, C2-C6, C2-C5, C2-C4, C2-C3, C3-C6, C3-C5, C3-C4, C 4 -C 6 , C4-C5, and C5-C6 alkyl.
  • treat means reducing the frequency or severin’ with which symptoms of a disease or condition are experienced by a subject by virtue of administering an agent or compound to the subject.
  • ranges throughout this disclosure, various aspects of the present disclosure can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the present disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4. from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6.
  • a range of “about 0.1% to about 5%” or “about 0.1% to 5%” should be interpreted to include not just about 0.1% to about 5%, but also the individual values (e.g, 1%, 2%, 3%, and 4%) and the sub-ranges (e.g., 0.1% to 0.5%, 1.1% to 2.2%, 3.3% to 4.4%) within the indicated range.
  • the statement “about X to Y” has the same meaning as “about X to about Y,” unless indicated otherwise. Tikewise, the statement “about X, Y, or about Z” has the same meaning as “about X, about Y, or about Z,” unless indicated otherwise. This applies regardless of the breadth of the range.
  • the disclosure includes a compound described herein, or a salt, solvate, isotopically labelled derivative, stereoisomer (such as, in a non-limiting example, an enantiomer or diastereoisomer, and/or any mixtures thereof, such as. in a non-limiting example, mixtures in any proportions of enantiomers and/or diastereoisomers thereof), tautomer and any mixtures thereof, and/or geometric isomer and any mixtures thereof:
  • the compound is a compound of formula (la) or (lb), or a salt, solvate, isotopically labelled derivative, stereoisomer, tautomer, or geometric isomer thereof, and any mixtures thereof: wherein in (la) or (lb):
  • R 1 is selected from the group consisting of H, -NR I; 'R 41 ’.
  • -NR 4c (optionally substituted phenyl), and optionally substituted N-linked heterocyclyl;
  • R 2 is selected from the group consisting of phenyl and -N(R 4c )-(CH2)i-3-(optionally substituted phenyl);
  • R a is independently H.
  • R 1 is H. In certain embodiments, R 1 is -NR 4a R 4b . In certain embodiments, R 1 is -NR 4c (optionally substituted phenyl). In certain embodiments, R 1 is optionally substituted N-linked heterocyclyl.
  • R 2 is optionally substituted phenyl. In certain embodiments, R 2 is -N(R 4c )-(CH2)i-3-(optionally substituted phenyl);
  • R 4b is H. In certain embodiments, R 4a is optionally substituted Ci-Ce alkyl. In certain embodiments, R 4b is optionally substituted Cs-Cs cycloalkyd.
  • R 4c is H. In certain embodiments, R 4c is optionally substituted Ci-Ce alkyl. In certain embodiments, R 4c is optionally substituted C3-C8 cycloalky l.
  • R 4d is H. In certain embodiments, R 4d is optionally substituted Ci-Ce alkyl. In certain embodiments, R 4d is optionally substituted C3-C8 cycloalky 1.
  • R 4e is H. In certain embodiments, R 4a is optionally substituted Ci-Ce alkyl. In certain embodiments, R 4e is optionally substituted C3-C8 cycloalky l.
  • R 4f is H. In certain embodiments, R 4f is optionally substituted Ci-Ce alkyl. In certain embodiments, R 4f is optionally substituted C3-C8 cycloalky l.
  • R 4g is H. In certain embodiments, R 4g is optionally substituted Ci-Ce alkyl. In certain embodiments, R 4g is optionally substituted C3-C8 cycloalky l.
  • R 4h is H. In certain embodiments, R 4b is optionally substituted Ci-Ce alkyl. In certain embodiments, R 4h is optionally substituted C3-C8 cycloalkyd.
  • R 1 is selected from the group consisting of H, -NR 4a R 4b , and optionally substituted N-linked heterocyclyl.
  • R 1 is NR 4c (optionally substituted phenyl).
  • R 3 is -NR 4d R 4e .
  • each phenyl in R 2 is independently substituted with at least one C2-C8 alky 1. In other embodiments, each phenyl in R 2 is independently substituted at the ortho-position with at least one C2-C8 alkyl. In other embodiments, each phenyl in R 2 is independently substituted at the meta-position with at least one C2-C8 alkyl. In other embodiments, each phenyl in R 2 is independently substituted at the para-position with at least one C2-C8 alkyl.
  • the N-linked heterocyclyl is aziridinyl. In certain embodiments, the N-linked heterocyclyl is azetidinyl. In certain embodiments, the N-linked heterocyclyl is pyrrolidinyl. In certain embodiments, the N-linked heterocyclyl is pyrazolidinyl. In certain embodiments, the N-linked heterocyclyl is piperidinyl. In certain embodiments, the N-linked heterocyclyl is 1,2,3,6-tetrahydropyridinyl. In certain embodiments, the N-linked heterocyclyl is 1,4-dihydropyridinyl. In certain embodiments, the N-linked heterocyclyl is piperazinyl.
  • the N-linked heterocyclyl is morpholinyl. In certain embodiments, the N-linked heterocyclyl is thiomorpholinyl. In certain embodiments, the N-linked heterocyclyl is homopiperazinyl. In certain embodiments, the N-linked heterocyclyl is or homopiperidinyl.
  • the heteroaryl is imidazolyl. In certain embodiments, the heteroaryl is pyridinyl. In certain embodiments, the heteroaryl is pyrimidinyl. In certain embodiments, the heteroaryl is pyrazinyl. In certain embodiments, the heteroaryl is thienyl. In certain embodiments, the heteroaryl is furyl. In certain embodiments, the heteroaryl is pyrrolyl. In certain embodiments, the heteroaryl is thiazolyl. In certain embodiments, the heteroar l is oxazolyl. In certain embodiments, the heteroaryl is pyrazolyl. In certain embodiments, the heteroaryl is is isothiazolyl.
  • the heteroaryl is 1,2,3- triazolyl. In certain embodiments, the heteroaryl is 1,2,4-triazolyl. In certain embodiments, the heteroaryl is 1.3,4-triazolyl. In certain embodiments, the heteroaryl is tetrazolyl. In certain embodiments, the heteroaryl is 1,2,3-thiadiazolyl. In certain embodiments, the heteroary l is 1,2,3-oxadiazolyl. In certain embodiments, the heteroaryl is 1,3,4-thiadiazolyl. In certain embodiments, the heteroaryl is 1,3,4-oxadiazolyl.
  • Non-limiting examples of the compound of formula (la) or (lb) include:
  • the compound is not: Compound A5: 2-(4-(tert- butyl)phenyl)-5-((4-methylpiperazin-l-yl)methyl)-[l,2,4]triazolo[l,5-a]pyrimidin-7(4H)-one, or a salt, solvate, isotopically labelled derivative, stereoisomer, tautomer, or geometric isomer thereof and any mixtures thereof
  • the compound is not: Compound A10: 5- ((isopropylamino)methyl)-2-((4-isopropylbenzyl)amino)-[l,2,4]triazolo[l,5-a]pyrimidin- 7(4H)-one. or a salt, solvate, isotopically labelled derivative, stereoisomer, tautomer, or geometric isomer thereof, and any mixtures thereof.
  • the compound is not: Compound Bl: 2-((4- isopropylbenzyl)amino)-5-(morpholinomethyl)-[l,2,4]triazolo[l,5-a]pyrimidin-7(4H)-one. or a salt, solvate, isotopically labelled derivative, stereoisomer, tautomer, or geometric isomer thereof, and any mixtures thereof.
  • the compound is not: Compound B2: 2-((4- isopropylbenzyl)amino)-5-methyl-[l,2,4]triazolo[l,5-a]pyrimidin-7(4H)-one, or a salt, solvate, isotopically labelled derivative, stereoisomer, tautomer, or geometric isomer thereof, and any mixtures thereof.
  • the compound is not: Compound B3: 5- ((phenylamino)methyl)-2-(o-tolyl)-[l,2,4]triazolo[l,5-a]pyrimidin-7(4H)-one, or a salt, solvate, isotopically labelled derivative, stereoisomer, tautomer, or geometric isomer thereof, and any mixtures thereof.
  • the compound is not: Compound B4: 2-((4- isopropylbenzyl)amino)-5-(pyrrolidin-l-ylmethyl)-[l,2,4]triazolo[l,5-a]pyrimidin-7(4H)-one, or a salt, solvate, isotopically labelled derivative, stereoisomer, tautomer, or geometric isomer thereof, and any mixtures thereof.
  • the compound is not 2-phenyl-5-((propylamino)methyl)-
  • the compound is not 5-((4-methylpiperidin-l-yl)methyl)-2-phenyl-
  • the compound is not 5-((benzyl(methyl)amino)methyl)-2-phenyl-
  • the compound is not 5-((benzyl(2-hydroxyethyl)amino)methyl)-2-phenyl-
  • the compound is not 2-(4-chlorophenyl)-5-((3,4-dihydroisoquinolin-2(lH)- yl)methyl)-[l,2,4]tnazolo[1.5-a]pyrimidin-7(4H)-one, or a salt, solvate, isotopically labelled derivative, stereoisomer, tautomer, or geometric isomer thereof, and any mixtures thereof.
  • the compound is not 5-(((2-methoxyphenyl)amino)methyl)-2-(p-tolyl)-
  • the compound is not 2-(p-tolyl)-5-((m-tolylamino)methyl)-[l,2,4]triazolo[l,5- a]pyrimidin-7(4H)-one, or a salt, solvate, isotopically labelled derivative, stereoisomer, tautomer, or geometric isomer thereof, and any mixtures thereof.
  • the compound is not 2-(p-tolyl)-5-((p-tolylamino)methyl)-[l,2,4]triazolo[l,5-a]pyrimidin-7(4H)- one, or a salt, solvate, isotopically labelled derivative, stereoisomer, tautomer, or geometric isomer thereof, and any mixtures thereof.
  • the compound is not 2-(p- tolyl)-5-((o-tolylamino)methyl)-[l,2,4]triazolo[l,5-a]pyrimidin-7(4H)-one.
  • the compound is not 2-(4-methoxyphenyl)-5- (piperidin-l-ylmethyl)-[l,2,4]triazolo[l,5-a]pyrimidin-7(4H)-one, or a salt, solvate, isotopically labelled derivative, stereoisomer, tautomer, or geometric isomer thereof, and any mixtures thereof.
  • the compound is not 2-(4-methoxyphenyl)-5- (morpholinomethyl)-[l,2,4]triazolo[l,5-a]pyrimidin-7(4H)-one, or a salt, solvate, isotopically labelled derivative, stereoisomer, tautomer, or geometric isomer thereof, and any mixtures thereof.
  • the compound is not 5-(((2,4- dimethoxyphenyl)amino)methyl)-2-(4-methoxyphenyl)-[l,2,4]triazolo[1.5-a]pyrimidin- 7(4H)-one, or a salt, solvate, isotopically labelled derivative, stereoisomer, tautomer, or geometric isomer thereof, and any mixtures thereof.
  • the compound is not 2-(4-(tert-buty l)phenyl)-5-((propylamino)methyl)-[l,2,4]triazolo[l,5-a]pyrimidin- 7(4H)-one, or a salt, solvate, isotopically labelled derivative, stereoisomer, tautomer, or geometric isomer thereof, and any mixtures thereof.
  • the compound is not 2-(3,4-dimethylphenyl)-5-((p-tolylamino)methyl)-[l ,2,4]triazolo[l ,5-a]pyrimidin- 7(4H)-one, or a salt, solvate, isotopically labelled derivative, stereoisomer, tautomer, or geometric isomer thereof, and any mixtures thereof.
  • the compound is not 5-methyl-2-(phenylamino)-[1.2.4]triazolo[l,5-a]pyrimidin-7-ol, or a salt, solvate, isotopically labelled derivative, stereoisomer, tautomer, or geometric isomer thereof, and any mixtures thereof.
  • the compound is not 5-methyl-2-phenyl-
  • the compound is not 2-(benzylamino)-5-(morpholinomethyl)-
  • the compound is not 2-((3,4-dimethoxybenzyl)amino)-5-(morpholinomethyl)-
  • the compound is not methyl 2-((2-((4-(tert-butyl)benzyl)amino)-5- (morpholinomethyl)-[l,2,4]triazolo[1.5-a]pyrimidin-7-yl)oxy)acetate, or a salt, solvate, isotopically labelled derivative, stereoisomer, tautomer, or geometric isomer thereof, and any mixtures thereof.
  • the compound is not 7-(benzyloxy)-N-(4-(tert- butyl)benzyl)-5-(morphohnomethyl)-[l,2,4]triazolo[l,5-a]pyrimidin-2-amine, or a salt, solvate, isotopically labelled derivative, stereoisomer, tautomer, or geometric isomer thereof, and any mixtures thereof.
  • the compound is not 2-((2-((4-(tert- butyl)benzyl)amino)-5-(morpholinomethyl)-[l,2,4]triazolo[l,5-a]pyrimidin-7-yl)oxy)-N,N- dimethylacetamide.
  • the compound of the disclosure is any compound disclosed herein, or a salt, solvate, prodrug, isotopically labelled, stereoisomer, any mixture of stereoisomers, tautomer, and/or any mixture of tautomers thereof.
  • the compounds of the disclosure may possess one or more stereocenters, and each stereocenter may exist independently in either the (R)- or ( ⁇ -configuration.
  • compounds described herein are present in optically active or racemic forms.
  • the compounds described herein encompass racemic, optically active, regioisomeric and stereoisomeric forms, or combinations thereof that possess the therapeutically useful properties described herein.
  • Preparation of optically active forms is achieved in any suitable manner, including, by way of non-limiting example, by resolution of the racemic form with recrystallization techniques, synthesis from optically active starting materials, chiral synthesis, or chromatographic separation using a chiral stationary phase.
  • a compound illustrated herein by the racemic formula further represents either of the two enantiomers or any mixtures thereof, or in the case where two or more chiral centers are present, all diastereomers or any mixtures thereof.
  • the compounds of the disclosure exist as tautomers. All tautomers are included within the scope of the compounds recited herein.
  • Compounds described herein also include isotopically labeled compounds wherein one or more atoms is replaced by an atom having the same atomic number, but an atomic mass or mass number different from the atomic mass or mass number usually found in nature.
  • isotopes suitable for inclusion in the compounds described herein include and are not limited to 2 H, 3 H, n C, 13 C, 14 C, 36 C1, 18 F, 123 I, 125 I, 1? N, 15 N, 15 O, 17 O, 18 0, 32 P, and 35 S.
  • substitution with heavier isotopes such as deuterium affords greater chemical stability.
  • Isotopically labeled compounds are prepared by any suitable method or by processes using an appropriate isotopically labeled reagent in place of the non-labeled reagent otherwise employed.
  • the compounds described herein are labeled by other means, including, but not limited to, the use of chromophores or fluorescent moieties, bioluminescent labels, or chemiluminescent labels.
  • the compounds described herein may form salts with acids or bases, and such salts are included in the present disclosure.
  • salts embraces addition salts of free acids or bases that are useful within the methods of the disclosure.
  • pharmaceutically acceptable salt refers to salts that possess toxicity profiles within a range that affords utility in pharmaceutical applications.
  • the salts are pharmaceutically acceptable salts.
  • Pharmaceutically unacceptable salts may nonetheless possess properties such as high crystallinity, which have utility in the practice of the present disclosure, such as for example utility in process of synthesis, purification or formulation of compounds useful within the methods of the disclosure.
  • Suitable pharmaceutically acceptable acid addition salts may be prepared from an inorganic acid or from an organic acid.
  • inorganic acids include sulfate, hydrogen sulfate, hydrochloric, hydrobromic, hydriodic, nitric, carbonic, sulfuric, and phosphoric acids (including hydrogen phosphate and dihydrogen phosphate).
  • Appropriate organic acids may be selected from aliphatic, cycloaliphatic, aromatic, araliphatic, heterocyclic, carboxylic and sulfonic classes of organic acids, examples of which include formic, acetic, propionic, succinic, glycolic, gluconic, lactic, malic, tartaric, citric, ascorbic, glucuronic, maleic, fumaric, pyruvic, aspartic, glutamic, benzoic, anthranilic, 4-hydroxybenzoic, phenylacetic, mandelic, embonic (or pamoic), methanesulfonic, ethanesulfonic, benzenesulfonic, pantothenic, sulfanilic.
  • Suitable pharmaceutically acceptable base addition salts of compounds of the disclosure include, for example, ammonium salts and metallic salts including alkali metal, alkaline earth metal and transition metal salts such as, for example, calcium, magnesium, potassium, sodium and zinc salts.
  • Pharmaceutically acceptable base addition salts also include organic salts made from basic amines such as, for example, A ⁇ V'-di benzyl ethylenediamine. chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (or A- methylglucamine) and procaine. All of these salts may be prepared from the corresponding compound by reacting, for example, the appropriate acid or base with the compound.
  • the compounds of the disclosure are useful within the methods of the disclosure in combination with one or more additional agents useful for treating, ameliorating, and/or preventing a disease or disorder contemplated herein.
  • additional agents may comprise compounds or compositions identified herein, or compounds (e. , commercially available compounds) known to treat, prevent, or reduce the symptoms of a disease or disorder contemplated herein.
  • a synergistic effect may be calculated, for example, using suitable methods such as, for example, the Sigmoid-Emax equation (Holford & Scheiner, 1981, Clin. Pharmacokinet. 6:429-453), the equation of Loewe additivity (Loewe & Muischnek, 1926, Arch. Exp. Pathol Pharmacol. 114: 313-326) and the median-effect equation (Chou & Talalay, 1984, Adv. Enzyme Regul. 22:27-55).
  • Each equation referred to elsewhere herein may be applied to experimental data to generate a corresponding graph to aid in assessing the effects of the drug combination.
  • the corresponding graphs associated with the equations referred to elsewhere herein are the concentration-effect curve, isobologram curve and combination index curve, respectively.
  • Preparation of the compounds can involve protection and deprotection of various chemical groups.
  • the need for protection and deprotection and the selection of appropriate protecting groups can be readily determined by one skilled in the art.
  • the chemistry of protecting groups can be found, for example, in Greene, et al., Protective Groups in Organic Synthesis, 2d. Ed. (Wiley & Sons, 1991), the entire disclosure of which is incorporated by reference herein for all purposes.
  • the disclosure provides a method of increasing antifungal activity of an antifungal agent used to treat, ameliorate, and/or prevent a fungal infection in a subject, wherein the antifungal agent disrupts synthesis and/or activity of ergosterol in the fungus, the method comprising administering a therapeutically effective amount of a compound of the disclosure to a subject being administered the antifungal agent.
  • the disclosure provides a method of reducing, minimizing, and/or eliminating at least one toxicity effect of an antifungal agent used to treat, ameliorate, and/or prevent a fungal infection in a subject, wherein the antifungal agent disrupts synthesis and/or activity of ergosterol in the fungus, the method comprising administering a therapeutically effective amount of a compound of the disclosure to a subject being administered the antifungal agent.
  • the disclosure provides a method of reducing, minimizing, and/or preventing drug resistance against an antifungal agent used to treat, ameliorate, and/or prevent a fungal infection in a subject, wherein the antifungal agent disrupts synthesis and/or activity of ergosterol in the fungus, the method comprising administering a therapeutically effective amount of a compound of the disclosure to a subject being administered the antifungal agent.
  • the disclosure provides a method of sensitizing a fungus to an antifungal agent administered to a subject to treat, ameliorate, and/or prevent the fungal infection in the subject, wherein the antifungal agent disrupts synthesis and/or activity of ergosterol in the fungus, the method comprising administering a therapeutically effective amount of a compound of the disclosure to a subject being administered the antifungal agent.
  • the fungus comprises at least one of Candida (such as but not limited to C. albicans, C. glabrata, C. auris, and C. parapsilosis'), Aspergillus (such as but not limited to A. fumigatus and A. terreus), Histoplasma, Cryptococcus, and Mucor.
  • the compound of the disclosure is administered to the subject in a pharmaceutically acceptable composition.
  • the compound of the disclosure and the antifungal agent are co-formulated. In certain embodiments, administration of the compound of the disclosure and the antifungal agent results in less likely drug resistance occurrence in the subject as compared to an equivalent subject that is administered the antifungal agent in the absence of the compound of the disclosure.
  • administration of the compound of the disclosure and the antifungal agent allows for administration of an amount of the antifungal agent that is lower than the corresponding amount of the antifungal agent that has to be administered in the absence of the compound of the disclosure to achieve equivalent treatment or prevention of the fungal infection.
  • the compound of the disclosure inhibits fungal PanK selectively over a human PanK enzyme.
  • the subject is a mammal. In other embodiments, the mammal is a human.
  • compositions comprising at least one compound of the disclosure or a salt or solvate thereof, which are useful to practice methods of the disclosure.
  • a pharmaceutical composition may consist of at least one compound of the disclosure or a salt or solvate thereof, in a form suitable for administration to a subject, or the pharmaceutical composition may comprise at least one compound of the disclosure or a salt or solvate thereof, and one or more pharmaceutically acceptable carriers, one or more additional ingredients, or any combinations of these.
  • At least one compound of the disclosure may be present in the pharmaceutical composition in the form of a physiologically acceptable salt, such as in combination with a physiologically acceptable cation or anion, as is well known in the art.
  • the pharmaceutical compositions useful for practicing the method of the disclosure may be administered to deliver a dose of between 1 ng/kg/day and 100 mg/kg/day. In other embodiments, the pharmaceutical compositions useful for practicing the disclosure may be administered to deliver a dose of between 1 ng/kg/day and 1,000 mg/kg/day.
  • compositions of the disclosure will vary, depending upon the identity, size, and condition of the subject treated and further depending upon the route by which the composition is to be administered.
  • the composition may comprise between 0.1% and 100% (w/w) active ingredient.
  • compositions that are useful in the methods of the disclosure may be suitably developed for nasal, inhalational, oral, rectal, vaginal, pleural, peritoneal, parenteral, topical, transdermal, pulmonary, intranasal, buccal, ophthalmic, epidural, intrathecal, intravenous, or another route of administration.
  • a composition useful within the methods of the disclosure may be directly administered to the brain, the brainstem, or any other part of the central nervous system of a mammal or bird.
  • Other contemplated formulations include projected nanoparticles, microspheres, liposomal preparations, coated particles, polymer conjugates, resealed erythrocytes containing the active ingredient, and immunologically- based formulations.
  • compositions of the disclosure are part of a pharmaceutical matrix, which allows for manipulation of insoluble materials and improvement of the bioavailability thereof, development of controlled or sustained release products, and generation of homogeneous compositions.
  • a pharmaceutical matrix may be prepared using hot melt extrusion, solid solutions, solid dispersions, size reduction technologies, molecular complexes (e.g., cyclodextrins, and others), microparticulate, and particle and formulation coating processes. Amorphous or cry stalline phases may be used in such processes.
  • the route(s) of administration will be readily apparent to the skilled artisan and will depend upon any number of factors including the type and severity of the disease being treated, the type and age of the veterinary or human patient being treated, and the like.
  • compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology' and pharmaceutics.
  • preparatory methods include the step of bringing the active ingredient into association with a carrier or one or more other accessory ingredients, and then, if necessary or desirable, shaping or packaging the product into a desired single-dose or multi-dose unit.
  • a “unit dose” is a discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient.
  • the amount of the active ingredient is generally equal to the dosage of the active ingredient that would be administered to a subject or a convenient fraction of such a dosage such as, for example, one-half or one- third of such a dosage.
  • the unit dosage fonn may be for a single daily dose or one of multiple daily doses (e.g. about 1 to 4 or more times per day). When multiple daily doses are used, the unit dosage form may be the same or different for each dose.
  • compositions suitable for ethical administration to humans are principally directed to pharmaceutical compositions suitable for ethical administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to animals of all sorts. Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and perform such modification with merely ordinary, if any, experimentation. Subjects to which administration of the pharmaceutical compositions of the disclosure is contemplated include, but are not limited to, humans and other primates, mammals including commercially relevant mammals such as cattle, pigs, horses, sheep, cats, and dogs.
  • compositions of the disclosure are formulated using one or more pharmaceutically acceptable excipients or carriers.
  • the pharmaceutical compositions of the disclosure comprise a therapeutically effective amount of at least one compound of the disclosure and a pharmaceutically acceptable carrier.
  • Pharmaceutically acceptable carriers include, but are not limited to, glycerol, water, saline, ethanol, recombinant human albumin (e.g., RECOMB UMIN®), solubilized gelatins (e.g., GELOFUSINE®), and other pharmaceutically acceptable salt solutions such as phosphates and salts of organic acids. Examples of these and other pharmaceutically acceptable carriers are described in Remington’s Pharmaceutical Sciences (1991, Mack Publication Co., New Jersey).
  • the carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), recombinant human albumin, solubilized gelatins, suitable mixtures thereof, and vegetable oils.
  • the proper fluidity may be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Prevention of the action of microorganisms may be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars, sodium chloride, or polyalcohols such as mannitol and sorbitol, are included in the composition.
  • Prolonged absorption of the injectable compositions may be brought about by including in the composition an agent that delays absorption, for example, aluminum monostearate or gelatin.
  • Formulations may be employed in admixtures with conventional excipients, i.e.. pharmaceutically acceptable organic or inorganic carrier substances suitable for oral, parenteral, nasal, inhalational, intravenous, subcutaneous, transdermal enteral, or any other suitable mode of administration, known to the art.
  • the pharmaceutical preparations may be sterilized and if desired mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure buffers, coloring, flavoring, and/or fragrance-conferring substances and the like.
  • additional ingredients include, but are not limited to, one or more ingredients that may be used as a pharmaceutical carrier.
  • the composition of the disclosure may comprise a preservative from about 0.005% to 2.0% by total weight of the composition.
  • the preservative is used to prevent spoilage in the case of exposure to contaminants in the environment.
  • Examples of preservatives useful in accordance with the disclosure include but are not limited to those selected from the group consisting of benzyl alcohol, sorbic acid, parabens, imidurea and any combinations thereof.
  • One such preservative is a combination of about 0.5% to 2.0% benzyl alcohol and 0.05-0.5% sorbic acid.
  • the composition may include an antioxidant and a chelating agent that inhibit the degradation of the compound.
  • Antioxidants for some compounds are BHT, BHA, alphatocopherol and ascorbic acid in the exemplary range of about 0.01% to 0.3%, or BHT in the range of 0.03% to 0.1% by weight by total weight of the composition.
  • the chelating agent may be present in an amount of from 0.01 % to 0.5% by weight by total weight of the composition.
  • Exemplary chelating agents include edetate salts (e.g. disodium edetate) and citric acid in the weight range of about 0.01% to 0.20%, or in the range of 0.02% to 0. 10% byweight by total weight of the composition.
  • the chelating agent is useful for chelating metal ions in the composition that may be detrimental to the shelf life of the formulation. While BHT and disodium edetate are exemplary antioxidant and chelating agent, respectively, for some compounds, other suitable and equivalent antioxidants and chelating agents may be substituted therefore as would be known to those skilled in the art.
  • Liquid suspensions may be prepared using conventional methods to achieve suspension of the active ingredient in an aqueous or oily vehicle.
  • Aqueous vehicles include, for example, water, and isotonic saline.
  • Oily vehicles include, for example, almond oil, oily esters, ethyl alcohol, vegetable oils such as arachis, olive, sesame, or coconut oil. fractionated vegetable oils, and mineral oils such as liquid paraffin.
  • Liquid suspensions may further comprise one or more additional ingredients including, but not limited to, suspending agents. dispersing or weting agents, emulsifying agents, demulcents, preservatives, buffers, salts, flavorings, coloring agents, and sweetening agents.
  • Oily suspensions may further comprise a thickening agent.
  • suspending agents include, but are not limited to, sorbitol syrup, hydrogenated edible fats, sodium alginate, polyvinylpyrrolidone, gum tragacanth, gum acacia, and cellulose derivatives such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethyl cellulose.
  • Known dispersing or weting agents include, but are not limited to, naturally-occurring phosphatides such as lecithin, condensation products of an alkylene oxide with a fatty acid, with a long chain aliphatic alcohol, with a partial ester derived from a fatty acid and a hexitol, or with a partial ester derived from a fatty' acid and a hexitol anhydride (e.g., polyoxyethylene stearate, heptadecaethyleneoxycetanol, polyoxyethylene sorbitol monooleate, and polyoxyethylene sorbitan monooleate, respectively).
  • naturally-occurring phosphatides such as lecithin
  • condensation products of an alkylene oxide with a fatty acid with a long chain aliphatic alcohol
  • with a partial ester derived from a fatty acid and a hexitol or with a partial ester derived from a fatty' acid and a hex
  • emulsifying agents include, but are not limited to, lecithin, acacia, and ionic or non-ionic surfactants.
  • Known preservatives include, but are not limited to, methyl, ethyl, or w-propyl para-hydroxybenzoates, ascorbic acid, and sorbic acid.
  • Known sweetening agents include, for example, glycerol, propylene glycol, sorbitol, sucrose, and saccharin.
  • Liquid solutions of the active ingredient in aqueous or oily solvents may be prepared in substantially the same manner as liquid suspensions, the primary difference being that the active ingredient is dissolved, rather than suspended in the solvent.
  • an “oily” liquid is one which comprises a carbon-containing liquid molecule and which exhibits a less polar character than water.
  • Liquid solutions of the pharmaceutical composition of the disclosure may comprise each of the components described with regard to liquid suspensions, it being understood that suspending agents will not necessarily aid dissolution of the active ingredient in the solvent.
  • Aqueous solvents include, for example, water, and isotonic saline.
  • Oily solvents include, for example, almond oil, oily esters, ethyl alcohol, vegetable oils such as arachis, olive, sesame, or coconut oil, fractionated vegetable oils, and mineral oils such as liquid paraffin.
  • Pow dered and granular formulations of a pharmaceutical preparation of the disclosure may be prepared using known methods. Such formulations may be administered directly to a subject, used, for example, to form tablets, to fill capsules, or to prepare an aqueous or oily suspension or solution by addition of an aqueous or oily vehicle thereto. Each of these formulations may further comprise one or more of dispersing or wheting agent, a suspending agent, ionic and non-ionic surfactants, and a preservative. Additional excipients, such as fillers and sweetening, flavoring, or coloring agents, may also be included in these formulations.
  • a pharmaceutical composition of the disclosure may also be prepared, packaged, or sold in the form of oil-in-water emulsion or a water-in-oil emulsion.
  • the oily phase may be a vegetable oil such as olive or arachis oil, a mineral oil such as liquid paraffin, or a combination of these.
  • compositions may further comprise one or more emulsifying agents such as naturally occurring gums such as gum acacia or gum tragacanth, naturally- occurring phosphatides such as soybean or lecithin phosphatide, esters or partial esters derived from combinations of fatty acids and hexitol anhydrides such as sorbitan monooleate, and condensation products of such partial esters with ethylene oxide such as polyoxyethylene sorbitan monooleate.
  • emulsions may also contain additional ingredients including, for example, sweetening or flavoring agents.
  • Methods for impregnating or coating a material with a chemical composition include, but are not limited to methods of depositing or binding a chemical composition onto a surface, methods of incorporating a chemical composition into the structure of a material during the synthesis of the material (i. e. , such as with a physiologically degradable material), and methods of absorbing an aqueous or oily solution or suspension into an absorbent material, with or without subsequent drying.
  • Methods for mixing components include physical milling, the use of pellets in solid and suspension formulations and mixing in a transdermal patch, as known to those skilled in the art.
  • the regimen of administration may affect what constitutes an effective amount.
  • the therapeutic formulations may be administered to the patient either prior to or after the onset of a disease or disorder. Further, several divided dosages, as well as staggered dosages may be administered daily or sequentially, or the dose may be continuously infused, or may be a bolus injection. Further, the dosages of the therapeutic formulations may be proportionally increased or decreased as indicated by the exigencies of the therapeutic or prophylactic situation.
  • compositions of the present disclosure may be carried out using known procedures, at dosages and for periods of time effective to treat a disease or disorder contemplated herein.
  • An effective amount of the therapeutic compound necessary to achieve a therapeutic effect may vary according to factors such as the activity of the particular compound employed; the time of administration; the rate of excretion of the compound; the duration of the treatment; other drugs, compounds or materials used in combination with the compound; the state of the disease or disorder, age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well-known in the medical arts. Dosage regimens may be adjusted to provide the optimum therapeutic response.
  • an effective dose range for a therapeutic compound of the disclosure is from about 0.01 mg/kg to 100 mg/kg of body weight/per day.
  • One of ordinary skill in the art would be able to study the relevant factors and make the determination regarding the effective amount of the therapeutic compound without undue experimentation.
  • the compound may be administered to an animal as frequently as several times daily, or it may be administered less frequently, such as once a day. once a week, once every two weeks, once a month, or even less frequently, such as once every several months or even once a year or less. It is understood that the amount of compound dosed per day may be administered, in non-limiting examples, every day, every other day, even,' 2 days, every' 3 days, every 4 days, or every 5 days. For example, with every other day administration, a 5 mg per day dose may be initiated on Monday with a first subsequent 5 mg per day dose administered on Wednesday, a second subsequent 5 mg per day dose administered on Friday, and so on.
  • the frequency of the dose is readily apparent to the skilled artisan and depends upon a number of factors, such as, but not limited to, type and severity of the disease being treated, and type and age of the animal.
  • Actual dosage levels of the active ingredients in the pharmaceutical compositions of this disclosure may be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.
  • a medical doctor e.g., physician or veterinarian, having ordinary skill in the art may readily determine and prescribe the effective amount of the pharmaceutical composition required.
  • physician or veterinarian could start doses of the compounds of the disclosure employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the patients to be treated; each unit containing a predetermined quantity of therapeutic compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical vehicle.
  • the dosage unit forms of the disclosure are dictated by and directly dependent on (a) the unique characteristics of the therapeutic compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding/formulating such a therapeutic compound for the treatment of a disease or disorder in a patient.
  • compositions of the disclosure are administered to the patient in dosages that range from one to five times per day or more.
  • the compositions of the disclosure are administered to the patient in range of dosages that include, but are not limited to, once every day, every two days, every three days to once a week, and once every' two weeks.
  • the frequency of administration of the various combination compositions of the disclosure will vary from subject to subject depending on many factors including, but not limited to, age, disease or disorder to be treated, gender, overall health, and other factors.
  • the disclosure should not be construed to be limited to any particular dosage regime and the precise dosage and composition to be administered to any patient will be determined by the attending physician taking all other factors about the patient into account.
  • Compounds of the disclosure for administration may be in the range of from about 1 pg to about 7,500 mg, about 20 pg to about 7,000 mg, about 40 pg to about 6,500 mg, about 80 pg to about 6.000 mg, about 100 pg to about 5,500 mg, about 200 pg to about 5,000 mg, about 400 pg to about 4.000 mg.
  • the dose of a compound of the disclosure is from about 0.5 pg and about 5,000 mg. In some embodiments, a dose of a compound of the disclosure used in compositions described herein is less than about 5,000 mg, or less than about 4,000 mg, or less than about 3,000 mg, or less than about 2.000 mg, or less than about 1.000 mg, or less than about 800 mg, or less than about 600 mg, or less than about 500 mg, or less than about 200 mg, or less than about 50 mg. Similarly, in some embodiments, a dose of a second compound as described herein is less than about 1,000 mg, or less than about 800 mg, or less than about 600 mg, or less than about 500 mg, or less than about 400 mg.
  • the present disclosure is directed to a packaged pharmaceutical composition
  • a packaged pharmaceutical composition comprising a container holding a therapeutically effective amount of a compound of the disclosure, alone or in combination with a second pharmaceutical agent; and instructions for using the compound to treat, prevent, or reduce one or more symptoms of a disease or disorder in a patient.
  • the term “container’' includes any receptacle for holding the pharmaceutical composition or for managing stability or water uptake.
  • the container is the packaging that contains the pharmaceutical composition, such as liquid (solution and suspension), semisolid, lyophilized solid, solution and powder or lyophilized formulation present in dual chambers.
  • the container is not the packaging that contains the pharmaceutical composition, i.e., the container is a receptacle, such as a box or vial that contains the packaged pharmaceutical composition or unpackaged pharmaceutical composition and the instructions for use of the pharmaceutical composition.
  • packaging techniques are well known in the art.
  • the instructions for use of the pharmaceutical composition may be contained on the packaging containing the pharmaceutical composition, and as such the instructions form an increased functional relationship to the packaged product.
  • the instructions may contain information pertaining to the compound’s abil i ty to perform its intended function, e.g., treating, preventing, or reducing a disease or disorder in a patient.
  • Routes of administration of any of the compositions of the disclosure include inhalational, oral, nasal, rectal, parenteral, sublingual, transdermal, transmucosal (e.g., sublingual, lingual, (trans)buccal, (trans)urethral, vaginal (e.g., trans- and perivaginally), (intra)nasal, and (trans)rectal).
  • compositions and dosage forms include, for example, tablets, capsules, caplets, pills, gel caps, troches, emulsions, dispersions, suspensions, solutions, syrups, granules, beads, transdermal patches, gels, powders, pellets, magmas, lozenges, creams, pastes, plasters, lotions, discs, suppositories, liquid sprays for nasal or oral administration, dry powder or aerosolized formulations for inhalation, compositions and formulations for intravesical administration and the like. It should be understood that the formulations and compositions that would be useful in the present disclosure are not limited to the particular formulations and compositions that are described herein.
  • compositions intended for oral use may be prepared according to any method known in the art and such compositions may contain one or more agents selected from the group consisting of inert, non-toxic, generally recognized as safe (GRAS) pharmaceutically excipients which are suitable for the manufacture of tablets.
  • GRAS inert, non-toxic, generally recognized as safe
  • excipients include, for example an inert diluent such as lactose: granulating and disintegrating agents such as cornstarch; binding agents such as starch; and lubricating agents such as magnesium stearate.
  • Tablets may be non-coated or they may be coated using known methods to achieve delayed disintegration in the gastrointestinal tract of a subject, thereby providing sustained release and absorption of the active ingredient.
  • a material such as glyceryl monostearate or glyceryl distearate may be used to coat tablets.
  • tablets may be coated using methods described in U.S. Patents Nos. 4,256,108; 4,160,452; and 4,265,874 to form osmotically controlled release tablets.
  • Tablets may further comprise a sweetening agent, a flavoring agent, a coloring agent, a preservative, or some combination of these in order to provide for pharmaceutically elegant and palatable preparation.
  • Hard capsules comprising the active ingredient may be made using a physiologically degradable composition, such as gelatin.
  • the capsules comprise the active ingredient, and may further comprise additional ingredients including, for example, an inert solid diluent such as calcium carbonate, calcium phosphate, or kaolin.
  • parenteral administration of a pharmaceutical composition includes any route of administration characterized by physical breaching of a tissue of a subject and administration of the pharmaceutical composition through the breach in the tissue.
  • Parenteral administration thus includes, but is not limited to. administration of a pharmaceutical composition by injection of the composition, by application of the composition through a surgical incision, by application of the composition through a tissue-penetrating non-surgical wound, and the like.
  • parenteral administration is contemplated to include, but is not limited to, subcutaneous, intravenous, intraperitoneal, intramuscular, intrastemal injection, and kidney dialytic infusion techniques.
  • Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations. Such formulations may further comprise one or more additional ingredients including, but not limited to. suspending, stabilizing, or dispersing agents.
  • the active ingredient is provided in dry (z.e., powder or granular) form for reconstitution with a suitable vehicle (e.g, sterile pyrogen-free water) prior to parenteral administration of the reconstituted composition.
  • compositions may be prepared, packaged, or sold in the form of a sterile injectable aqueous or oily suspension or solution.
  • This suspension or solution may be formulated according to the known art, and may comprise, in addition to the active ingredient, additional ingredients such as the dispersing agents, wetting agents, or suspending agents described herein.
  • Such sterile injectable formulations may be prepared using a nontoxic parenterally acceptable diluent or solvent, such as water or 1,3-butanediol, for example.
  • Other acceptable diluents and solvents include, but are not limited to, Ringer’s solution, isotonic sodium chloride solution, and fixed oils such as synthetic mono- or di-glycerides.
  • compositions for sustained release or implantation may comprise pharmaceutically acceptable polymeric or hydrophobic materials such as an emulsion, an ion exchange resin, a sparingly soluble polymer, or a sparingly soluble salt.
  • stratum comeum layer of the epidermis An obstacle for topical administration of pharmaceuticals is the stratum comeum layer of the epidermis.
  • the stratum comeum is a highly resistant layer comprised of protein, cholesterol, sphingolipids, free fatty acids and various other lipids, and includes cornified and living cells.
  • One of the factors that limit the penetration rate (flux) of a compound through the stratum comeum is the amount of the active substance that can be loaded or applied onto the skin surface. The greater the amount of active substance which is applied per unit of area of the skin, the greater the concentration gradient between the skin surface and the lower layers of the skin, and in turn the greater the diffusion force of the active substance through the skin. Therefore, a formulation containing a greater concentration of the active substance is more likely to result in penetration of the active substance through the skin, and more of it, and at a more consistent rate, than a formulation having a lesser concentration, all other things being equal.
  • Formulations suitable for topical administration include, but are not limited to, liquid or semi-liquid preparations such as liniments, lotions, oil-in-water or water-in-oil emulsions such as creams, ointments or pastes, and solutions or suspensions.
  • Topically administrable formulations may, for example, comprise from about 1% to about 10% (w/w) active ingredient, although the concentration of the active ingredient may be as high as the solubility limit of the active ingredient in the solvent.
  • Formulations for topical administration may further comprise one or more of the additional ingredients described herein.
  • One acceptable vehicle for topical deliver ⁇ ’ of some of the compositions of the disclosure may contain liposomes.
  • the composition of the liposomes and their use are known in the art (z.e., U.S. Patent No. 6,323,219).
  • the topically active pharmaceutical composition may be optionally combined with other ingredients such as adjuvants, anti-oxidants, chelating agents, surfactants, foaming agents, wetting agents, emulsifying agents, viscosifiers. buffering agents, preservatives, and the like.
  • a permeation or penetration enhancer is included in the composition and is effective in improving the percutaneous penetration of the active ingredient into and through the stratum comeum with respect to a composition lacking the permeation enhancer.
  • the topically active pharmaceutical composition should be applied in an amount effective to affect desired changes.
  • amount effective shall mean an amount sufficient to cover the region of skin surface where a change is desired.
  • An active compound should be present in the amount of from about 0.0001% to about 15% by weight volume of the composition. For example, it should be present in an amount from about 0.0005% to about 5% of the composition; for example, it should be present in an amount of from about 0.001% to about 1% of the composition.
  • Such compounds may be synthetically-or naturally derived.
  • a pharmaceutical composition of the disclosure may be prepared, packaged, or sold in a formulation suitable for buccal administration.
  • Such formulations may, for example, be in the form of tablets or lozenges made using conventional methods, and may contain, for example, 0.1 to 20% (w/w) of the active ingredient, the balance comprising an orally dissolvable or degradable composition and, optionally, one or more of the additional ingredients described herein.
  • formulations suitable for buccal administration may comprise a powder or an aerosolized or atomized solution or suspension comprising the active ingredient.
  • Such powdered, aerosolized, or aerosolized formulations, when dispersed may have an average particle or droplet size in the range from about 0.
  • a pharmaceutical composition of the disclosure may be prepared, packaged, or sold in a formulation suitable for rectal administration.
  • a composition may be in the form of, for example, a suppository, a retention enema preparation, and a solution for rectal or colonic irrigation.
  • Suppository formulations may be made by combining the active ingredient with a non-irritating pharmaceutically acceptable excipient which is solid at ordinary room temperature (i.e., about 20°C) and which is liquid at the rectal temperature of the subject (z.e., about 37°C in a healthy human).
  • Suitable pharmaceutically acceptable excipients include, but are not limited to, cocoa butter, polyethylene glycols, and various glycerides.
  • Suppository formulations may further comprise various additional ingredients including, but not limited to, antioxidants, and preservatives.
  • Retention enema preparations or solutions for rectal or colonic irrigation may be made by combining the active ingredient with a pharmaceutically acceptable liquid earner.
  • enema preparations may be administered using, and may be packaged within, a delivery device adapted to the rectal anatomy of the subject.
  • Enema preparations may further comprise various additional ingredients including, but not limited to. antioxidants, and preservatives.
  • Additional dosage forms of this disclosure include dosage forms as described in U.S. Patents Nos. 6,340,475, 6,488,962, 6,451,808. 5,972,389, 5,582,837, and 5,007,790. Additional dosage forms of this disclosure also include dosage forms as described in U.S. Patent Applications Nos. 20030147952, 20030104062, 20030104053, 20030044466, 20030039688, and 20020051820. Additional dosage forms of this disclosure also include dosage forms as described in PCT Applications Nos.
  • compositions and/or formulations of the present disclosure may be, but are not limited to, short-term, rapid-offset, as well as controlled, for example, sustained release, delayed release and pulsatile release formulations.
  • sustained release is used in its conventional sense to refer to a drug formulation that provides for gradual release of a drug over an extended period of time, and that may. although not necessarily, result in substantially constant blood levels of a drug over an extended time period.
  • the period of time may be as long as a month or more and should be a release which is longer that the same amount of agent administered in bolus form.
  • the compounds may be formulated with a suitable polymer or hydrophobic material which provides sustained release properties to the compounds.
  • the compounds for use the method of the disclosure may be administered in the form of microparticles, for example, by injection or in the form of wafers or discs by implantation.
  • the compounds useful within the disclosure are administered to a subject, alone or in combination with another pharmaceutical agent, using a sustained release formulation.
  • delayed release is used herein in its conventional sense to refer to a drug formulation that provides for an initial release of the drug after some delay following drug administration and that may. although not necessarily, include a delay of from about 10 minutes up to about 12 hours.
  • pulsatile release is used herein in its conventional sense to refer to a drug formulation that provides release of the drug in such a way as to produce pulsed plasma profiles of the drug after drug administration.
  • immediate release is used in its conventional sense to refer to a drug formulation that provides for release of the drug immediately after drug administration.
  • short-term refers to any period of time up to and including about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40 minutes, about 20 minutes, or about 10 minutes and any or all whole or partial increments thereof after drug administration after drug administration.
  • rapid-offset refers to any period of time up to and including about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40 minutes, about 20 minutes, or about 10 minutes, and any and all whole or partial increments thereof after drug administration.
  • reaction conditions including but not limited to reaction times, reaction size/volume, and experimental reagents, such as solvents, catalysts, pressures, atmospheric conditions, e.g.. nitrogen atmosphere, and reducing/oxi dizing agents, with art- recognized alternatives and using no more than routine experimentation, are within the scope of the present application.
  • range such as from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4. from 1 to 5, from 2 to 4, from 2 to 6. from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.
  • Yeast strains (Saccharomyces cerevisiae) used in this study are JS91. 15-23 MATa his3 leu2 trpl ura3), JS91. 14-24 ⁇ MATa ura3 his3 cab Its) (Olzhausen. et al. , 2009, Curr Genet 55, 163-173), W303B, cablA+ pFL38-CABl + pFL39, cablA+ pFL39-cabl G351S + pFL38, cablA+ pFL39-cabl° 51S + pFL38-CABl (Ceccatelli Berti et al., 2020, Int. J. Mol. Sci).
  • Candida strains used in the study include C. albicans and C. parapsilosis . Wild type and mutant strains were propagated either in YPD medium (2% bacto-peptone, 2% D-(+)-glucose and 1% yeast extract) or defined pantothenic acid-free (minimal) medium composed of Yeast Nitrogen Base (MP Biomedicals), supplemented with Complete Supplement Mixture (MP Biomedicals) and all vitamins except pantothenic acid. Where indicated, media were supplemented with appropriate concentrations of pantothenic acid. Growth assays on solid and liquid media
  • Spoting assays were performed as follows. Pre-cultures of WT and mutant yeast strains were prepared in YPD medium overnight at 30 °C. Cells were harvested, washed, and diluted to 10 8 cells in 500 pL sterile water. Subsequent serial dilutions were made and 5 pL of cell suspensions were spoted on YPD agar plates lacking or supplemented with amorolfme 10 ng/mL, amphotericin B 1 pg/mL. fluconazole 10 pg/mL or terbinafme 10 pg/mL to achieve 10 6 , 10 5 , 10 4 , 10 3 , 10 2 , and 10 1 cells per spot.
  • Relative time to mid-log phase was calculated by first graphing grow th curves of the varying concentrations of terbinafme for each concentration of pantothenic acid, and then determining the value at which the untreated curve reached saturation, and dividing it by 2 to represent the mid-log value. Dividing the time to mid-log phase of treated cells by the time to mid-log phase of untreated gave the relative time to mid-log phase.
  • the data from the time-point where the 0 pg/mL drug A curve reached saturation was used to generate percent inhibition values, representing growth inhibition based on the concentration of drug A. This was done by dividing the value of interest by the untreated (0 pg/mL drug A) value, multiplying by 100 to get a percentage, and then subtracting that percentage from 100, representing 100% inhibition. The resulting values were used to create an inhibition curve, using the best fit function to generate an MICso. This was done for both drug A in drug B and drug B in drug A. The resulting MIC50 values were plugged into the SFIC50 equation, and the SFICso values were used to generate the curve.
  • Example 1 Inhibition of Cablp activity leads to enhanced susceptibility to antifungal drugs
  • cahl is mutant which was obtained through random mutagenesis by Olhauzen et al (1989) (FIGs. 1 A-1H) and a well-defined genetic background cablA+CABl, which lacks the chromosomal CAB1 gene but expresses either the wild type CAB1 or various alleles of the CAB1 gene on a plasmid.
  • the growth of cablts cells at 30 °C can be significantly improved in media supplemented with increased concentrations of pantothenic acid, as substrate availability compensates for the weak activity of the mutant enzyme.
  • PanK pantothenate kinase
  • Example 3 Compounds of interest were assayed for their activity as inhibitors of fungal pantothenate kinase (PanK), modulators of human PanK, and general toxicity against HeLa cells. All compounds in Table 2 were found to be activators of human PanK. Table 2.
  • the cabl ⁇ cabl 3G - G mutant displayed reduced MICso values with fold reductions compared to the wild type determined to be ⁇ 7, ⁇ 9, and ⁇ 5 for fluconazole, terbinafine, and amphotericin, respectively (FIG. 13 A).
  • the cabl mutant alleles showed higher susceptibility to caspofungin, hygromycin and cycloheximide compared to the wild type (FIG. 12B).
  • the cabl G351s mutation resulted in the highest drug susceptibility with the MIC50 values of caspofungin, hygromycin, and cycloheximide determined to be ⁇ 5, ⁇ 23, and ⁇ 16- fold lower compared to the wild type (FIG. 13 A).
  • cabl SI58A mutation resulted in reduced MIC50 values for caspofungin, hygromycin, and cycloheximide by ⁇ 6, ⁇ 13, and ⁇ 4- fold compared to the wild type (FIG. 13 A). All complemented strains carrying the wild-ty pe CAB1 gene displayed susceptibility' levels comparable to those of the wild-type (WT) strain FIG. 13B.
  • PanK-deficient cells have altered vacuole biogenesis and xenobiotic detoxification mechanism
  • cab ID+cabl 1 ⁇ 2901 and cablD+cabl sl ⁇ 8A mutants 'as dramatically reduced in media supplemented with FeSOi or CuSO4 compared to the wild type and complemented strains (FIG. 14).
  • cablD+cabl N2901 and cablD+cabl SI58A mutants were all found to have enlarged vacuoles compared to the wild type and complemented strains (FIG. 15 A and FIG.
  • cablD+cabl N2901 and cablD+cabl SI58A mutants showed severe growth defects on non-fermentable carbon sources (glycerol, ethanol, and lactate-based media) (FIG. 17) and altered oxygen consumption rates (OCR) (FIG. 18 A, FIG. 18B, FIG. 18C, and FIG 18D).
  • OCR oxygen consumption rates
  • ROS reactive oxygen species
  • pantothenate utilization level ( ⁇ 2% that of the wild type) was measured for the cabl sl58A mutant followed by 25% for the cabl G35IS mutant and 34% for the cab I ' 22901 mutant.
  • Expression of the wild-type CAB1 gene in these strains restored PA utilization to levels similar or above those in the isogenic wild type-strain.
  • cellular CoA levels in the mutants were also significantly lower compared to the wild-type and complemented strains (FIG. 21B, FIG. 30A, and FIG. 30B).
  • genes in each of the mutants decreased dramatically (between 75% and -95%) compared to the wild type strain (FIG. 23B and FIG. 31).
  • a yeast mutant carrying the ACS2 gene under the regulatory control of the tet- off promoter oc.s2-tct-olT
  • the MICso for caspofungin shifted from 16 ng/ml in the absence of doxycycline to 10 ng/ml in the presence of the compound; that for fluconazole from 4.8 pg/ml to 0.06 pg/ml; and that for terbinafme from 4.5 pg/ml to 0.005 pg/ml.
  • the celecoxib derivative AR.-12 which is also a potent inhibitor of fungal AcCoA synthetases increased yeast susceptibility to caspofungin (MICso shift from 16 ng/ml to 3 ng/ml in the absence vs presence of AR-12).
  • fluconazole MICso shift from 14.6 pg/ml to 0.9 pg/ml
  • terbinafme MICso shift from 3.6 pg/ml to 0.07 pg/ml
  • the compound had no antifungal activity against S. cerevisiae, C. albicans or A. fumigatus at concentrations up to 50 pM (FIG. 26A. FIG. 26B, FIG. 27A, FIG. 27B, FIG. 28A, FIG. 28B, FIG. 35 A, FIG. 35B, FIG. 36, and FIG. 39).
  • pantothenate phosphorylation unraveled major alterations in vacuolar and mitochondrial biogenesis and enhanced susceptibility to xenobiotics including metals and commonly used antifungal drugs including both drugs that target ergosterol biosynthesis inhibitors (terbinafine, fluconazole and Amphotericin B) and unrelated pathways (caspofungin. hygromycin. cycloheximide).
  • ergosterol biosynthesis inhibitors terbinafine, fluconazole and Amphotericin B
  • unrelated pathways caspofungin. hygromycin. cycloheximide
  • Such broad-spectrum drug susceptibility is possible if major mechanisms used by fungi for drug detoxification, such as those mediated by the vacuole, are altered when the PCA pathway is inhibited.
  • yeast mutants with altered PanK activity can present with enlarged vacuoles.
  • the PCA pathway controls fungal mechanisms of detoxification both through direct effect on vacuolar biogenesis and indirectly through inhibition of ergosterol biosynthesis.
  • restricted CoA and consequently reduced AcCoA levels resulting from disruptions to the PCA pathway set off a cascade of events, leading to a deficiency in ergosterol and impaired V-ATPase function. This results in vacuolar dysfunction and subsequent loss of its ability to detoxify xenobiotics (FIG. 29).
  • yeast cells altered in CoA biosynthesis from pantothenic acid to a broad spectrum of antifungals can be recapitulated through genetic and pharmacological inhibition of specific enzymes downstream of the PCA pathway.
  • AcCoA can be formed from CoA through multiple routes including by the AcCoA synthetases, Acsl and Acs2 (See FIG. 11).
  • Cells lacking bothACST and ACS2 genes are inviable, as are cells lacking ACS2 in glucose medium since ACS1 is subject to glucose repression.
  • pantazine PZ-2891 Unlike its function as an activator of the human pantothenate kinases (PANK3), it was found that the pantazine PZ-2891 has major drug potentiation activity in fungal cells. Growth assays demonstrated potentiation of both caspofungin and amphotericin B in both A. cerevlsicie and C. albicans at concentrations far below their MICso’s (FIG. 26A and FIG. 26B). As the potentiation applies broadly to antifungals with varied mechanisms of action and to metals, one can reason that this is consistent with a broad-based disruption of the ability of fungal cells to detoxify drugs. The initial metabolic studies showing that CoA levels are increased (FIG.
  • cablA/pFL38-CAS7 wild type and mutant strains were generated using plasmid shuffling as from the parent strain cab 1 A/pFI.39-co/> 1 G351S .
  • Add-back strains were generated by introducing pFL39-C4 A/ vector into yeast recipient strains.
  • Yeast strains (WT and cabl mutants) were grown overnight at 30°C in YPD medium and harvested (700 x g for 5 mins at 4°C), washed with water, and resuspended in 0.9% NaCl solution at ODeoo of 0.5. Serial 10-fold dilutions were made and 5 pL of cell suspensions were spoted on YPD agar plates containing various antifungals (amphotericin B, caspofungin, fluconazole, terbinafine, hygromycin and cycloheximide).
  • YP medium supplemented with ethanol, lactic acid or glycerol were used.
  • GMM 1% glucose, 6 g/L NaNOs, 0.52 g/L KC1, 0.52 g/L MgSO 4 «7H 2 O, 1.52 g/L KH2PO4 monobasic, 2.2 mg/L ZnSO4»7H2O, 1.1 mg/L H3BO3, 0.5 mg/L MnCl 2 , 4H 2 O, 0.5 mg/L FeSO 4 *7H 2 O, 0.16 mg/L COC1 2 *5H 2 O, 0.16 mg/L CuSO4 «5H 2 O, 0.11 mg/L (NH 4 )6MO7O24 «4H 2 O, and 5 mg/L Na4EDTA; pH 6.5).
  • Yeast strains (WT and cabl mutants) were grown overnight at 30°C in YPD medium, harvested, and refreshed in YP media with 2% glycerol until reached ODeoo of 1. The cells were harvested, washed, and used for high pressure freezing and freeze substitution for electron microscopy analysis. Unfixed samples were high pressure frozen using a Leica HMP100 at 2000 psi. The frozen samples were then freeze substituted using a Leica Freeze AFS unit starting at -95°C using 0.1% uranyl acetate in acetone for 50 h to -60°C, then rinsed in 100% acetone and infiltrated over 24 h to -45°C with Lowicryl HM20 resin (Electron Microscopy Science).
  • Samples were placed in gelatin capsules and UV hardened at -45°C for 48 h.
  • the blocks were allowed to cure for a further few days before trimmed and cut using a Leica UltraCut UC7.
  • the 60nm sections were collected on formvar/carbon coated nickel grids and contrast stained using 2% uranyl acetate and lead citrate.
  • the 60nm sections on grids were viewed FEI Tecnai Biotwin TEM at 80Kv. Images were taken using AMT NanoSprintl5 MK2 sCMOS camera.
  • Pantothenate utilization assay using labeled PA was performed as summarized below: briefly, cell-free extracts from yeast producing Cabl variants were obtained by homogenization, followed by centrifugation at 700 x g for 5 min.
  • the 40 pL enzyme reaction contained reaction buffer (100 mM Tris HC1, 2.5 mM MgCb, 2.5 mM ATP, pH 7.4), D-[l - 14 C] pantothenate (2 nmol, 0.1 pCi), and 144 pg cell-free extracts.
  • the lysates total protein content was determined using the Bradford assay.
  • the reaction was done at 30 °C for 10 min following the addition of 4 pL of 10% acetic acid to stop the reaction.
  • the reaction mixture was spotted on a DE-81 filter (0.6 mm in diameter) placed within a spin column with a 2 rnL collection tube. Following 5 min incubation, the spotted filters were centrifuged for 20 s at 700 x g, washed twice with 1% acetic acid in ethanol, and collected for liquid scintillation spectrometry.
  • RNA samples from yeast strain producing different Cabl variants were extracted using YeaStar RNA kit (Zymo Research).
  • Total RNA quality is determined by estimating the A260/A280 and A260/A230 ratios by nanodrop.
  • the RNA integrity is determined by resolving an aliquot of the extracted RNA on Agilent Bioanalyzer gel, which measures the ratio of the ribosomal peaks. Samples with RNA integrity number (RIN) values of 7 or greater are recommended for library preparation.
  • the mRNAs are purified from approximately 200ng of total RNA with oligo-dT beads and sheared by incubation at 94 °C in the presence of Mg (Kapa mRNA Hyper Prep). Following first-strand synthesis with random primers, second strand synthesis and A-tailing are performed with dUTP for generating strand-specific sequencing libraries. Adapter ligation with 3’ dTMP overhangs are ligated to library' insert fragments. Library amplification amplifies fragments carry ing the appropriate adapter sequences at both ends. Strands marked with dUTP are not amplified.
  • Indexed libraries that meet appropriate cut-offs for both are quantified by qRT-PCR using a commercially available kit (KAPA Biosystems) and insert size distribution determined with the LabChip GX or Agilent Bioanalyzer. Samples with a yield of >0.5 ng/pL are used for sequencing.
  • Flow Cell Preparation and Sequencing Sample concentrations are normalized to 1.2 nM and loaded onto an Illumina NovaSeq flow cell at a concentration that yields 25 million passing filter clusters per sample. Samples are sequenced using lOObp paired-end sequencing on an Illumina NovaSeq according to Illumina protocols. The 1 Obp unique dual index is read during additional sequencing reads that automatically follow the completion of read 1. Data generated during sequencing runs are simultaneously transferred to the YCGA high-performance computing cluster.
  • a positive control (prepared bacteriophage Phi X library) provided by Illumina is spiked into every lane at a concentration of 0.3% to monitor sequencing quality in real time.
  • RTA Real Time Analysis
  • Base calls are transferred from the machine’s dedicated personal computer to the Yale High Performance Computing cluster via a 1 Gigabit network mount for downstream analysis.
  • Primary analysis - sample de-multiplexing and alignment to the human genome - is performed using Illumina’s CAS AV A 1.8.2 software suite.
  • the data are returned to the user if the sample error rate is less than 2% and the distribution of reads per sample in a lane is within reasonable tolerance. Data is retained on the cluster for at least 6 months, after which it is transferred to a tape backup system.
  • Partek Flow was used to organize and process fastq files from paired-end sequencing. Paired-end reads were trimmed for quality' (Q-Score > 20, Min read length 25) and adapters were removed using FastQC, then aligned to the S. cerevisiae W303-1B genome (Accession: JRIU00000000; ATCC Number: 200060, downloaded from SGD) using STAR v2.7.8a. Reads were normalized via trimmed mean of M values (TMM). and further processed in Excel, where the three biological replicates per condition (WT, three mutants, and three mutants with addback) were grouped to generate means and standard deviations for all genes in each of the 7 conditions. Log fold changes in FIG.
  • OCR oxygen consumption rate
  • the seeded plate was centrifuged at 500 rpm for 5 min to promote yeast adhesion and the plate was rested for 30 min at RT.
  • a soaked and calibrated Seahorse XF96 Sensor Cartridge was prepared before loading into the Seahorse XF96 analyzer (Agilent) which determined the cells basal OCR and following the injection of mitochondrial uncoupling drugs; oligomycin (5 pM), carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone (FCCP) (10 pM), antimycin A (10 pM), and rotenone (5 pM).
  • the readouts were normalized using nuclear Hoechst staining for the immobilized yeast cells.
  • yeast growth in the presence of antifungal drugs, inhibitors, and potentiators To investigate the effect of common AFDs (amphotericin B, caspofungin, fluconazole, and terbinafine) in combination with compounds and potentiators (concanamycin A, doxycycline, PZ-2891 , a-Pan Am, AR- 12) the yeast growth was monitored using liquid assay in a 96-well plate. Overnight yeast precultures (WT strains or acs-Tet-Off when mentioned) were prepared in YPD medium at 30°C. Cells were w ashed and refreshed in YPD until reaching ODeoo of 0.6.
  • AFDs amphotericin B, caspofungin, fluconazole, and terbinafine
  • ROS Reactive Oxygen Species
  • ROS Reactive oxygen species
  • His-tagged Cabl recombinant enzyme was produced and purified as was previously described.
  • a Kinase-Glo (Promega) assay kit for kinase activity was used to determine the activity of the purified PanK under different conditions.
  • the radial growth measurements of A. fumigatus were performed by taking 2 pL of a 2.5 X IO 6 mL-1 conidial suspension of wild-type CEA10 A. fumigatus was point inoculated onto the center of a solid GMM in the absence or presence of 50 pM PZ-2891, 20 pg/mL caspofungin, and their combination. Plates were incubated for 96 h at 35°C, with colony diameters measured and photographed taken each day.
  • the ACS assay was performed by monitoring formation of the adenyl acetate, the intermediate of the enzyme reaction, utilizing 5. cerevisiae acetyl Coa synthetase (Sigma, A1765), following established protocols with some modifications.
  • a 100 pL reaction volume composed of 100 mM potassium phosphate at pH7.5, 5 mM MgCh, 2 mM ATP, 50 mM potassium fluoride, 10 mM reduced glutathione, 0.35 mM CoA, 10 mM potassium acetate, 200 mM neutralized hydroxylamine adjusted to pH 7.3, 0.005 units of the enzy me, and the inhibitors (in 1% DMSO), the components were combined.
  • Embodiment 1 A method of: increasing antifungal activity' of an antifungal agent used to treat, ameliorate, and/or prevent a fungal infection in a subject; reducing, minimizing, and/or eliminating at least one toxicity effect of an antifungal agent used to treat, ameliorate, and/or prevent a fungal infection in a subject; reducing, minimizing, and/or preventing drug resistance against an antifungal agent used to treat, ameliorate, and/or prevent a fungal infection in a subject; and/or sensitizing a fungus to an antifungal agent administered to a subject to treat, ameliorate, and/or prevent the fungal infection in the subject; the method comprising administering a therapeutically effective amount of a compound which is a pantothenate kinase (PanK) inhibitor and/or modulator to a subject being administered the antifungal agent.
  • PanK pantothenate kinase
  • Embodiment 2 The method of Embodiment 1, wherein the compound comprises a pantazine and/or pantothenic acid analogue and/or derivative.
  • Embodiment 3 The method of Embodiment 1 , wherein the compound comprises a compound of formula (la) or (lb), wherein in (la) or (lb):
  • R 1 is selected from the group consisting of H, -NR 4a R 4b , -NR 4c (optionally substituted phenyl), and optionally substituted N-linked heterocyclyl;
  • R 2 is selected from the group consisting of phenyl and -N(R 4c )-(CH2)i-3-(optionally substituted phenyl);
  • R 3 is selected from the group consisting of F, Cl, Br, I, -OR 4d .
  • R a is independently H, optionally substituted Ci-Ce alkyl, optionally substituted Cs-Cs cycloalkyd, optionally substituted phenyl, or optionally substituted heteroaryl, or two R a groups combine with the N to which they are bound to form Cs-Cs heterocyclyl.
  • Embodiment 4 The method of any one of Embodiments 1-3, wherein the fungal infection is caused by at least one of C. albicans, C. parapsilosis, and/or A. fumigatus.
  • Embodiment 5 The method of Embodiment 3, wherein R 1 is selected from the group consisting ofH, -NR 4a R 4b , and optionally substituted N-linked heterocyclyl.
  • Embodiment 6 The method of any one of Embodiments 1 and 5, wherein R 1 is NR 4c (optionally substituted phenyl).
  • Embodiment 8 The method of any one of Embodiments 1 and 5-7, wherein each phenyl in R 2 is independently substituted with at least one C2-C8 alkyl.
  • Embodiment 9 The method of any one of Embodiments 1 and 5-8. wherein each phenyl in R 2 is independently substituted at the para-position with at least one C2-C8 alkyl.
  • Embodiment 10 The method of any one of Embodiments 1 and 5-9, wherein the N- linked heterocyclyl comprises aziridinyl, azetidinyl, pyrrolidinyl, pyrazolidinyl, piperidinyl, 1,2,3,6-tetrahydropyridinyl, 1,4-dihydropyridinyl, piperazinyl, morpholinyl, thiomorpholinyl, homopiperazinyl, or homopiperidinyl.
  • the N- linked heterocyclyl comprises aziridinyl, azetidinyl, pyrrolidinyl, pyrazolidinyl, piperidinyl, 1,2,3,6-tetrahydropyridinyl, 1,4-dihydropyridinyl, piperazinyl, morpholinyl, thiomorpholinyl, homopiperazinyl, or homopiperidinyl.
  • Embodiment 11 The method of any one of Embodiments 1 and 5-10, wherein the heteroaryl comprises imidazolyl, pyridinyl, pyrimidinyl, pyrazinyl, thienyl, furyl, pyrrolyl, thiazolyl, oxazolyl, pyrazolyl, isothiazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, 1,3,4-triazolyl, tetrazolyl, 1,2,3-thiadiazolyl, 1,2,3-oxadiazolyl, 1,3.4-thiadiazolyl, or 1,3,4-oxadiazolyl.
  • the heteroaryl comprises imidazolyl, pyridinyl, pyrimidinyl, pyrazinyl, thienyl, furyl, pyrrolyl, thiazolyl, oxazolyl, pyrazolyl, isothiazolyl, 1,2,3-triazoly
  • Embodiment 12 The method of any one of Embodiments 1 and 5-11, wherein the compound is at least one of:
  • Embodiment 13 The method of any one of Embodiments 1-12, wherein the compound is administered to the subject as part of a pharmaceutical composition.
  • Embodiment 14 The method of any one of Embodiments 1-13, wherein the compound and the antifungal agent are co-administered to the subject.
  • Embodiment 15 The method of Embodiment 14, wherein the compound and the antifungal agent are co-formulated.
  • Embodiment 16 The method of any one of Embodiments 1-15, wherein the compound is not: Compound -(tert-butjl)phenyl)-5-((4- methylpiperazin-l-yl)methyl)-[l,2,4]triazolo[l,5-a]pyrimidin-7(4H)-one;
  • Embodiment 17 The method of any one of Embodiments 1-16, wherein the fungus is on the skin of the subject or within the subject.
  • Embodiment 18 The method of any one of Embodiments 1-17, wherein administration of the compound and the antifungal agent results in less likely drug resistance occurrence in the subject as compared to an equivalent subject that is administered the antifungal agent in the absence of the compound.
  • Embodiment 19 The method of any one of Embodiments 1-18, wherein administration of the compound and the antifungal agent allows for administration of an amount of the antifungal agent that is lower than the corresponding amount of the antifungal agent that has to be administered in the absence of the compound to achieve equivalent treatment, amelioration, and/or prevention of the fungal infection.
  • Embodiment 20 The method of any one of Embodiments 1-19, wherein the antifungal agent comprises an agent that disrupts synthesis and/or activity or ergosterol, an inhibitor of any other metabolic pathway, and/or an inhibitor of nucleic acid and/or protein synthesis.
  • the antifungal agent comprises an agent that disrupts synthesis and/or activity or ergosterol, an inhibitor of any other metabolic pathway, and/or an inhibitor of nucleic acid and/or protein synthesis.
  • Embodiment 21 The method of Embodiment 20, wherein the agent is selected from an azole antifungal agent, morpholine antifungal agent, allylamine antifungal agent, and/or polyene antifungal agent.
  • the agent is selected from an azole antifungal agent, morpholine antifungal agent, allylamine antifungal agent, and/or polyene antifungal agent.
  • Embodiment 22 The method of Embodiment 21, wherein at least one of the following applies:
  • the azole antifungal agent comprises an imidazole antifungal agent selected from bifonazole, butoconazole, clotrimazole, econazole, fenticonazole, isoconazole, ketoconazole, luliconazole, miconazole, omoconazole, oxiconazole, sertaconazole, sulconazole, and tioconazole;
  • the azole antifungal agent comprises a triazole antifungal agent selected from albaconazole, efinaconazole, epoxiconazole, fluconazole, isavuconazole, itraconazole, posaconazole, propiconazole, ravuconazole, terconazole, and voriconazole;
  • the azole antifungal agent comprises a thiazole antifungal agent selected from abafungin;
  • the azole antifungal agent comprises a tetrazole antifungal agent selected from oteseconazole;
  • the allylamine antifungal agent is selected from butenafme, naftifine, and terbinafine;
  • the polyene antifungal agent is selected from amphotericin B, candicidin, filipin, hamycin. natamycin, nystatin, and rimocidin;
  • the morpholine antifungal agent is selected from amorolfine and fenpropimorph;
  • the inhibitor is cycloheximide;
  • the inhibitor is an echinocandin, optionally selected from anidulafungin, caspofungin, and micafungin;
  • the inhibitor is a cytosine analogue, optionally 5-flucytosine;
  • the inhibitor is an aminoglycoside, optionally hygromycin B.
  • Embodiment 23 The method of any of Embodiments 1-22. wherein the compound inhibits fungal PanK selectively over a human PanK enzyme.
  • Embodiment 24 The method of any of Embodiments 1-23, wherein the subject is a mammal.
  • Embodiment 25 The method of Embodiment 24, wherein the mammal is a human.

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Abstract

The disclosure relates, in certain aspects, to certain compounds that are useful to treat, ameliorate, and/or prevent fungal infections in a subject. The disclosure further relates, in certain aspects, to certain compounds that are useful for treating, ameliorating, and/or preventing diseases or disorders associated with reduced and/or deficient PanK activity in a subject.

Description

TITLE
PanK Modulators and Methods of Treatment Using Same
CROSS REFERENCE TO RELATED APPLICATIONS
This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 63/476,521 filed December 21, 2022, which is incorporated herein by reference in its entirety.
BACKGROUND
In all living organisms, coenzyme A (CoA) plays a fundamental role in cellular metabolism, including synthesis and oxidation of fatty acids, and oxidation of pyruvate in the citric acid cycle. Its unique chemical structure, with a reactive thiol group and a nucleotide moiety, allows it to serve as a cofactor in critical biochemical and regulatory functions through activation of carboxylic acids and production of thioester derivatives.
In the yeast Saccharomyces cerevisiae, CoA is synthesized from vitamin B5 (pantothenic acid) either imported into the cell via the Fen2p pantothenate transporter or produced de novo in the cell from P-alanine and pantoate catalyzed by the enzy me pantothenate synthase Pan6p. Pantothenic acid utilization is absolutely essential for yeast survival as a double mutant lacking both the FEN2 and PAN6 genes is inviable. The first step in pantothenate utilization is its phosphorylation to 4'-phosphopantothenate by the pantothenate kinase (PanK) Cab Ip, encoded by a single copy gene GABI. This gene is required for yeast viability since deletion of GABI results in cell death. A mutant cablG3:,IS, which produces a kinase enzyme with glycine 351 substituted to serine, was found to be viable at 30 °C but is completely inviable at 37 °C. Biochemical assays using purified wild type and mutated Cablp enzymes showed that the G351S mutation results in 67 to 94% loss of enzy me activity. The pantothenate analog a-PanAm inhibits yeast growth in a pantothenic acid dose-dependent manner. The MICso of the compound was found to be about 4 times higher in medium supplemented with 1 pM pantothenic acid (MICso -7.6 pg/mL) compared to medium with 100 nM pantothenic acid (MICso ~1.9 pg/mL). In vitro pantothenate phosphorylation assays and mass spectrometry analysis showed that a-PanAm is also phosphorylated by Cablp and acts as a competitor of pantothenic acid at the enz me catalytic site. a-PanAm has also been proposed to act on downstream steps in CoA biosynthesis and ultimately exerts its activity by reducing cellular CoA levels. In fungi, acetylation of CoA by acetyl-CoA synthetases generates acetyl-CoA, a key node in multiple metabolic and cellular processes.
Figure imgf000004_0001
a-PanAm
Fungal diseases are a major global health problem and are particularly threatening as opportunistic infections in immunosuppressed individuals such as AIDS and cancer patients. Despite major advances in understanding the biology of fungi and in antifungal drug discovery’, fungal infections continue to cause significant mortality and morbidity’ worldwide. Commonly used antifungal drugs include azoles, echinocandins, polyenes, and allylamines. However, due to widespread resistance to some of these drugs and their lack of efficacy against a diverse array of pathogens such as Candida albicans, Candida auris and Aspergillus fumigaius. both new drugs and alternative strategies to modulate the efficacy and safety of current drugs and reverse resistance are urgently needed.
In mammals (including humans) there are at least four closely related active PanK isoforms — PanKia, PanKip, PanK2, and PanK3 — which are encoded by three genes. Regulation of cellular CoA occurs through feedback inhibition of PanK enzyme activity by CoA itself or CoA thioesters, and each isoform responds to inhibition with a distinct sensitivity. The PanK isoform expression profiles differ among individual cell types, tissues, and organs, and the relative abundance of one or more isoforms determines the respective CoA levels.
There is thus a need in the art for the identification of novel compounds that can be used to treat, ameliorate, and/or prevent fungal infections. The present disclosure addresses these needs.
BRIEF SUMMARY
In one aspect, the disclosure provides a method of increasing antifungal activity of an antifungal agent used to treat, ameliorate, and/or prevent a fungal infection in a subject.
In one aspect, the disclosure provides a method of reducing, minimizing, and/or eliminating at least one toxicity effect of an antifungal agent used to treat, ameliorate, and/or prevent a fungal infection in a subject.
In one aspect, the disclosure provides a method of reducing, minimizing, and/or preventing drug resistance against an antifungal agent used to treat, ameliorate, and/or prevent a fungal infection in a subject. In one aspect, the disclosure provides a method of sensitizing a fungus to an antifungal agent administered to a subject to treat, ameliorate, and/or prevent the fungal infection in the subject.
In certain embodiments, the method comprises administering a therapeutically effective amount of a compound which is a pantothenate kinase (PanK) inhibitor and/or modulator to a subject being administered the antifungal agent.
In certain embodiments, the compound comprises a pantazine and/or pantothenic acid analogue and/or derivative.
In certain embodiments, the compound comprises any of the compounds contemplated within the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
The following detailed description of selected embodiments of the disclosure will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating the disclosure, non-limiting embodiments are shown in the drawings. It should be understood, however, that the disclosure is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.
FIGs. 1 A-1H illustrate relationship between pantothenate utilization and yeast susceptibility to amphotericin B. FIGs. 1 A-1F depict Growth curves of wild type (WT) and a mutant cablts (which carries the G35 IS mutation) without or with 1 pg/mL amphotericin B (amphB) in liquid minimal medium supplemented with varying concentrations of pantothenic acid (1, 10 and 100 pM PA). FIG. 1G depicts relative times for treated cells to reach mid-log phase compared to untreated cells, calculated from FIG. 1A. Time for untreated cells to grow to mid-log was normalized to 1, and is represented by the dashed line. FIG. 1H shows isobologram of the interaction between amphotericin B and the pantothenate analog a- PanAm (n=2). The solid line represents the theoretical curve of an additive effect. Data below the solid line (dashed curve) shows synergism between the two compounds.
FIG. 2 illustrates a non-limiting model of the mechanism by which inhibition of Cab 1 activity and CoA biosynthesis leads to enhanced susceptibility to antifungal drugs. Without wishing to be limited by theory, inhibition or reduction of Cab Ip activity through genetic mutation (use of Cabl mutants) or enzyme inhibition (using Cabl inhibitors or analogs of pantothenic acid such as a-PanAm) results in altered vacuolar function (wherein the vacuole plays a key role in drug detoxification in fungi), which increases susceptibility of fungi to antifungals.
FIGs. 3A-3B illustrate the finding that reduced fungal PanK activity’ results in increased sensitivity to the ergosterol biosynthesis inhibitors amphotericin B and fluconazole. In this figure, reduced PanK activity is achieved through expression of the CablG351s allele on a plasmid in a strain lacking the chromosomal CAB1 gene (cabl A). The assays were conducted using limiting dilution on YPD-based agar plates or in liquid YPD medium lacking or supplemented with fluconazole or amphotericin B.
FIG. 4 illustrates the finding that reduced fungal PanK activity in 6 S. cerevisiae mutants carrying different Cabl alleles leads to enhanced susceptibility to a large panel of antifungal drugs. Addition of the wild type CAB1 gene restores drug susceptibility to wild ty pe levels. Drug susceptibility was assessed using a limiting dilution assay on YPD-based agar plates lacking or supplemented with each of the 8 drugs at the indicated concentrations.
FIG. 5 shows the MIC50 values of wild type and cablG351s strains for 7 antifungal drugs and the ratio of WT MICso/ cablG351s MICso to represent the fold increase in susceptibility to these antifungal drugs caused by the G351S mutation.
FIG. 6 is a graphical representation of the data in FIG. 5.
FIG. 7 illustrates the finding that susceptibility’ of the S. cerevisiae wild type strain to terbinafine (5 and 10 pg/ml) is significantly enhanced by the commercially available drug PZ-2891 at 2 different concentrations (50 pM and 100 pM). Addition of PZ -2891 further increased the susceptibility' of this strain to terbinafine. The compound (PZ-2891) itself had no effect on fungal growth at concentrations up to 200 pM.
FIG. 8 illustrates the finding that susceptibility of the S', cerevisiae “Monster” strain (that lacks several genes involved in drug transport and detoxification) to terbinafine (5 and 10 pg/ml) is significantly enhanced by the commercially available drug PZ-2891 at 2 different concentrations (50 pM and 100 pM). This strain is more sensitive to terbinafine compared to the wild type parent strain. Addition of PZ-2891 further increased the susceptibility of this strain to terbinafine. The compound (PZ-2891) itself had no effect on fungal growth at concentrations up to 200 pM.
FIG. 9 illustrates the finding that susceptibility’ of a Candida parapsilosis clinical isolate to terbinafine (5 pg/ml) is significantly enhanced by the commercially available drug PZ-2891 at 2 different concentrations (50 pM and 100 pM). The compound (PZ-2891) itself had no effect on fungal growth at concentrations up to 200 pM.
FIG. 10 illustrates the finding that susceptibility of a Candida albicans clinical isolate to terbinafine (20 pg/ml) is significantly enhanced by the commercially available drug PZ- 2891 at 2 different concentrations (50 pM and 100 pM). The compound (PZ-2891) itself had no effect on fungal growth at concentrations up to 200 pM.
FIG. 11 illustrates how Cabl mutants display growth defects and increased susceptibility toward ergosterol targeting drugs. (A) Schematic diagram of Coenzyme A synthetic pathway and biological roles of the Acetyl-CoA. Pantothenate kinase (PanK) catalyzes the phosphorylation of pantothenic acid to form 4?-phosphopantothenic acid, the first step in the biosynthesis of CoA. Phosphopantothenoylcysteine synthase (PPCS) converts 4’ -phosphopantothenic acid to phosphopantothenoylcysteine. Phosphopantothenoylcysteine decarboxylase (PPCD) catalyzes the decarboxylation of phosphopantothenoylcysteine to form pantetheine. Pantetheine kinase (PANK) catalyzes the phosphorylation of pantetheine to form dephospho-CoA. Dephospho-CoA kinase (DPCK) catalyzes the transfer of a phosphate group from ATP to dephospho-CoA, resulting in the formation of CoA.
FIGs. 12A-12B shows the cabl mutants display susceptibility toward drugs targeting ergosterol pathway and non-ergosterol targeting pathway. Yeast liquid growth curve assay with known AFDs targeting the ergosterol biosynthesis pathway was done using yeast cells harboring different CAB1 mutants. Cells were inoculated into 100 pL of YPD liquid media containing the antifungals in serial dilutions at 10 cells per pL ratio and incubated at 30°C while cell grow th was monitored by ODeoo. % of cell growth of individual mutant strain in presence of AFDs was obtained compared to the cell growth in the vehicle control.
FIG. 13A illustrates normalized relative MICso of yeast strains producing Cabl variants against AFDs. MICso values w ere determined on MICso data obtained from the liquid growth assays presented in FIG. 12A and 12B. These values were calculated by dividing the MICso value of the cabl mutant or the wild type strain for the specified AFD by the MICso value of the wild type strain.
FIG. 13B depicts complementation of the w ild type CAB1 gene restores AFD resistance in the cabl mutants to the similar level as that observed in wild-type cells. The cab 1 \ w303-l strain harboring 1) wild type CABL 2) cablmutar,t, or 3) cablmutant + wild t pe CAB were inoculated into synthetic glucose medium and grown overnight. Cells were harvested and re-suspended in 0.9% NaCl. Ten-fold serial dilutions of cells were spotted onto the YPD plates containing fluconazole (5 pg/mL), amphotericin B (0.25 pg/mL), and terbinafine (10 pg/mL), caspofungin (30 ng/mL), hygromycin (20 pg/mL), or cycloheximide (100 ng/mL). and incubated at 30°C for 3 days
FIG. 14 illustrates how yeast strains expressing various CAB1 mutations have defects in detoxification and vacuolar function and structure. The cabl mutations altered yeast capacity to overcome metal toxicity. Solid growth assays were performed with the yeast strains described above in glucose media supplemented with 7 mM FeSC or with 10 mM CuSO4.
FIG. 15A shows morphological analysis of vacuolar defects shows that cabl mutant strains have unusually enlarged vacuoles. Immuno-fluorescence microscopy images of cablX + cad7mutant strains described above show enlarged and/or fragmented vacuoles, while the cablX + CAB!''''2 and the cablX + cabl mutant+ CABl^1 strains have typical vacuolar morphology.
FIG. 15B shows quantitative analysis of vacuolar area size (as a proportion of total cell area) as a function of CAB1 status.
FIG. 16 illustrates Electron microscopy images of cab \ + CABllVT and cab 1 X+cab 7G351S single cells confirm enlargement of vacuole induced by G351S mutation.
FIG. 17 illustrates how yeast cablX strains harboring various CAB1 mutations display defects in mitochondrial function. Solid growth assays reveal that mutations in CAB1 alter strains’ ability to utilize non-fermentable carbon sources. Solid growth assays using ten-fold serial dilutions plated onto YPD (glucose), YPL (lactate), YPG (glycerol), or YPE (ethanol) media and observed over 3-4 days.
FIGs. 18A-18B show oxygen consumption rate (OCR) of yeast cells harboring CAB1 variants. OCR profile of cablX strains using Seahorse 96X and Mito Stress kit. Dashed lines represent the injections of mitochondrial uncoupling drugs; oligomycin, FCCP, antimycin A, and rotenone.
FIGs. 18C-18D show basal respiration and maximal respiration of cablX strains (t- tests performed for each group comparing mutants to the wild type strain at p=0.05
FIG. 19 illustrates cablX strains exhibit mitochondrial structural defects. Immunofluorescence microscopy images of cablX strains reveal aberrant mitochondria structures.
FIG. 20 shows cablX strains have increased levels of reactive oxygen species (ROS). ROS analysis was performed using dihydrorodamine 123.
FIG. 21 A illustrates metabolic defects in yeast strains harboring cabl mutations. (A) PA utilization in the cablX strains harboring various CABl mutations. Cell free extracts of yeast cabl mutants were used to measure the endogenous PA utilization of cablX + CAB1, cablX + cablG25IS, cablX + cablG25IS+ CABL cablX + cablSI58A, cablX + cablSI5SA + CABL and cablX + cabl812901. cablX + cablN2901 + CAB1 strains using D-[1-14C] pantothenate as a substrate for 10 min at 30°C. FIG. 21B depicts cellular levels of CoA in cablS strains. CoA levels were measured using metabolite extracts from the yeast strains mentioned above grown in the presence of 0.2 pM PA.
FIG. 22 depicts cysteine cellular levels of cahl \ strains harboring various CABl mutations. Cellular cysteine levels were measured using the metabolite extracts from the yeast strain mentioned above grown in the presence of 0.2 pM PA. For these assays, t-test was done per each group compared to the parent CABl wild-type strain (p=0.05
FIG. 23 A illustrates a non-limiting schematic of the connection between the PCA pathway and the SUL/MET pathways. Transcription of the SUIAMET genes, responsible for synthesizing the crucial sulfur-containing amino acids methionine and cysteine, is mediated by the transcription factor Met4. This regulatory’ process is sensitive to changes in cellular cysteine levels. When cysteine levels increase, Met4 undergoes ubiquitination by the ubiquitin ligase Met30, leading to Met4’s inactivation and subsequent repression of the SUL/MET genes.
FIG. 23B shows RNA-Seq analysis for cysteine and sulfur homeostasis expressed in cablS strains. Expression values are TMM normalized and compared using a double-sided t- test. Large circles in corresponds to genes for which the p-value (WT vs. mutant) <0.05, but the p-value (WT vs. complement) >0.05, indicating the null hypothesis of equal expression was rejected for the mutant, but not the addback, at p=0.05. Small circles correspond to genes for which either of these criteria were not met. The gene list with annotations shown in Table 3.
FIG. 24A illustrates chemical inhibition of Acs2 and V-type ATPase increases susceptibility to a variety' of AFDs via a schematic of the relevant portions of the PCA pathway.
FIG. 24B shows the growth of acs2 tet-off strain is inhibited by increasing concentrations of doxycycline. S. cerevisiae acs2 tet-off strain was inoculated in the presence or absence of doxycycline and normalized to DMSO treated wells (no drug=100% growth) and 200 pM amorolfine (0% growth).
FIG. 25 A illustrates AR- 12 potentiates caspofungin. fluconazole, and terbinafine by factors of ~100x against S. cerevisiae WT.
FIG. 25B illustrates Concanamycin A increases yeast susceptibility7 to antifungal drugs. S. cerevisiae WT was inoculated in the presence or absence of concanamycin A at 30°C for 48h. The growth was normalized to DMSO treated wells (no drug=100% growth) and 200 pM amorolfine well (0% growth). FIG. 26A illustrates PZ-2891 increases AFD susceptibility in A cerevisiae. C. albicans and A. fumigatus . Solid growth assays showing increased S', cerevisiae and C. albicans susceptibility to amphotericin B when potentiated by PZ-2891.
FIG. 26B shows liquid growth assays showing increased C. albicans susceptibility to amphotericin B (125 ng/mL) and caspofungin (3.9 ng/mL) is potentiated by PZ-2891. The growth was normalized to DMSO treated wells (no drug=100% growth) and 200 pM amorolfine well (0% growth).
FIGs. 27A-27B illustrates average colony diameter rate and captures of solid media assay (up to 72h of growth) with A. fumigatus cells under caspofungin treatment (20 pg/ml) in the presence or absence of PZ-2891 (50 pM).
FIG. 28A illustrates cellular CoA levels in S cerevisiae following treatment with PZ- 2891 . CoA levels were measured using the metabolite extracts from the S cerevisiae cells grown in minimal glucose medium supplemented with 1 pM PA in the presence or absence of 50 pM PZ-2891.
FIG. 28B shows yeast acetyl CoA synthetase activity. The in vitro activity of purified enzyme from S cerevisiae was measured using a standard hydroxylamine-coupled assay, at 37 °C for 30 minutes, in the presence of absence of 18 pM of AR- 12 or PZ-2891
FIG. 29 illustrates a model for PCA pathway-mediated regulation of vacuolar detoxification and susceptibility to antifungal drugs in fungi.
FIGs. 30A-30B illustrate cellular CoA (FIG. 30A) and cysteine (FIG. 30B) levels in cab IX strains harboring various CAB1 mutations. CoA and cysteine levels were measured using the metabolite extracts from the yeast strains grown in the presence of 1 pM PA
FIG. 31 illustrates RNA-Seq analysis for cysteine and sulfur homeostasis genes expressed in cab IX strains. The results are based on normalized TMM compared with the expression profile of the WT parent strain. The gene list with annotations shown in Table 3.
FIG. 32 illustrates RNA-Seq analysis for PCA pathway genes expressed in cab IX strains harboring various CABl mutations. Large circles in corresponds to genes for which the p-value (WT vs. mutant) <0.05, but the p-value (WT vs. addback) >0.05, indicating the null hypothesis of equal expression was rejected for the mutant, but not rejected for the addback at p=0.05. Small circles correspond to genes for which either of these criteria were not met. The full gene list is shown in Table 3.
FIG. 33A illustrates the effect of modulation of downstream steps from the PCA pathway on the growth of S. cerevisiae. S. cerevisiae (WT or acs2-tetoff mutant, as mentioned) cells w ere inoculated in the presence or absence of rising concentration of doxycycline, FIG. 33B shows AR-12, and FIG. 33C shows concanamycin A, at 30°C for 24- 48 h. The growth was normalized to DMSO treated wells (no drug=100% growth) and 200 pM amorolfine well (0% growth).
FIG. 34 illustrates PA utilization in the cabl ' strains harboring various CAB1 mutations. Cell free extracts of yeast expressing cabl mutants w ere used to measure the endogenous PA utilization of cabl variants using D-[1-14C] pantothenate as a substrate for 10 min at 30°C. The extracts PA utilization was measured in the absence or presence of 20 pM PZ-2891 (hPanK3 activator), a-PanAm (known Cabl inhibitor), and YU385599 (reported Cabl inhibitor).
FIG. 35A illustrates potentiation of PZ-2891 on antifungal susceptibility' in different yeast species. Caspofungin efficacy in S. cerevisiae with PZ-2891. S', cerevisiae cells were inoculated in the presence or absence PZ-2891, in combination with caspofungin treatment at 30°C for 24-48 h. The growth w as normalized to DMSO treated wells (no drug=100% growth) and 200 pM amorolfine well (0% growth). For these assays, t-test was done among the mentioned groups (p=0.05
FIG. 35B shows potentiation of terbinafine efficacy in C. albicans with PZ-2891. C. albicans spotting growth assays w ere performed when cells were inoculated into YPD overnight, harvested, w ashed, and re-suspended in 0.9% NaCl. Serial dilutions of cells were spotted onto YPD plates containing terbinafine (20 pg/mL) in the presence or absence of PZ- 2891 (50 pM) at 30°C for 4 days.
FIG. 36 show s the average growth rate (based on colony diameter) of A. fumigatus colonies in the presence or absence of PZ-2891 (50 pM) in combination with caspofungin treatment (20 pg/ml). The results were calculated after 72 h of growth.
FIG. 37A illustrates PZ-2891 does not have inhibitory effect on either . cerevisiae Cabl enzymatic activity or S. cerevisiae cell growth as shown in the dose-response curve for AcCoA effect on recombinant Cabl enzyme activity.
FIG. 37B shows the dose-response curve for PZ-2891 effect on recombinant Cabl enzyme activity.
FIG. 38 shows a dose-response curve for AcCoA effect on recombinant Cabl enzyme activity in the absence of presence of PZ-2891 or YU385599 (inhibitor control).
FIG. 39 illustrates liquid growth assay for cables strains harboring various CAB1 mutations in the presence or absence of 1-100 pM PZ-2891.
DETAILED DESCRIPTION The disclosure relates, in certain aspects, to certain compounds that are useful to treat, ameliorate, and/or prevent fungal infections in a subject.
In certain embodiments, the fungus causing the fungal infection comprises C. albicans, C. parapsilosis, and/or A. fumigatus.
In certain embodiments, the compounds of the disclosure comprise any pantothenate kinase (PanK) inhibitor and/or modulator known in the art.
In certain embodiments, the compounds of the disclosure comprise any pantazine know n in the art. Non-limiting examples of such pantazines include PZ-2891, any PanK modulator disclosed in WO2017/223474, WO2019/133635, US20190300499 Al, US20210246113 Al, and US20210061788 Al (each of which is incorporated herein in its entirety by reference), any PanK modulator disclosed in Sharma, el al.. 2018, "A therapeutic approach to pantothenate kinase associated neurodegeneration ” , Nature Comm., 9(1):4399, doi: 10. 1038/s41467-018-06703-2; and/or Sharma et al., 2021, “LipE guided discovery of isopropylphenyl pyridazines as pantothenate kinase modulators ”, Bioorg Med Chem. 52: 116504. doi: 10. 1016/j.bmc.2021.116504 (which is incorporated herein in its entirety by reference). In certain embodiments, the compound is PZ-2891. In certain embodiments, the compound is not PZ-2891.
In certain embodiments, the compounds of the disclosure comprise any pantothenic acid analogue and/or derivative. Non-limiting examples of such pantothenic acid analogues and/or derivatives include a-PanAm, any pantothenamide analogue disclosed in WO2016/072854 and US20180282279 Al (all of which are incorporated herein in their entireties by reference), any pantothenamide analogue disclosed in de Vries, et al., 2022, “Preclinical characterization and target validation of the antimalarial pantothenamide MMV693183”, ^aX Covarr. 13(1):2158, doi: 10.1038/s41467-022-29688-5; de Vries, et al., 2021, "Pantothenate and CoA biosynthesis in Apicomplexa and their promise as antiparasitic drug targets ” , PLoS Pathog. 17(12):el010124, doi: 10.1371/joumal.ppat.l010124; Guan, et al., 2021, "Exploring Heteroaromatic Rings as a Replacement for the Labile Amide of Antiplasmodial Pantothenamides ”, J Med Chem. 64(8):4478-4497, doi: 10.1021/acs.jmedchem.0c01755; Schalkwijk, et l., 2019. "Antimalarial pantothenamide metabolites target acetyl-coenzyme A biosynthesis in Plasmodium falciparum ”, Sci Transl Med. 11(510) - Erratum published: Sci Transl Med. 2021, 13(580):eabg8900. doi: 10.1126/scitranslmed.abg8900: Spry, et al., 2020, "Towarda Stable and Potent Coenzyme A-Targeting Antiplasmodial Agent: Structure-Activity Relationship Studies ofN-Phenethyl-a-methyl-pantothenamide ”, ACS Infect Dis. 6(7): 1844- 1854, doi: 10.1021/acsinfecdis.0c00075 (all of which are incorporated herein in their entireties by reference). In certain embodiments, the compound is a-PanAm. In certain embodiments, the compound is not a-PanAm.
In certain embodiments, the compounds of the disclosure comprise certain compounds disclosed herein, such as but not limited to a compound of formula (la) or (lb).
In certain embodiments, the compounds of the disclosure potentiate the biological activity of known antifungal drugs. In certain embodiments, the compounds of the disclosure have synergistic inhibitory effects against fungi when used in combination with other antifungal agents. In certain embodiments, the contemplated antifungal agents include but are not limited to agents that disrupt synthesis and/or activity of ergosterol, such as but not limited to azoles, morpholines, allylamines, and polyenes, as well as inhibitors of other metabolic pathways such as cell wall metabolism, as well as nucleic acid and protein synthesis.
Non-limiting examples of azole antifungal agents contemplated herein include:
(a) imidazoles: bifonazole, butoconazole. clotrimazole, econazole, fenticonazole, isoconazole, ketoconazole, luliconazole, miconazole, omoconazole, oxiconazole, sertaconazole, sulconazole, tioconazole.
(b) triazoles: albaconazole, efinaconazole, epoxiconazole, fluconazole, isavuconazole, itraconazole, posaconazole, propiconazole, ravuconazole, terconazole, voriconazole.
(c) thiazoles: abafungin.
(d) tetrazoles: oteseconazole.
Non-limiting examples of allylamine antifungal agents contemplated herein include butenafme, naftifme, and terbinafme.
Non-limiting examples of polyene antifungal agents contemplated herein include amphotericin B, candicidin, filipin, hamycin, natamycin, nystatin, and rimocidin.
Non-limiting examples of morpholine antifungal agents contemplated herein include amorolfme and fenpropimorph.
Non-limiting examples of inhibitors of other metabolic pathways include cycloheximide (a protein synthesis inhibitor), echinocandins (inhibitors of fungal cell wall synthesis; non-limiting examples include anidulafungin, caspofungin, and/or micafungin), cytosine analogues (such as but not limited to 5-flucytosine), aminoglycosides (such as but not limited to hygromy cin B).
In certain embodiments, the compounds of the disclosure increase potency of an antifungal agent against a fungal infection in a subject. In certain embodiments, the antifungal agent comprises for example agents that disrupt synthesis and/or activity of ergosterol, such as but not limited to azoles, morpholines, allylamines, and polyenes, as well as inhibitors of other metabolic pathways.
In certain embodiments, the compounds of the disclosure reduce at least one toxicity effect of an antifungal agent against a fungal infection in a subject when a compound of the disclosure is co-administered with the antifungal agent. In certain embodiments, administration of a compound of the disclosure and the antifungal agent allows for a lower dose of the antifungal agent as compared to the dose of the antifungal agent alone required to achieve equivalent antifungal effect. In certain embodiments, the antifungal agent comprises for example agents that disrupt synthesis and/or activity of ergosterol, such as but not limited to azoles, morpholines, allylamines, and polyenes, as well as inhibitors of other metabolic pathways.
In certain embodiments, the compounds of the disclosure reduce drug resistance against an antifungal agent administered to a subject to treat, ameliorate, and/or prevent a fungal infection in a subject when a compound of the disclosure is co-administered with the antifungal agent. In certain embodiments, administration of a compound of the disclosure and the antifungal agent allows for a lower dose/lower dosing frequency /lower overall dosing period of the antifungal agent as compared to the dose of the antifungal agent alone required to achieve equivalent antifungal effect. In certain embodiments, the antifungal agent comprises for example agents that disrupt synthesis and/or activity of ergosterol, such as but not limited to azoles, morpholines, allylamines, and polyenes, as well as inhibitors of other metabolic pathways.
In certain embodiments, the compounds of the disclosure sensitize fungal pathogens to an antifungal agent administered to a subject to treat, ameliorate, and/or prevent a fungal infection in a subject when a compound of the disclosure is co-administered with the antifungal agent. In certain embodiments, the antifungal agent comprises for example agents that disrupt synthesis and/or activity of ergosterol, such as but not limited to azoles, morpholines, allylamines, and polyenes, as well as inhibitors of other metabolic pathways.
Definitions
As used herein, each of the following terms has the meaning associated with it in this section. Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Generally, the nomenclature used herein and the laboratory procedures in animal pharmacology, pharmaceutical science, separation science, and organic chemistry are those well-known and commonly employed in the art. It should be understood that the order of steps or order for performing certain actions is immaterial, so long as the present teachings remain operable. Any use of section headings is intended to aid reading of the document and is not to be interpreted as limiting; information that is relevant to a section heading may occur within or outside of that particular section. All publications, patents, and patent documents referred to in this document are incorporated by reference herein in their entirety, as though individually incorporated by reference.
In the application, where an element or component is said to be included in and/or selected from a list of recited elements or components, it should be understood that the element or component can be any one of the recited elements or components and can be selected from a group consisting of two or more of the recited elements or components.
In the methods described herein, the acts can be carried out in any order, except when a temporal or operational sequence is explicitly recited. Furthermore, specified acts can be carried out concurrently unless explicit claim language recites that they be carried out separately. For example, a claimed act of doing X and a claimed act of doing Y can be conducted simultaneously within a single operation, and the resulting process will fall within the literal scope of the claimed process.
In this document, the terms "a." "an." or “the’' are used to include one or more than one unless the context clearly dictates otherwise. The term “or” is used to refer to a nonexclusive “or” unless otherw ise indicated. The statement “at least one of A and B” or “at least one of A or B” has the same meaning as “A, B, or A and B.”
As used herein, the term “about” will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which it is used. As used herein, “about” when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20%, ±10%, ±5%, ±1%, or ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.
As used herein, the term “alkenyl,” employed alone or in combination with other terms, means, unless otherwise stated, a stable monounsaturated or diunsaturated straight chain or branched chain hydrocarbon group having the stated number of carbon atoms. Examples include vinyl, propenyl (or allyl), crotyl, isopentenyl, butadienyl, 1,3-pentadienyl, 1,4-pentadienyl, and the higher homologs and isomers. A functional group representing an alkene is exemplified by -CH2-CH=CH2.
As used herein, the term “alkoxy” employed alone or in combination with other terms means, unless otherwise stated, an alkyl group having the designated number of carbon atoms, as defined elsewhere herein, connected to the rest of the molecule via an oxygen atom, such as, for example, methoxy, ethoxy, 1 -propoxy, 2-propoxy (or isopropoxy) and the higher homologs and isomers. A specific example is (Ci-Cs)alkoxy, such as, but not limited to, ethoxy and methoxy.
As used herein, the term “alkyl” by itself or as part of another substituent means, unless otherwise stated, a straight or branched chain hydrocarbon having the number of carbon atoms designated (i.e., C1-C10 means one to ten carbon atoms) and includes straight, branched chain, or cyclic substituent groups. Examples include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, neopentyl, hexyl, and cyclopropylmethyl. A specific embodiment is (Ci-Ce)alkyl, such as, but not limited to, ethyl, methyl, isopropyl, isobutyl, /7-pentyl, n-hexyl, and cyclopropylmethyl.
As used herein, the term “alkynyl” employed alone or in combination with other terms means, unless otherwise stated, a stable straight chain or branched chain hydrocarbon group with a triple carbon-carbon bond, having the stated number of carbon atoms. Nonlimiting examples include ethynyl and propynyl, and the higher homologs and isomers. The term “propargylic” refers to a group exemplified by -CH2-C=CH. The term "homopropargylic" refers to a group exemplified by -CH2CH2-C=CH.
As used herein, the term “aromatic” refers to a carbocycle or heterocycle with one or more polyunsaturated rings and having aromatic character, z.e., having (4n+2) delocalized n (pi) electrons, where ‘n’ is an integer.
As used herein, the term “aryl” employed alone or in combination with other terms means, unless otherwise stated, a carbocyclic aromatic system containing one or more rings (typically one, two or three rings) wherein such rings may be attached together in a pendent manner, such as a biphenyl, or may be fused, such as naphthalene. Examples include phenyl, anthracyl and naphthyl. Aryl groups also include, for example, phenyl or naphthyl rings fused with one or more saturated or partially saturated carbon rings (e.g, bicyclo [4.2.0] octa-1, 3,5- trienyl, or indanyl), which can be substituted at one or more carbon atoms of the aromatic and/or saturated or partially saturated rings.
As used herein, the term “aryl-(Ci-C6)alkyl” refers to a functional group wherein a one-to-six carbon alky lene chain is attached to an aryl group, e.g., -CEECEb-phenyl or -CH2- phenyl (or benzyl). Specific examples are aryl-CEE- and ary 1-CH(CH3)-. The term “substituted aryl-(Ci-C6)alkyl” refers to an aryl-(Ci-C6)alkyl functional group in which the aryl group is substituted. A specific example is substituted aryl(CH2)-. Similarly, the term “heteroaryl-(Ci-C6)alkyl” refers to a functional group wherein a one-to-three carbon alkylene chain is attached to a heteroaryl group, e.g., -CH2CH2-pyridyl. A specific example is heteroaryl-(CH2>-. The term '‘substituted heteroaryl-(Ci-C6)alky refers to a heteroaryl-(Ci- Cejalkyl functional group in which the heteroaryl group is substituted. A specific example is substituted heteroar 1 -(CH2)-.
In one aspect, the terms “co-administered” and “co-administration” as relating to a subject refer to administering to the subject a compound and/or composition of the disclosure along with a compound and/or composition that may also treat or prevent a disease or disorder contemplated herein. In certain embodiments, the co-administered compounds and/or compositions are administered separately , or in any kind of combination as part of a single therapeutic approach. The co-administered compound and/or composition may be formulated in any kind of combinations as mixtures of solids and liquids under a variety of solid, gel, and liquid formulations, and as a solution.
As used herein, the term “cycloalkyl” by itself or as part of another substituent refers to, unless otherwise stated, a cyclic chain hydrocarbon having the number of carbon atoms designated (i.e., C3-C6 refers to a cyclic group comprising a ring group consisting of three to six carbon atoms) and includes straight, branched chain or cyclic substituent groups. Examples of (Cs-Cejcycloalkyl groups are cyclopropyl, cyclobutyl. cyclopentyl and cyclohexyl. Cycloalkyl rings can be optionally substituted. Non-limiting examples of cycloalkyl groups include: cyclopropyl, 2-methyl-cyclopropyl, cyclopropenyl, cyclobutyl, 2,3-dihydroxycyclobutyl, cyclobutenyl, cyclopentyl, cyclopentenyl, cyclopentadienyl, cyclohexyl, cyclohexenyl, cycloheptyl, cyclooctanyl, decalinyl, 2.5-dimethylcyclopentyl. 3,5- dichlorocyclohexyl, 4-hydroxy cyclohexyl, 3,3,5-trimethylcyclohex-l-yl, octahydropental enyl. octahydro- I/7-indenyl, 3a.4.5.6.7.7a-hexahydro-3//-inden-4-yl. decahydroazulenyk bicyclo[6.2.0]decanyl, decahydronaphthalenyl, and dodecahydro- 1/7- fluorenyl. The term “cycloalky l” also includes bicyclic hydrocarbon rings, non-limiting examples of which include, bicyclo[2.1.1]hexanyl, bicyclo[2.2.1]heptanyl, bicyclo[3.1.1]heptanyl, l,3-dimethyl[2.2.1]heptan-2-yl, bicyclo[2.2.2]octanyl, and bicyclo[3.3.3]undecanyl.
As used herein, a “disease” is a state of health of a subject wherein the subject cannot maintain homeostasis, and wherein if the disease is not ameliorated then the subject’s health continues to deteriorate.
As used herein, a “disorder” in a subject is a state of health in which the subject is able to maintain homeostasis, but in which the subject’s state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the subject's state of health.
As used herein, the term ’‘halide” refers to a halogen atom bearing a negative charge. The halide anions are fluoride (F-), chloride (CL), bromide (Br ), and iodide (I ).
As used herein, the term “halo” or “halogen” alone or as part of another substituent refers to, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom.
As used herein, the term “heteroalkenyl” by itself or in combination with another term refers to, unless otherwise stated, a stable straight or branched chain monounsaturated or diunsaturated hydrocarbon group consisting of the stated number of carbon atoms and one or two heteroatoms selected from the group consisting of O, N, and S, and wherein the nitrogen and sulfur atoms may optionally be oxidized and the nitrogen heteroatom may optionally be quatemized. Up to two heteroatoms may be placed consecutively. Examples include - CH=CH-O-CH3, -CH=CH-CH2-OH, -CH2-CH=N-OCH3, -CH=CH-N(CH3)-CH3, and -CH2- CH=CH-CH2-SH.
As used herein, the term “heteroalkyl” by itself or in combination with another term refers to, unless otherwise stated, a stable straight or branched chain alkyl group consisting of the stated number of carbon atoms and one or two heteroatoms selected from the group consisting of O, N, and S, and wherein the nitrogen and sulfur atoms may be optionally oxidized and the nitrogen heteroatom may be optionally quatemized. The heteroatom(s) maybe placed at any position of the heteroalkyl group, including between the rest of the heteroalkyl group and the fragment to which it is attached, as well as attached to the most distal carbon atom in the heteroalkyl group. Examples include: -OCH2CH2CH3, - CH2CH2CH2OH. -CH2CH2NHCH3, -CH2SCH2CH3, and -CH2CH2S(=O)CH3. Up to two heteroatoms may be consecutive, such as, for example, -CH2NH-OCH3, or -CH2CH2SSCH3.
As used herein, the term “heteroaryl” or “heteroaromatic” refers to a heterocycle having aromatic character. A polycyclic heteroaryl may- include one or more rings that are partially saturated. Examples include tetrahydroquinoline and 2,3-dihydrobenzofuryl.
As used herein, the term “heterocycle” or “heterocyclyl” or “heterocyclic” by itself or as part of another substituent refers to. unless otherwise stated, an unsubstituted or substituted, stable, mono- or multi-cyclic heterocyclic ring system that comprises carbon atoms and at least one heteroatom selected from the group consisting of N, O, and S, and wherein the nitrogen and sulfur heteroatoms may be optionally oxidized, and the nitrogen atom may be optionally quatemized. The heterocyclic system may be attached, unless otherwise stated, at any heteroatom or carbon atom that affords a stable structure. A heterocycle may be aromatic or non-aromatic in nature. In certain embodiments, the heterocycle is a heteroaryl.
Examples of non-aromatic heterocycles include monocyclic groups such as aziridine, oxirane, thiirane, azetidine, oxetane, thietane, pyrrolidine, pyrroline, imidazoline, pyrazolidine, dioxolane, sulfolane, 2,3-dihydrofuran, 2,5-dihydrofuran, tetrahydrofuran, thiophane, piperidine, 1,2,3,6-tetrahydropyridine, 1.4-dihydropyridine, piperazine, morpholine, thiomorpholine, pyran, 2.3-dihydropyran, tetrahydropyran, 1,4-di oxane. 1,3- dioxane, homopiperazine, homopiperidine, 1,3-dioxepane, 4,7-dihydro-l,3-dioxepin, and hexamethyleneoxide.
Examples of heteroaryl groups include pyridyl, pyrazinyl, pyrimidinyl (such as, but not limited to, 2- and 4-pyrimidinyl), pyridazinyl. thienyl, furyl, pyrrolyl. imidazolyl, thiazolyl, oxazolyl, pyrazolyl, isothiazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, 1,3,4-triazolyl, tetrazolyl, 1,2,3-thiadiazolyl, 1,2,3-oxadiazolyl, 1,3,4-thiadiazolyl, and 1,3,4-oxadiazolyl.
Examples of polycyclic heterocycles include indolyl (such as, but not limited to, 3-, 4- , 5-, 6- and 7-indolyl), indolinyl. quinolyl, tetrahydroquinolyl, isoquinolyl (such as, but not limited to, 1- and 5-isoquinolyl), 1,2,3,4-tetrahydroisoquinolyl, cinnohnyl, quinoxalinyl (such as, but not limited to, 2- and 5 -quinoxalinyl), quinazolinyl, phthalazinyl, 1,8-naphthyridinyl, 1,4-benzodioxanyl, coumarin, dihydrocoumarin, 1,5-naphthyridinyl, benzo fury I (such as, but not limited to, 3-, 4-, 5-, 6- and 7-benzofuryl), 2,3-dihydrobenzofuryl, 1 ,2-benzisoxazolyl, benzothienyl (such as, but not limited to, 3-. 4-, 5-, 6-, and 7-benzothienyl), benzoxazolyl, benzothiazolyl (such as, but not limited to, 2-benzothiazolyl and 5-benzothiazolyl), purinyl, benzimidazolyl, benztriazolyl, thioxanthinyl, carbazolyl, carbolinyl, acridinyl, pyrrolizidinyl, and quinolizidinyl.
The aforementioned listing of heterocyclyl and heteroaryl moi eties is intended to be representative and not limiting.
As used herein, the term “pharmaceutical composition” or “composition” refers to a mixture of at least one compound useful within the disclosure with a pharmaceutically acceptable carrier. The pharmaceutical composition facilitates administration of the compound to a subject.
As used herein, the term “pharmaceutically acceptable” refers to a material, such as a carrier or diluent, which does not abrogate the biological activity or properties of the compound useful within the disclosure, and is relatively non-toxic, z.e., the material may be administered to a subject without causing undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained. As used herein, the term “pharmaceutically acceptable carrier” means a pharmaceutically acceptable material, composition or earner, such as a liquid or solid filler, stabilizer, dispersing agent, suspending agent, diluent, excipient, thickening agent, solvent or encapsulating material, involved in carrying or transporting a compound useful within the disclosure within or to the subject such that it may perform its intended function. Typically, such constructs are carried or transported from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation, including the compound useful within the disclosure, and not injurious to the subject. Some examples of materials that may serve as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as com starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, com oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; surface active agents; alginic acid; pyrogen-free water; isotonic saline; Ringer’s solution; ethyl alcohol; phosphate buffer solutions; and other non-toxic compatible substances employed in pharmaceutical formulations. As used herein, “pharmaceutically acceptable carrier” also includes any and all coatings, antibacterial and antifungal agents, and absorption delaying agents, and the like that are compatible with the activity of the compound useful within the disclosure, and are physiologically acceptable to the subject. Supplementary active compounds may also be incorporated into the compositions. The “pharmaceutically acceptable carrier” may further include a pharmaceutically acceptable salt of the compound useful within the disclosure. Other additional ingredients that may be included in the pharmaceutical compositions used in the practice of the disclosure are known in the art and described, for example in Remington’s Pharmaceutical Sciences (Genaro, Ed., Mack Publishing Co., 1985. Easton, PA), which is incorporated herein by reference.
As used herein, the language “pharmaceutically acceptable salt” refers to a salt of the administered compound prepared from pharmaceutically acceptable non-toxic acids and/or bases, including inorganic acids, inorganic bases, organic acids, inorganic bases, solvates (including hydrates) and clathrates thereof.
As used herein, a “pharmaceutically effective amount,” “therapeutically effective amount,’' or “effective amount” of a compound is that amount of compound that is sufficient to provide a beneficial effect to the subject to which the compound is administered.
The term “prevent,” “preventing,” or “prevention” as used herein means avoiding or delaying the onset of symptoms associated with a disease or condition in a subject that has not developed such symptoms at the time the administering of an agent or compound commences. Disease, condition and disorder are used interchangeably herein.
As used herein. “PZ2891” corresponds to the compound 6-(4-(2-(4- isopropylphenyl)acetyl)piperazin-l-yl)pyridazine-3-carbonitrile or
Figure imgf000021_0001
salt or solvate thereof. PZ-2891 is a pantothenate kinase (PANK) modulator with ICso values of 40.2nM, 0.7nM and 1.3nM for human pantothenate kinases PANK10, PANK2, and PANK3, respectively.
By the term “specifically bind” or “specifically binds” as used herein is meant that a first molecule preferentially binds to a second molecule (e.g., a particular receptor or enzyme), but does not necessarily bind only to that second molecule.
As used herein, the terms “subj ect” and “individual” and “patient” can be used interchangeably and may refer to a human or non-human mammal or a bird. Non-human mammals include, for example, livestock and pets, such as ovine, bovine, porcine, canine, feline and murine mammals. In certain embodiments, the subject is human.
As used herein, the term “substituted” refers to that an atom or group of atoms has replaced hydrogen as the substituent attached to another group.
As used herein, the term “substituted alkyl,” “substituted cycloalkyl,” “substituted alkenyl,” or “substituted alkynyl” refers to alkyl, cycloalkyl, alkenyl, or alkynyl, as defined elsewhere herein, substituted by one, two or three substituents independently selected from the group consisting of halogen, -OH, alkoxy, tetrahydro-2 -H-pyranyl, -NH2. -NH(Ci-Ce alkyl), -N(Ci-Ce alkyl)2. l-methyl-imidazol-2-yl, pyridin-2-yl, pyridin-3-yl, pyndm-4-yl, - C(=O)OH, -C(=O)O(Ci-C6)alkyl, trifluoromethyl, -C=N, -C(=O)NH2, -C(=O)NH(Ci- C6)alkyl, -C(=O)N((Ci-C6)alkyl)2, -SO2NH2, -SO2NH(CI-C6 alkyl), -SO2N(CI-C6 alky 1)2, - C(=NH)NH2, and -NO2, in certain embodiments containing one or two substituents independently selected from halogen, -OH, alkoxy, -NH2. trifluoromethyl, -N(CH3)2. and - C(=O)OH, in certain embodiments independently selected from halogen, alkoxy and -OH. Examples of substituted alkyls include, but are not limited to, 2,2-difluoropropyl, 2- carboxy cyclopentyl and 3-chloropropyl.
For aryl, aryl-(Ci-C3)alkyl and heterocyclyl groups, the term ‘'substituted’’ as applied to the rings of these groups refers to any level of substitution, namely mono-, di-, tri-, tetra-, or penta-substitution, where such substitution is permitted. The substituents are independently selected, and substitution may be at any chemically accessible position. In certain embodiments, the substituents vary in number between one and four. In other embodiments, the substituents vary in number between one and three. In yet other embodiments, the substituents vary7 in number between one and two. In yet other embodiments, the substituents are independently selected from the group consisting of Ci-Ce alkyl, -OH, Ci-Ce alkoxy, halo, amino, acetamido and nitro. As used herein, where a substituent is an alkyl or alkoxy group, the carbon chain may be branched, straight or cyclic.
In certain embodiments, each occurrence of alkyl, alkenyl, alkynyl, or cycloalkyl is independently optionally substituted with at least one substituent selected from the group consisting of Ci-Ce alkyl, C3-C8 cycloalkyl, halo, cyano (-CN), -ORa. optionally substituted phenyl (thus yielding, in non-limiting examples, optionally substituted phenyl-(Ci-C3 alkyl), such as, but not limited to, benzyd or substituted benzyl), optionally7 substituted heteroary l, optionally substituted heterocyclyl, -C(=O)ORa, -OC(=O)Ra, -SRa, -S(=O)Ra, -S(=O)2Ra, - S(=O)2NRaRa, -N(Ra)S(=O)2Ra, -N(Ra)C(=O)Ra, -C(=O)NRaRa, and -N(Ra)(Ra), wherein each occurrence of Ra is independently H. optionally substituted Ci-Ce alky l, optionally substituted C3-C8 cycloalkyl, optionally substituted aryl, or optionally substituted heteroaryl, or two Ra groups combine with the N to which they are bound to form a heterocycle.
In certain embodiments, each occurrence of aryl or heteroaryl is independently optionally substituted with at least one substituent selected from the group consisting of Ci- Ce alkyl, C3-C8 cycloalkyl, phenyl, Ci-Ce hydroxyalkyl, (Ci-Ce alkoxy )-Ci-Ce alkyd, Ci-Ce haloalkyl, Ci-C6 haloalkoxy, halogen, -CN, -ORb, -N(Rb)(Rb), -NO2, -C(=O)N(Rb)(Rb), - C(=O)ORb, -OC(=O)Rb, -SRb, -S(=O)Rb, -S(=O)2Rb, -N(Rb)S(=O)2Rb, -S(=O)2N(Rb)(Rb), acyl, and Ci-Ce alkoxycarbonyl, wherein each occurrence of Rb is independently H, Ci-Ce alkyl, or Cs-Cs cycloalkyl. wherein in Rb the alkyl or cycloalkyl is optionally substituted with at least one selected from the group consisting of halogen, -OH, Ci-Ce alkoxy, and heteroaryd; or substituents on two adjacent carbon atoms combine to form -O(CH2)i-3O-.
In certain embodiments, each occurrence of aryl or heteroaryl is independently optionally substituted with at least one substituent selected from the group consisting of Ci- Ce alkyl, C3-Cs cycloalkyl, phenyl, Ci-Ce hydroxyalkyl, (Ci-Ce alkoxy )-Ci-Ce alkyl, Ci-Ce haloalkyl, Ci-C6 haloalkoxy, halogen, -ORb. -C(=O)N(Rb)(Rb), -C(=O)ORb, -OC(=O)Rb. - SRb, -S(=O)Rb, -S(=O)2Rb, and -N(Rb)S(=O)2Rb, wherein each occurrence of Rb is independently H, Ci-Ce alkyl, or Cs-Cs cycloalkyl, wherein in Rb the alkyd or cycloalkyl is optionally substituted with at least one selected from the group consisting of halogen, -OH, Ci-Ce alkoxy, and heteroaryl; or substituents on two adjacent carbon atoms combine to form - O(CH2)I-3O-.
In certain embodiments, the alkyl, alkenyl, alkynyl, cycloalkyl, heteroaryl, heterocyclyl, aryl, or benzyl group is optionally independently substituted with at least one group selected from the group consisting of Ci-Ce alkyd; Ci-Ce alkoxy; Ci-Ce haloalky l; Ci- C6 haloalkoxy; -NH2, -NH(CI-C6 alkyl), -N(Ci-C& alkyl)(Ci-C6 alk d), halogen, -OH; -CN; phenoxy. -NHC(=O)H, -NHC(=O)CI-C6 alkyl, -C(=O)NH2, -C(=O)NHCI-C6 alkyl, - C(=O)N(CI-C6 alkyl)(C 1-C6 alkyd), tetrahydropyranyl, morpholinyl, -C(=O)CH3, - C(=O)CH2OH, -C(=O)NHCH3, -C(=O)CH2OMe, or an Woxide thereof.
In certain embodiments, each occurrence of the heteroary 1 is independently selected from the group consisting of quinolinyl, imidazo[l,2-a]pyridyl, pyridyl, pyrimidyl, pyrazinyl, imidazolyl, thiazolyl, pyrazolyl. isoxazolyl. oxadiazolyl (including 1.2.3-, 1,2,4-. 1,2,5-. and 1,3,4-oxadiazole), and triazolyl (such as 1 ,2,3-triazolyl and 1,2,4-triazolyl).
In certain embodiments, each occurrence of the heterocycly 4 group is independently selected from the group consisting of tetrahy drofuranyl. tetrahy dropyranyl, piperidinyl, piperazinyl, pyrrolidinyl, morpholinyl, thiomorpholinyl, 1-oxido-thiomorpholinyl, 1.1- dioxido-thiomorpholinyl, oxazolidinyl, azetidinyl, and the corresponding oxo analogues (where a methylene ring group is replaced with a carbonyl) thereof.
Unless otherwise noted, when two substituents are taken together to form a ring having a specified number of ring atoms (e.g.. R’ and R taken together with the nitrogen to w hich they are attached to form a ring having from 3 to 7 ring members), the ring can have carbon atoms and optionally7 one or more (e.g, 1 to 3) additional heteroatoms independently7 selected from nitrogen, oxygen, or sulfur. The ring can be saturated or partially saturated, and can be optionally substituted.
Whenever a term or either of their prefix roots appear in a name of a substituent the name is to be interpreted as including those limitations provided herein. For example, whenever the term “alkyl” or "aryl" or either of their prefix roots appear in a name of a substituent (e.g., arylalkyd, alkydamino) the name is to be interpreted as including those limitations given elsewhere herein for “alkyl” and “aryl” respectively.
In certain embodiments, substituents of compounds are disclosed in groups or in ranges. It is specifically intended that the description include each and every individual subcombination of the members of such groups and ranges. For example, the term “Ci-6 alkyl” is specifically intended to individually disclose Ci, C2, C3, C4, Cs, Ce, Ci-Ce, C1-C5, C1-C4, C1-C3, C1-C2, C2-C6, C2-C5, C2-C4, C2-C3, C3-C6, C3-C5, C3-C4, C4-C6, C4-C5, and C5-C6 alkyl.
The terms “treat,” “treating” and “treatment.” as used herein, means reducing the frequency or severin’ with which symptoms of a disease or condition are experienced by a subject by virtue of administering an agent or compound to the subject.
Ranges: throughout this disclosure, various aspects of the present disclosure can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the present disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4. from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. For example, a range of “about 0.1% to about 5%” or “about 0.1% to 5%” should be interpreted to include not just about 0.1% to about 5%, but also the individual values (e.g, 1%, 2%, 3%, and 4%) and the sub-ranges (e.g., 0.1% to 0.5%, 1.1% to 2.2%, 3.3% to 4.4%) within the indicated range. The statement “about X to Y” has the same meaning as “about X to about Y,” unless indicated otherwise. Tikewise, the statement “about X, Y, or about Z” has the same meaning as “about X, about Y, or about Z,” unless indicated otherwise. This applies regardless of the breadth of the range.
Compounds
The disclosure includes a compound described herein, or a salt, solvate, isotopically labelled derivative, stereoisomer (such as, in a non-limiting example, an enantiomer or diastereoisomer, and/or any mixtures thereof, such as. in a non-limiting example, mixtures in any proportions of enantiomers and/or diastereoisomers thereof), tautomer and any mixtures thereof, and/or geometric isomer and any mixtures thereof:
In certain embodiments, the compound is a compound of formula (la) or (lb), or a salt, solvate, isotopically labelled derivative, stereoisomer, tautomer, or geometric isomer thereof, and any mixtures thereof:
Figure imgf000025_0001
wherein in (la) or (lb):
R1 is selected from the group consisting of H, -NR I;'R41’. -NR4c(optionally substituted phenyl), and optionally substituted N-linked heterocyclyl;
R2 is selected from the group consisting of phenyl and -N(R4c)-(CH2)i-3-(optionally substituted phenyl);
R3 is selected from the group consisting of F, Cl, Br, I, -OR4d. -NR4dR4e, -C(=O)OR4d,- C(=O)NR4eR4f. -C(=O)N(R4g)-(CH2)i-3-(optionally substituted phenyl), and -C(=O)N(R4h)- (CH2)i-3-(optionally substituted heteroaryl); each occurrence of R4a-R4h is independently selected from the group consisting of H, optionally substituted Ci-Ce alkyl, and optionally substituted Cs-Cs cycloalkyl; each occurrence of alkyl, cycloalkyl, phenyl, heterocyclyl, and heteroaryl is optionally independently substituted with at least one of Ci-Ce alkyl, C3-C8 cycloalkyl, halogen, -CN, - ORa, optionally substituted phenyl, optionally substituted heteroaryl, optionally substituted heterocyclyl, -C(=O)ORa, -OC(=O)Ra, -SRa, -S(=O)Ra, -S(=O)2Ra, -S(=O)2NRaRa, - N(Ra)S(=O)2Ra, -N(Ra)C(=O)Ra, -C(=O)NRaRa. and -N(Ra)(Ra); and each occurrence of Ra is independently H. optionally substituted Ci-Ce alkyl, optionally substituted C3-C8 cycloalkyl, optionally substituted phenyl, or optionally substituted heteroaryl, or two Ra groups combine with the N to which they are bound to form Cs-Cs heterocyclyl.
In certain embodiments, R1 is H. In certain embodiments, R1 is -NR4aR4b. In certain embodiments, R1 is -NR4c(optionally substituted phenyl). In certain embodiments, R1 is optionally substituted N-linked heterocyclyl.
In certain embodiments, R2 is optionally substituted phenyl. In certain embodiments, R2 is -N(R4c)-(CH2)i-3-(optionally substituted phenyl);
In certain embodiments, R3 is F. In certain embodiments, R3 is Cl. In certain embodiments, R3 is Br. In certain embodiments, R3 is I. In certain embodiments, R3 is - OR4d. In certain embodiments, R3 is -NR4dR4e. In certain embodiments, R3 is -C(=O)OR4d. In certain embodiments, R3 is -C(=O)NR4eR4f. In certain embodiments, R3 is -C(=O)N(R4g)- (CH2)i-3-optionally substituted phenyl). In certain embodiments, R3 is -C(=O)N(R4h)-(CH2)i- 3-(optionally substituted heteroaryl). In certain embodiments, R4a is H. In certain embodiments, R4a is optionally substituted Ci-Ce alkyl. In certain embodiments, R4a is optionally substituted Cs-Cs cycloal kyl.
In certain embodiments, R4b is H. In certain embodiments, R4a is optionally substituted Ci-Ce alkyl. In certain embodiments, R4b is optionally substituted Cs-Cs cycloalkyd.
In certain embodiments, R4c is H. In certain embodiments, R4c is optionally substituted Ci-Ce alkyl. In certain embodiments, R4c is optionally substituted C3-C8 cycloalky l.
In certain embodiments, R4d is H. In certain embodiments, R4d is optionally substituted Ci-Ce alkyl. In certain embodiments, R4d is optionally substituted C3-C8 cycloalky 1.
In certain embodiments, R4e is H. In certain embodiments, R4a is optionally substituted Ci-Ce alkyl. In certain embodiments, R4e is optionally substituted C3-C8 cycloalky l.
In certain embodiments, R4f is H. In certain embodiments, R4f is optionally substituted Ci-Ce alkyl. In certain embodiments, R4f is optionally substituted C3-C8 cycloalky l.
In certain embodiments, R4g is H. In certain embodiments, R4g is optionally substituted Ci-Ce alkyl. In certain embodiments, R4g is optionally substituted C3-C8 cycloalky l.
In certain embodiments, R4h is H. In certain embodiments, R4b is optionally substituted Ci-Ce alkyl. In certain embodiments, R4h is optionally substituted C3-C8 cycloalkyd.
In certain embodiments, R1 is selected from the group consisting of H, -NR4aR4b, and optionally substituted N-linked heterocyclyl.
In certain embodiments, R1 is NR4c(optionally substituted phenyl).
In certain embodiments, R3 is -NR4dR4e.
In certain embodiments, R3 is -C(=O)OR4d.
In certain embodiments, each phenyl in R2 is independently substituted with at least one C2-C8 alky 1. In other embodiments, each phenyl in R2 is independently substituted at the ortho-position with at least one C2-C8 alkyl. In other embodiments, each phenyl in R2 is independently substituted at the meta-position with at least one C2-C8 alkyl. In other embodiments, each phenyl in R2 is independently substituted at the para-position with at least one C2-C8 alkyl.
In certain embodiments, the N-linked heterocyclyl is aziridinyl. In certain embodiments, the N-linked heterocyclyl is azetidinyl. In certain embodiments, the N-linked heterocyclyl is pyrrolidinyl. In certain embodiments, the N-linked heterocyclyl is pyrazolidinyl. In certain embodiments, the N-linked heterocyclyl is piperidinyl. In certain embodiments, the N-linked heterocyclyl is 1,2,3,6-tetrahydropyridinyl. In certain embodiments, the N-linked heterocyclyl is 1,4-dihydropyridinyl. In certain embodiments, the N-linked heterocyclyl is piperazinyl. In certain embodiments, the N-linked heterocyclyl is morpholinyl. In certain embodiments, the N-linked heterocyclyl is thiomorpholinyl. In certain embodiments, the N-linked heterocyclyl is homopiperazinyl. In certain embodiments, the N-linked heterocyclyl is or homopiperidinyl.
In certain embodiments, the heteroaryl is imidazolyl. In certain embodiments, the heteroaryl is pyridinyl. In certain embodiments, the heteroaryl is pyrimidinyl. In certain embodiments, the heteroaryl is pyrazinyl. In certain embodiments, the heteroaryl is thienyl. In certain embodiments, the heteroaryl is furyl. In certain embodiments, the heteroaryl is pyrrolyl. In certain embodiments, the heteroaryl is thiazolyl. In certain embodiments, the heteroar l is oxazolyl. In certain embodiments, the heteroaryl is pyrazolyl. In certain embodiments, the heteroaryl is isothiazolyl. In certain embodiments, the heteroaryl is 1,2,3- triazolyl. In certain embodiments, the heteroaryl is 1,2,4-triazolyl. In certain embodiments, the heteroaryl is 1.3,4-triazolyl. In certain embodiments, the heteroaryl is tetrazolyl. In certain embodiments, the heteroaryl is 1,2,3-thiadiazolyl. In certain embodiments, the heteroary l is 1,2,3-oxadiazolyl. In certain embodiments, the heteroaryl is 1,3,4-thiadiazolyl. In certain embodiments, the heteroaryl is 1,3,4-oxadiazolyl.
Non-limiting examples of the compound of formula (la) or (lb) include:
Figure imgf000027_0001
5-((4-acetylpiperazin-l-yl)methyl)-2-((4-(tert-butyl)benzyl)amino)- [1, 2, 4]tri azolof l,5-a]pyrimidin-7(4H)-one;
Figure imgf000027_0002
tert-buty l 4-((2-((4-(tert-butyl)benzyl)amino)-7-oxo-4,7-dihydro-[1.2.4]triazolo[l,5- a]pyrimidin-5-yl)methyl)piperazine-l-carboxylate;
Compound
Figure imgf000028_0001
2-((4-(tert-butyl)benzyl)amino)-5-methyl-[l,2,4]triazolo[l,5-a]pyrimidine-7- carboxylic acid;
Compound
Figure imgf000028_0002
N-(2-amino-2-oxoethyl)-2-((4-(tert-butyl)benzyl)amino)-5-methyl-[l,2,4]triazolo[l,5- a]pyrimidine-7 -carboxamide;
Compound
Figure imgf000028_0003
2-(4-(tert-butyl)phenyl)-5-((4-methylpiperazin-l-yl)methyl)-[l,2,4]triazolo[l,5- a]pyrimidin-7(4H)-one;
Compound
Figure imgf000028_0004
N-(4-(tert-butyl)benzyl)-7-methoxy-5-(morpholinomethyl)-[l,2,4]triazolo[l,5- a]pyrimidin-2-amine;
Compound
Figure imgf000028_0005
2-((4-(tert-butyl)benzyl)arruno)-5-methyl-N-(pyridin-2-yl)-[l,2,4]triazolo[l,5- a]pyrimidine-7 -carboxamide;
Compound
Figure imgf000029_0001
methyl (2-((4-(tert-butyl)benzyl)amino)-5-methyl-[l,2,4]triazolo[1.5-a]pyrimidine-7- carbonyl)glycinate; Compound
Figure imgf000029_0002
2-((4-(tert-butyl)benzyl)amino)-N-(2-hydroxyethyl)-5-methyl-[l,2,4]triazolo[l,5- a]pyrimidine-7-carboxamide;
Compound
Figure imgf000029_0003
5-((isopropylamino)methyl)-2-((4-isopropylbenzyl)amino)-[l,2,4]triazolo[l,5- a]pyrimidin-7(4H)-one;
Compound
Figure imgf000029_0004
2-((2-((4-(tert-butyl)benzyl)amino)-5-(morpholinomethyl)-[l,2,4]triazolo[l,5- a]pyrimidin-7-yl)oxy)acetamide:
Compound
Figure imgf000029_0005
2-((2-((4-(tert-butyl)benzyl)amino)-5-(morpholinomethyl)-[l,2,4]triazolo[l,5- a]pyrimidin-7-yl)oxy )ethan- 1 -ol;
Compound
Figure imgf000030_0001
N2-(4-(tert-butyl)benzyl)-5-(morpholinomethyl)-[l,2,4]triazolo[l,5-a]pyrimidine-2,7- diamine;
Compound
Figure imgf000030_0002
2-((2-((4-(tert-butyl)benzyl)amino)-5-(morpholinomethyl)-[l, 2, 4]tri azolof 1,5- a]pyrimi din-7 -yl)oxy)acetic acid;
Compound
Figure imgf000030_0003
N-(4-(tert-butyl)benzyl)-7-ethoxy-5-(morpholinomethyl)-[l,2,4]triazolo[l,5- a]pyrimidin-2-amine;
Compound
Figure imgf000030_0004
N-(4-(tert-butyl)benzyl)-7-chloro-5-(morpholinomethyl)-[l,2,4]triazolo[l,5-
Figure imgf000030_0005
2-((4-(tert-butyl)benzyl)amino)-5-methyl-N-(pyridin-3-ylmethyl)-[l,2,4]triazolo[1.5- a]pyrimidine-7-carboxamide; Compound
Figure imgf000031_0001
(2-((4-(tert-butyl)benz d)amino)-5-methyl-[l,2,4]triazololl.5-aJpyrimidine-7- carbonyl)glycine.
Compound
Figure imgf000031_0002
2-((4-isopropylbenzyl)amino)-5-(morpholinomethyl)-[l,2,4]triazolo[l,5-a]pyrimidin-
7(4H)-one;
Compound
Figure imgf000031_0003
2-((4-isopropylbenzyl)amino)-5-methyl-[l,2,4]triazolo[l,5-a]pyrimidin-7(4H)-one;
Compound
Figure imgf000031_0004
5-((phenylamino)methyl)-2-(o-tolyl)-[l,2,4]triazolo[l,5-a]pyrimidin-7(4H)-one;
Compound
Figure imgf000031_0005
2-((4-isopropylbenzyl)amino)-5-(pyrrolidin-l-ylmethyl)-[ 1.2,4]tri azolof 1,5- aJpyrimidin-7(4H)-one;
Compound
Figure imgf000031_0006
N2-(4-(tert-butyl)benzyl)-N7-methyl-5-(morpholinomethyl)-[l,2,4]triazolo[l,5- a]pyrimidine-2,7-diamine; Compound
Figure imgf000032_0001
2-((4-(tert-butyl)benzyl)amino)-5-(morpholinomethyl)-[l,2,4]triazolo[l,5- a]pyrimi din-7 (4H)-one;
Compound
Figure imgf000032_0002
N2-(4-(tert-butyl)benzyl)-N7,N7-dimethyl-5-(morpholinomethyl)-[1.2.4]triazolofl,5- a]pyrimidine-2,7-diamine;
Compound
Figure imgf000032_0003
2-((4-(tert-butyl)benzyl)amino)-5-((4-methylpiperazin-l-yl)methyl)-
[l,2,4]triazolo[l,5-a]pyrimidin-7(4H)-one;
Compound
Figure imgf000032_0004
2-((4-(tert-butyl)benzyl)amino)-5-(piperidin-l-ylmethyl)-[l,2,4]triazolo[l,5- a]pyrimidin-7(4H)-one; or a salt, solvate, isotopically labelled derivative, stereoisomer, tautomer, or geometric isomer thereof, and any mixtures thereof.
In certain embodiments, the compound is not: Compound A5: 2-(4-(tert- butyl)phenyl)-5-((4-methylpiperazin-l-yl)methyl)-[l,2,4]triazolo[l,5-a]pyrimidin-7(4H)-one, or a salt, solvate, isotopically labelled derivative, stereoisomer, tautomer, or geometric isomer thereof and any mixtures thereof
In certain embodiments, the compound is not: Compound A10: 5- ((isopropylamino)methyl)-2-((4-isopropylbenzyl)amino)-[l,2,4]triazolo[l,5-a]pyrimidin- 7(4H)-one. or a salt, solvate, isotopically labelled derivative, stereoisomer, tautomer, or geometric isomer thereof, and any mixtures thereof.
In certain embodiments, the compound is not: Compound Bl: 2-((4- isopropylbenzyl)amino)-5-(morpholinomethyl)-[l,2,4]triazolo[l,5-a]pyrimidin-7(4H)-one. or a salt, solvate, isotopically labelled derivative, stereoisomer, tautomer, or geometric isomer thereof, and any mixtures thereof.
In certain embodiments, the compound is not: Compound B2: 2-((4- isopropylbenzyl)amino)-5-methyl-[l,2,4]triazolo[l,5-a]pyrimidin-7(4H)-one, or a salt, solvate, isotopically labelled derivative, stereoisomer, tautomer, or geometric isomer thereof, and any mixtures thereof.
In certain embodiments, the compound is not: Compound B3: 5- ((phenylamino)methyl)-2-(o-tolyl)-[l,2,4]triazolo[l,5-a]pyrimidin-7(4H)-one, or a salt, solvate, isotopically labelled derivative, stereoisomer, tautomer, or geometric isomer thereof, and any mixtures thereof.
In certain embodiments, the compound is not: Compound B4: 2-((4- isopropylbenzyl)amino)-5-(pyrrolidin-l-ylmethyl)-[l,2,4]triazolo[l,5-a]pyrimidin-7(4H)-one, or a salt, solvate, isotopically labelled derivative, stereoisomer, tautomer, or geometric isomer thereof, and any mixtures thereof.
In certain embodiments, the compound is not 2-phenyl-5-((propylamino)methyl)-
[1.2.4]triazolo[l,5-a]pyrimidin-7(4H)-one, or a salt, solvate, isotopically labelled derivative, stereoisomer, tautomer, or geometric isomer thereof, and any mixtures thereof. In certain embodiments, the compound is not 5-((4-methylpiperidin-l-yl)methyl)-2-phenyl-
[1.2.4]triazolo[ 1.5-a]pyrimidin-7(4H)-one, or a salt, solvate, isotopically labelled derivative, stereoisomer, tautomer, or geometric isomer thereof, and any mixtures thereof. In certain embodiments, the compound is not 5-((benzyl(methyl)amino)methyl)-2-phenyl-
[1.2.4]triazolo[l,5-a]pyrimidin-7(4H)-one, or a salt, solvate, isotopically labelled derivative, stereoisomer, tautomer, or geometric isomer thereof, and any mixtures thereof. In certain embodiments, the compound is not 5-((benzyl(2-hydroxyethyl)amino)methyl)-2-phenyl-
[l,2,4]triazolo[l,5-a]pyrimidin-7(4H)-one, or a salt, solvate, isotopically labelled derivative, stereoisomer, tautomer, or geometric isomer thereof, and any mixtures thereof. In certain embodiments, the compound is not 2-(4-chlorophenyl)-5-((3,4-dihydroisoquinolin-2(lH)- yl)methyl)-[l,2,4]tnazolo[1.5-a]pyrimidin-7(4H)-one, or a salt, solvate, isotopically labelled derivative, stereoisomer, tautomer, or geometric isomer thereof, and any mixtures thereof. In certain embodiments, the compound is not 5-(((2-methoxyphenyl)amino)methyl)-2-(p-tolyl)-
[l,2,4]triazolo[l,5-a]pyrimidin-7(4H)-one, or a salt, solvate, isotopically labelled derivative, stereoisomer, tautomer, or geometric isomer thereof, and any mixtures thereof. In certain embodiments, the compound is not 2-(p-tolyl)-5-((m-tolylamino)methyl)-[l,2,4]triazolo[l,5- a]pyrimidin-7(4H)-one, or a salt, solvate, isotopically labelled derivative, stereoisomer, tautomer, or geometric isomer thereof, and any mixtures thereof. In certain embodiments, the compound is not 2-(p-tolyl)-5-((p-tolylamino)methyl)-[l,2,4]triazolo[l,5-a]pyrimidin-7(4H)- one, or a salt, solvate, isotopically labelled derivative, stereoisomer, tautomer, or geometric isomer thereof, and any mixtures thereof. In certain embodiments, the compound is not 2-(p- tolyl)-5-((o-tolylamino)methyl)-[l,2,4]triazolo[l,5-a]pyrimidin-7(4H)-one. or a salt, solvate, isotopically labelled derivative, stereoisomer, tautomer, or geometric isomer thereof, and any mixtures thereof. In certain embodiments, the compound is not 2-(4-methoxyphenyl)-5- (piperidin-l-ylmethyl)-[l,2,4]triazolo[l,5-a]pyrimidin-7(4H)-one, or a salt, solvate, isotopically labelled derivative, stereoisomer, tautomer, or geometric isomer thereof, and any mixtures thereof. In certain embodiments, the compound is not 2-(4-methoxyphenyl)-5- (morpholinomethyl)-[l,2,4]triazolo[l,5-a]pyrimidin-7(4H)-one, or a salt, solvate, isotopically labelled derivative, stereoisomer, tautomer, or geometric isomer thereof, and any mixtures thereof. In certain embodiments, the compound is not 5-(((2,4- dimethoxyphenyl)amino)methyl)-2-(4-methoxyphenyl)-[l,2,4]triazolo[1.5-a]pyrimidin- 7(4H)-one, or a salt, solvate, isotopically labelled derivative, stereoisomer, tautomer, or geometric isomer thereof, and any mixtures thereof. In certain embodiments, the compound is not 2-(4-(tert-buty l)phenyl)-5-((propylamino)methyl)-[l,2,4]triazolo[l,5-a]pyrimidin- 7(4H)-one, or a salt, solvate, isotopically labelled derivative, stereoisomer, tautomer, or geometric isomer thereof, and any mixtures thereof. In certain embodiments, the compound is not 2-(3,4-dimethylphenyl)-5-((p-tolylamino)methyl)-[l ,2,4]triazolo[l ,5-a]pyrimidin- 7(4H)-one, or a salt, solvate, isotopically labelled derivative, stereoisomer, tautomer, or geometric isomer thereof, and any mixtures thereof. In certain embodiments, the compound is not 5-methyl-2-(phenylamino)-[1.2.4]triazolo[l,5-a]pyrimidin-7-ol, or a salt, solvate, isotopically labelled derivative, stereoisomer, tautomer, or geometric isomer thereof, and any mixtures thereof. In certain embodiments, the compound is not 5-methyl-2-phenyl-
[1.2.4]triazolo[l,5-a]pyrimidin-7-ol, or a salt, solvate, isotopically labelled derivative, stereoisomer, tautomer, or geometric isomer thereof, and any mixtures thereof. In certain embodiments, the compound is not 2-(benzylamino)-5-(morpholinomethyl)-
[1.2.4]triazolo[l,5-a]pyrimidin-7(4H)-one, or a salt, solvate, isotopically labelled derivative, stereoisomer, tautomer, or geometric isomer thereof, and any mixtures thereof. In certain embodiments, the compound is not 2-((3,4-dimethoxybenzyl)amino)-5-(morpholinomethyl)-
[1.2.4]triazolo[1.5-a]pyrimidin-7(4H)-one, or a salt, solvate, isotopically labelled derivative, stereoisomer, tautomer, or geometric isomer thereof, and any mixtures thereof. In certain embodiments, the compound is not methyl 2-((2-((4-(tert-butyl)benzyl)amino)-5- (morpholinomethyl)-[l,2,4]triazolo[1.5-a]pyrimidin-7-yl)oxy)acetate, or a salt, solvate, isotopically labelled derivative, stereoisomer, tautomer, or geometric isomer thereof, and any mixtures thereof. In certain embodiments, the compound is not 7-(benzyloxy)-N-(4-(tert- butyl)benzyl)-5-(morphohnomethyl)-[l,2,4]triazolo[l,5-a]pyrimidin-2-amine, or a salt, solvate, isotopically labelled derivative, stereoisomer, tautomer, or geometric isomer thereof, and any mixtures thereof. In certain embodiments, the compound is not 2-((2-((4-(tert- butyl)benzyl)amino)-5-(morpholinomethyl)-[l,2,4]triazolo[l,5-a]pyrimidin-7-yl)oxy)-N,N- dimethylacetamide.
In certain embodiments, the compound of the disclosure is any compound disclosed herein, or a salt, solvate, prodrug, isotopically labelled, stereoisomer, any mixture of stereoisomers, tautomer, and/or any mixture of tautomers thereof.
The compounds of the disclosure may possess one or more stereocenters, and each stereocenter may exist independently in either the (R)- or (^-configuration. In certain embodiments, compounds described herein are present in optically active or racemic forms. The compounds described herein encompass racemic, optically active, regioisomeric and stereoisomeric forms, or combinations thereof that possess the therapeutically useful properties described herein. Preparation of optically active forms is achieved in any suitable manner, including, by way of non-limiting example, by resolution of the racemic form with recrystallization techniques, synthesis from optically active starting materials, chiral synthesis, or chromatographic separation using a chiral stationary phase. A compound illustrated herein by the racemic formula further represents either of the two enantiomers or any mixtures thereof, or in the case where two or more chiral centers are present, all diastereomers or any mixtures thereof.
In certain embodiments, the compounds of the disclosure exist as tautomers. All tautomers are included within the scope of the compounds recited herein.
Compounds described herein also include isotopically labeled compounds wherein one or more atoms is replaced by an atom having the same atomic number, but an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes suitable for inclusion in the compounds described herein include and are not limited to 2H, 3H, nC, 13C, 14C, 36C1, 18F, 123I, 125I, 1?N, 15N, 15O, 17O, 180, 32P, and 35S. In certain embodiments, substitution with heavier isotopes such as deuterium affords greater chemical stability. Isotopically labeled compounds are prepared by any suitable method or by processes using an appropriate isotopically labeled reagent in place of the non-labeled reagent otherwise employed.
In certain embodiments, the compounds described herein are labeled by other means, including, but not limited to, the use of chromophores or fluorescent moieties, bioluminescent labels, or chemiluminescent labels.
In all of the embodiments provided herein, examples of suitable optional substituents are not intended to limit the scope of the claimed disclosure. The compounds of the disclosure may contain any of the substituents, or combinations of substituents, provided herein.
Salts
The compounds described herein may form salts with acids or bases, and such salts are included in the present disclosure. The term "salts" embraces addition salts of free acids or bases that are useful within the methods of the disclosure. The term ‘'pharmaceutically acceptable salt’’ refers to salts that possess toxicity profiles within a range that affords utility in pharmaceutical applications. In certain embodiments, the salts are pharmaceutically acceptable salts. Pharmaceutically unacceptable salts may nonetheless possess properties such as high crystallinity, which have utility in the practice of the present disclosure, such as for example utility in process of synthesis, purification or formulation of compounds useful within the methods of the disclosure.
Suitable pharmaceutically acceptable acid addition salts may be prepared from an inorganic acid or from an organic acid. Examples of inorganic acids include sulfate, hydrogen sulfate, hydrochloric, hydrobromic, hydriodic, nitric, carbonic, sulfuric, and phosphoric acids (including hydrogen phosphate and dihydrogen phosphate). Appropriate organic acids may be selected from aliphatic, cycloaliphatic, aromatic, araliphatic, heterocyclic, carboxylic and sulfonic classes of organic acids, examples of which include formic, acetic, propionic, succinic, glycolic, gluconic, lactic, malic, tartaric, citric, ascorbic, glucuronic, maleic, fumaric, pyruvic, aspartic, glutamic, benzoic, anthranilic, 4-hydroxybenzoic, phenylacetic, mandelic, embonic (or pamoic), methanesulfonic, ethanesulfonic, benzenesulfonic, pantothenic, sulfanilic. 2-hydroxyethanesulfonic, trifluoromethanesulfonic, p-toluenesulfonic, cyclohexylaminosulfonic, stearic, alginic. P-hydroxybutyric, salicylic, galactaric. galacturonic acid, glycerophosphonic acids and saccharin (e g., saccharinate, saccharate). Salts may be comprised of a fraction of one, one or more than one molar equivalent of acid or base with respect to any compound of the disclosure.
Suitable pharmaceutically acceptable base addition salts of compounds of the disclosure include, for example, ammonium salts and metallic salts including alkali metal, alkaline earth metal and transition metal salts such as, for example, calcium, magnesium, potassium, sodium and zinc salts. Pharmaceutically acceptable base addition salts also include organic salts made from basic amines such as, for example, A^V'-di benzyl ethylenediamine. chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (or A- methylglucamine) and procaine. All of these salts may be prepared from the corresponding compound by reacting, for example, the appropriate acid or base with the compound.
Combination Therapies
In one aspect, the compounds of the disclosure are useful within the methods of the disclosure in combination with one or more additional agents useful for treating, ameliorating, and/or preventing a disease or disorder contemplated herein. These additional agents may comprise compounds or compositions identified herein, or compounds (e. , commercially available compounds) known to treat, prevent, or reduce the symptoms of a disease or disorder contemplated herein.
A synergistic effect may be calculated, for example, using suitable methods such as, for example, the Sigmoid-Emax equation (Holford & Scheiner, 1981, Clin. Pharmacokinet. 6:429-453), the equation of Loewe additivity (Loewe & Muischnek, 1926, Arch. Exp. Pathol Pharmacol. 114: 313-326) and the median-effect equation (Chou & Talalay, 1984, Adv. Enzyme Regul. 22:27-55). Each equation referred to elsewhere herein may be applied to experimental data to generate a corresponding graph to aid in assessing the effects of the drug combination. The corresponding graphs associated with the equations referred to elsewhere herein are the concentration-effect curve, isobologram curve and combination index curve, respectively.
Synthesis
The present disclosure further provides methods of preparing compounds of the present disclosure. Compounds of the present teachings can be prepared in accordance with the procedures outlined herein, from commercially available starting materials, compounds known in the literature, or readily prepared intermediates, by employing standard synthetic methods and procedures known to those skilled in the art. Standard synthetic methods and procedures for the preparation of organic molecules and functional group transformations and manipulations can be readily obtained from the relevant scientific literature or from standard textbooks in the field.
Preparation of the compounds can involve protection and deprotection of various chemical groups. The need for protection and deprotection and the selection of appropriate protecting groups can be readily determined by one skilled in the art. The chemistry of protecting groups can be found, for example, in Greene, et al., Protective Groups in Organic Synthesis, 2d. Ed. (Wiley & Sons, 1991), the entire disclosure of which is incorporated by reference herein for all purposes.
Methods
The disclosure provides a method of increasing antifungal activity of an antifungal agent used to treat, ameliorate, and/or prevent a fungal infection in a subject, wherein the antifungal agent disrupts synthesis and/or activity of ergosterol in the fungus, the method comprising administering a therapeutically effective amount of a compound of the disclosure to a subject being administered the antifungal agent.
The disclosure provides a method of reducing, minimizing, and/or eliminating at least one toxicity effect of an antifungal agent used to treat, ameliorate, and/or prevent a fungal infection in a subject, wherein the antifungal agent disrupts synthesis and/or activity of ergosterol in the fungus, the method comprising administering a therapeutically effective amount of a compound of the disclosure to a subject being administered the antifungal agent.
The disclosure provides a method of reducing, minimizing, and/or preventing drug resistance against an antifungal agent used to treat, ameliorate, and/or prevent a fungal infection in a subject, wherein the antifungal agent disrupts synthesis and/or activity of ergosterol in the fungus, the method comprising administering a therapeutically effective amount of a compound of the disclosure to a subject being administered the antifungal agent.
The disclosure provides a method of sensitizing a fungus to an antifungal agent administered to a subject to treat, ameliorate, and/or prevent the fungal infection in the subject, wherein the antifungal agent disrupts synthesis and/or activity of ergosterol in the fungus, the method comprising administering a therapeutically effective amount of a compound of the disclosure to a subject being administered the antifungal agent.
In certain embodiments, the fungus comprises at least one of Candida (such as but not limited to C. albicans, C. glabrata, C. auris, and C. parapsilosis'), Aspergillus (such as but not limited to A. fumigatus and A. terreus), Histoplasma, Cryptococcus, and Mucor.
In certain embodiments, the compound of the disclosure is administered to the subject in a pharmaceutically acceptable composition.
In certain embodiments, the compound of the disclosure and the antifungal agent are co-formulated. In certain embodiments, administration of the compound of the disclosure and the antifungal agent results in less likely drug resistance occurrence in the subject as compared to an equivalent subject that is administered the antifungal agent in the absence of the compound of the disclosure.
In certain embodiments, administration of the compound of the disclosure and the antifungal agent allows for administration of an amount of the antifungal agent that is lower than the corresponding amount of the antifungal agent that has to be administered in the absence of the compound of the disclosure to achieve equivalent treatment or prevention of the fungal infection.
In certain embodiments, the compound of the disclosure inhibits fungal PanK selectively over a human PanK enzyme.
In certain embodiments, the subject is a mammal. In other embodiments, the mammal is a human.
Pharmaceutical Compositions and Formulations
The disclosure provides pharmaceutical compositions comprising at least one compound of the disclosure or a salt or solvate thereof, which are useful to practice methods of the disclosure. Such a pharmaceutical composition may consist of at least one compound of the disclosure or a salt or solvate thereof, in a form suitable for administration to a subject, or the pharmaceutical composition may comprise at least one compound of the disclosure or a salt or solvate thereof, and one or more pharmaceutically acceptable carriers, one or more additional ingredients, or any combinations of these. At least one compound of the disclosure may be present in the pharmaceutical composition in the form of a physiologically acceptable salt, such as in combination with a physiologically acceptable cation or anion, as is well known in the art.
In certain embodiments, the pharmaceutical compositions useful for practicing the method of the disclosure may be administered to deliver a dose of between 1 ng/kg/day and 100 mg/kg/day. In other embodiments, the pharmaceutical compositions useful for practicing the disclosure may be administered to deliver a dose of between 1 ng/kg/day and 1,000 mg/kg/day.
The relative amounts of the active ingredient, the pharmaceutically acceptable carrier, and any additional ingredients in a pharmaceutical composition of the disclosure will vary, depending upon the identity, size, and condition of the subject treated and further depending upon the route by which the composition is to be administered. By way of example, the composition may comprise between 0.1% and 100% (w/w) active ingredient.
Pharmaceutical compositions that are useful in the methods of the disclosure may be suitably developed for nasal, inhalational, oral, rectal, vaginal, pleural, peritoneal, parenteral, topical, transdermal, pulmonary, intranasal, buccal, ophthalmic, epidural, intrathecal, intravenous, or another route of administration. A composition useful within the methods of the disclosure may be directly administered to the brain, the brainstem, or any other part of the central nervous system of a mammal or bird. Other contemplated formulations include projected nanoparticles, microspheres, liposomal preparations, coated particles, polymer conjugates, resealed erythrocytes containing the active ingredient, and immunologically- based formulations.
In certain embodiments, the compositions of the disclosure are part of a pharmaceutical matrix, which allows for manipulation of insoluble materials and improvement of the bioavailability thereof, development of controlled or sustained release products, and generation of homogeneous compositions. By way of example, a pharmaceutical matrix may be prepared using hot melt extrusion, solid solutions, solid dispersions, size reduction technologies, molecular complexes (e.g., cyclodextrins, and others), microparticulate, and particle and formulation coating processes. Amorphous or cry stalline phases may be used in such processes.
The route(s) of administration will be readily apparent to the skilled artisan and will depend upon any number of factors including the type and severity of the disease being treated, the type and age of the veterinary or human patient being treated, and the like.
The formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology' and pharmaceutics. In general, such preparatory methods include the step of bringing the active ingredient into association with a carrier or one or more other accessory ingredients, and then, if necessary or desirable, shaping or packaging the product into a desired single-dose or multi-dose unit.
As used herein, a “unit dose” is a discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient that would be administered to a subject or a convenient fraction of such a dosage such as, for example, one-half or one- third of such a dosage. The unit dosage fonn may be for a single daily dose or one of multiple daily doses (e.g. about 1 to 4 or more times per day). When multiple daily doses are used, the unit dosage form may be the same or different for each dose. Although the descriptions of pharmaceutical compositions provided herein are principally directed to pharmaceutical compositions suitable for ethical administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to animals of all sorts. Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and perform such modification with merely ordinary, if any, experimentation. Subjects to which administration of the pharmaceutical compositions of the disclosure is contemplated include, but are not limited to, humans and other primates, mammals including commercially relevant mammals such as cattle, pigs, horses, sheep, cats, and dogs.
In certain embodiments, the compositions of the disclosure are formulated using one or more pharmaceutically acceptable excipients or carriers. In certain embodiments, the pharmaceutical compositions of the disclosure comprise a therapeutically effective amount of at least one compound of the disclosure and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers, which are useful, include, but are not limited to, glycerol, water, saline, ethanol, recombinant human albumin (e.g., RECOMB UMIN®), solubilized gelatins (e.g., GELOFUSINE®), and other pharmaceutically acceptable salt solutions such as phosphates and salts of organic acids. Examples of these and other pharmaceutically acceptable carriers are described in Remington’s Pharmaceutical Sciences (1991, Mack Publication Co., New Jersey).
The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), recombinant human albumin, solubilized gelatins, suitable mixtures thereof, and vegetable oils. The proper fluidity may be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms may be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, isotonic agents, for example, sugars, sodium chloride, or polyalcohols such as mannitol and sorbitol, are included in the composition. Prolonged absorption of the injectable compositions may be brought about by including in the composition an agent that delays absorption, for example, aluminum monostearate or gelatin.
Formulations may be employed in admixtures with conventional excipients, i.e.. pharmaceutically acceptable organic or inorganic carrier substances suitable for oral, parenteral, nasal, inhalational, intravenous, subcutaneous, transdermal enteral, or any other suitable mode of administration, known to the art. The pharmaceutical preparations may be sterilized and if desired mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure buffers, coloring, flavoring, and/or fragrance-conferring substances and the like. They may also be combined where desired with other active agents, e.g., other analgesic, anxiolytics or hypnotic agents. As used herein, “additional ingredients” include, but are not limited to, one or more ingredients that may be used as a pharmaceutical carrier.
The composition of the disclosure may comprise a preservative from about 0.005% to 2.0% by total weight of the composition. The preservative is used to prevent spoilage in the case of exposure to contaminants in the environment. Examples of preservatives useful in accordance with the disclosure include but are not limited to those selected from the group consisting of benzyl alcohol, sorbic acid, parabens, imidurea and any combinations thereof. One such preservative is a combination of about 0.5% to 2.0% benzyl alcohol and 0.05-0.5% sorbic acid.
The composition may include an antioxidant and a chelating agent that inhibit the degradation of the compound. Antioxidants for some compounds are BHT, BHA, alphatocopherol and ascorbic acid in the exemplary range of about 0.01% to 0.3%, or BHT in the range of 0.03% to 0.1% by weight by total weight of the composition. The chelating agent may be present in an amount of from 0.01 % to 0.5% by weight by total weight of the composition. Exemplary chelating agents include edetate salts (e.g. disodium edetate) and citric acid in the weight range of about 0.01% to 0.20%, or in the range of 0.02% to 0. 10% byweight by total weight of the composition. The chelating agent is useful for chelating metal ions in the composition that may be detrimental to the shelf life of the formulation. While BHT and disodium edetate are exemplary antioxidant and chelating agent, respectively, for some compounds, other suitable and equivalent antioxidants and chelating agents may be substituted therefore as would be known to those skilled in the art.
Liquid suspensions may be prepared using conventional methods to achieve suspension of the active ingredient in an aqueous or oily vehicle. Aqueous vehicles include, for example, water, and isotonic saline. Oily vehicles include, for example, almond oil, oily esters, ethyl alcohol, vegetable oils such as arachis, olive, sesame, or coconut oil. fractionated vegetable oils, and mineral oils such as liquid paraffin. Liquid suspensions may further comprise one or more additional ingredients including, but not limited to, suspending agents. dispersing or weting agents, emulsifying agents, demulcents, preservatives, buffers, salts, flavorings, coloring agents, and sweetening agents. Oily suspensions may further comprise a thickening agent. Known suspending agents include, but are not limited to, sorbitol syrup, hydrogenated edible fats, sodium alginate, polyvinylpyrrolidone, gum tragacanth, gum acacia, and cellulose derivatives such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethyl cellulose. Known dispersing or weting agents include, but are not limited to, naturally-occurring phosphatides such as lecithin, condensation products of an alkylene oxide with a fatty acid, with a long chain aliphatic alcohol, with a partial ester derived from a fatty acid and a hexitol, or with a partial ester derived from a fatty' acid and a hexitol anhydride (e.g., polyoxyethylene stearate, heptadecaethyleneoxycetanol, polyoxyethylene sorbitol monooleate, and polyoxyethylene sorbitan monooleate, respectively). Known emulsifying agents include, but are not limited to, lecithin, acacia, and ionic or non-ionic surfactants. Known preservatives include, but are not limited to, methyl, ethyl, or w-propyl para-hydroxybenzoates, ascorbic acid, and sorbic acid. Known sweetening agents include, for example, glycerol, propylene glycol, sorbitol, sucrose, and saccharin.
Liquid solutions of the active ingredient in aqueous or oily solvents may be prepared in substantially the same manner as liquid suspensions, the primary difference being that the active ingredient is dissolved, rather than suspended in the solvent. As used herein, an “oily” liquid is one which comprises a carbon-containing liquid molecule and which exhibits a less polar character than water. Liquid solutions of the pharmaceutical composition of the disclosure may comprise each of the components described with regard to liquid suspensions, it being understood that suspending agents will not necessarily aid dissolution of the active ingredient in the solvent. Aqueous solvents include, for example, water, and isotonic saline. Oily solvents include, for example, almond oil, oily esters, ethyl alcohol, vegetable oils such as arachis, olive, sesame, or coconut oil, fractionated vegetable oils, and mineral oils such as liquid paraffin.
Pow dered and granular formulations of a pharmaceutical preparation of the disclosure may be prepared using known methods. Such formulations may be administered directly to a subject, used, for example, to form tablets, to fill capsules, or to prepare an aqueous or oily suspension or solution by addition of an aqueous or oily vehicle thereto. Each of these formulations may further comprise one or more of dispersing or wheting agent, a suspending agent, ionic and non-ionic surfactants, and a preservative. Additional excipients, such as fillers and sweetening, flavoring, or coloring agents, may also be included in these formulations. A pharmaceutical composition of the disclosure may also be prepared, packaged, or sold in the form of oil-in-water emulsion or a water-in-oil emulsion. The oily phase may be a vegetable oil such as olive or arachis oil, a mineral oil such as liquid paraffin, or a combination of these. Such compositions may further comprise one or more emulsifying agents such as naturally occurring gums such as gum acacia or gum tragacanth, naturally- occurring phosphatides such as soybean or lecithin phosphatide, esters or partial esters derived from combinations of fatty acids and hexitol anhydrides such as sorbitan monooleate, and condensation products of such partial esters with ethylene oxide such as polyoxyethylene sorbitan monooleate. These emulsions may also contain additional ingredients including, for example, sweetening or flavoring agents.
Methods for impregnating or coating a material with a chemical composition are know n in the art, and include, but are not limited to methods of depositing or binding a chemical composition onto a surface, methods of incorporating a chemical composition into the structure of a material during the synthesis of the material (i. e. , such as with a physiologically degradable material), and methods of absorbing an aqueous or oily solution or suspension into an absorbent material, with or without subsequent drying. Methods for mixing components include physical milling, the use of pellets in solid and suspension formulations and mixing in a transdermal patch, as known to those skilled in the art.
Administration/Dosing
The regimen of administration may affect what constitutes an effective amount. The therapeutic formulations may be administered to the patient either prior to or after the onset of a disease or disorder. Further, several divided dosages, as well as staggered dosages may be administered daily or sequentially, or the dose may be continuously infused, or may be a bolus injection. Further, the dosages of the therapeutic formulations may be proportionally increased or decreased as indicated by the exigencies of the therapeutic or prophylactic situation.
Administration of the compositions of the present disclosure to a patient, such as a mammal, such as a human, may be carried out using known procedures, at dosages and for periods of time effective to treat a disease or disorder contemplated herein. An effective amount of the therapeutic compound necessary to achieve a therapeutic effect may vary according to factors such as the activity of the particular compound employed; the time of administration; the rate of excretion of the compound; the duration of the treatment; other drugs, compounds or materials used in combination with the compound; the state of the disease or disorder, age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well-known in the medical arts. Dosage regimens may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation. A non-limiting example of an effective dose range for a therapeutic compound of the disclosure is from about 0.01 mg/kg to 100 mg/kg of body weight/per day. One of ordinary skill in the art would be able to study the relevant factors and make the determination regarding the effective amount of the therapeutic compound without undue experimentation.
The compound may be administered to an animal as frequently as several times daily, or it may be administered less frequently, such as once a day. once a week, once every two weeks, once a month, or even less frequently, such as once every several months or even once a year or less. It is understood that the amount of compound dosed per day may be administered, in non-limiting examples, every day, every other day, even,' 2 days, every' 3 days, every 4 days, or every 5 days. For example, with every other day administration, a 5 mg per day dose may be initiated on Monday with a first subsequent 5 mg per day dose administered on Wednesday, a second subsequent 5 mg per day dose administered on Friday, and so on. The frequency of the dose is readily apparent to the skilled artisan and depends upon a number of factors, such as, but not limited to, type and severity of the disease being treated, and type and age of the animal.
Actual dosage levels of the active ingredients in the pharmaceutical compositions of this disclosure may be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.
A medical doctor, e.g., physician or veterinarian, having ordinary skill in the art may readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of the compounds of the disclosure employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.
In particular embodiments, it is especially advantageous to formulate the compound in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the patients to be treated; each unit containing a predetermined quantity of therapeutic compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical vehicle. The dosage unit forms of the disclosure are dictated by and directly dependent on (a) the unique characteristics of the therapeutic compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding/formulating such a therapeutic compound for the treatment of a disease or disorder in a patient.
In certain embodiments, the compositions of the disclosure are administered to the patient in dosages that range from one to five times per day or more. In other embodiments, the compositions of the disclosure are administered to the patient in range of dosages that include, but are not limited to, once every day, every two days, every three days to once a week, and once every' two weeks. It will be readily apparent to one skilled in the art that the frequency of administration of the various combination compositions of the disclosure will vary from subject to subject depending on many factors including, but not limited to, age, disease or disorder to be treated, gender, overall health, and other factors. Thus, the disclosure should not be construed to be limited to any particular dosage regime and the precise dosage and composition to be administered to any patient will be determined by the attending physician taking all other factors about the patient into account.
Compounds of the disclosure for administration may be in the range of from about 1 pg to about 7,500 mg, about 20 pg to about 7,000 mg, about 40 pg to about 6,500 mg, about 80 pg to about 6.000 mg, about 100 pg to about 5,500 mg, about 200 pg to about 5,000 mg, about 400 pg to about 4.000 mg. about 800 pg to about 3.000 mg, about 1 mg to about 2,500 mg, about 2 mg to about 2,000 mg, about 5 mg to about 1 ,000 mg, about 10 mg to about 750 mg, about 20 mg to about 600 mg, about 30 mg to about 500 mg, about 40 mg to about 400 mg, about 50 mg to about 300 mg, about 60 mg to about 250 mg, about 70 mg to about 200 mg, about 80 mg to about 150 mg. and any and all whole or partial increments there-in- between.
In some embodiments, the dose of a compound of the disclosure is from about 0.5 pg and about 5,000 mg. In some embodiments, a dose of a compound of the disclosure used in compositions described herein is less than about 5,000 mg, or less than about 4,000 mg, or less than about 3,000 mg, or less than about 2.000 mg, or less than about 1.000 mg, or less than about 800 mg, or less than about 600 mg, or less than about 500 mg, or less than about 200 mg, or less than about 50 mg. Similarly, in some embodiments, a dose of a second compound as described herein is less than about 1,000 mg, or less than about 800 mg, or less than about 600 mg, or less than about 500 mg, or less than about 400 mg. or less than about 300 mg, or less than about 200 mg, or less than about 100 mg, or less than about 50 mg, or less than about 40 mg, or less than about 30 mg, or less than about 25 mg, or less than about 20 mg, or less than about 15 mg. or less than about 10 mg, or less than about 5 mg. or less than about 2 mg, or less than about 1 mg, or less than about 0.5 mg, and any and all whole or partial increments thereof.
In certain embodiments, the present disclosure is directed to a packaged pharmaceutical composition comprising a container holding a therapeutically effective amount of a compound of the disclosure, alone or in combination with a second pharmaceutical agent; and instructions for using the compound to treat, prevent, or reduce one or more symptoms of a disease or disorder in a patient.
The term “container’' includes any receptacle for holding the pharmaceutical composition or for managing stability or water uptake. For example, in certain embodiments, the container is the packaging that contains the pharmaceutical composition, such as liquid (solution and suspension), semisolid, lyophilized solid, solution and powder or lyophilized formulation present in dual chambers. In other embodiments, the container is not the packaging that contains the pharmaceutical composition, i.e., the container is a receptacle, such as a box or vial that contains the packaged pharmaceutical composition or unpackaged pharmaceutical composition and the instructions for use of the pharmaceutical composition. Moreover, packaging techniques are well known in the art. It should be understood that the instructions for use of the pharmaceutical composition may be contained on the packaging containing the pharmaceutical composition, and as such the instructions form an increased functional relationship to the packaged product. However, it should be understood that the instructions may contain information pertaining to the compound’s abil i ty to perform its intended function, e.g., treating, preventing, or reducing a disease or disorder in a patient.
Administration
Routes of administration of any of the compositions of the disclosure include inhalational, oral, nasal, rectal, parenteral, sublingual, transdermal, transmucosal (e.g., sublingual, lingual, (trans)buccal, (trans)urethral, vaginal (e.g., trans- and perivaginally), (intra)nasal, and (trans)rectal). intravesical, intrapulmonary. intraduodenal. intragastrical, intrathecal, epidural, intrapleural, intraperitoneal, subcutaneous, intramuscular, intradermal, intra-arterial, intravenous, intrabronchial, inhalation, and topical administration.
Suitable compositions and dosage forms include, for example, tablets, capsules, caplets, pills, gel caps, troches, emulsions, dispersions, suspensions, solutions, syrups, granules, beads, transdermal patches, gels, powders, pellets, magmas, lozenges, creams, pastes, plasters, lotions, discs, suppositories, liquid sprays for nasal or oral administration, dry powder or aerosolized formulations for inhalation, compositions and formulations for intravesical administration and the like. It should be understood that the formulations and compositions that would be useful in the present disclosure are not limited to the particular formulations and compositions that are described herein.
Oral Administration
For oral application, particularly suitable are tablets, dragees, liquids, drops, capsules, caplets and gelcaps. Other formulations suitable for oral administration include, but are not limited to, a powdered or granular formulation, an aqueous or oily suspension, an aqueous or oily solution, a paste, a gel, toothpaste, a mouthwash, a coating, an oral rinse, or an emulsion. The compositions intended for oral use may be prepared according to any method known in the art and such compositions may contain one or more agents selected from the group consisting of inert, non-toxic, generally recognized as safe (GRAS) pharmaceutically excipients which are suitable for the manufacture of tablets. Such excipients include, for example an inert diluent such as lactose: granulating and disintegrating agents such as cornstarch; binding agents such as starch; and lubricating agents such as magnesium stearate.
Tablets may be non-coated or they may be coated using known methods to achieve delayed disintegration in the gastrointestinal tract of a subject, thereby providing sustained release and absorption of the active ingredient. By way of example, a material such as glyceryl monostearate or glyceryl distearate may be used to coat tablets. Further by way of example, tablets may be coated using methods described in U.S. Patents Nos. 4,256,108; 4,160,452; and 4,265,874 to form osmotically controlled release tablets. Tablets may further comprise a sweetening agent, a flavoring agent, a coloring agent, a preservative, or some combination of these in order to provide for pharmaceutically elegant and palatable preparation. Hard capsules comprising the active ingredient may be made using a physiologically degradable composition, such as gelatin. The capsules comprise the active ingredient, and may further comprise additional ingredients including, for example, an inert solid diluent such as calcium carbonate, calcium phosphate, or kaolin.
Parenteral Administration
As used herein, “parenteral administration” of a pharmaceutical composition includes any route of administration characterized by physical breaching of a tissue of a subject and administration of the pharmaceutical composition through the breach in the tissue. Parenteral administration thus includes, but is not limited to. administration of a pharmaceutical composition by injection of the composition, by application of the composition through a surgical incision, by application of the composition through a tissue-penetrating non-surgical wound, and the like. In particular, parenteral administration is contemplated to include, but is not limited to, subcutaneous, intravenous, intraperitoneal, intramuscular, intrastemal injection, and kidney dialytic infusion techniques.
Formulations of a pharmaceutical composition suitable for parenteral administration comprise the active ingredient combined with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations may be prepared, packaged, or sold in a form suitable for bolus administration or for continuous administration. Injectable formulations may be prepared, packaged, or sold in unit dosage form, such as in ampules or in multidose containers containing a preservative. Injectable formulations may also be prepared, packaged, or sold in devices such as patient-controlled analgesia (PCA) devices. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations. Such formulations may further comprise one or more additional ingredients including, but not limited to. suspending, stabilizing, or dispersing agents. In certain embodiments of a formulation for parenteral administration, the active ingredient is provided in dry (z.e., powder or granular) form for reconstitution with a suitable vehicle (e.g, sterile pyrogen-free water) prior to parenteral administration of the reconstituted composition.
The pharmaceutical compositions may be prepared, packaged, or sold in the form of a sterile injectable aqueous or oily suspension or solution. This suspension or solution may be formulated according to the known art, and may comprise, in addition to the active ingredient, additional ingredients such as the dispersing agents, wetting agents, or suspending agents described herein. Such sterile injectable formulations may be prepared using a nontoxic parenterally acceptable diluent or solvent, such as water or 1,3-butanediol, for example. Other acceptable diluents and solvents include, but are not limited to, Ringer’s solution, isotonic sodium chloride solution, and fixed oils such as synthetic mono- or di-glycerides. Other parentally-administrable formulations which are useful include those which comprise the active ingredient in microcrystalline form in a recombinant human albumin, a fluidized gelatin, in a liposomal preparation, or as a component of a biodegradable polymer system. Compositions for sustained release or implantation may comprise pharmaceutically acceptable polymeric or hydrophobic materials such as an emulsion, an ion exchange resin, a sparingly soluble polymer, or a sparingly soluble salt.
Topical Administration
An obstacle for topical administration of pharmaceuticals is the stratum comeum layer of the epidermis. The stratum comeum is a highly resistant layer comprised of protein, cholesterol, sphingolipids, free fatty acids and various other lipids, and includes cornified and living cells. One of the factors that limit the penetration rate (flux) of a compound through the stratum comeum is the amount of the active substance that can be loaded or applied onto the skin surface. The greater the amount of active substance which is applied per unit of area of the skin, the greater the concentration gradient between the skin surface and the lower layers of the skin, and in turn the greater the diffusion force of the active substance through the skin. Therefore, a formulation containing a greater concentration of the active substance is more likely to result in penetration of the active substance through the skin, and more of it, and at a more consistent rate, than a formulation having a lesser concentration, all other things being equal.
Formulations suitable for topical administration include, but are not limited to, liquid or semi-liquid preparations such as liniments, lotions, oil-in-water or water-in-oil emulsions such as creams, ointments or pastes, and solutions or suspensions. Topically administrable formulations may, for example, comprise from about 1% to about 10% (w/w) active ingredient, although the concentration of the active ingredient may be as high as the solubility limit of the active ingredient in the solvent. Formulations for topical administration may further comprise one or more of the additional ingredients described herein.
Enhancers of permeation may be used. These materials increase the rate of penetration of drugs across the skin. Typical enhancers in the art include ethanol, glycerol monolaurate, PGML (polyethylene glycol monolaurate), dimethylsulfoxide, and the like. Other enhancers include oleic acid, oleyl alcohol, ethoxydiglycol, laurocapram, alkanecarboxylic acids, dimethylsulfoxide, polar lipids, or JV-methyl-2-pyrrolidone.
One acceptable vehicle for topical deliver}’ of some of the compositions of the disclosure may contain liposomes. The composition of the liposomes and their use are known in the art (z.e., U.S. Patent No. 6,323,219).
In alternative embodiments, the topically active pharmaceutical composition may be optionally combined with other ingredients such as adjuvants, anti-oxidants, chelating agents, surfactants, foaming agents, wetting agents, emulsifying agents, viscosifiers. buffering agents, preservatives, and the like. In other embodiments, a permeation or penetration enhancer is included in the composition and is effective in improving the percutaneous penetration of the active ingredient into and through the stratum comeum with respect to a composition lacking the permeation enhancer. Various permeation enhancers, including oleic acid, oleyl alcohol, ethoxydiglycol, laurocapram, alkanecarboxylic acids, dimethylsulfoxide, polar lipids, or A-methyl-2-pyrrolidone. are known to those of skill in the art. In another aspect, the composition may further comprise a hydrotropic agent, which functions to increase disorder in the structure of the stratum comeum, and thus allows increased transport across the stratum comeum. Various hydrotropic agents such as isopropyl alcohol, propylene glycol, or sodium xylene sulfonate, are known to those of skill in the art.
The topically active pharmaceutical composition should be applied in an amount effective to affect desired changes. As used herein “amount effective” shall mean an amount sufficient to cover the region of skin surface where a change is desired. An active compound should be present in the amount of from about 0.0001% to about 15% by weight volume of the composition. For example, it should be present in an amount from about 0.0005% to about 5% of the composition; for example, it should be present in an amount of from about 0.001% to about 1% of the composition. Such compounds may be synthetically-or naturally derived.
Buccal Administration
A pharmaceutical composition of the disclosure may be prepared, packaged, or sold in a formulation suitable for buccal administration. Such formulations may, for example, be in the form of tablets or lozenges made using conventional methods, and may contain, for example, 0.1 to 20% (w/w) of the active ingredient, the balance comprising an orally dissolvable or degradable composition and, optionally, one or more of the additional ingredients described herein. Alternately, formulations suitable for buccal administration may comprise a powder or an aerosolized or atomized solution or suspension comprising the active ingredient. Such powdered, aerosolized, or aerosolized formulations, when dispersed, may have an average particle or droplet size in the range from about 0. 1 to about 200 nanometers, and may further comprise one or more of the additional ingredients described herein. The examples of formulations described herein are not exhaustive and it is understood that the disclosure includes additional modifications of these and other formulations not described herein, but which are know n to those of skill in the art.
Rectal Administration
A pharmaceutical composition of the disclosure may be prepared, packaged, or sold in a formulation suitable for rectal administration. Such a composition may be in the form of, for example, a suppository, a retention enema preparation, and a solution for rectal or colonic irrigation.
Suppository formulations may be made by combining the active ingredient with a non-irritating pharmaceutically acceptable excipient which is solid at ordinary room temperature (i.e., about 20°C) and which is liquid at the rectal temperature of the subject (z.e., about 37°C in a healthy human). Suitable pharmaceutically acceptable excipients include, but are not limited to, cocoa butter, polyethylene glycols, and various glycerides. Suppository formulations may further comprise various additional ingredients including, but not limited to, antioxidants, and preservatives.
Retention enema preparations or solutions for rectal or colonic irrigation may be made by combining the active ingredient with a pharmaceutically acceptable liquid earner. As is well known in the art, enema preparations may be administered using, and may be packaged within, a delivery device adapted to the rectal anatomy of the subject. Enema preparations may further comprise various additional ingredients including, but not limited to. antioxidants, and preservatives.
Additional Administration Forms
Additional dosage forms of this disclosure include dosage forms as described in U.S. Patents Nos. 6,340,475, 6,488,962, 6,451,808. 5,972,389, 5,582,837, and 5,007,790. Additional dosage forms of this disclosure also include dosage forms as described in U.S. Patent Applications Nos. 20030147952, 20030104062, 20030104053, 20030044466, 20030039688, and 20020051820. Additional dosage forms of this disclosure also include dosage forms as described in PCT Applications Nos. WO 03/35041, WO 03/35040, WO 03/35029, WO 03/35177, WO 03/35039, WO 02/96404, WO 02/32416, WO 01/97783, WO 01/56544, WO 01/32217, WO 98/55107, WO 98/11879, WO 97/47285, WO 93/18755, and WO 90/1 1757.
Controlled Release Formulations and Drug Delivery Systems:
In certain embodiments, the compositions and/or formulations of the present disclosure may be, but are not limited to, short-term, rapid-offset, as well as controlled, for example, sustained release, delayed release and pulsatile release formulations.
The term sustained release is used in its conventional sense to refer to a drug formulation that provides for gradual release of a drug over an extended period of time, and that may. although not necessarily, result in substantially constant blood levels of a drug over an extended time period. The period of time may be as long as a month or more and should be a release which is longer that the same amount of agent administered in bolus form.
For sustained release, the compounds may be formulated with a suitable polymer or hydrophobic material which provides sustained release properties to the compounds. As such, the compounds for use the method of the disclosure may be administered in the form of microparticles, for example, by injection or in the form of wafers or discs by implantation.
In certain embodiments of the disclosure, the compounds useful within the disclosure are administered to a subject, alone or in combination with another pharmaceutical agent, using a sustained release formulation.
The term delayed release is used herein in its conventional sense to refer to a drug formulation that provides for an initial release of the drug after some delay following drug administration and that may. although not necessarily, include a delay of from about 10 minutes up to about 12 hours.
The term pulsatile release is used herein in its conventional sense to refer to a drug formulation that provides release of the drug in such a way as to produce pulsed plasma profiles of the drug after drug administration.
The term immediate release is used in its conventional sense to refer to a drug formulation that provides for release of the drug immediately after drug administration.
As used herein, short-term refers to any period of time up to and including about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40 minutes, about 20 minutes, or about 10 minutes and any or all whole or partial increments thereof after drug administration after drug administration.
As used herein, rapid-offset refers to any period of time up to and including about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40 minutes, about 20 minutes, or about 10 minutes, and any and all whole or partial increments thereof after drug administration.
Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific procedures, embodiments, claims, and examples described herein. Such equivalents were considered to be within the scope of this disclosure and covered by the claims appended hereto. For example, it should be understood, that modifications in reaction conditions, including but not limited to reaction times, reaction size/volume, and experimental reagents, such as solvents, catalysts, pressures, atmospheric conditions, e.g.. nitrogen atmosphere, and reducing/oxi dizing agents, with art- recognized alternatives and using no more than routine experimentation, are within the scope of the present application.
It is to be understood that, wherever values and ranges are provided herein, the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, all values and ranges encompassed by these values and ranges are meant to be encompassed within the scope of the present disclosure. Moreover, all values that fall within these ranges, as well as the upper or lower limits of a range of values, are also contemplated by the present application. The description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range and, when appropriate, partial integers of the numerical values within ranges. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4. from 1 to 5, from 2 to 4, from 2 to 6. from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.
The following examples further illustrate aspects of the present disclosure. However, they are in no way a limitation of the teachings or disclosure of the present disclosure as set forth herein.
EXAMPLES
The disclosure is now described with reference to the following Examples. These Examples are provided for the purpose of illustration only, and the disclosure is not limited to these Examples, but rather encompasses all variations that are evident as a result of the teachings provided herein.
Materials & Methods
Yeast strains:
Yeast strains (Saccharomyces cerevisiae) used in this study are JS91. 15-23 MATa his3 leu2 trpl ura3), JS91. 14-24 {MATa ura3 his3 cab Its) (Olzhausen. et al. , 2009, Curr Genet 55, 163-173), W303B, cablA+ pFL38-CABl + pFL39, cablA+ pFL39-cablG351S + pFL38, cablA+ pFL39-cabl° 51S + pFL38-CABl (Ceccatelli Berti et al., 2020, Int. J. Mol. Sci). Candida strains used in the study include C. albicans and C. parapsilosis . Wild type and mutant strains were propagated either in YPD medium (2% bacto-peptone, 2% D-(+)-glucose and 1% yeast extract) or defined pantothenic acid-free (minimal) medium composed of Yeast Nitrogen Base (MP Biomedicals), supplemented with Complete Supplement Mixture (MP Biomedicals) and all vitamins except pantothenic acid. Where indicated, media were supplemented with appropriate concentrations of pantothenic acid. Growth assays on solid and liquid media
Spoting assays were performed as follows. Pre-cultures of WT and mutant yeast strains were prepared in YPD medium overnight at 30 °C. Cells were harvested, washed, and diluted to 108 cells in 500 pL sterile water. Subsequent serial dilutions were made and 5 pL of cell suspensions were spoted on YPD agar plates lacking or supplemented with amorolfme 10 ng/mL, amphotericin B 1 pg/mL. fluconazole 10 pg/mL or terbinafme 10 pg/mL to achieve 106, 105, 104, 103, 102, and 101 cells per spot. Plates were incubated at 30 °C or 37 °C and imaged every 24 hours. For liquid assay in 96-well plate format, cells were pre-cultured overnight in liquid YPD medium at 30 °C, washed three times in water and diluted to achieve 104 cells per well in 150 pL of minimal medium supplemented with 1 pM, 10 pM or 100 pM pantothenic acid and either lacking or supplemented with terbinafme (160 pg/ml). All plates were incubated at 30 °C. Optical density measurements were taken with a BioTek Synergy Mx microplate reader (OD630) every' 8 hours for a total of 96 hours. Relative time to mid-log phase was calculated by first graphing grow th curves of the varying concentrations of terbinafme for each concentration of pantothenic acid, and then determining the value at which the untreated curve reached saturation, and dividing it by 2 to represent the mid-log value. Dividing the time to mid-log phase of treated cells by the time to mid-log phase of untreated gave the relative time to mid-log phase.
Drug-drug interactions and isobologram calculations
Drug-drug interactions were determined using liquid growth assays in 96-well plates using the checkerboard method as described in Orhan, et al., 2005, J. Clin. Microbiol. 43: 140-143. The isobologram was produced by solving for the sum 50% fractional inhibitory' concentration (LFICso) using the equation:
Figure imgf000055_0001
The data from the time-point where the 0 pg/mL drug A curve reached saturation was used to generate percent inhibition values, representing growth inhibition based on the concentration of drug A. This was done by dividing the value of interest by the untreated (0 pg/mL drug A) value, multiplying by 100 to get a percentage, and then subtracting that percentage from 100, representing 100% inhibition. The resulting values were used to create an inhibition curve, using the best fit function to generate an MICso. This was done for both drug A in drug B and drug B in drug A. The resulting MIC50 values were plugged into the SFIC50 equation, and the SFICso values were used to generate the curve.
Example 1: Inhibition of Cablp activity leads to enhanced susceptibility to antifungal drugs
To investigate the link between pantothenate utilization and antifungal susceptibility, analysis on the effect of inhibition of Cablp activity and susceptibility to antifungal drugs was conducted in two different genetic backgrounds: cahlis mutant, which was obtained through random mutagenesis by Olhauzen et al (1989) (FIGs. 1 A-1H) and a well-defined genetic background cablA+CABl, which lacks the chromosomal CAB1 gene but expresses either the wild type CAB1 or various alleles of the CAB1 gene on a plasmid. The growth of cablts cells at 30 °C can be significantly improved in media supplemented with increased concentrations of pantothenic acid, as substrate availability compensates for the weak activity of the mutant enzyme. Supplementation of PA did not significantly affect WT grow th patterns, as the time to mid-log phase was ~26, 26, and 28 hours at 1 pM, 10 pM, and 100 pM PA, respectively (FIGs. 1A, 1C. IE), while cablis cells demonstrated improved growth with higher exogenous PA concentrations, with time to mid-log phases ~34, 30, and 29 hours, respectively (FIGs. IB, ID, IF). Consistent w ith the effect of amphotericin B on YPD agar, the growth of WT cells was not significantly affected at 100 pM PA in the presence of 1 pg/mL amphotericin B, but was increasingly and significantly affected as the exogenous PA concentration was lowered to 1 pM (relative time to mid-log phase of 2. 1 in the presence vs. absence of the drug) (FIG. 1G). The cablts cells show ed a similar trend as the WT cells, displaying more sensitivity to amphotericin B as exogenous PA was reduced. However, it is apparent that the mutant strain was more sensitive to the drug, especially noting that the strain did not grow whatsoever in the presence of the drug when only 1 pM PA medium was used (FIG. 3G). Analysis of the combined effect of amphotericin B and a-PanAm showed a ty pical drug-drug synergism pattern in WT cells, demonstrating that inhibition of Cablp activity7 results in increased susceptibility to amphotericin B (FIG. 1H).
Example 2:
Efforts w ere made to identify inhibitors of pantothenate kinase (PanK), which plays an important role in fungal viability. Initially, cloning and expression of PanKs from different fungal pathogens {Candida albicans, Candida auris. Aspergillus fumigatu and Histoplasma capsiilatum) were implemented. Screening of compounds against the A. fumigatus enzyme (AfPanK) allowed for the identification of various active compounds, which are exemplified in Table 1.
In Table 1, '‘mixed’’ indicates a compound that is an activator at lower concentrations and is an inhibitor at higher concentrations. % growth inhibition S. cerevisicte monster w as determined at 200 pM of compound in VFM medium with no pantothenic acid at 24h (105 cells/ml).
Table 1.
Figure imgf000057_0001
Example 3: Compounds of interest were assayed for their activity as inhibitors of fungal pantothenate kinase (PanK), modulators of human PanK, and general toxicity against HeLa cells. All compounds in Table 2 were found to be activators of human PanK. Table 2.
Figure imgf000058_0001
Example 4: Genetic study of PanK mutant susceptibility to broad-range susceptibility to antifungal drugs
To gain further insights into the link between Cabl activity, the PCA pathway (FIG. 11), and fungal susceptibility to antifungal drugs, a detailed characterization of 5. cerevisiae drug susceptibility using strains cartying various mutations in the CAB1 gene was conducted. The yeast strains used in these studies carry a chromosomal deletion of the CAB1 gene but express on a centromeric plasmid either a wild type CAB1 (cablA+CABl'). mutant alleles of CAB1 (cab \ cablr" with m= cablG331s, cablN29()I , and cablsl58A) or both the mutant alleles and the wild type CAB1 (add-back: cabl/^+cablm+CABl).
As shown FIG. 12B, all the yeast strains expressing different CAB1 alleles exhibited increased susceptibility to fluconazole, amphotericin B, and terbinafine, compared to the isogenic strain carrying the wild type CABl. The cab! +cablG35IS mutant was found to be highly susceptible to all ergosterol biosynthesis inhibitors examined with a reduction in MICso values determined to be ~12, ~15, and ~3- fold for fluconazole, terbinafine, and amphotericin, respectively, compared to the wild type strains (FIG. 13 A). Similarly, the cabl \ cabl3G-G mutant displayed reduced MICso values with fold reductions compared to the wild type determined to be ~7, ~9, and ~5 for fluconazole, terbinafine, and amphotericin, respectively (FIG. 13 A).
The broad-range susceptibility of cabl mutants to antifungal drugs led to examination of whether the underlying mechanism could be linked to disruption of a particular metabolic process such as ergosterol biosynthesis by the PCA pathway or to a broader disruption of yeast’s ability to detoxify xenobiotics. Therefore, the susceptibility of the mutants to drugs that target unrelated pathways was examined, including hygromycin and cycloheximide, which target protein synthesis, and caspofungin, which targets cell wall integrity (FIG. 11). Yeast strains used in this study are s own in Table 3. Table 3: Yeast strain and their vectors used in the Example 4 study.
Figure imgf000059_0001
Figure imgf000060_0001
Similar to their susceptibility to ergosterol biosynthesis inhibitors, the cabl mutant alleles showed higher susceptibility to caspofungin, hygromycin and cycloheximide compared to the wild type (FIG. 12B). The cablG351s mutation resulted in the highest drug susceptibility with the MIC50 values of caspofungin, hygromycin, and cycloheximide determined to be ~5, ~23, and ~16- fold lower compared to the wild type (FIG. 13 A). Similarly, cablSI58A mutation resulted in reduced MIC50 values for caspofungin, hygromycin, and cycloheximide by ~6, ~13, and ~4- fold compared to the wild type (FIG. 13 A). All complemented strains carrying the wild-ty pe CAB1 gene displayed susceptibility' levels comparable to those of the wild-type (WT) strain FIG. 13B.
These data demonstrate that inhibition of PanK activity leads to enhanced susceptibility’ to a wide variety' of antifungal drugs.
PanK-deficient cells have altered vacuole biogenesis and xenobiotic detoxification mechanism
The overall enhanced drug susceptibility of Cabl defective mutants led to investigation of a possible role of the vacuole in this mechanism. In fungi, the vacuole plays a critical role in the detoxification of xenobiotics such as drugs and metals. Therefore, testing was performed on whether the cabl mutants might also be susceptible to metals such as FeSOi and CuSC>4. which are detoxified in the vacuole. Consistent with an altered vacuolar detoxification in the mutants, the growth of the cabl I) cabl''3:AA. cab ID+cabl1^2901 and cablD+cablsl^8A mutants 'as dramatically reduced in media supplemented with FeSOi or CuSO4 compared to the wild type and complemented strains (FIG. 14). Vacuolar integrity' w as further investigated by measuring the accumulation of the cell-tracker dye, CMAC, using fluorescence microscopy. The cabl D cab 1G 51A. cablD+cablN2901 and cablD+cablSI58A mutants were all found to have enlarged vacuoles compared to the wild type and complemented strains (FIG. 15 A and FIG. 15B), with the vacuoles in the mutants occupying 60 to 70% more of the total cell area compared to the vacuoles of the wild-type and complemented strains. Vacuolar enlargement in the mutants was further confirmed by electron microscopy as shown in FIG. 16. Taken together, these data show that pantothenate phosphorylation regulates vacuolar homeostasis and xenobiotic detoxification.
It was surmised that altered vacuolar function in the cabl mutants could also result in altered mitochondrial function. Accordingly, the cablD+cablG35Is. cablD+cablN2901 and cablD+cablSI58A mutants showed severe growth defects on non-fermentable carbon sources (glycerol, ethanol, and lactate-based media) (FIG. 17) and altered oxygen consumption rates (OCR) (FIG. 18 A, FIG. 18B, FIG. 18C, and FIG 18D). Consistent with these findings, immunofluorescence assay s aimed to localize the mitochondrial outer membrane protein Porlp revealed that unlike the wild-type and complemented strains, the cabl mutants exhibited fragmented mitochondria (FIG. 19). Since the overproduction of reactive oxygen species (ROS) is associated with dysfunctional mitochondria, cellular ROS levels of the cabl mutants were also determined by measuring the conversion of the non-fluorescent dihydrorhodamine 123 (DHR-123) to the fluorescent rhodamine 123. As shown in FIG. 20, ROS levels in the cablD+cablG3:,IS, cab lD+cablN2901 and cab!D+cablSI:,8A mutants were found to be 10.1, 13.0 and 4.1-fold higher than in the wild-type and complemented strains, respectively.
Altered pantothenate phosphorylation leads to reduced pantothenate utilization and CoA biosynthesis and increased cysteine levels in yeast
To gain further insights into the mechanism by which altered pantothenate phosphorylation leads to defective vacuolar biogenesis, the effect of the Cabl mutations on the activity of the PCA pathway was assessed. Endogenous pantothenate utilization of cabl \+cablG:':G:. cab l/y+cablsl98A , and cab l \-cab l '220! was determined by measuring following metabolism of 14C-pantothenate (FIG. 21 A). As shown in FIG. 21A, all cabl mutants show ed significantly lower levels of 14C-pantothenate utilization compared to the isogenic strain carrying the wild type CABl. The lowest pantothenate utilization level (~2% that of the wild type) was measured for the cablsl58A mutant followed by 25% for the cablG35IS mutant and 34% for the cab I '22901 mutant. Expression of the wild-type CAB1 gene in these strains restored PA utilization to levels similar or above those in the isogenic wild type-strain. Consistent with the reduced pantothenate utilization in the mutants, cellular CoA levels in the mutants were also significantly lower compared to the wild-type and complemented strains (FIG. 21B, FIG. 30A, and FIG. 30B). Because reduced pantothenate phosphorylation results in less phosphopantothenate available for the second step in CoA biosynthesis catalyzed by phosphopantothenoylcysteine synthetase (Cab2) to form phosphopantothenoylcysteine from phosphopantothenate and cysteine (FIG. 23 A), it was reasoned that altered Cabl activity would also result in accumulation of cysteine. As shown in FIG. 22, cysteine levels increased by 2.7, 3.3, and 2.2-fold in the cabl''3'12. cablSI58A, and cablN2901 mutants, respectively, compared to the wild-type or complemented strains.
Consistent with these findings, analysis of the transcription profile of the cabIG32ls cablsl58f and cablN2901 mutants show ed a significant downregulation of the genes involved in sulfur assimilation and the cysteine/methionine biosynthetic pathway compared to the wild type and mutant strains carrying a wild type CAB1 gene FIG. 23 A, FIG. 23B, FIG. 31, and FIG. 32. Among these genes, the expression of the ATP sulfurylase-encoding gene, MET3, a- subunit of sulfite reductase, MetlO, cystathionine P-synthase, Cys4, bifunctional dehydrogenase and ferrochelatase, MET8, and sulfate permease, SUL2, genes in each of the mutants decreased dramatically (between 75% and -95%) compared to the wild type strain (FIG. 23B and FIG. 31).
Inhibition of fungal ACS2 or V-type ATPase enzymes increases susceptibility to antifungals
The genetic data described above demonstrated a direct role of pantothenate utilization and CoA biosynthesis in yeast susceptibility7 to antifungals through alteration of vacuolar detoxification. Considering the potential implication of these findings on fungal therapy and reversal of multidrug resistance, an assessment whether a pharmacological approach using compounds that inhibit Cabl activity or downstream steps such as AcCoA synthesis or vacuolar V-ATPase could mimic these genetic findings and usher in a new antifungal treatment modality7. Analysis of the transcription profile of the cabl mutants showed a dramatic decrease (12.6%. 12.9%. and 44.6% of that of the wild type) in the expression of the ACS1 gene encoding one of two yeast AcCoA synthetases (FIG. 32). Interestingly, a yeast mutant carrying the ACS2 gene under the regulatory control of the tet- off promoter (oc.s2-tct-olT) w as highly susceptible to caspofungin, fluconazole and terbinafme following addition of doxycycline (FIG. 24B). The MICso for caspofungin shifted from 16 ng/ml in the absence of doxycycline to 10 ng/ml in the presence of the compound; that for fluconazole from 4.8 pg/ml to 0.06 pg/ml; and that for terbinafme from 4.5 pg/ml to 0.005 pg/ml. Consistent with these data, the celecoxib derivative AR.-12, which is also a potent inhibitor of fungal AcCoA synthetases increased yeast susceptibility to caspofungin (MICso shift from 16 ng/ml to 3 ng/ml in the absence vs presence of AR-12). fluconazole (MICso shift from 14.6 pg/ml to 0.9 pg/ml), and terbinafme (MICso shift from 3.6 pg/ml to 0.07 pg/ml) (FIG. 25 A, FIG. 33A, FIG. 33B, and FIG. 33C). Finally, because of the major alteration in vacuolar function and integrity in mutants altered in Cabl activity’, the susceptibility of wildtype S. cerevisiae to caspofungin, fluconazole, and terbinafine in the absence or presence of concanamycin A, a known inhibitor of the vacuole V-Type ATPase, was examined. As shown in FIG. 25B, treatment of yeast cells with concanamycin-A potentiates their susceptibility to caspofungin (MICso shift from 36 pg/ml to 6.5 ng/ml), fluconazole (MICso shift from 8.1 pg/ml to 5.7 pg/ml). and terbinafine (MICso shift from 3.9 pg/ml to 0.9 pg/ml). Small molecule modulation of the PCA pathway leads to increased susceptibility of pathogenic fungi to antifungal drugs
The genetic and pharmacological data described above suggest that inhibition of specific steps in the CoA biosynthesis pathway or downstream steps leading to the regulation of vacuolar detoxification could be a promising therapeutic strategy for the treatment of fungal infections to enhance the potency of approved drugs while reducing their toxicity. Therefore, screening was performed with a library of PanK and CoA biosynthesis modulators to search for compounds that could render pathogenic fungi susceptible to clinically approved antifungal drugs. The pantazine analog, PZ-2891. an orthostenc activator of human PANK.3. showed the highest potentiation among all compounds tested. Unlike other Cabl inhibitors, PZ-2891 did not inhibit pantothenate utilization of both WT and cabl mutant enzy mes (FIG. 34). Furthermore, the compound had no antifungal activity against S. cerevisiae, C. albicans or A. fumigatus at concentrations up to 50 pM (FIG. 26A. FIG. 26B, FIG. 27A, FIG. 27B, FIG. 28A, FIG. 28B, FIG. 35 A, FIG. 35B, FIG. 36, and FIG. 39).
Interestingly, combinations of PZ-2891 with either amphotericin B, caspofungin or terbinafine at sublethal concentrations resulted in dramatic increases in the susceptibility of S'. cerevisiae and C. albicans to these drugs (FIG. 26A, FIG. 26B, FIG. 35A and FIG. 35B). Similarly, PZ-2891 was found to increase the susceptibility of A. fumigatus to caspofungin (FIG. 1K, FIG. 27B, and FIG. 36). Unlike its inhibitory activity of human PanK3 in the absence or low levels of AcCoA, the results showed that PZ-2891 had little to no effect on Cabl activity in vitro in the absence or presence of AcCoA (FIG. 37A, FIG. 37B, FIG. 38, FIG. 39). Instead, the steady state levels of CoA following treatment with the compound increased by ~ 1.7-fold (FIG. 28 A). These findings indicate that the mechanism of drug potentiation mediated by PZ-2891 in yeast can be in certain embodiments through inhibition of CoA utilization, potentially by blocking the conversion of CoA to AcCoA by Acsl, which is not essential for yeast viability on glucose-based media.
Therefore, the direct inhibition of Acsl activity by PZ-2891 was examined using a hydroxylamine-coupled assay as described in Berg, Paul. Journal of Biological Chemistry 222.2 (1956): 991-1013 and Koselny, Kristy, et al. ACS infectious diseases 2.4 (2016): 268-280. As shown in Fig. 6F, Acsl activity was inhibited by PZ-2891 with 32.7% inhibition of the enzyme activity at 18.8 pM (FIG. 28B). As a control the activity of purified yeast Acsl in vitro was inhibited by AR-12 with a calculated ICso of ~18 pM (FIG. 28B). Together these data demonstrate that the mechanism of drug potentiation of the pantazine analog PZ-2891 is through alteration of a critical downstream step in the PCA pathway catalyzed by Acsl.
In this study, it was demonstrated that the biosynthesis of CoA from pantothenic acid and the subsequent conversion of CoA to AcCoA (the PCA pathway) play a crucial role in the regulation of vacuolar homeostasis and xenobiotic detoxification. Consequently, inhibition of the PCA pathway confers increased susceptibility to antifungal drugs, thus revealing a novel therapeutic strategy for potentiation of frontline antifungal drugs to prevent fungal infections. Examining the implications of altered PCA pathway is important to the understanding of the biology of fungal pathogens, and this study unveiled new cell biological mechanisms regulated by this pathway. In S. cerevisiae. genetic modulation of pantothenate phosphorylation unraveled major alterations in vacuolar and mitochondrial biogenesis and enhanced susceptibility to xenobiotics including metals and commonly used antifungal drugs including both drugs that target ergosterol biosynthesis inhibitors (terbinafine, fluconazole and Amphotericin B) and unrelated pathways (caspofungin. hygromycin. cycloheximide). Such broad-spectrum drug susceptibility is possible if major mechanisms used by fungi for drug detoxification, such as those mediated by the vacuole, are altered when the PCA pathway is inhibited. The data also showed that yeast mutants with altered PanK activity can present with enlarged vacuoles. Thus, in certain embodiments the PCA pathway controls fungal mechanisms of detoxification both through direct effect on vacuolar biogenesis and indirectly through inhibition of ergosterol biosynthesis. In certain embodiments, restricted CoA and consequently reduced AcCoA levels resulting from disruptions to the PCA pathway set off a cascade of events, leading to a deficiency in ergosterol and impaired V-ATPase function. This results in vacuolar dysfunction and subsequent loss of its ability to detoxify xenobiotics (FIG. 29).
The susceptibility of yeast cells altered in CoA biosynthesis from pantothenic acid to a broad spectrum of antifungals can be recapitulated through genetic and pharmacological inhibition of specific enzymes downstream of the PCA pathway. In yeast, AcCoA can be formed from CoA through multiple routes including by the AcCoA synthetases, Acsl and Acs2 (See FIG. 11). Cells lacking bothACST and ACS2 genes are inviable, as are cells lacking ACS2 in glucose medium since ACS1 is subject to glucose repression. The studies demonstrated that repression of the ACS2 gene results in increased susceptibility to commonly used antifungal drugs. This phenotype was further replicated using the Acs 1/2 inhibitor AR- 12 and the V-type ATPase inhibitor concanamycin A.
Unlike its function as an activator of the human pantothenate kinases (PANK3), it was found that the pantazine PZ-2891 has major drug potentiation activity in fungal cells. Growth assays demonstrated potentiation of both caspofungin and amphotericin B in both A. cerevlsicie and C. albicans at concentrations far below their MICso’s (FIG. 26A and FIG. 26B). As the potentiation applies broadly to antifungals with varied mechanisms of action and to metals, one can reason that this is consistent with a broad-based disruption of the ability of fungal cells to detoxify drugs. The initial metabolic studies showing that CoA levels are increased (FIG. 28A) following treatment with PZ-2891 suggesting that the compound’s activity is achieved not through direct inhibition of Cabl activity but through inhibition of either Acsl or Acs2. Meanwhile, since treatment with PZ-2891 does not inhibit growth in glucose-rich media, and ACS2 knockouts or mutants are inviable in such conditions, this leaves Acsl as the primary candidate for PZ-2891’s target in fungi. The biochemical studies using purified yeast Acsl demonstrated that PZ-2891, like AR-12, inhibits this enzyme's activity (FIG. 28B). In clinical studies, an analog of PZ-2891. BBP-671 (NCT04836494), has been found to be largely safe with limited adverse events reported in humans. Thus, this class of small molecules constitutes the first antifungal adjuvants to enhance the potency of current drugs against drug-sensitive and -resistant strains while also lowering their toxicity.
This study shows that modulation of PanK activity results in impaired vacuolar homeostasis and xenobiotic detoxification, which in turns leads to enhanced fungal susceptibility to antifungal drugs.
Cabl mutation study in selection of yeast strains carrying cabl mutations
Methods cablA/pFL38-CAS7 wild type and mutant strains were generated using plasmid shuffling as from the parent strain cab 1 A/pFI.39-co/> 1G351S . Add-back strains were generated by introducing pFL39-C4 A/ vector into yeast recipient strains.
Growth assays
Yeast strains (WT and cabl mutants) were grown overnight at 30°C in YPD medium and harvested (700 x g for 5 mins at 4°C), washed with water, and resuspended in 0.9% NaCl solution at ODeoo of 0.5. Serial 10-fold dilutions were made and 5 pL of cell suspensions were spoted on YPD agar plates containing various antifungals (amphotericin B, caspofungin, fluconazole, terbinafine, hygromycin and cycloheximide). For the respiratory growth assay, YP medium supplemented with ethanol, lactic acid or glycerol were used. Plates were incubated at 30°C and the growth was monitored by image scan using the device ChemiDoc MP (Bio-Rad) every 24 hours. For the liquid growth assay, the yeast strains were pre-grown as above and then diluted into 3 mL of yeast rich media supplemented with either 2% glucose (YPD), 2% glycerol (YPG) or 2% lactate (YPL) liquid media at the concentrations of 10 cells per pL and incubated at 30°C by shaking at 230 rpm and the cell growth was monitored by optical density’ (ODeoo). A. fumigatus growth was examined on GMM (1% glucose, 6 g/L NaNOs, 0.52 g/L KC1, 0.52 g/L MgSO4«7H2O, 1.52 g/L KH2PO4 monobasic, 2.2 mg/L ZnSO4»7H2O, 1.1 mg/L H3BO3, 0.5 mg/L MnCl2 ,4H2O, 0.5 mg/L FeSO4*7H2O, 0.16 mg/L COC12*5H2O, 0.16 mg/L CuSO4«5H2O, 0.11 mg/L (NH4)6MO7O24«4H2O, and 5 mg/L Na4EDTA; pH 6.5).
Electron Microscopy Analysis
Yeast strains (WT and cabl mutants) were grown overnight at 30°C in YPD medium, harvested, and refreshed in YP media with 2% glycerol until reached ODeoo of 1. The cells were harvested, washed, and used for high pressure freezing and freeze substitution for electron microscopy analysis. Unfixed samples were high pressure frozen using a Leica HMP100 at 2000 psi. The frozen samples were then freeze substituted using a Leica Freeze AFS unit starting at -95°C using 0.1% uranyl acetate in acetone for 50 h to -60°C, then rinsed in 100% acetone and infiltrated over 24 h to -45°C with Lowicryl HM20 resin (Electron Microscopy Science). Samples were placed in gelatin capsules and UV hardened at -45°C for 48 h. The blocks were allowed to cure for a further few days before trimmed and cut using a Leica UltraCut UC7. The 60nm sections were collected on formvar/carbon coated nickel grids and contrast stained using 2% uranyl acetate and lead citrate. The 60nm sections on grids were viewed FEI Tecnai Biotwin TEM at 80Kv. Images were taken using AMT NanoSprintl5 MK2 sCMOS camera.
Pantothenate utilization assay using 14C -labeled PA
Pantothenate utilization assay using labeled PA was performed as summarized below: briefly, cell-free extracts from yeast producing Cabl variants were obtained by homogenization, followed by centrifugation at 700 x g for 5 min. The 40 pL enzyme reaction contained reaction buffer (100 mM Tris HC1, 2.5 mM MgCb, 2.5 mM ATP, pH 7.4), D-[l - 14C] pantothenate (2 nmol, 0.1 pCi), and 144 pg cell-free extracts. The lysates total protein content was determined using the Bradford assay. The reaction was done at 30 °C for 10 min following the addition of 4 pL of 10% acetic acid to stop the reaction. The reaction mixture was spotted on a DE-81 filter (0.6 mm in diameter) placed within a spin column with a 2 rnL collection tube. Following 5 min incubation, the spotted filters were centrifuged for 20 s at 700 x g, washed twice with 1% acetic acid in ethanol, and collected for liquid scintillation spectrometry.
Cellular CoA determination
The determination of cellular CoA levels in yeast strains producing harboring different Cabl variants was done using soluble metabolites extractions from S', cerevisiae as previously described. Metabolites extracts were then used in Coenzyme A detection kit (Sigma) to quantify cellular CoA.
Cellular cysteine determination
The determination of cellular cysteine levels in yeast strain producing different Cabl variants was done using soluble metabolites extractions from S', cerevisiae as previously described. Metabolites extracts were then used in fluorometric cysteine assay kit (Abeam).
RNA sequencing and data analysis
RNA samples from yeast strain producing different Cabl variants were extracted using YeaStar RNA kit (Zymo Research).
RNA Seq Quality Control
Total RNA quality is determined by estimating the A260/A280 and A260/A230 ratios by nanodrop. The RNA integrity is determined by resolving an aliquot of the extracted RNA on Agilent Bioanalyzer gel, which measures the ratio of the ribosomal peaks. Samples with RNA integrity number (RIN) values of 7 or greater are recommended for library preparation.
RNA Seq Library Prep
The mRNAs are purified from approximately 200ng of total RNA with oligo-dT beads and sheared by incubation at 94 °C in the presence of Mg (Kapa mRNA Hyper Prep). Following first-strand synthesis with random primers, second strand synthesis and A-tailing are performed with dUTP for generating strand-specific sequencing libraries. Adapter ligation with 3’ dTMP overhangs are ligated to library' insert fragments. Library amplification amplifies fragments carry ing the appropriate adapter sequences at both ends. Strands marked with dUTP are not amplified. Indexed libraries that meet appropriate cut-offs for both are quantified by qRT-PCR using a commercially available kit (KAPA Biosystems) and insert size distribution determined with the LabChip GX or Agilent Bioanalyzer. Samples with a yield of >0.5 ng/pL are used for sequencing. Flow Cell Preparation and Sequencing: Sample concentrations are normalized to 1.2 nM and loaded onto an Illumina NovaSeq flow cell at a concentration that yields 25 million passing filter clusters per sample. Samples are sequenced using lOObp paired-end sequencing on an Illumina NovaSeq according to Illumina protocols. The 1 Obp unique dual index is read during additional sequencing reads that automatically follow the completion of read 1. Data generated during sequencing runs are simultaneously transferred to the YCGA high-performance computing cluster. A positive control (prepared bacteriophage Phi X library) provided by Illumina is spiked into every lane at a concentration of 0.3% to monitor sequencing quality in real time.
Data Analysis and Storage
Signal intensities are converted to individual base calls during a run using the system’s Real Time Analysis (RTA) software. Base calls are transferred from the machine’s dedicated personal computer to the Yale High Performance Computing cluster via a 1 Gigabit network mount for downstream analysis. Primary analysis - sample de-multiplexing and alignment to the human genome - is performed using Illumina’s CAS AV A 1.8.2 software suite. The data are returned to the user if the sample error rate is less than 2% and the distribution of reads per sample in a lane is within reasonable tolerance. Data is retained on the cluster for at least 6 months, after which it is transferred to a tape backup system.
Data analysis
Partek Flow was used to organize and process fastq files from paired-end sequencing. Paired-end reads were trimmed for quality' (Q-Score > 20, Min read length 25) and adapters were removed using FastQC, then aligned to the S. cerevisiae W303-1B genome (Accession: JRIU00000000; ATCC Number: 200060, downloaded from SGD) using STAR v2.7.8a. Reads were normalized via trimmed mean of M values (TMM). and further processed in Excel, where the three biological replicates per condition (WT, three mutants, and three mutants with addback) were grouped to generate means and standard deviations for all genes in each of the 7 conditions. Log fold changes in FIG. 23 correspond to the log base 2 of the ratio between one of 6 experimental conditions and the WT control condition. To quantify the degree of confidence of rescue of a gene differentially expressed in a mutant by its addback, the following decision matrix was utilized. First, the p-value between the wild type and mutant expression was computed using the standard deviations and means of the WT (N=3) and mutant (N=3 for each mutant) expression. Then, the p-value between wild type and complemented mutant was computed. ‘‘Large” circles in FIG. 23B corresponds to genes for which the first p-value<0.05, but the second p-value >0.05, indicating the null hypothesis of equal expression was rejected for the mutant, but not rejected for the addback at p=0.05. “Small” circles correspond to genes for which either of these criteria were not met.
Seahorse analysis
The oxygen consumption rate (OCR) of yeast strains expressing different CAB1 variants was determined using Seahorse 96X and Mito Stress kit. Yeast strains (WT and cabl mutants) were grown overnight at 30°C in YPD medium, harvested, and refreshed in SC medium supplemented with 2% glucose until reached ODeoo of 0.6. Then, cells were harvested, washed and seeded (6 X 104 cells per well) in Seahorse XFp plates coated with poly-Lysine (50 pL of 0.1 mg/mL). A minimum of 8 technical replicates were performed for each experiment at 30°C. The seeded plate was centrifuged at 500 rpm for 5 min to promote yeast adhesion and the plate was rested for 30 min at RT. A soaked and calibrated Seahorse XF96 Sensor Cartridge was prepared before loading into the Seahorse XF96 analyzer (Agilent) which determined the cells basal OCR and following the injection of mitochondrial uncoupling drugs; oligomycin (5 pM), carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone (FCCP) (10 pM), antimycin A (10 pM), and rotenone (5 pM). The readouts were normalized using nuclear Hoechst staining for the immobilized yeast cells.
Yeast growth in the presence of antifungal drugs, inhibitors, and potentiators To investigate the effect of common AFDs (amphotericin B, caspofungin, fluconazole, and terbinafine) in combination with compounds and potentiators (concanamycin A, doxycycline, PZ-2891 , a-Pan Am, AR- 12) the yeast growth was monitored using liquid assay in a 96-well plate. Overnight yeast precultures (WT strains or acs-Tet-Off when mentioned) were prepared in YPD medium at 30°C. Cells were w ashed and refreshed in YPD until reaching ODeoo of 0.6. In a 96-well plate, cells (103 cells/mL, 100 pL final volume) were treated with decreasing concentrations (two-fold dilutions) of AFDs, and different dosages of compounds and potentiators. For reference, amorolfine (200 pM) and DMSO (0.6%) were used as positive and negative controls to determine 100% and 0% growth inhibition, respectively. Plates were incubated at 30°C. Optical density measurements were taken using a BioTek Synergy Mx microplate reader every 12 h. Data are shown as mean ± SD of four independent experiments. Growth curves where visualized and determined from a sigmoidal dose-response curve using GraphPad Prism version 9.5. 1 (GraphPad Softw are, San Diego, CA). Statistical significance w as determined using t-test (p=0.05) with GraphPad Prism.
Fluorescence microscopy of cell organelles To visualize vacuolar structure, the yeast strains were grown to OD of 1 at 600 nm, harvested and resuspend cells at 106 cells/mL in 10 mM HEPES buffer, pH 7.4, containing 5% glucose. CellTracker™ Blue CMAC was added to the cell suspension to a final concentration of 100 pM. The cells were incubated at room temperature for 30 minutes and the stained cells were visualized by fluorescence microscopy.
Reactive Oxygen Species (ROS) content
Reactive oxygen species (ROS) in the cabl mutants was determined by change of oxidative status of fluorescence dye caused by ROS inside of the cell. ROS oxidize dihydrorhodamine 123 (DHR123; Sigma-Aldrich®, Darmstadt, Germany), which in turn produces green, fluorescent R123. To monitor ROS, cells were pre-grown overnight at 30 °C in YPD to the ODeoo of 0.5 ~1.0 and the cells were diluted to the OD of 0.4 and loaded with 1.25 pg/mL of DHR123 for 2 h at 30 °C. At the end of the incubation time, cells were harvested (2 min at 9,000 x g) and re-suspended in water at the ODeoo of 0.05, and the fluorescence was quantified by a plate reader. For each sample, 100 pL of cell suspension was added into each well and the fluorescence was measured (excitation/emission spectra of 488/530 nm). Emission values from the control cells untreated with the dye were used as background for each strain. ROS generation for each cabl strains was measured as the percentage of fluorescence emission obtained from the cab IS strain harboring WT CAB1 gene.
PanK activity^ of recombinant Cabl in the presence ofPZ-2891 andAcCoA
His-tagged Cabl recombinant enzyme was produced and purified as was previously described. A Kinase-Glo (Promega) assay kit for kinase activity was used to determine the activity of the purified PanK under different conditions.
Vacuolar visualization and cell size determination
To determine the ratio of vacuolar area over cell area, different yeast strains were stained with CellTracker™ Blue CMAC as explained in the methods above. Images were captured using fluorescence microscope and analyzed using Image J software. The cell surface area (in square pixel) and vacuolar surface area (in square pixel) were calculated in Image J and percentage of vacuolar area/cell area was calculated. A total of 100 cells were analyzed from each yeast strain. The data was plotted and analyzed in GraphPad Prism version 9.5.1 (GraphPad Software, San Diego, CA). Statistical significance was determined using Welch's t-test with GraphPad Prism.
Radial growth assay and AFD sensitivity assays with A. fumigatus
The radial growth measurements of A. fumigatus were performed by taking 2 pL of a 2.5 X IO6 mL-1 conidial suspension of wild-type CEA10 A. fumigatus was point inoculated onto the center of a solid GMM in the absence or presence of 50 pM PZ-2891, 20 pg/mL caspofungin, and their combination. Plates were incubated for 96 h at 35°C, with colony diameters measured and photographed taken each day.
Acetyl CoA synthetase (ACS) activity’ assay
The ACS assay was performed by monitoring formation of the adenyl acetate, the intermediate of the enzyme reaction, utilizing 5. cerevisiae acetyl Coa synthetase (Sigma, A1765), following established protocols with some modifications. In a 100 pL reaction volume, composed of 100 mM potassium phosphate at pH7.5, 5 mM MgCh, 2 mM ATP, 50 mM potassium fluoride, 10 mM reduced glutathione, 0.35 mM CoA, 10 mM potassium acetate, 200 mM neutralized hydroxylamine adjusted to pH 7.3, 0.005 units of the enzy me, and the inhibitors (in 1% DMSO), the components were combined. The mixture was then incubated for 30 minutes at 37 °C. Termination of the reaction was achieved by addition of 50 pL of a solution containing ferric chloride (12 M) and trichloroacetic acid (12%). The resultant product, acethydroxamic acid, was quantified using a BioTek Synergy Mx microplate reader at OD540. The background correction was performed by utilizing a blank reaction comprising all the reaction components, which was subsequently terminated using acidified ferric chloride solution, without undergoing any incubation time.
Enumerated Embodiments
The following enumerated embodiments are provided, the numbering of which is not be construed as designating levels of importance.
Embodiment 1 : A method of: increasing antifungal activity' of an antifungal agent used to treat, ameliorate, and/or prevent a fungal infection in a subject; reducing, minimizing, and/or eliminating at least one toxicity effect of an antifungal agent used to treat, ameliorate, and/or prevent a fungal infection in a subject; reducing, minimizing, and/or preventing drug resistance against an antifungal agent used to treat, ameliorate, and/or prevent a fungal infection in a subject; and/or sensitizing a fungus to an antifungal agent administered to a subject to treat, ameliorate, and/or prevent the fungal infection in the subject; the method comprising administering a therapeutically effective amount of a compound which is a pantothenate kinase (PanK) inhibitor and/or modulator to a subject being administered the antifungal agent.
Embodiment 2: The method of Embodiment 1, wherein the compound comprises a pantazine and/or pantothenic acid analogue and/or derivative. Embodiment 3 : The method of Embodiment 1 , wherein the compound comprises a compound of formula (la) or (lb),
Figure imgf000072_0001
wherein in (la) or (lb):
R1 is selected from the group consisting of H, -NR4aR4b, -NR4c(optionally substituted phenyl), and optionally substituted N-linked heterocyclyl;
R2 is selected from the group consisting of phenyl and -N(R4c)-(CH2)i-3-(optionally substituted phenyl);
R3 is selected from the group consisting of F, Cl, Br, I, -OR4d. -NR4dR4e, -C(=O)OR4d,- C(=O)NR4eR4t, -C(=O)N(R4g)-(CH2)i-3-(optionally substituted phenyl), and -C(=O)N(R4h)- (CH2)i-3-(optionally substituted heteroar l): each occurrence of R4a-R4h is independently selected from the group consisting of H, optionally substituted Ci-Ce alkyl, and optionally substituted Cs-Cs cycloalkyl; each occurrence of alkyl, cycloalkyl, phenyl, heterocyclyl, and heteroaryl is optionally independently substituted with at least one of Ci-Ce alkyl, Cs-Cs cycloalkyl, halogen, -CN, - ORa, optionally substituted phenyl, optionally substituted heteroaryl, optionally substituted heterocyclyl, -C(=O)ORa, -OC(=O)Ra, -SRa, -S(=O)Ra, -S(=O)2Ra, -S(=O)2NRaRa, - N(Ra)S(=O)2Ra, -N(Ra)C(=O)Ra, -C(=O)NRaRa. and -N(Ra)(Ra); and each occurrence of Ra is independently H, optionally substituted Ci-Ce alkyl, optionally substituted Cs-Cs cycloalkyd, optionally substituted phenyl, or optionally substituted heteroaryl, or two Ra groups combine with the N to which they are bound to form Cs-Cs heterocyclyl.
Embodiment 4: The method of any one of Embodiments 1-3, wherein the fungal infection is caused by at least one of C. albicans, C. parapsilosis, and/or A. fumigatus.
Embodiment 5: The method of Embodiment 3, wherein R1 is selected from the group consisting ofH, -NR4aR4b, and optionally substituted N-linked heterocyclyl.
Embodiment 6: The method of any one of Embodiments 1 and 5, wherein R1 is NR4c(optionally substituted phenyl).
Embodiment 7: The method of any one of Embodiments 1 and 5-6, wherein R3 is - NR4dR4e or -C(=O)OR4d.
Embodiment 8: The method of any one of Embodiments 1 and 5-7, wherein each phenyl in R2 is independently substituted with at least one C2-C8 alkyl. Embodiment 9: The method of any one of Embodiments 1 and 5-8. wherein each phenyl in R2 is independently substituted at the para-position with at least one C2-C8 alkyl.
Embodiment 10: The method of any one of Embodiments 1 and 5-9, wherein the N- linked heterocyclyl comprises aziridinyl, azetidinyl, pyrrolidinyl, pyrazolidinyl, piperidinyl, 1,2,3,6-tetrahydropyridinyl, 1,4-dihydropyridinyl, piperazinyl, morpholinyl, thiomorpholinyl, homopiperazinyl, or homopiperidinyl.
Embodiment 11: The method of any one of Embodiments 1 and 5-10, wherein the heteroaryl comprises imidazolyl, pyridinyl, pyrimidinyl, pyrazinyl, thienyl, furyl, pyrrolyl, thiazolyl, oxazolyl, pyrazolyl, isothiazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, 1,3,4-triazolyl, tetrazolyl, 1,2,3-thiadiazolyl, 1,2,3-oxadiazolyl, 1,3.4-thiadiazolyl, or 1,3,4-oxadiazolyl.
Embodiment 12: The method of any one of Embodiments 1 and 5-11, wherein the compound is at least one of:
Compound
Figure imgf000073_0001
acetylpiperazin-l - yl)methyl)-2-((4-(tert-butyl)benzyl)amino)-[l,2,4]triazolo[l,5-a]pyrimidin-7(4H)-one;
Figure imgf000073_0002
butyl)benzyl)amino)-7-oxo-4,7-dihydro-[l,2,4]triazolo[l,5-a]pyrimidin-5- yl)methyl)piperazine-l-carboxylate;
Compound
Figure imgf000073_0003
-(tert-butyl)benzyl)amino)-5-methyl-
[l,2,4]triazolo[l,5-a]pyrimidine-7-carboxylic acid;
Compound
Figure imgf000073_0004
-amino-2-oxoethyl)-2-((4-(tert- butyl)benzyl)amino)-5-methyl-[l,2,4]triazolo[l,5-a]pyrimidine-7-carboxamide; Compound
Figure imgf000074_0001
-(tert-butjl)phenyl)-5-((4- methylpiperazin-l-yl)methyl)-[l,2,4]triazolo[l,5-a]pyrimidin-7(4H)-one;
Compound
Figure imgf000074_0002
-(tert-butyl)benzyl)-7- methoxy-5-(morpholinomethyl)-[ 1.2.41 triazolo| 1.5-a|pyrimidin-2-amine:
Figure imgf000074_0003
(tert-butyl)benzyl)arnino)-5-methyl-
N-(pyridin-2-yl)-[l,2,4]triazolo[l,5-a]pyrimidine-7-carboxamide;
Compound
Figure imgf000074_0004
methyl (2-((4-(tert-butyl)benzyl)amino)-5- methyl-[l,2,4]triazololl.5-aJpyrimidine-7-carbonyl)glycinate;
Compound
Figure imgf000074_0005
-(tert-butyl)benzyl)amino)-N-(2- hydro\yethyl)-5- ethyl-| l.2.4|triazolo| l.5-a|pyrirnidine-7-carboxarnide:
Compound
Figure imgf000074_0006
(isopropylamino)methyl)-2-((4- isopropylbenzyl)amino)-[l,2,4]triazolo[1.5-a]pyrimidin-7(4H)-one; Compound
Figure imgf000075_0001
(tert- butyl)benzyl)amino)-5-(morpholinomethyl)-[l,2,4]triazolo[l,5-a]pyrimidin-7- yl)oxy)acetamide;
Compound
Figure imgf000075_0002
-(tert- butyl)benzyl)amino)-5-(morpholinomethyl)-[ 1 ,2,4]triazolo[ 1 ,5-a]pyrimi din-7 -yl)oxy)ethan- l-ol;
Compound
Figure imgf000075_0003
-(tert-butyl)benzyl)-5-
(morpholinomethyl)-[l,2,4]triazolo[l,5-a]pyrimidine-2,7-diarnine;
Compound
Figure imgf000075_0004
-(tert- butyl)benzyl)amino)-5-(morpholinomethyl)-[l,2,4]triazolo[ 1.5-a]pyrimidin-7-yl)oxy)acetic acid;
Compound
Figure imgf000075_0005
-(tert-butyl)benzyl)-7- ethoxy-5-(morpholinomethyl)-[l,2,4]triazolo[1.5-a]pyrimidin-2-amine:
Compound
Figure imgf000075_0006
-(tert-butyl)benzyl)-7- chloro-5-(morpholinomethyl)-[l,2,4]triazolo[l,5-a]pyrimidin-2-arnine;
Compound
Figure imgf000076_0001
(tert-butyl)benzyl)amino)-5- methyl-N-(pyridin-3-ylmethyl)-[l?2.4]triazolo[l,5-a]pyrimidine-7-carboxamide;
Compound
Figure imgf000076_0002
-(tert-butyl)benzyl)amino)-5- methyl-[ 1 ,2,4]triazolo[ 1 ,5-a]pyrimidine-7 -carbonyl)gly cine.
Compound
Figure imgf000076_0003
-isopropylbenzyl)amino)-5-
(morpholinomethyl)-[l ,2,4]triazolo[ 1 ,5-a]pyrimidin-7(4H)-one;
Compound
Figure imgf000076_0004
-isopropylbenzyl)amino)-5-methyl-
[l,2,4]triazolo[1.5-a]pyrimidin-7(4H)-one;
Compound
Figure imgf000076_0005
(phenylamino)methyl)-2-(o-tolyl)-
[l,2,4]triazolo[1.5-a]pyrimidin-7(4H)-one;
Compound
Figure imgf000076_0006
isopropylbenzyl)amino)-5-
(pyrrolidin-l-ylmethyl)-[l,2?4]triazolo[l,5-a]pyrimidin-7(4H)-one; Compound
Figure imgf000077_0001
-(tert-butyl)benzyl)-N7- methyl-5-(morpholinomethyl)-[l,2,4]triazolo[l,5-a]pyrimidine-2,7-diamine;
Compound
Figure imgf000077_0002
(tert-butyl)benzyl)amino)-5-
(morpholinomethyl)-[l,2,4]triazolo[l,5-a]pyrimidin-7(4H)-one;
Compound
Figure imgf000077_0003
-(tert-bubt'l)benzjd)-N7,N7- dmrethyl-5-(morpholinomelhyl)-| 1.2.4 |triazolo| 1.5-a|pynmidine-2.7-di amine;
Compound
Figure imgf000077_0004
-(tert-butyl)benzyl)amino)-
5-((4-methylpiperazin-l-yl)methyl)-[l,2,4]triazolo[l,5-a]pyrimidin-7(4H)-one; and
Compound
Figure imgf000077_0005
(tert-butyl)benzyl)amino)-5-
(piperidin-l-ylmethyl)-[l,2,4]triazolo[1.5-a]pyrimidin-7(4H)-one. Embodiment 13: The method of any one of Embodiments 1-12, wherein the compound is administered to the subject as part of a pharmaceutical composition.
Embodiment 14: The method of any one of Embodiments 1-13, wherein the compound and the antifungal agent are co-administered to the subject.
Embodiment 15: The method of Embodiment 14, wherein the compound and the antifungal agent are co-formulated.
Embodiment 16: The method of any one of Embodiments 1-15, wherein the compound is not: Compound
Figure imgf000078_0001
-(tert-butjl)phenyl)-5-((4- methylpiperazin-l-yl)methyl)-[l,2,4]triazolo[l,5-a]pyrimidin-7(4H)-one;
Compound
Figure imgf000078_0002
(isopropylamino)methyl)-2-((4- isopropylbenzyl)amino)-[l,2,4]triazolo[l,5-a]pyrimidin-7(4H)-one;
Compound
Figure imgf000078_0003
-isopropylbenzyl)amino)-5-
(morpholinomethyl)-[l,2,4]triazolo[l,5-a]pyrimidin-7(4H)-one;
Compound
Figure imgf000078_0004
-isopropylbenzyl)amino)-5-methyl-
[l,2,4]triazolo[l,5-a]pyrimidin-7(4H)-one;
Figure imgf000078_0005
(phenylamino)methyl)-2-(o-tolyl)- [l,2,4]triazolo[l,5-a]pyrimidin-7(4H)-one; or
Compound
Figure imgf000078_0006
isopropylbenzyl)amino)-5-
(pyrrolidin-1 -ylmethyl)-[l ,24]triazolo[l ,5-a]pyrimidin-7(4H)-one;
Figure imgf000078_0007
-phenyl-5-((propylamino)methyl)-[l,2,4]triazolo[l,5- a]pyrimidin-7(4H)-one;
Figure imgf000079_0001
methylpiperidin-l -yl)methyl)-2-phenyl-
[l,2,4]triazolo[l,5-a]pyrimidin-7(4H)-one;
Figure imgf000079_0002
(benzyl(methyl)amino)methyl)-2-phenyl-
[l,2,4]triazolo[1.5-a]pyrimidin-7(4H)-one;
Figure imgf000079_0003
(benzyl(2-hydroxyethyl)amino)methyl)-2-phenyl-
[l,2,4]triazolo[l,5-a]pyrimidin-7(4H)-one;
Figure imgf000079_0004
-chlorophenyl)-5-((3,4-dihydroisoquinolin-
2(lH)-yl)methyl)-[l,2,4]triazolo[l,5-a]pyrimidin-7(4H)-one;
Figure imgf000079_0005
-methoxyphenyl)amino)methyl)-2-(p-tolyl)- [1 ,2,4]triazolo[ 1.5-a]pyrimidin-7(4H)-one;
Figure imgf000079_0006
tolyl)-5-((m-tolylamino)methyl)-
[ 1 ,2,4]triazolo[ 1.5-a]pyrimidin-7(4H)-one;
Figure imgf000079_0007
tolyl)-5-((p-tolylamino)methyl)- [l,2,4]triazolo[l,5-a]pyrimidin-7(4H)-one;
Figure imgf000080_0006
[ 1 ,2,4]triazolo[ 1.5-a]pyrimidin-7(4H)-one;
Figure imgf000080_0001
-methoxyphenyl)-5-(morpholinomethyl)-
[l,2,4]triazolo[l,5-a]pyrimidin-7(4H)-one;
Figure imgf000080_0002
-dimethoxyphenyl)amino)methyl)-2-(4- methoxyphenyl)-[l,2,4]triazolo[l,5-a]pyrimidin-7(4H)-one;
Figure imgf000080_0003
-(tert-butyl)phenyl)-5-((propylamino)methyl)-
[l,2,4]triazolo[1.5-a]pyrimidin-7(4H)-one;
Figure imgf000080_0004
dimethylphenyl)-5-((p-tolylamino)methyl)-
[l,2,4]triazolo[1.5-a]pyrimidin-7(4H)-one;
Figure imgf000080_0005
-methyl-2-(phenylamino)-[l,2,4]triazolo[l,5-a]pyrimidin-7-ol;
Figure imgf000081_0005
[l,2,4]triazolo[l,5-a]pyrimidin-7(4H)-one;
Figure imgf000081_0001
dimethoxybenzyl)amino)-5- (morpholinomethyl)-[ 1 ,2,4]triazolo[ 1.5-a]pyrimidin-7(4H)-one;
Figure imgf000081_0002
methyl 2-((2-((4-(tert-butyl)benzyl)amino)-5-
(morpholinomethyl)-[l ,2,4]tnazolo[ 1 ,5-a]pyrimidin-7-yl)oxy)acetate;
Figure imgf000081_0003
(morpholinomethyl)-[l ,2,4]triazolo[ 1.5-a]pyrimidin-2-amine: and
Figure imgf000081_0004
-(tert-butyl)benzyl)amino)-5-
(morpholinomethyl)-[l,2,4]triazolo[L5-a]pyrimidin-7-yl)oxy)-N,N-dimethylacetamide.
Embodiment 17: The method of any one of Embodiments 1-16, wherein the fungus is on the skin of the subject or within the subject.
Embodiment 18: The method of any one of Embodiments 1-17, wherein administration of the compound and the antifungal agent results in less likely drug resistance occurrence in the subject as compared to an equivalent subject that is administered the antifungal agent in the absence of the compound.
Embodiment 19: The method of any one of Embodiments 1-18, wherein administration of the compound and the antifungal agent allows for administration of an amount of the antifungal agent that is lower than the corresponding amount of the antifungal agent that has to be administered in the absence of the compound to achieve equivalent treatment, amelioration, and/or prevention of the fungal infection.
Embodiment 20: The method of any one of Embodiments 1-19, wherein the antifungal agent comprises an agent that disrupts synthesis and/or activity or ergosterol, an inhibitor of any other metabolic pathway, and/or an inhibitor of nucleic acid and/or protein synthesis.
Embodiment 21 : The method of Embodiment 20, wherein the agent is selected from an azole antifungal agent, morpholine antifungal agent, allylamine antifungal agent, and/or polyene antifungal agent.
Embodiment 22: The method of Embodiment 21, wherein at least one of the following applies:
(a) the azole antifungal agent comprises an imidazole antifungal agent selected from bifonazole, butoconazole, clotrimazole, econazole, fenticonazole, isoconazole, ketoconazole, luliconazole, miconazole, omoconazole, oxiconazole, sertaconazole, sulconazole, and tioconazole;
(b) the azole antifungal agent comprises a triazole antifungal agent selected from albaconazole, efinaconazole, epoxiconazole, fluconazole, isavuconazole, itraconazole, posaconazole, propiconazole, ravuconazole, terconazole, and voriconazole;
(c) the azole antifungal agent comprises a thiazole antifungal agent selected from abafungin;
(d) the azole antifungal agent comprises a tetrazole antifungal agent selected from oteseconazole;
(e) the allylamine antifungal agent is selected from butenafme, naftifine, and terbinafine;
(1) the polyene antifungal agent is selected from amphotericin B, candicidin, filipin, hamycin. natamycin, nystatin, and rimocidin;
(g) the morpholine antifungal agent is selected from amorolfine and fenpropimorph; (h) the inhibitor is cycloheximide;
(i) the inhibitor is an echinocandin, optionally selected from anidulafungin, caspofungin, and micafungin;
(j) the inhibitor is a cytosine analogue, optionally 5-flucytosine; (k) the inhibitor is an aminoglycoside, optionally hygromycin B.
Embodiment 23: The method of any of Embodiments 1-22. wherein the compound inhibits fungal PanK selectively over a human PanK enzyme.
Embodiment 24: The method of any of Embodiments 1-23, wherein the subject is a mammal. Embodiment 25 : The method of Embodiment 24, wherein the mammal is a human.
The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated herein by reference in their entirety. While this disclosure has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of this disclosure may be devised by others skilled in the art without departing from the true spirit and scope of the disclosure. The appended claims are intended to be construed to include all such embodiments and equivalent variations.

Claims

CLAIMS What is claimed is:
1. A method of:
(a) increasing antifungal activity of an antifungal agent used to treat, ameliorate, and/or prevent a fungal infection in a subject;
(b) reducing, minimizing, and/or eliminating at least one toxicity effect of an antifungal agent used to treat, ameliorate, and/or prevent a fungal infection in a subject;
(c) reducing, minimizing, and/or preventing drug resistance against an antifungal agent used to treat, ameliorate, and/or prevent a fungal infection in a subject; and/or
(d) sensitizing a fungus to an antifungal agent administered to a subject to treat, ameliorate, and/or prevent the fungal infection in the subject; the method comprising administering a therapeutically effective amount of a compound which is a pantothenate kinase (PanK) inhibitor and/or modulator to a subject being administered the antifungal agent.
2. The method of claim 1, wherein the compound comprises a pantazine and/or pantothenic acid analogue and/or derivative.
3. The method of claim 1 , wherein the compound comprises a compound of formula (la) or (lb),
Figure imgf000084_0001
wherein in (la) or (lb):
R1 is selected from the group consisting ofH, -NR4aR4b, -NR4c(optionally substituted phenyl), and optionally substituted N-linked heterocyclyl;
R2 is selected from the group consisting of phenyl and -N(R4C)-(CH2)I-3- (optionally substituted phenyl);
R3 is selected from the group consisting of F, Cl, Br, I, -OR4d, -NR4dR4e, - C(=O)OR4d,-C(=O)NR4eR4f, -C(=O)N(R4g)-(CH2)i-3-(optionally substituted phenyl), and -C(=O)N(R4h)-(CH2)i-3-(optionally substituted heteroaryl); each occurrence of R4a-R4h is independently selected from the group consisting of H, optionally substituted Ci-Ce alkyl, and optionally substituted C'3-Cs cycloalkyl; each occurrence of alkyl, cycloalkyl, phenyl, heterocyclyl, and heteroaryl is optionally independently substituted with at least one of Ci-Cs alkyl, C3-C8 cycloalkyl, halogen, -CN, -ORa, optionally substituted phenyl, optionally substituted heteroaryl, optionally substituted heterocyclyl, -C(=O)ORa, -OC(=O)Ra, -SRa, - S(=O)Ra, -S(=O)2Ra, -S(=O)2NRaRa, -N(Ra)S(=O)2Ra. -N(Ra)C(=O)Ra, -C(=O)NRaRa, and -N(Ra)(Ra); and each occurrence of Ra is independently H, optionally substituted Ci-Ce alkyl, optionally substituted C3-C8 cycloalkyl, optionally substituted phenyl, or optionally substituted heteroaryl, or two Ra groups combine with the N to which they are bound to form C3-C8 heterocyclyl.
4. The method of any one of claims 1-3, wherein the fungal infection is caused by at least one of C. albicans, C. parapsilosis, and/or A. fumi gains.
5. The method of claim 3, wherein R1 is selected from the group consisting of H, -
NR4aR4b, and optionally substituted N-linked heterocyclyl.
6. The method of any one of claims 1 and 5, wherein R1 is NR4c(optionally substituted phenyl).
7. The method of any one of claims 1 and 5-6, wherein R3 is -NR4dR4e or -C(=O)OR4d.
8. The method of any one of claims 1 and 5-7, wherein each phenyl in R2 is independently substituted with at least one C2-C8 alkyl.
9. The method of any one of claims 1 and 5-8, wherein each phenyl in R2 is independently substituted at the para-position with at least one C2-C8 alky l.
10. The method of any one of claims 1 and 5-9, wherein the N-linked heterocyclyl comprises aziridinyl, azetidinyl, pyrrolidinyl, pyrazolidinyl, piperidinyl, 1 ,2,3,6- tetrahydropyridinyl, 1,4-dihydropyridinyl, piperazinyl, morpholinyl, thiomorpholinyl, homopiperazinyL or homopiperidinyl.
11. The method of any one of claims 1 and 5-10, wherein the heteroaryl comprises imidazolyl, pyridinyl, pyrimidinyl. pyrazinyl, thienyl, furyl, pyrrolyl, thiazolyl, oxazolyL pyrazolyl, isothiazolyl. 1,2,3-triazolyl. 1 ,2,4-triazolyl. 1,3,4-triazolyl. tetrazolyl, 1,2,3-thiadiazolyl, 1,2,3-oxadiazolyl, 1,3,4-thiadiazolyl, or 1,3,4- oxadiazolyl.
12. The method of any one of claims 1 and 5-11, wherein the compound is at least one of:
Compound
Figure imgf000086_0001
5-((4-acetylpiperazin-l-yl)methyl)-2-((4-(tert-butyl)benzyl)amino)- [1, 2, 4]tri azolof l,5-a]pyrimidin-7(4H)-one;
Figure imgf000086_0002
tert-butyl 4-((2-((4-(tert-butyl)benzyl)amino)-7-oxo-4,7-dihydro-[l,2,4]triazolo[l,5- a]pyrimidin-5-yl)methyl)piperazine-l-carboxylate;
Compound
Figure imgf000086_0003
2-((4-(tert-butyl)benzyl)amino)-5-methyl-[l,2,4]triazolo[l,5-a]pyrimidine-7- carboxylic acid;
Compound
Figure imgf000086_0004
N-(2-amino-2-oxoethyl)-2-((4-(tert-butyl)benzyl)amino)-5-methyl-[l,2,4]triazolo[l,5- a]pyrimidine-7-carboxamide;
Compound
Figure imgf000087_0001
2-(4-(tert-butyl)phenyl)-5-((4-methylpiperazin-l-yl)methyl)-[l,2,4]triazolo[l,5- a]pyrimi din-7 (4H)-one;
Compound
Figure imgf000087_0002
N-(4-(tert-butyl)benzyl)-7-methoxy-5-(morpholinomethyl)-[l,2,4]triazolo[l,5- a]pyrimidin-2-amine;
Compound
Figure imgf000087_0003
2-((4-(tert-butyl)benzyl)amino)-5-methyl-N-(pyridin-2-yl)-[1.2.4]triazolo[l,5- a]pyrimidine-7-carboxamide;
Compound
Figure imgf000087_0004
methyl (2-((4-(tert-butyl)benzyl)amino)-5-methyl-[l,2,4]triazolo[l,5-a]pyrimidine-7- carbonyl)glycinate;
Compound
Figure imgf000087_0005
2-((4-(tert-butyl)benzyl)amino)-N-(2-hydroxyethyl)-5-methyl-[l,2,4]triazolo[l,5- a]pyrimidine-7-carboxamide;
Compound
Figure imgf000088_0001
5-((isopropylamino)methyl)-2-((4-isopropylbenzyl)amino)-[l,2,4]tri azolof 1,5- a]pyrimi din-7 (4H)-one;
Compound
Figure imgf000088_0002
2-((2-((4-(tert-butyl)benzyl)amino)-5-(morpholinomethyl)-[l,2,4]triazolo[l,5- a]pyrimidin-7-yl)oxy)acetamide:
Compound
Figure imgf000088_0003
2-((2-((4-(tert-butyl)benzyl)amino)-5-(morpholinomethyl)-[l,2,4]triazolo[l,5- a]pyrimi din-7 -yl)oxy )ethan- 1 -ol;
Compound
Figure imgf000088_0004
N2-(4-(tert-butyl)benzyl)-5-(morpholinomethyl)-[1.2.4]triazolo[l,5-a]pyrimidine-2,7- diamine;
Compound
Figure imgf000088_0005
2-((2-((4-(tert-butyl)benzyl)amino)-5-(morpholinomethyl)-[l,2,4]triazolo[l,5- a]pyrimi din-7 -yl)oxy)acetic acid; Compound
Figure imgf000089_0001
N-(4-(tert-butyl)benzyl)-7-ethoxy-5-(morpholinomethyl)-[l, 2,4]tri azolof 1,5- a]pyrimidin-2-amine;
Compound
Figure imgf000089_0002
N-(4-(tert-butyl)benzyl)-7-chloro-5-(morpholinomethyl)-[l,2,4]triazolo[l,5- a]pyrimidin-2-amine;
Compound
Figure imgf000089_0003
2-((4-(tert-butyl)benzyl)amino)-5-methyl-N-(pyridin-3-ylmethyl)-[l,2,4]triazolo[1.5- a]pyrimidine-7 -carboxamide;
Compound
Figure imgf000089_0004
(2-((4-(tert-butyl)benzyl)arnino)-5-methyl-[l,2,4]triazolo[l,5-a]pyrimidine-7- carbonyl)glycine.
Compound
Figure imgf000089_0005
2-((4-isopropylbenzyl)amino)-5-(morpholinomethyl)-[l,2,4]triazolo[l,5-a]pyrimidin-
7(4H)-one; Compound
Figure imgf000090_0001
2-((4-isopropylbenzyl)amino)-5-methyl-[l,2,4]triazolo[l,5-a]pyrimidin-7(4H)-one;
Compound
Figure imgf000090_0002
5-((phenylamino)methyl)-2-(o-tolyl)-[l, 2, 4]tri azolof l,5-a]pyrimidin-7(4H)-one;
Compound
Figure imgf000090_0003
2-((4-isopropylbenzyl)amino)-5-(pyrrolidin-l-ylmethyl)-[l, 2, 4]tri azolof 1,5- a]pyrimi din-7 (4H)-one;
Compound
Figure imgf000090_0004
N2-(4-(tert-butyl)benzyl)-N7-methyl-5-(morpholinomethyl)-[ 1.2.4]tri azoIofl, 5- a]pyrimidine-2,7-diamine;
Compound
Figure imgf000090_0005
2-((4-(tert-butyl)benzyl)amino)-5-(morpholinomethyl)-[l, 2, 4]tri azolof 1,5- a]pyrimi din-7 (4H)-one;
Compound
Figure imgf000090_0006
N2-(4-(tert-buty4)benzy l)-N7,N7-dimethyl-5-(morpholinomethyl)-[l,2,4]triazolo[l,5- a]pyrimidine-2,7-diamine; Compound
Figure imgf000091_0001
2-((4-(tert-butyl)benzyl)amino)-5-((4-methylpiperazin-l-yl)methyl)-
[l,2,4]triazolo[l,5-a]pyrimidin-7(4H)-one; and
Compound
Figure imgf000091_0002
2-((4-(tert-butyl)benzyl)amino)-5-(piperidin-l-ylmethyl)-[l,2,4]triazolo[l,5- a]pyrimidin-7(4H)-one.
13. The method of any one of claims 1-12, wherein the compound is administered to the subject as part of a pharmaceutical composition.
14. The method of any one of claims 1-13, wherein the compound and the antifungal agent are co-administered to the subject.
15. The method of claim 14, wherein the compound and the antifungal agent are coformulated.
16. The method of any one of claims 1-15, wherein the compound is not:
Compound
Figure imgf000091_0003
2-(4-(tert-butyl)phenyl)-5-((4-methylpiperazin-l-yl)methyl)-[l,2,4]triazolo[l,5- a]pyrimidin-7(4H)-one;
Compound
Figure imgf000091_0004
5-((isopropylamino)methyl)-2-((4-isopropylbenzyl)amino)-[l,2,4]triazolo[l,5- a]pyrimi din-7 (4H)-one; Compound
Figure imgf000092_0001
2-((4-isopropylbenzyl)arnino)-5-(morpholinomethyl)-[l,2,4]triazolo[l,5-a]pyrimidin- 7(4H)-one;
Compound
Figure imgf000092_0002
2-((4-isopropylbenzyl)amino)-5-methyl-[l,2,4]triazolo[l,5-a]pyrimidin-7(4H)-one;
Compound
Figure imgf000092_0003
5-((phenylamino)methyl)-2-(o-tolyl)-[h2,4]triazolo[l,5-a]pyrimidin-7(4H)-one; or
Figure imgf000092_0004
2-((4-isopropylbenzyl)amino)-5-(pyrrolidin-l-ylmethyl)-[1.2.4]triazolo[l,5- a]pyrimidin-7(4H)-one;
Figure imgf000092_0006
5-((4-methylpiperidin-l-yl)methyl)-2-phenyl-[l,2.4]triazolo[l,5-a]pyrimidin-7(4H)- one;
Figure imgf000092_0005
5-((benzyl(methyl)amino)methyl)-2-phenyl-[l,2,4]triazolo[l,5-a]pyrirnidin-7(4H)- one;
Figure imgf000093_0001
5-((benzyl(2-hydroxyethyl)amino)methyl)-2-phenyl-[1.2.4]triazolo[l,5-a]pyrimidin- 7(4H)-one;
Figure imgf000093_0002
2-(4-chlorophenyl)-5-((3,4-dihydroisoquinolin-2(lH)-yl)methyl)-[l,2,4]triazolo[l,5- a]pyrimi din-7 (4H)-one;
Figure imgf000093_0003
5-(((2-methoxyphenyl)amino)methyl)-2-(p-tolyl)-[l,2,4]triazolo[l,5-a]pyrimidin-
7(4H)-one;
Figure imgf000093_0005
2-(p-tolyl)-5-((p-tolylamino)methyl)-[l,2,4]triazolo[l,5-a]pyrimidin-7(4H)-one;
Figure imgf000093_0004
2-(p-tolyl)-5-((o-tolylamino)methyl)-[l,2,4]triazolo[l,5-a]pyrimidin-7(4H)-one;
Figure imgf000094_0002
2-(4-methoxyphenyl)-5-(morpholinomethyl)-[l,2,4]triazolo[l,5-a]pyrimidin-7(4H)- one;
Figure imgf000094_0001
5-(((2,4-dimethoxyphenyl)amino)rnethyl)-2-(4-rnethoxyphenyl)-[ 1 ,2,4]triazolo[l ,5- a]pyrimi din-7 (4H)-one;
Figure imgf000094_0003
5-methyl-2-(phenylamino)-[ l,2,4]triazolo[ 1.5-a]pyrimidin-7-ol:
Figure imgf000095_0004
2-(benzylamino)-5-(morpholinomethyl)-[l,2,4]triazolo[1.5-a]pyrimidin-7(4H)-one;
Figure imgf000095_0001
2-((3,4-dimethoxybenzyl)amino)-5-(morpholinomethyl)-[l,2,4]triazolo[l,5- a]pyrimi din-7 (4H)-one;
Figure imgf000095_0002
methyl 2-((2-((4-(tert-butyl)benzyl)amino)-5-(morpholinomethyl)-[l,2,4]triazolo[l,5- a]pyrimidin-7-yl)oxy)acetate;
Figure imgf000095_0003
7-(benzyloxy)-N-(4-(tert-butyl)benzyl)-5-(morpholinomethyl)-[l,2,4]triazolo[l,5- a]pyrimidin-2-amine; and
Figure imgf000096_0001
2-((2-((4-(tert-butyl)benzyl)amino)-5-(morpholinomethyl)-[l, 2, 4]tri azolof 1,5- a]pyrimidin-7-yl)oxy)-N,N-dimethylacetamide.
17. The method of any one of claims 1-16, wherein the fungus is on the skin of the subject or within the subject.
18. The method of any one of claims 1-17, wherein administration of the compound and the antifungal agent results in less likely drug resistance occurrence in the subject as compared to an equivalent subject that is administered the antifungal agent in the absence of the compound.
19. The method of any one of claims 1-18, wherein administration of the compound and the antifungal agent allows for administration of an amount of the antifungal agent that is lower than the corresponding amount of the antifungal agent that has to be administered in the absence of the compound to achieve equivalent treatment, amelioration, and/or prevention of the fungal infection.
20. The method of any one of claims 1-19, wherein the antifungal agent comprises an agent that disrupts synthesis and/or activity or ergosterol, an inhibitor of any other metabolic pathway, and/or an inhibitor of nucleic acid and/or protein synthesis.
21. The method of claim 20. wherein the agent is selected from an azole antifungal agent, morpholine antifungal agent, allylamine antifungal agent, and/or polyene antifungal agent.
22. The method of claim 21, wherein at least one of the following applies:
(a) the azole antifungal agent comprises an imidazole antifungal agent selected from bifonazole, butoconazole, clotrimazole, econazole, fenticonazole, isoconazole, ketoconazole, luliconazole, miconazole, omoconazole, oxiconazole, sertaconazole, sulconazole, and tioconazole;
(b) the azole antifungal agent comprises a triazole antifungal agent selected from albaconazole, efinaconazole, epoxiconazole, fluconazole, isavuconazole, itraconazole, posaconazole, propiconazole, ravuconazole, terconazole, and voriconazole;
(c) the azole antifungal agent comprises a thiazole antifungal agent selected from abafungin;
(d) the azole antifungal agent comprises a tetrazole antifungal agent selected from oteseconazole;
(e) the allylamine antifungal agent is selected from butenafme, naftifme, and terbinafine;
(f) the polyene antifungal agent is selected from amphotericin B, candicidin, filipin, hamycin. natamycin, nystatin, and rimocidin;
(g) the morpholine antifungal agent is selected from amorolfme and fenpropimorph;
(h) the inhibitor is cycloheximide;
(i) the inhibitor is an echinocandin, optionally selected from anidulafungin, caspofungin, and micafungin;
(j) the inhibitor is a cytosine analogue, optionally 5-flucytosine;
(k) the inhibitor is an aminoglycoside, optionally hygromycin B.
23. The method of any of claims 1-22, wherein the compound inhibits fungal PanK selectively over a human PanK enzyme.
24. The method of any of claims 1-23, wherein the subject is a mammal.
25. The method of claim 24, wherein the mammal is a human.
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Publication number Priority date Publication date Assignee Title
WO2009062746A2 (en) * 2007-11-17 2009-05-22 Bayer Consumer Care Ag Topical drugs for use in antifungal therapy
WO2021262786A1 (en) * 2020-06-24 2021-12-30 Yale University Pank modulators and methods using same

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009062746A2 (en) * 2007-11-17 2009-05-22 Bayer Consumer Care Ag Topical drugs for use in antifungal therapy
WO2021262786A1 (en) * 2020-06-24 2021-12-30 Yale University Pank modulators and methods using same

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