WO2022261069A1 - Procédés et traitement d'infection virale avec des pyrazolo-pyrimidines substituées - Google Patents

Procédés et traitement d'infection virale avec des pyrazolo-pyrimidines substituées Download PDF

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WO2022261069A1
WO2022261069A1 PCT/US2022/032465 US2022032465W WO2022261069A1 WO 2022261069 A1 WO2022261069 A1 WO 2022261069A1 US 2022032465 W US2022032465 W US 2022032465W WO 2022261069 A1 WO2022261069 A1 WO 2022261069A1
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optionally substituted
4alkyl
compound
alkyl
methyl
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PCT/US2022/032465
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English (en)
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Jane RHODES
Michelle MIGHDOLL
Irene Y. Choi
Brian KOPEC
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Verge Analytics, Inc.
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    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses

Definitions

  • the present disclosure relates to methods of i) blocking alpha-coronavirus, beta- coronavirus lineage B, and/or beta-coronavirus lineage A into a host cell; and ii) preventing and treating an infection caused by alpha-coronavirus, beta-coronavirus lineage B, and/or beta- coronavirus lineage A with compounds that are FYVE-type finger-containing phosphoinositide kinase (“PIKfyve”) inhibitors.
  • PIKfyve FYVE-type finger-containing phosphoinositide kinase
  • the SARS-CoV-2 which is responsible for the COVID-19 (2019 novel coronavirus (2019-nCoV) disease, is an enveloped, positive-sense, RNA virus that belongs to the Betacoronavirus genus. Bouhaddou et al, Cell 182: 1-28 (2020). Improved understanding of key steps in viral entry and ways to disrupt them can lead to the development of effective antiviral drugs, not only for COVID-19, but for future viral outbreaks as well. Treatments for COVID-19 are greatly needed. [0004] Coronavirus entry into susceptible cells is a complex process that requires the concerted action of receptor-binding and proteolytic processing of the coronavirus S protein to promote virus-cell fusion.
  • viral entry into cells may be mediated by a viral glycoprotein (GP), which attaches viral particles to the cell surface, delivers them to endosomes, and catalyzes fusion between viral and endosomal membranes.
  • GP viral glycoprotein
  • S protein binds to an ACE2 receptor on the target cell and is subsequently primed by a serine protease, TMPRSS2, that cleaves the S protein and allows fusion of viral and lysosomal membranes.
  • TMPRSS2 serine protease
  • Coronaviruses are known to interact with phosphatidylinositol (PI) kinases, which are distributed across various b ll l B i l Vi 12 1124 d i 103390/ 12101124 (2020) Th acidification is required for SARS-CoV-2 to enter the cell. Ou, et al, Nat. Commun. 11(1620):1- 12 (2020). PIKfyve inhibition with small molecule inhibitors has been shown to inhibit SARS- CoV-2 infection. Kang et al, PNAS 117(34):20803-20813 (2020); Nelson, et al, PLoS Negl. Trop. Dis. 11(4):e0005540 (2017).
  • PI phosphatidylinositol
  • Embodiment 1 is a method of blocking alpha-coronavirus, beta-coronavirus lineage B, and/or beta-coronavirus lineage A entry into a host cell and preventing an infection caused by alpha-coronavirus, beta-coronavirus lineage B, and/or beta-coronavirus lineage A, comprising administering to a subject in need thereof a compound of (i) Formula (I): wherein: X is N or CH, Y is N or CR a provided that when X is N, then Y is CR a , when Y is N, then X is CH; one of R a and R 1 is H, and the other is phenyl or heteroaryl, each optionally substituted with one, two, or three R d substituents; wherein each R d substituent is independently C1-4alkyl, C1-4alkenyl, C1-4alkynyl, -O-C1-4alkyl,
  • Embodiment 2 is the method of embodiment 1, wherein a. the alpha-coronavirus is HCoV 229E; b. the beta-coronavirus lineage B is SARS-CoV2; and c. the beta-coronavirus lineage A is HCoV OC43.
  • Embodiment 3 is the method of embodiment 2, wherein the infection is caused by SARS-CoV-2, and wherein the infection is COVID-19.
  • Embodiment 4 is the method of any one of the preceding embodiments, wherein X is CH and Y is N.
  • Embodiment 5 is the method of any one of embodiments 1-3, wherein X is N and Y is CR a .
  • Embodiment 6 is the method of any one of the preceding embodiments, wherein R a is H methyl ethyl propyl isopropyl butyl isobutyl sec butyl or tert butyl
  • Embodiment 7 is the method of any one of embodiments 1-5, wherein R a is H or methyl.
  • Embodiment 8 is the method of any one of embodiments 1-5, wherein R a is H.
  • Embodiment 9 is the method of any one of embodiments 1-5, wherein R a or R 1 is optionally substituted phenyl.
  • Embodiment 10 is the method of any one of embodiments 1-5, wherein R a or R 1 is optionally substituted monocyclic heteroaryl.
  • Embodiment 11 is the method of any one of embodiments 1-5, wherein R a or R 1 is optionally substituted pyrrole, imidazole, pyrazole, triazole, tetrazole, furan, oxazole, isoxazole, thiazole, isothiazole, pyridine, pyrimidine, pyrazine, or pyridazine.
  • Embodiment 12 is the method of any one of embodiments 1-5, wherein R a or R 1 is optionally substituted pyridine or pyrimidine.
  • Embodiment 13 is the method of any one of embodiments 1-5, wherein R a or R 1 is optionally substituted pyridine.
  • Embodiment 14 is the method of any of the preceding embodiments, wherein R a or R 1 is substituted with one R d and the R d is C1-4alkyl, -NR g R h , or halo.
  • Embodiment 15 is the method of any one of embodiments 1-14, wherein R a or R 1 is substituted with one R d and the R d is methyl, NH 2 , fluoro, chloro, or bromo.
  • Embodiment 16 is the method of any one of embodiments 1-5, R a or R 1 is phenyl or pyridyl, each optionally substituted with one or two substituents selected from C1-4alkyl, -CF3, fluoro, chloro, bromo, -OCH 3 , and -OCF 3 .
  • Embodiment 17 is the method of any one of embodiments 1-5, wherein R a or R 1 is unsubstituted phenyl or tolyl.
  • Embodiment 18 is the method of any one of embodiments 1-5, wherein R a or R 1 is unsubstituted phenyl or m-tolyl.
  • Embodiment 19 is the method of any one of embodiments 1-5, wherein R a or R 1 is unsubstituted pyridyl.
  • Embodiment 20 is the method of any one of embodiments 1-5, wherein R a or R 1 is 4-pyridyl.
  • Embodiment 21 is the method of any one of the preceding embodiments, wherein R 2 and R 3 taken together with the nitrogen to which they are attached form pyrrolidine, piperidine, piperazine, morpholine, thiomorpholine, thiomorpholine-1,1-dioxide, or 2-oxa-6- azaspiro[3.3]heptane, each optionally substituted with one, two, or three R j substituents.
  • Embodiment 22 is the method of any one of embodiments 1-21, wherein R 2 and R 3 taken together with the nitrogen to which they are attached form morpholine, optionally substituted with one or two R j substituents.
  • Embodiment 23 is the method of any one of the embodiments 1-22, wherein each R j substituent is independently methyl, hydroxy, -OCH 3 , oxo, halo, -CF 3 , or -OCF 3 .
  • Embodiment 24 is the method of any one of the preceding embodiments, wherein R k and R l are each independently H or methyl.
  • Embodiment 25 is the method of any one of the embodiments 1-24, wherein R 4 is optionally substituted phenyl.
  • Embodiment 26 is the method of any one of embodiments 1-24, wherein R 4 is optionally substituted heteroaryl.
  • Embodiment 27 is the method of any one of embodiments 1-24, wherein R 4 is optionally substituted pyrazole, thiazole, oxazole, pyridine or pyrimidine.
  • Embodiment 28 is the method of any one of embodiments 1-24, wherein R 4 is optionally substituted pyridine.
  • Embodiment 29 is the method of any one of embodiments 1-24, wherein R 4 is pyridine.
  • Embodiment 30 is the method of any one of embodiments 1-24, wherein R 4 is 4- pyridyl.
  • Embodiment 31 is the method of any one of embodiments 1-24, wherein R 4 is optionally substituted with one or two R z substituents.
  • Embodiment 32 is the method of any one of embodiments 1-24, wherein R 4 is phenyl or pyridyl, each optionally substituted with one or two substituents selected from C1-4alkyl, - CF3, fluoro, chloro, -OCH3, and -OCF3.
  • Embodiment 33 is the method of any one of embodiments 1-24, wherein R 4 is -C(O)NR x R y .
  • Embodiment 34 is the method of any one of embodiments 1-33, wherein R x is H.
  • Embodiment 35 is the method of any one of embodiments 1-33, wherein R x is methyl or ethyl, optionally substituted with one, two, or three R o substituents.
  • Embodiment 36 is the method of any one of embodiments 1-33, wherein R x is methyl.
  • Embodiment 37 is the method of any one of the preceding embodiments, wherein R y is H.
  • Embodiment 38 is the method of any one of embodiments 1-36, wherein R y is C1-4alkyl, -C1-4alkyl(monocyclic cycloalkyl), monocyclic cycloalkyl, monocyclic h l lk l li h l l O li h l l O C lk l SO C
  • Embodiment 39 is the method of any one of embodiments 1-36, wherein R y is C1- 4 alkyl, optionally substituted with one, two, or three R o substituents.
  • Embodiment 40 is the method of any one of embodiments 1-36, wherein R y is methyl, ethyl, propyl, or isopropyl, each optionally substituted with one, two, or three R o substituents.
  • Embodiment 41 is the method of any one of embodiments 1-36, wherein R y is methyl, ethyl, isopropyl, methoxyethyl, dimethoxypropanyl, (dimethylamino)ethyl, or (dimethylamino)butyl.
  • Embodiment 42 is the method of any one of embodiments 1-36, wherein R y is methoxy.
  • Embodiment 43 is the method of any one of embodiments 1-36, wherein R y is -SO2- methyl.
  • Embodiment 44 is the method of any one of embodiments 1-36, wherein R y is monocyclic cycloalkyl or -C1-2alkyl(monocyclic cycloalkyl), each optionally substituted with one, two, or three R o substituents.
  • Embodiment 45 is the method of any one of embodiments 1-36, wherein R y is monocyclic cycloalkyl, optionally substituted with one, two, or three R o substituents.
  • Embodiment 46 is the method of any one of embodiments 1-36, wherein R y is cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl, each optionally substituted with one, two, or three R o substituents.
  • Embodiment 47 is the method of any one of embodiments 1-36, wherein R y is cyclopropyl.
  • Embodiment 48 is the method of any one of embodiments 1-36, wherein R y is cyclopentyl.
  • Embodiment 49 is the method of any one of embodiments 1-36, wherein R y is cyclopropyl, cyclobutyl, cyclopentyl, cyclopropylmethyl, 1-cyclopropylethyl, 2- cyclopropylethyl, cyclobutylmethyl, or cyclopentylmethyl.
  • Embodiment 50 is the method of any one of embodiments 1-36, wherein R y is monocyclic heterocyclyl or -O-monocyclic heterocyclyl, optionally substituted with one, two, or three R o substituents.
  • Embodiment 51 is the method of any one of embodiments 1-36, wherein R y is optionally substituted tetrahydrofuranyl, tetrahydropyranyl, oxetanyl, azetidinyl, pyrrolidinyl, piperidinyl, morpholinyl, piperazinyl, or oxetanyloxy.
  • Embodiment 52 is the method of any one of embodiments 1-36, wherein R y is l l
  • Embodiment 53 is the method of any one of embodiments 1-36, wherein R y is monocyclic heterocycloalkyl, optionally substituted with one, two, or three R o substituents.
  • Embodiment 54 is the method of any one of embodiments 1-36, wherein R y is optionally substituted with one or two R o substituents, and R o is methyl.
  • Embodiment 55 is the method of any one of embodiments 1-33, wherein R x is methyl and R y is methyl, ethyl, cyclopropyl, methoxy, or cyclopentyl.
  • Embodiment 56 is the method of any one of embodiments 1-33, wherein R x and R y taken together with the nitrogen to which they are attached form a monocyclic heterocycloalkyl, optionally substituted with C 1-4 alkyl.
  • Embodiment 57 is the method of any one of embodiments 1-33, wherein R x and R y taken together with the nitrogen to which they are attached form a monocyclic heterocyclyl, optionally substituted with C 1-4 alkyl.
  • Embodiment 58 is the method of any one of embodiments 1-33, wherein R x and R y are taken together with the nitrogen to which they are attached to form azetidinyl, pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, 6-oxa-1-azaspiro[3.3]heptanyl, or 2-oxa- 6-azaspiro[3.3]heptanyl, each optionally substituted with methyl.
  • Embodiment 59 is the method of any one of the preceding embodiments, wherein each R z is independently C 1-4 alkyl, halo, -OH, -OC 1-4 alkyl, C 1-4 alkylNR m R n or -NR m R n .
  • Embodiment 60 is the method of any one of embodiments 1-58, wherein each R z is independently methyl, -OH, halo, or -OCH3.
  • Embodiment 61 is the method of any one of embodiments 1-58, wherein R z is C 2-3 alkyl substituted with -NR m R n .
  • Embodiment 62 is the method of any one of the preceding embodiments, wherein R m and R n are each independently H or C 1-4 alkyl.
  • Embodiment 63 is the method of any one of embodiments 1-61, wherein R m and R n are each methyl.
  • Embodiment 64 is the method of any one of embodiments 1-61, wherein R m and R n taken together with the nitrogen to which they are attached form a monocyclic heterocyclyl, optionally substituted with one or two R o substituents.
  • Embodiment 65 is the method of any one of embodiments 1-61, wherein R m and R n taken together with the nitrogen to which they are attached form pyrrolidine, piperidine, piperazine, morpholine, thiomorpholine, or thiomorpholine-1,1-dioxide, each optionally substituted with one or two R o substituents.
  • Embodiment 66 is the method of any one of embodiments 1-61, wherein R m and R n taken together with the nitrogen to which they are attached form pyrrolidine, piperidine, piperazine, or morpholine, each optionally substituted with one or two R o substituents.
  • Embodiment 67 is the method of any one of the preceding embodiments, wherein R o substituent is C 1-4 alkyl.
  • Embodiment 68 is the method of any one of embodiments 1-66, wherein R o substituent is -OH.
  • Embodiment 69 is the method of any one of embodiments 1-66, wherein R o substituent is -NR p R q .
  • Embodiment 70 is the method of any one of the preceding embodiments, wherein R p and R q are each independently H or methyl.
  • Embodiment 71 is the method of any one of embodiments 1-69, wherein R p and R q taken together with the nitrogen to which they are attached form a heterocyclyl.
  • Embodiment 72 is the method of any one of embodiments 1-69, wherein R p and R q taken together with the nitrogen to which they are attached form azetidinyl, pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl 6-oxa-1-azaspiro[3.3]heptanyl, or 2-oxa- 6-azaspiro[3.3]heptanyl.
  • Embodiment 73 is the method of any one of the preceding embodiments, wherein R 5 is H, methyl, ethyl, chloro, bromo, fluoro, -OH, or -OCH3.
  • Embodiment 74 is the method of any one of embodiments 1-72, wherein R 5 is H.
  • Embodiment 75 is the method of any one of the preceding embodiments, wherein the compound is selected from a compound of Table 1 or a pharmaceutically acceptable salt thereof.
  • Embodiment 76 is the method of any one of embodiments 1 to 75, wherein one or more hydrogen atoms attached to carbon atoms of the compound are replaced by deuterium atoms
  • Embodiment 77 is the method of any one of the preceding embodiments, wherein the compound and/or the pharmaceutically acceptable salt is in a pharmaceutical composition.
  • Embodiment 78 is the method of embodiment 77, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.
  • Figures 1A-C show antiviral effect and cell toxicity data for Compound 22, Compound 18, and Compound 17 in Vero-E6 cells.
  • Concentration-dependent antiviral effect i.e., antiviral activity
  • percent cell survival i.e., percent inhibition
  • C i d d ll i i i h shows data for Compound 22.
  • Figure 1B shows data for Compound 18.
  • Figure 1C shows data for Compound 17.
  • Figures 2A-C show antiviral effects of Compound 14, Compound 17, and Compound 43 in A549-ACE2 cells.
  • Viral titer is shown as Log10 of plaque forming units per mL (PFU/mL) on the left axis (data represented by circles).
  • FIG. 1 Percent cell viability is shown on the right axis (data represented by squares).
  • Figure 2A shows data for Compound 14.
  • Figure 2B shows data for Compound 17.
  • Figure 2C shows data for Compound 43.
  • Figures 3A-B show the effects of Compounds 17 and 18 on human alphacoronavirus strain 229E (HCoV 229E).
  • Figure 3A shows data for Compound 17;
  • Figure 3B shows data for Compound 18.
  • Figures 4A-B show the effects of Compounds 17 and 18 on human betacoronavirus strain OC43 (HCoV OC43).
  • Figure 4A shows data for Compound 17;
  • Figure 4B shows data for Compound 18.
  • Figures 5A-B shows level of infection in hamster lung tissue after treatment with Compound 17.
  • Figure 5A shows the number of copies of viral RNA per mg of lung tissue after treatment of a hamster with Compound 17 at 20 and 50 mg/kg doses compared to the vehicle control.
  • Figure 5B shows the viral load per mg of lung tissue after treatment of a hamster with Compound 17 at 20 and 50 mg/kg doses compared to the vehicle control.
  • Figure 6 shows the improved cumulative lung scores in hamsters after treatment with Compound 17 at 20 and 50 mg/kg doses compared to the vehicle control.
  • Figures 7A-B show the activity of Compound 17 (Fig. 17A) and the remdesivir control (Fig.
  • the present disclosure provides methods and compositions for the treatment of coronavirus infections.
  • the present disclosure provides methods and compositions for blocking alpha-coronavirus, beta-coronavirus lineage B, or beta-coronavirus lineage A into a host cell with compounds that are phosphoinositide kinase inhibitors, in particular FYVE-type finger-containing phosphoinositide kinase (“PIKfyve”) inhibitors.
  • PIKfyve FYVE-type finger-containing phosphoinositide kinase
  • “about” or “approximately” can mean a range of up to 10% (i.e., ⁇ 10%) or more depending on the limitations of the measurement system. For example, about 5 mg can include any number between 4.5 mg and 5.5 mg. Furthermore, particularly with respect to biological systems or processes, the terms can mean up to an order of magnitude or up to 5-fold of a value. When particular values or compositions are provided in the instant disclosure, unless otherwise stated, the meaning of “about” or “approximately” should be assumed to be within an acceptable error range for that particular value or composition. “Or” is used in the inclusive sense, i.e., equivalent to “and/or,” unless the context requires otherwise.
  • the term “and/or” as used in a phrase such as “A, B, and/or C” is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).
  • the terms “or a combination thereof” and “or combinations thereof” as used herein refers to any and all permutations and combinations of the listed terms preceding the term.
  • A, B, C, or combinations thereof is intended to include at least one of: A, B, C, AB, AC, BC, or ABC, and if order is important in a particular context, also BA, CA, CB, ACB, CBA, BCA, BAC, or CAB.
  • the subject can be human, non-human primates, simian, ape, murine (e.g., mice and rats), bovine, porcine, equine, canine, feline, caprine, lupine, ranine or piscine.
  • administering refers to the physical introduction of an agent to a subject, using any of the various methods and delivery systems known to those skilled in the art.
  • Exemplary routes of administration for the formulations disclosed herein include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral routes of administration, for example by injection or infusion.
  • parenteral administration means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion, as well as in vivo electroporation.
  • the formulation is administered via a non-parenteral route, e.g., orally.
  • non-parenteral routes include a topical, epidermal or mucosal route of administration, for example, intranasally, vaginally, rectally, sublingually or topically.
  • Administering can also be performed, for example, once, a plurality of times, and/or over one or more extended periods.
  • the terms “treat,” “treating,” and “treatment,” as used herein, covers any administration or application of a therapeutic for disease/disorder in a subject, and includes inhibiting the disease/disorder, arresting its development, relieving one or more symptoms of the disease/disorder, or curing the disease/disorder.
  • prevent means inhibiting or arresting development of a disease/disorder in a subject deemed to be disease/disorder free.
  • block and “blocking” as used herein with reference to viral entry into a host cell refers to stopping the entry of some or all of a virus, such as a coronavirus, into a host cell or host cells. The blocking may be complete or partial. Partial blocking includes preventing at least some virus from entering host cells, for example, enough virus to prevent the subject from displaying at least one symptom associated with such viral infection.
  • a “pharmaceutically acceptable vehicle” for therapeutic purposes is a physical embodiment that can be administered to a subject.
  • Pharmaceutically acceptable vehicles include pills, capsules, caplets, tablets, oral fluids, injection fluids, sprays, aerosols, troches, dietary supplements, creams, lotions, oils, solutions, pastes, powders, steam, or a liquid, but is not limited to these.
  • a pharmaceutically acceptable vehicle is a buffered isotonic solution such as phosphate buffered saline (PBS).
  • PBS phosphate buffered saline
  • Alkyl means a linear saturated monovalent hydrocarbon radical of one to six carbon atoms or a branched saturated monovalent hydrocarbon radical of three to six carbon atoms, e.g., methyl, ethyl, propyl, 2-propyl, butyl (including all isomeric forms), pentyl (including all isomeric forms), and the like.
  • Alkylene means a linear saturated divalent hydrocarbon radical of one to six carbon atoms or a branched saturated divalent hydrocarbon radical of three to six carbon atoms unless otherwise stated e.g., methylene, ethylene, propylene, 1-methylpropylene, 2-methylpropylene, butylene, pentylene, and the like.
  • Alkylsulfonyl means a –SO 2 R radical where R is alkyl as defined above, e.g., methylsulfonyl, ethyl sulfonyl, and the like.
  • Amino means a -NH2.
  • Alkoxy means a -OR radical where R is alkyl as defined above, e.g., methoxy, ethoxy, propoxy, or 2-propoxy, n-, iso-, or tert-butoxy, and the like.
  • Alkoxyalkyl means a linear monovalent hydrocarbon radical of one to six carbon atoms or a branched monovalent hydrocarbon radical of three to six carbons substituted with an alkoxy group, (in one embodiment one or two alkoxy groups), as defined above, e.g., 2- methoxyethyl, 1-, 2-, or 3-methoxypropyl, 2-ethoxyethyl, and the like.
  • Alkoxycarbonyl means a -C(O)OR radical where R is alkyl as defined above, e.g., methoxycarbonyl, ethoxycarbonyl, and the like.
  • Acyl means a -COR radical where R is alkyl, haloalkyl, or cycloalkyl, e.g., acetyl, propionyl, cyclopropylcarbonyl, and the like. When R is alkyl, the radical is also referred to herein as alkylcarbonyl.
  • Cycloalkyl means a cyclic saturated monovalent hydrocarbon radical of three to ten carbon atoms wherein one or two carbon atoms may be replaced by an oxo group, e.g., cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl, and the like.
  • Carboxy means –COOH.
  • a “coronavirus,” “corona respiratory virus,” and “CoV” are used interchangeably h i f i b l i h f il C i id C i l d RNA viruses.
  • Coronaviruses infect a variety of host species, including humans and several other vertebrates. These viruses predominantly cause respiratory and intestinal tract infections and induce a wide range of clinical manifestations. In general, coronaviruses can be classified into low pathogenic CoVs (including human CoVs (hCoVs)) and highly pathogenic CoVs, such as severe acute respiratory syndrome CoV (SARS-CoV) and Middle East respiratory syndrome CoV (MERS-CoV). Low pathogenic hCoV infect upper airways and cause seasonal mild to moderate cold-like respiratory illnesses in healthy individuals.
  • hCoVs human CoVs
  • SARS-CoV severe acute respiratory syndrome CoV
  • MERS-CoV Middle East respiratory syndrome CoV
  • hCoV pathogenic hCoV
  • ALI acute lung injury
  • ARDS acute respiratory distress syndrome
  • Haloalkyl means alkyl radical as defined above, which is substituted with one or one to five halogen atoms (in one embodiment fluorine or chlorine,) including those substituted with different halogens, e.g., -CH2Cl, -CF3, -CHF2, -CH2CF3, -CF2CF3, -CF(CH3)2, and the like. When the alkyl is substituted with only fluoro, it can be referred to in this disclosure as fluoroalkyl.
  • Haloalkoxy means a –OR radical where R is haloalkyl as defined above e.g., -OCF3, -OCHF 2 , and the like.
  • R is haloalkyl where the alkyl is substituted with only fluoro, it can be referred to in this disclosure as fluoroalkoxy.
  • “Hydroxyalkyl” means a linear monovalent hydrocarbon radical of one to six carbon atoms or a branched monovalent hydrocarbon radical of three to six carbons substituted with one or two hydroxy groups, provided that if two hydroxy groups are present they are not both on the same carbon atom.
  • Representative examples include, but are not limited to, hydroxymethyl, 2- hydroxyethyl, 2-hydroxypropyl, 3-hydroxypropyl, 1-(hydroxymethyl)-2-methylpropyl, 2- hydroxybutyl, 3-hydroxybutyl, 4-hydroxybutyl, 2,3-dihydroxypropyl, 1-(hydroxymethyl)-2- hydroxyethyl, 2,3-dihydroxybutyl, 3,4-dihydroxybutyl and 2-(hydroxymethyl)-3-hydroxypropyl.
  • Further examples include, but are not limited to, 2-hydroxyethyl, 2,3-dihydroxypropyl, and 1- (hydroxymethyl)-2-hydroxyethyl.
  • Heterocyclyl means a saturated or unsaturated monovalent monocyclic or bi-cyclic group (fused bi-cyclic or bridged bi-cyclic or spiro compounds) of 4 to 10 ring atoms in which one or two ring atoms are heteroatom selected from N, O, and S(O) n , where n is an integer from 0 to 2, the remaining ring atoms being C. Additionally, one or two ring carbon atoms in the heterocyclyl ring can optionally be replaced by a –CO- group.
  • heterocyclylalkyl or “heterocycloalkyl” means a –(alkylene)-R radical where R is heterocyclyl ring as defined above e.g., tetraydrofuranylmethyl, piperazinylmethyl, morpholinylethyl, and the like.
  • Heterocycloamino means a saturated or unsaturated monovalent monocyclic group of 4 to 8 ring atoms in which one or two ring atoms are heteroatom selected from N, O, or S(O)n, where n is an integer from 0 to 2, the remaining ring atoms being C provided that at least one of the ring atoms is N. Additionally, one or two ring carbon atoms in the heterocycloamino ring can optionally be replaced by a –CO- group. When the heterocycloamino ring is unsaturated it can contain one or two ring double bonds provided that the ring is not aromatic.
  • Heterocycloaminoalkyl means a –(alkylene)-R radical where R is heterocycloamino as described above.
  • Heteroaryl means a monovalent monocyclic or bicyclic aromatic radical of 5 to 10 ring atoms where one or more, (in one embodiment one, two, or three), ring atoms are heteroatom selected from N, O, and S, the remaining ring atoms being carbon.
  • Representative examples include, but are not limited to, pyrrolyl, thienyl, thiazolyl, imidazolyl, furanyl, indolyl, isoindolyl, oxazolyl, isoxazolyl, benzothiazolyl, benzoxazolyl, quinolinyl, isoquinolinyl, pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl, triazolyl, tetrazolyl, and the like.
  • heterocyclyl group optionally substituted with an alkyl group means that the alkyl may but need not be present, and the description includes situations where the heterocyclyl group is substituted with an alkyl group and situations where the heterocyclyl group is not substituted with alkyl.
  • phrases “pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, i h bl b fi / i k i
  • pharmaceutically acceptable excipient or “pharmaceutically acceptable carrier” as used herein means a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material.
  • Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient.
  • materials which can serve as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and eth
  • PIKfyve inhibitor refers to a molecule that inhibits phosphatidylinositol 3-phosphate 5-kinase (PIKfyve).
  • salt or “pharmaceutically acceptable salt” refers to salts derived from a variety of organic and inorganic counter ions well known in the art. Pharmaceutically acceptable acid addition salts can be formed with inorganic acids and organic acids. Inorganic acids from which salts can be derived include, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like.
  • Organic acids from which salts can be derived include, for example, acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, and the like.
  • Pharmaceutically acceptable base addition salts can be formed with inorganic and organic bases.
  • Inorganic bases from which salts can be derived include, for example, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum, and the like.
  • Organic bases from which salts can be derived include, for example, primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, basic ion exchange resins, and the like, specifically such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, and ethanolamine.
  • I b di h h i ll bl b ddi i l i h f pharmaceutically acceptable salts are non-toxic.
  • compositions [0134] Provided herein are methods of treating an infection caused by a virus, inhibiting entry of a virus into a host cell, or inhibiting fusion of viral membrane with a host cell membrane comprising administering to a subject in need thereof a compound of Formula (I) or any of the embodiments thereof (e.g., compounds of Table 1) described herein.
  • the disclosure relates to a method of blocking alpha-coronavirus entry into a host cell and preventing an infection caused by alpha-coronavirus, comprising administering to a subject in need thereof a compound of Formula (I) or any of the embodiments thereof described herein.
  • the alpha-coronavirus is HCoV 229E.
  • the disclosure relates to a method of blocking beta-coronavirus lineage B entry into a host cell and preventing an infection caused by beta-coronavirus lineage B, comprising administering to a subject in need thereof a compound of Formula (I) or any of the embodiments thereof described herein.
  • the beta-coronavirus lineage B is SARS-CoV2.
  • the infection caused by SARS-CoV-2 is COVID-19.
  • the methods treat or prevent COVID-19 infection.
  • the disclosure relates to a method of blocking beta-coronavirus lineage A entry into a host cell and preventing an infection caused by beta-coronavirus lineage A, comprising administering to a subject in need thereof a compound of Formula (I) or any of the embodiments thereof described herein.
  • the beta-coronavirus lineage A is HCoV OC43.
  • a method of inhibiting coronavirus viral membrane fusion with an early endosomal membrane comprising administering a compound of Formula (I) or any of the embodiments thereof (e.g., compounds of Table 1) described herein.
  • a method of inhibiting coronavirus viral membrane fusion with a maturing endosomal membrane comprising administering a compound of Formula (I) or any of the embodiments thereof (e.g., compounds of Table 1) described herein.
  • a method of inhibiting coronavirus viral membrane fusion with a late endosomal membrane comprising administering a compound of Formula (I) or any of the embodiments thereof (e.g., compounds of Table 1) described herein.
  • a method of inhibiting coronavirus viral membrane fusion with an endo-lysosomal membrane is id d i i d i i i d f F l (I) f h b di inhibiting coronavirus viral membrane fusion with a lysosomal membrane is provided comprising administering a compound of Formula (I) or any of the embodiments thereof (e.g., compounds of Table 1) described herein.
  • a method of inhibiting coronavirus viral membrane fusion with an early macropinosomal membrane comprising administering a compound of Formula (I) or any of the embodiments thereof (e.g., compounds of Table 1) described herein.
  • a method of inhibiting coronavirus viral membrane fusion with a macropinosomal membrane comprising administering a compound of Formula (I) or any of the embodiments thereof (e.g., compounds of Table 1) described herein.
  • a method of inhibiting coronavirus viral membrane fusion with a late macropinosomal membrane comprising administering a compound of Formula (I) or any of the embodiments thereof (e.g., compounds of Table 1) described herein.
  • a method of inhibiting coronavirus viral membrane fusion with the endoplasmic reticulum (ER) comprising administering a compound of Formula (I) or any of the embodiments thereof (e.g., compounds of Table 1) described herein.
  • a method of inhibiting coronavirus viral membrane fusing with the plasma membrane directly comprising administering a compound of Formula (I) or any of the embodiments thereof (e.g., compounds of Table 1) described herein.
  • the coronavirus may be an alpha-coronavirus, a beta-coronavirus lineage B, or a beta-coronavirus lineage A.
  • the compounds of this disclosure will be administered in a therapeutically effective amount by any of the accepted modes of administration for agents that serve similar utilities.
  • compounds of this disclosure will be administered as pharmaceutical compositions by any one of the following routes: oral, systemic (e.g., transdermal, intranasal or by suppository), or parenteral (e.g., intramuscular, intravenous, or subcutaneous) administration.
  • the manner of administration is nasal using a convenient daily dosage regimen, which can be adjusted according to the degree of affliction.
  • compositions can take the form of tablets, pills, capsules, semisolids, powders, sustained release formulations, solutions, suspensions, elixirs, aerosols, or any other appropriate compositions.
  • Pharmaceutical compositions can be formulated using one or more pharmaceutically acceptable carriers comprising excipients and auxiliaries. The formulation can be modified depending upon the route of administration chosen.
  • the pharmaceutical compositions can also include the compounds described herein in a free base form or a pharmaceutically acceptable l f
  • Methods for formulation of the pharmaceutical compositions can include formulating any of the compounds described herein with one or more inert, pharmaceutically acceptable excipients or carriers to form a solid, semi-solid, or liquid composition.
  • Solid compositions can include, for example, powders, tablets, dispersible granules and capsules, and in some aspects, the solid compositions further contain nontoxic, auxiliary substances, for example wetting or emulsifying agents, pH buffering agents, and other pharmaceutically acceptable additives.
  • the compositions described herein can be lyophilized or in powder form for re- constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
  • the active ingredients can be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization (e.g., hydroxymethylcellulose or gelatin microcapsules and poly-(methylmethacylate) microcapsules, respectively), in colloidal drug- delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions.
  • the pharmaceutical compositions and formulations can be sterilized. Sterilization can be accomplished by filtration through sterile filtration.
  • the pharmaceutical compositions described herein can be formulated for administration as an injection.
  • Non-limiting examples of formulations for injection can include a sterile suspension, solution, or emulsion in oily or aqueous vehicles.
  • Suitable oily vehicles can include, but are not limited to, lipophilic solvents or vehicles such as fatty oils, synthetic fatty acid esters, or liposomes.
  • Aqueous injection suspensions can contain substances which increase the viscosity of the suspension.
  • the suspension can also contain suitable stabilizers.
  • Injections can be formulated for bolus injection or continuous infusion.
  • the compounds can be formulated in a unit dosage injectable form (e.g., solution, suspension, emulsion) in association with a pharmaceutically acceptable parenteral vehicle.
  • Such vehicles can be inherently nontoxic, and non-therapeutic.
  • a vehicle can be water, saline, Ringer’s solution, dextrose solution, and 5% human serum albumin.
  • Nonaqueous vehicles such as fixed oils and ethyl oleate can also be used.
  • Liposomes can be used as carriers.
  • the vehicle can contain minor amounts of additives such as substances that enhance isotonicity and chemical stability (e.g., buffers and preservatives).
  • Sustained-release preparations can also be prepared.
  • sustained-release matrices can include polyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides, copolymers of L-JOXWDPLF ⁇ DFLG ⁇ DQG ⁇ HWK ⁇ O-L-glutamate, non- degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPO TM (i i j bl i h d f l i id l li id [0147]
  • Pharmaceutical formulations of the compositions described herein can be prepared for storage by mixing a compound with a pharmaceutically acceptable carrier, excipient, and/or a stabilizer.
  • This formulation can be a lyophilized formulation or an aqueous solution.
  • Acceptable carriers, excipients, and/or stabilizers can be nontoxic to recipients at the dosages and concentrations used.
  • Acceptable carriers, excipients, and/or stabilizers can include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives, polypeptides; proteins, such as serum albumin or gelatin; hydrophilic polymers; amino acids; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes; and/or non- ionic surfactants or polyethylene glycol.
  • Compounds of the present disclosure may be used in methods of treating in combination with one or more other combination agents (e.g., one, two, or three other drugs) that are used in the prevention, treatment, control, amelioration, or reduction of risk of the diseases or conditions for which compounds of the present disclosure are useful.
  • the combination of the drugs together is safer or more effective than either drug alone.
  • the compounds disclosed herein and the one or more combination agents have complementary activities that do not adversely affect each other. Such molecules can be present in combination in amounts that are effective for the purpose intended.
  • Such other drug(s) may be administered, by a route and in an amount commonly used therefore, contemporaneously or sequentially with a compound of the present disclosure.
  • the agents are administered together in a single pharmaceutical composition in unit dosage form.
  • the pharmaceutical compositions of the present disclosure also include those that contain one or more other active ingredients, in addition to a compound of the present disclosure.
  • the weight ratio of the compound of the present disclosure to the second active agent may be varied and will depend upon the effective dose of each ingredient. Generally, an effective dose of each will be used.
  • combination therapy includes therapies in which the compound of the present disclosure and one or more other drugs are administered separately, and in some cases, the two or more agents are administered on different, overlapping schedules.
  • the compounds of the present disclosure and the other active ingredients may be used in lower doses than when each is used singly.
  • Th d h i l i i d h d f h di l mammal a human, a non-human mammal, a domesticated animal (e.g., laboratory animals, household pets, or livestock), a non-domesticated animal (e.g., wildlife), a dog, a cat, a rodent, a mouse, a hamster, a cow, a bird, a chicken, a fish, a pig, a horse, a goat, a sheep, or a rabbit.
  • compounds, pharmaceutical compositions, and methods of the present disclosure are used for treating a human.
  • the compounds, pharmaceutical compositions, and methods described herein can be useful as a therapeutic or preventative, for example a treatment or preventative that can be administered to a subject in need thereof.
  • a therapeutic or preventative effect can be obtained in a subject by prevention (complete or partial), reduction, suppression, remission, or eradication of a disease state, including, but not limited to, a symptom thereof.
  • a therapeutic effect in a subject having a disease or condition, or pre-disposed to have or is beginning to have the disease or condition can be obtained by a reduction, a suppression, a prevention, a remission, or an eradication of the condition or disease, or pre-condition or pre-disease state.
  • therapeutically effective amounts of the compounds or pharmaceutical compositions described herein can be administered to a subject in need thereof, often for treating and/or preventing a condition or progression thereof.
  • a pharmaceutical composition can affect the physiology of the subject, such as the immune system, inflammatory response, or other physiologic affect.
  • Treat and/or treating can refer to any indicia of success in the treatment or amelioration of the disease or condition. Treating can include, for example, reducing, delaying or alleviating the severity of one or more symptoms of the disease or condition, or it can include reducing the frequency with which symptoms of a disease, defect, disorder, or adverse condition, and the like, are experienced by a patient. Treat can be used herein to refer to a method that results in some level of treatment or amelioration of the disease or condition and can contemplate a range of results directed to that end, including but not restricted to prevention of the condition entirely.
  • Prevent, preventing, and the like can refer to the prevention of the disease or condition in the patient. For example, if an individual at risk of contracting a disease is treated with the methods of the present disclosure and does not later contract the disease, then the disease has been prevented, at least over a period of time, in that individual.
  • the PIKfyve inhibitors described herein can prevent coronavirus infection. In some embodiments the PIKfyve inhibitors described herein can treat a coronavirus infection.
  • a h i ll ff i b h f d effect or to otherwise reduce a detrimental non-beneficial event to the individual to whom the composition is administered.
  • a therapeutically effective dose can be a dose that produces one or more desired or desirable (e.g., beneficial) effects for which it is administered, such administration occurring one or more times over a given period of time.
  • An exact dose can depend on the purpose of the treatment and can be ascertainable by one skilled in the art using known techniques.
  • the compounds or pharmaceutical compositions described herein that can be used in the methods and uses described herein can be formulated and dosages established in a fashion consistent with good medical practice taking into account the disorder to be treated, the condition of the individual patient, the site of delivery of the compound or pharmaceutical composition, the method of administration and other factors known to practitioners.
  • the compounds or pharmaceutical compositions can be prepared according to the description of preparation described herein.
  • administration of the compounds or pharmaceutical compositions can include routes of administration, non-limiting examples of administration routes include intravenous, intraarterial, subcutaneous, subdural, intramuscular, intracranial, intrasternal, intratumoral, or intraperitoneally.
  • a pharmaceutical composition or compound can be administered to a subject by additional routes of administration, for example, by inhalation, oral, dermal, intranasal, or intrathecal administration.
  • Pharmaceutical compositions or compounds of the present disclosure can be administered to a subject in need thereof in a first administration, and in one or more additional administrations. The one or more additional administrations can be administered to the subject in need thereof minutes, hours, days, weeks, or months following the first administration.
  • any one of the additional administrations can be administered to the subject in need thereof less than 21 days, or less than 14 days, less than 10 days, less than 7 days, less than 4 days or less than 1 day after the first administration.
  • the one or more administrations can occur more than once per d h k h h Th d h i l compositions can be administered to the subject in need thereof in cycles of 21 days, 14 days, 10 days, 7 days, 4 days, or daily over a period of one to seven days.
  • the compounds, pharmaceutical compositions, and methods provided herein can be useful for the treatment of a plurality of diseases or conditions or preventing a disease or a condition in a subject, or other therapeutic applications for subjects in need thereof. III.
  • the PIKfyve inhibitors described herein can be administered to treat and/or prevent certain coronavirus infections.
  • the coronaviruses belong to the order Nidovirales, family Coronaviridae, and the subfamily Coronavirinae. They are genetically categorized into four genera: the Alphacoronavirus, Betacoronavirus, Gammacoronavirus, and Deltacoronavirus. Mahendra et al., Cureus, 12(3):e7423 (2020).
  • Alphacoronaviruses and the Betacoronaviruses typically infect mammals, whereas the Gammacoronaviruses and the Deltacoronaviruses predominantly infect birds.
  • the coronavirus is an Alphacoronavirus, Betacoronavirus, Gammacoronavirus, or Deltacoronavirus.
  • Alphacoronavirus strains that are associated with human disease include HCoV-229E and HCoV-NL63.
  • alphacoronavirus strains include feline infectious peritonitis virus (FIPV), canine coronavirus (CCoV), porcine respiratory coronavirus (PRCV), porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), Rhinolophus bat coronavirus (Rh-BatCoV) HKU2, Miniopterus bat coronavirus (Mi-BatCoV) HKU8, Mi-BatCoV 1A, Mi-BatCoV 1B, Rousettus bat coronavirus (Ro-BatCoV) HKU10183A, Hipposideros bat coronavirus (Hi-BatCoV) HKU10 LSH5A, and Scotiphilus bat coronavirus (Sc-BatCoV) 512.
  • FIPV feline infectious peritonitis virus
  • CoV canine coronavirus
  • PRCV porcine respiratory coronavirus
  • PEDV porcine epidemic diarrhea virus
  • TGEV transmissible gastroenteriti
  • the coronavirus is HCoV-229E or HCoV-NL63.
  • the coronavirus is FIPV, CCoV, PRCV, PEDV, TGEV, Rh-BatCoV HKU2, Mi-BatCoV HKU8, Mi-BatCoV 1A, Mi-BatCoV 1B, Ro-BatCoV HKU10183A, Hi- BatCoV HKU10 LSH5A, and Sc-BatCoV 512.
  • Betacoronaviruses are divided into four subgroups: Embecovirus (lineage A), Sarbecovirus (lineage B), Merbecovirus (lineage C), and Nobecovirus (lineage D).
  • Embecovirus (lineage A) include bovine coronavirus (BCoV), human coronavirus OC43 (HCoV-OC43), HCoV-HKU1, porcine hemagglutinating encephalomyelitis virus (PHEV), giraffe coronavirus (GiCoV), and murine hepatitis virus (MHV).
  • Sarbecovirus examples include SARS-CoV-1 and SARS Rh B C V HKU3 Id E l f M b i (li C) i l d MERS C V HKU5. Id.
  • Nobecovirus examples include Ro-BatCoV HKU9. Id. Betacoronavirus strains that are associated with human disease include SARS-CoV-1, HCoV- OC43, HCoV-HKU1, and MERS-CoV.
  • the coronavirus causes upper respiratory tract disease, lower respiratory tract disease, fever, sore throat, swollen adenoids, colds with minor or major symptoms, malaise, muscle and joint pains, nausea, vomiting, loss of appetite, pneumonia, secondary bacterial infection, bronchitis, dyspnea, diarrhea, shortness of breath, acute respiratory distress syndrome, cytokine storm, multi-organ failure, septic shock, blood clots, loss of smell, or loss of taste.
  • the coronavirus causes no symptoms at all.
  • the disclosed compounds are administered to block an alpha- coronavirus entry into a host cell.
  • the disclosed compounds are administered to block a beta-coronavirus lineage B entry into a host cell. In some embodiments, the disclosed compounds are administered to block a beta-coronavirus lineage A entry into a host cell. In some embodiments, the disclosed compounds are administered to prevent an alpha- coronavirus infection in a subject. In some embodiments, the disclosed compounds are administered to prevent a beta-coronavirus lineage B infection in a subject. In some embodiments, the disclosed compounds are administered to prevent a beta-coronavirus lineage A infection in a subject. In some embodiments, the disclosed compounds are administered to block an alpha-coronavirus entry into a host cell and prevent an alpha-coronavirus infection in a subject.
  • the disclosed compounds are administered to block an beta- coronavirus lineage B entry into a host cell prevent a beta-coronavirus lineage B infection in a subject. In some embodiments, the disclosed compounds are administered to block a beta- coronavirus lineage A entry into a host cell prevent a beta-coronavirus lineage A infection in a subject.
  • the alpha-coronavirus is HCoV 229E.
  • the beta-coronavirus lineage B is SARS-CoV2. .
  • the beta-coronavirus lineage A is HCoV OC43.
  • the infection is caused by SARS-CoV-2, and the infection is COVID-19. IV.
  • PIKfyve inhibitor compounds [0166]
  • the compounds described herein may in some cases exist as diastereomers, enantiomers, or other stereoisomeric forms. All chiral, diastereomeric, racemic forms, as individual forms and mixtures thereof, are within the scope of this disclosure, unless the specific stereochemistry or isomeric form is specifically indicated.
  • Compounds of the present disclosure containing an asymmetrically substituted atom may be isolated in optically active, optically i h d i ll i f I i ll k i h h i ll by chromatography or by forming diastereomers and separating by recrystallization, or chromatography, or any combination thereof.
  • Stereoisomers may also be obtained by stereoselective synthesis.
  • Certain compounds of Formula (I) (or any of the embodiments thereof described herein) and/or a pharmaceutically acceptable salt thereof may exist as tautomers and/or geometric isomers. All possible tautomers and cis and trans isomers, as individual forms and mixtures thereof, are within the scope of this disclosure.
  • pyrazole tautomers as shown below are equivalent structures. The depiction of one such structure is intended to encompass both structures.
  • alkyl includes all the possible isomeric forms of said alkyl group albeit only a few examples are set forth. Furthermore, when the cyclic groups such as heteroaryl, heterocyclyl are substituted, they include all the positional isomers.
  • Pharmaceutically acceptable salts of the compounds of Formula (I) are within the scope of this disclosure. In addition, the compounds described herein include hydrates and solvates of the compounds or pharmaceutically acceptable salts thereof.
  • the present disclosure also includes the prodrugs of compounds of Formula (I) (or any of the embodiments thereof described herein) and/or a pharmaceutically acceptable salt thereof.
  • prodrug is intended to represent covalently bonded carriers, which are capable of releasing the active ingredient of Formula (I) (or any of the embodiments thereof described herein) when the prodrug is administered to a mammalian subject. Release of the active ingredient occurs in vivo.
  • Prodrugs can be prepared by techniques known to one skilled in the art. These techniques generally modify appropriate functional groups in a given compound. These modified functional groups however regenerate original functional groups in vivo or by routine manipulation.
  • Prodrugs of compounds of Formula (I) (or any of the embodiments thereof described herein) include compounds wherein a hydroxy, amino, carboxylic, or a similar group is modified.
  • prodrugs include, but are not limited to esters (e.g., acetate, formate, and benzoate derivatives), carbamates (e.g., N,N-dimethylaminocarbonyl) of hydroxy or amino functional groups in compounds of Formula (I)), amides (e.g., trifluoroacetylamino, embodiments thereof described herein) and/or a pharmaceutically acceptable salt thereof are also within the scope of this disclosure.
  • the present disclosure also includes polymorphic forms (amorphous as well as crystalline) and deuterated forms of compounds of Formula (I) (or any of the embodiments thereof described herein) and/or a pharmaceutically acceptable salt thereof.
  • the compounds disclosed herein are used in different enriched isotopic forms, e.g., enriched in the content of 2 H, 3 H, 11 C, 13 C and/or 14 C.
  • the compound is deuterated in at least one position.
  • deuterated forms can be made by the procedure described in U.S. Patent Nos. 5,846,514 and 6,334,997.
  • deuteration can improve the metabolic stability and or efficacy, thus increasing the duration of action of drugs.
  • structures depicted herein are intended to include compounds which differ only in the presence of one or more isotopically enriched atoms.
  • compounds having the present structures except for the replacement of a hydrogen by a deuterium or tritium, or the replacement of a carbon by 13 C- or 14 C-enriched carbon are within the scope of the present disclosure.
  • the compounds of the present disclosure optionally contain unnatural proportions of atomic isotopes at one or more atoms that constitute such compounds.
  • the compounds may be labeled with isotopes, such as for example, deuterium ( 2 H), tritium ( 3 H), iodine-125 ( 125 I) or carbon-14 ( 14 C).
  • X is N or CH
  • Y is N or CR a provided that when X is N, then Y is CR a , when Y is N, then X is CH
  • X is CH and Y is N. In some embodiments, X is N and Y is CR a . In some embodiments, R a is H, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, or tert-butyl. In some embodiments, R a is H or methyl. In some embodiments, R a is H. [0180] In some embodiments, R a or R 1 is optionally substituted phenyl. In some embodiments, R a or R 1 is tolyl. In some embodiments, R a or R 1 is m-tolyl.
  • R a or R 1 is optionally substituted monocyclic heteroaryl. In some embodiments, R a or R 1 is optionally substituted pyrrole, imidazole, pyrazole, triazole, tetrazole, furan, oxazole, isoxazole, thiazole, isothiazole, pyridine, pyrimidine, pyrazine, or pyridazine. In some embodiments, R a or R 1 is optionally substituted pyridine or pyrimidine.
  • each R d substituent is independently C 1- 4alkyl, halo-C1-4alkyl, phenyl, -C1-4alkyl-phenyl, pyridyl, thiophenyl, cycloalkyl, or -C1-4alkyl- cycloalkyl, wherein the phenyl, pyridyl, and thiophenyl are each optionally substituted with one or two substituents R e .
  • each R d substituent is independently methyl, ethyl, isopropyl, -CF3, -OCH3, -OCF3, phenyl, pyridyl, thiophenyl, benzyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclopropylmethyl, cyclobutylmethyl, or cyclopentylmethyl, wherein the phenyl, cycloalkyl, and heteroaryl of R d are each optionally substituted with one or two substituents R e .
  • each R d is independently selected from C1-4alkyl, -CF3, fluoro, chloro, -OCH3, and -OCF3.
  • R a or R 1 is substituted with one R d and the R d is C 1-4 alkyl.
  • R a or R 1 is substituted with one R d and the R d is methyl.
  • R a or R 1 is phenyl or pyridyl, each optionally substituted with one or two substituents selected from C1-4alkyl, -CF3, fluoro, chloro, -OCH3, and -OCF3.
  • R a or R 1 is unsubstituted phenyl or tolyl. In some embodiments, R a or R 1 is unsubstituted phenyl or m-tolyl. In some embodiments, R a or R 1 is unsubstituted pyridyl. In some embodiments, R a or R 1 is 4-pyridyl. [0182] In some embodiments, each R e substituent is independently C 1-4 alkyl, halo, halo-C 1- 4 alkyl, -O-C 1-4 alkyl, or -O-C 1-4 -haloalkyl.
  • each R e substituent is independently methyl, -CF3, fluoro, chloro, -OCH3, or -OCF3.
  • each R d substituent is independently methyl, ethyl, isopropyl, -CF 3 , phenyl, pyridyl, thiophenyl, benzyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclopropylmethyl, cyclobutylmethyl, or cyclopentylmethyl, wherein each R e is independently methyl, -CF3, fluoro, chloro, -OCH3, or -OCF3.
  • R g and R h are each independently H or methyl.
  • R 2 and R 3 taken together with the nitrogen to which they are attached form pyrrolidine, piperidine, piperazine, morpholine, thiomorpholine, orhi h li 11 di id h i ll b i d i h h R j b i
  • R 2 and R 3 taken together with the nitrogen to which they are attached form morpholine, optionally substituted with one or two R j substituents.
  • each R j substituent is independently methyl, hydroxy, -OCH3, halo, -CF3, or -OCF3.
  • R k and R l are each independently H or methyl.
  • R 4 is optionally substituted phenyl. In some embodiments, R 4 is optionally substituted heteroaryl. In some embodiments, R 4 is optionally substituted monocyclic heteroaryl.
  • R 4 is optionally substituted pyrrole, imidazole, pyrazole, triazole, tetrazole, furan, oxazole, isoxazole, thiazole, isothiazole, pyridine, pyrimidine, pyrazine, or pyridazine.
  • R 4 is optionally substituted pyrazole, pyridine or pyrimidine.
  • R 4 is optionally substituted pyridine.
  • R 4 is pyridine.
  • R 4 is 4-pyridyl.
  • R 4 is optionally substituted with one or two R z substituents.
  • R 4 is phenyl or pyridyl, each optionally substituted with one or two substituents selected from C 1-4 alkyl, -CF 3 , fluoro, chloro, -OCH 3 , and -OCF 3 .
  • R 4 is -C(O)NR x R y .
  • R x is H.
  • R x is methyl or ethyl, optionally substituted with one, two, or three R o substituents.
  • R x is methyl.
  • R y is H.
  • R y is C1-4alkyl, -C1-4alkyl(monocyclic cycloalkyl), monocyclic cycloalkyl, monocyclic heterocycloalkyl, monocyclic heterocyclyl, -O-monocyclic heterocyclyl, -O-C1- 4 alkyl, -SO 2 -C 1-4 alkyl, optionally substituted with one, two, or three R o substituents. In some embodiments, R y is C1-4alkyl, optionally substituted with one, two, or three R o substituents.
  • R y is methyl, ethyl, propyl, or isopropyl, each optionally substituted with one, two, or three R o substituents.
  • R y is methyl, ethyl, isopropyl, methoxyethyl, dimethoxypropanyl, (dimethylamino)ethyl, or (dimethylamino)butyl.
  • R y is methoxy.
  • R y is -SO2-methyl.
  • R y is monocyclic cycloalkyl or -C 1-2 alkyl(monocyclic cycloalkyl), each optionally substituted with one, two, or three R o substituents. In some embodiments, R y is monocyclic cycloalkyl, optionally substituted with one, two, or three R o substituents. In some embodiments, R y is cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl, each optionally substituted with one, two, or three R o substituents. In some embodiments, R y is cyclopropyl. In some embodiments, R y is cyclopentyl.
  • R y is cyclopropyl, cyclobutyl, cyclopentyl, cyclopropylmethyl, 1-cyclopropylethyl, 2-cyclopropylethyl, l b l h l l l h l I b di R y i li h l l
  • R y is optionally substituted tetrahydrofuranyl, tetrahydropyranyl, oxetanyl, azetidinyl, pyrrolidinyl, piperidinyl, morpholinyl, piperazinyl, or oxetanyloxy.
  • R y is oxetanyl, or oxetanyloxy.
  • R y is monocyclic heterocycloalkyl, optionally substituted with one, two, or three R o substituents.
  • R y is optionally substituted oxetanylmethyl, or (3- (hydroxymethyl)oxetan-3-yl)methyl.
  • R x is methyl and R y is methyl, ethyl, cyclopropyl, methoxy, or cyclopentyl.
  • R x and R y taken together with the nitrogen to which they are attached form a monocyclic heterocycloalkyl, optionally substituted with C 1-4 alkyl. In some embodiments, R x and R y taken together with the nitrogen to which they are attached form a monocyclic heterocyclyl, optionally substituted with C 1-4 alkyl.
  • R x and R y are taken together with the nitrogen to which they are attached to form azetidinyl, pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl 6-oxa-1-azaspiro[3.3]heptanyl, or 2-oxa- 6-azaspiro[3.3]heptanyl, each optionally substituted with methyl.
  • each R z is independently C 1-4 alkyl, halo, -OH, -OC 1-4 alkyl, C1-4alkylNR m R n or -NR m R n , wherein each alkyl is optionally substituted with -NR m R n .
  • each R z is independently methyl, -OH, halo, or -OCH3.
  • R z is C 2-3 alkyl substituted with -NR m R n .
  • R m and R n are each independently H or C1-4alkyl.
  • R m and R n are each methyl.
  • R m and R n taken together with the nitrogen to which they are attached form a monocyclic heterocycloalkyl, optionally substituted with one or two R o substituents.
  • R m and R n taken together with the nitrogen to which they are attached form pyrrolidine, piperidine, piperazine, morpholine, thiomorpholine, or thiomorpholine-1,1-dioxide, each optionally substituted with one or two R o substituents.
  • R m and R n taken together with the nitrogen to which they are attached form pyrrolidine, piperidine, piperazine, or morpholine, each optionally substituted with one or two R o substituents.
  • each R o substituent is C 1-4 alkyl. In some embodiments, each R o substituent is methyl. In some embodiments, each R o substituent is -OH. In some embodiments, each R o substituent is -NR p R q . In some embodiments, R p and R q are each independently H or methyl. [0195] In some embodiments, R p and R q taken together with the nitrogen to which they are attached form a heterocyclyl.
  • R p and R q taken together with the nitrogen hi h h h d f idi l lidi l i idi l i i l h li l [0196]
  • R 5 is H, methyl, ethyl, chloro, bromo, fluoro, -OH, or -OCH3. In some embodiments, R 5 is H.
  • the compound of Formula (I) or the pharmaceutically acceptable salt thereof is a compound of Formula (II): wherein R 1a is phenyl or pyridyl, each optionally substituted with one or two substituents selected from C1-4alkyl, CO2R p , -C(O)NR p R q , fluoro, chloro, bromo, NH2, and -OCH3, ; and R 4a is -C(O)NR x R y , or is a phenyl or pyridyl, each optionally substituted with one or two substituents selected from C 1-4 alkyl, -CF 3 , fluoro, chloro, -OCH 3 , and -OCF 3 ; wherein R x is H or C1-4alkyl and R y is H, C1-4alkyl, -O-C1-4alkyl, -SO2-C1-4alkyl, C1-4alkyl- SO2- R
  • R 1a is phenyl or pyridyl, each optionally substituted with methyl or -CF3. In some embodiments, R 1a is phenyl or m-tolyl. In some embodiments, R 1a is pyridyl. In some embodiments, R 1a is 4-pyridyl. In some embodiments, R 4a is phenyl or pyridyl, each optionally substituted with methyl or -CF3. In some embodiments, R 4a is phenyl or m-tolyl. In some embodiments, R 4a is pyridyl. In some embodiments, R 4a is 4-pyridyl.
  • one or more hydrogen atoms attached to carbon atoms of R d , R e R g , R h , R j , R k , R l , R m , R n , R o , R p , R q , R r , R x , R y , or R z are replaced by deuterium atoms.
  • one or more R d , R e R g , R h , R j , R k , R l , R m , R n , R o , R p , R q , R r , R x , R y , or R z group is a C1-4alkyl group wherein one or more hydrogen atoms attached to carbon atoms are replaced by deuterium atoms.
  • one or more R d , R e R g , R h , R j , R k , R l , R m , R n , R o , R p , R q , R r , R x , R y , or R z group is a methyl group wherein one or more hydrogen atoms attached to the carbon atom are replaced by deuterium atoms.
  • one or more R d , R e R g , R h , R j , R k , R l , R m , R n , R o , R p , R q , R r , R x , R y , or R z group is -CD3.
  • the compound of Formula (I) or Formula (II) comprises a -D in place of at least one -H, or a -CD 3 substituent in place of at least one CH 3 .
  • kits for the treatment of a coronavirus infection are SARS-CoV-1, SARS-CoV-2, MERS-CoV, HCoV-OC43, HCoV-HKU1, HCoV-229E, or HCoV-NL63.
  • the kit may contain a compound of Formula I (or any of the embodiments thereof described herein), a pharmaceutically acceptable carrier, a physiologically acceptable carrier, instructions for use, a container, a vessel for administration, or any combination thereof.
  • the disclosure further provides any compounds disclosed herein for use in a method of disclosed herein, such as inhibiting, reducing, or reducing progression of the diseases disclosed herein.
  • the disclosure further provides any compound disclosed herein for prevention or treatment of any condition disclosed herein.
  • the disclosure also provides any compound or pharmaceutical composition thereof disclosed herein for obtaining any clinical outcome disclosed herein for any condition disclosed herein.
  • the disclosure also provides use of any compound disclosed herein in the manufacture of a medicament for preventing or treating any disease or condition disclosed herein.
  • Example 1 7-morpholino-2-(pyridin-4-yl)-N-(3-(p-tolyl)-1H-pyrazol-5- yl)pyrazolo[1,5-a]pyrimidin-5-amine, hydrogen chloride salt
  • Step 1 Preparation of 3-(4-fluorophenyl)-N,N-dimethyl-5- ((7-morpholino-2- (pyridin-4-yl)pyrazolo[1,5-a]pyrimidin-5-yl)amino)-1H-pyrazole-1- sulfonamide
  • Step 2 Preparation of 7-morpholino-2-(pyridin-4-yl)-N-(3-(p-tolyl)-1H-pyrazol-5- yl)pyrazolo[1,5-a]pyrimidin-5-amine, hydrogen chloride salt
  • HCl/Et2O 1 mL
  • Example 2 7-morpholino-N-(5-phenyl-1H-pyrazol-3-yl)-2-(4- pyridyl)pyrazolo[1,5-a]pyrimidin-5-amine
  • Compound 2 was prepared from Intermediate A and a protected pyrazole according to the same two-step procedure used for Example 1.
  • the protected pyrazole was prepared from 3- phenyl-1H-pyrazol-5-amine according to the procedure used to prepare Intermediate B.
  • Example 3 7-morpholino-N-[5-(o-tolyl)-1H-pyrazol-3-yl]-2-(4- pyridyl)pyrazolo[1,5-a]pyrimidin-5-amine
  • Compound 3 was prepared from Intermediate A and a protected pyrazole according to the same two-step procedure used for Example 1.
  • the protected pyrazole was prepared from 3- (2-methylphenyl)-1H-pyrazol-5-amine according to the procedure used to prepare Intermediate B.
  • Example 4 7-morpholino-N-[3-(m-tolyl)-1H-pyrazol-5-yl]-2-(4- pyridyl)pyrazolo[1,5-a]pyrimidin-5-amine, hydrogen chloride salt
  • Compound 4 was prepared from Intermediate A and a protected pyrazole according to the same two-step procedure used for Example 1.
  • the protected pyrazole was prepared from 3- (3-methylphenyl)-1H-pyrazol-5-amine according to the procedure used to prepare Intermediate B.
  • Example 5 N-(5-methyl-1H-pyrazol-3-yl)-7-morpholino-2-(4- pyridyl)pyrazolo[1,5-a]pyrimidin-5-amine [0237]
  • Compound 5 was prepared from Intermediate A and a protected pyrazole according to the same two-step procedure used for Example 1.
  • the protected pyrazole was prepared from 3- (3-methyl)-1H-pyrazol-5-amine according to the procedure used to prepare Intermediate B.
  • Example 6 7-morpholino-2-(4-pyridyl)-N-[5-(4-pyridyl)-1H-pyrazol-3- yl]pyrazolo[1,5-a]pyrimidin-5-amine [0239]
  • Compound 6 was prepared from Intermediate A and a protected pyrazole according to the same two-step procedure used for Example 1.
  • the protected pyrazole was prepared from 3- (pyrid-4-yl)-1H-pyrazol-5-amine according to the procedure used to prepare Intermediate B.
  • Example 7 1-(7-morpholino-2-(pyridin-4-yl)pyrazolo[1,5-a]pyrimidin-5-yl)-3- phenyl-1H-pyrazol-5-amine
  • reaction mixture was concentrated directly and purified by silica gel column chromatography by eluting with a gradient of 2% MeOH/DCM to 3% MeOH/DCM to provide 1-(7-morpholino-2-(pyridin-4-yl)pyrazolo[1,5-a]pyrimidin-5-yl)-3-phenyl-1H-pyrazol-5-amine (Compound 7, 14.2 mg, 0.03 mmol) as a white solid; LC-MS (ESI+): m/z 439 (MH + ).
  • Example 8 1-(7-morpholino-2-(pyridin-4-yl)pyrazolo[1,5-a]pyrimidin-5-yl)-3-(o- tolyl)-1H-pyrazol-5-amine [0243]
  • Compound 8 was prepared from Intermediate A and 3-(2-methylphenyl)-1H-pyrazol-5- amine according to the procedure used for Example 7; LC-MS (ESI+): m/z 453 (MH + ).
  • Example 9 4-[5-(4-phenylpyrazol-1-yl)-2-(4-pyridyl)pyrazolo[1,5-a]pyrimidin-7- yl]morpholine [0245] To a solution of 4-phenyl-1H-pyrazole (29.4 mg, 0.20 mmol) in DMF (5 mL) at 0 °C was added NaH (14 mg, 0.34 mmol). The mixture was stirred for 30 minutes. Then to the mixture was added 4-(5-chloro-2-(pyridin-4-yl)pyrazolo[1,5-a]pyrimidin-7-yl)morpholine (Intermediate A, 62 mg, 0.19 mmol).
  • the reaction mixture was heated to 80 °C and stirred overnight. The progress of the reaction was monitored by TLC.
  • the reaction mixture was quenched with water (10 mL).
  • the aqueous solution was extracted with ethyl acetate (3 x 10 mL).
  • the combined organic phase was dried over anhydrous Na2SO4, filtrated and concentrated under reduce pressure.
  • Step 1 Synthesis of tert-butyl 4-(3-methylphenyl)-1H-pyrazole-1-carboxylate
  • Step 2 Synthesis of 4-(3-methylphenyl)-1H-pyrazole hydrochloride, Intermediate C [0250] To a solution of tert-butyl 4-(3-methylphenyl)-1H-pyrazole-1-carboxylate (117 mg, 0.45 mmol) in DCM was added HCl/Et 2 O (1 mL). The reaction was stirred at ambient temperature overnight. A large amount of solid was precipitated.
  • Example 10 4-[5-[4-(m-tolyl)pyrazol-1-yl]-2-(4-pyridyl)pyrazolo[1,5-a]pyrimidin- 7-yl]morpholine [0252] T l ti f 4 (3 th l h l) 1H l h d hl id (I t di t C 40 was stirred for 30 min. To this mixture was added 4-(5-chloro-2-(pyridin-4-yl)pyrazolo[1,5- a]pyrimidin-7-yl)morpholine (Intermediate A, 65 mg, 0.20 mmol). The resulting reaction mixture was heated to 80 °C overnight.
  • reaction was confirmed complete by TLC.
  • the reaction mixture was quenched with water (10 mL) and the aqueous solution was extracted with ethyl acetate (3 x 10 mL). The combined organic phase was dried over anhydrous Na 2 SO 4 , filtrated and concentrated under reduce pressure.
  • Example 11 4-[2-(4-pyridyl)-5-[4-(4-pyridyl)pyrazol-1-yl]pyrazolo[1,5- a]pyrimidin-7-yl]morpholine
  • Compound 11 was prepared by reaction of Intermediate A with 4-(4-pyridyl)-1H- pyrazole according to the conditions used for Examples 9 and 10.
  • Example 12 4-[5-(4-methylpyrazol-1-yl)-2-(4-pyridyl)pyrazolo[1,5-a]pyrimidin-7- [0256]
  • Compound 12 was prepared by reaction of Intermediate A with 4-methylpyrazole according to the conditions used for Examples 9 and 10.
  • Example 13 4-[5-(3-phenylpyrazol-1-yl)-2-(4-pyridyl)pyrazolo[1,5-a]pyrimidin-7- yl]morpholine [0258] To a solution of 3-phenylpyrazole (29.4 mg, 0.20 mmol) in DMF (5 mL) at 0 °C was added NaH (14 mg, 0.34 mmol). The mixture was stirred for 30 minutes. To the mixture was added 4-(5-chloro-2-(pyridin-4-yl)pyrazolo[1,5-a]pyrimidin-7-yl)morpholine (Intermediate A, 62 mg, 0.19 mmol).
  • the reaction mixture was heated to 80 °C and stirred overnight. The progress of the reaction was monitored by TLC.
  • the reaction mixture was quenched with water (10 mL).
  • the aqueous solution was extracted with ethyl acetate (3 x 10 mL).
  • the combined organic phase was dried over anhydrous Na 2 SO 4 , filtrated and concentrated under reduce pressure.
  • Example 14 4-[5-[3-(m-tolyl)pyrazol-1-yl]-2-(4-pyridyl)pyrazolo[1,5-a]pyrimidin- 7-yl]morpholine 4-[5-[3-(3-methylphenyl)pyrazol-1-yl]-2-(4-pyridyl)pyrazolo[1,5- a]pyrimidin-7-yl]morpholine [0260] Compound 14 was prepared by reaction of Intermediate A with 3-(3- methylphenyl)pyrazole according to the conditions used for Example 13.
  • Example 15 4-[2-(4-pyridyl)-5-[3-(4-pyridyl)pyrazol-1-yl]pyrazolo[1,5- a]pyrimidin-7-yl]morpholine [0262]
  • Compound 15 was prepared by reaction of Intermediate A with 3-(4-pyridyl)pyrazole according to the conditions used for Example 13.
  • Example 16 4-[5-(3-methylpyrazol-1-yl)-2-(4-pyridyl)pyrazolo[1,5-a]pyrimidin-7- yl]morpholine [0264] Compound 16 was prepared by reaction of Intermediate A with 3-(4-pyridyl)pyrazole according to the conditions used for Examples 13.
  • D.4 Synthesis of 5,7-dichloro-2-(pyridin-2-yl)pyrazolo[1,5-a]pyrimidine
  • a solution of crude 2-(pyridin-2-yl)pyrazolo[1,5-a]pyrimidine-5,7(4H,6H)-dione (3.89 g, 17.1 mmol) in phenylphosphonic dichloride (20 mL) was heated to 110 °C overnight. The reaction mixture was quenched with saturated NaHCO3 solution and made basic to pH 8. The aqueous solution was extracted with DCM/MeOH (15:1, 6 x 50 mL). The combined organic phase was dried over anhydrous Na2SO4, filtered and concentrated under reduce pressure.
  • Intermediate E 4-(5-chloro-2-(pyridin-3-yl)pyrazolo[1,5-a]pyrimidin-7- yl)morpholine
  • Intermediate E was prepared by the same method used for the preparation of Intermediate D except that the starting material used in the first step is 3-oxo-3-(pyridin-3- yl)propanenitrile, not 3-oxo-3-(pyridin-2-yl)propanenitrile.
  • Example 17 4-(2-(pyridin-3-yl)-5-(3-(m-tolyl)-1H-pyrazol-1-yl)pyrazolo[1,5- a]pyrimidin-7-yl)morpholine.
  • Example 18 4-(2-(pyridin-2-yl)-5-(3-(m-tolyl)-1H-pyrazol-1-yl)pyrazolo[1,5- a]pyrimidin-7-yl)morpholine.
  • F.5 ethyl 7-morpholino-5-(3-(m-tolyl)-1H-pyrazol-1-yl)pyrazolo[1,5- a]pyrimidine-2-carboxylate
  • Example 19 N-ethyl-7-morpholino-5-(3-(m-tolyl)-1H-pyrazol-1-yl)pyrazolo[1,5- a]pyrimidine-2-carboxamide.
  • Example 20 N-cyclopropyl-7-morpholino-5-(3-(m-tolyl)-1H-pyrazol-1- yl)pyrazolo[1,5-a]pyrimidine-2-carboxamide.
  • Compound 20 was prepared from Intermediate F and aminocyclopropane according to the method used for Example 19. LC-MS (ESI+): m/z 444 (MH + ).
  • Example 21 (R)-N-(1-cyclopropylethyl)-7-morpholino-5-(3-(m-tolyl)-1H-pyrazol- 1-yl)pyrazolo[1,5-a]pyrimidine-2-carboxamide [0296]
  • Compound 21 was prepared from Intermediate F and (R)-1-cyclopropylethylamine according to the procedure used for Example 19.
  • Example 23 N-(2-methoxyethyl)-7-morpholino-5-(3-(m-tolyl)-1H-pyrazol-1- yl)pyrazolo[1,5-a]pyrimidine-2-carboxamide [0300]
  • Compound 23 was prepared from Intermediate F and 3-(methoxy)ethylamine according to the procedure used for Example 19.
  • Example 24 N,N-dimethyl-7-morpholino-5-(3-(m-tolyl)-1H-pyrazol-1- yl)pyrazolo[1,5-a]pyrimidine-2-carboxamide
  • Example 25 N-ethyl-N-methyl-7-morpholino-5-(3-(m-tolyl)-1H-pyrazol-1- yl)pyrazolo[1,5-a]pyrimidine-2-carboxamide [0304]
  • Compound 25 was prepared by N-methylation of Compound 19 according to the procedure used for Example 37.
  • Example 26 N-cyclopropyl-N-methyl-7-morpholino-5-(3-(m-tolyl)-1H-pyrazol-1- yl)pyrazolo[1,5-a]pyrimidine-2-carboxamide [0306]
  • Compound 26 w as prepared from Intermediate F and N-methyl(cyclopropyl)amine according to the method used for Example 19.
  • Example 27 N-(cyclopropylmethyl)-7-morpholino-5-(3-(m-tolyl)-1H-pyrazol-1- yl)pyrazolo[1,5-a]pyrimidine-2-carboxamide [0308]
  • Compound 27 was prepared by reaction of Intermediate F with N- (cyclopropylmethyl)amine according to the coupling procedure used for Example 19.
  • Example 28 azetidin-1-yl(7-morpholino-5-(3-(m-tolyl)-1H-pyrazol-1- yl)pyrazolo[1,5-a]pyrimidin-2-yl)methanone [0310]
  • Compound 28 was prepared from azetidine and Intermediate F according to the coupling procedure used for Example 19.
  • Example 29 (7-morpholino-5-(3-(m-tolyl)-1H-pyrazol-1-yl)pyrazolo[1,5- a]pyrimidin-2-yl)(pyrrolidin-1-yl)methanone [0312]
  • Compound 29 was prepared by the same procedure used for Example 19 by reaction of Intermediate F with pyrrolidine.
  • Example 30 (7-morpholino-5-(3-(m-tolyl)-1H-pyrazol-1-yl)pyrazolo[1,5- a]pyrimidin-2-yl)(piperidin-1-yl)methanone [0314]
  • Compound 30 was prepared by the same procedure used for Example 19 by reaction of Intermediate F with piperidine.
  • Example 31 morpholino(7-morpholino-5-(3-(m-tolyl)-1H-pyrazol-1- yl)pyrazolo[1,5-a]pyrimidin-2-yl)methanone [0316] Compound 31 was prepared by the same procedure used for Example 19 by reaction of Intermediate F with morpholine. LC-MS (ESI+): m/z 474 (MH + ).
  • Example 32 (4-methylpiperazin-1-yl)(7-morpholino-5-(3-(m-tolyl)-1H-pyrazol-1- yl)pyrazolo[1,5-a]pyrimidin-2-yl)methanone [0318]
  • Compound 32 was prepared by the same procedure used for Example 19 by reaction of Intermediate F with N-(methyl)piperidine.
  • Example 33 N-methoxy-N-methyl-7-morpholino-5-(3-(m-tolyl)-1H-pyrazol-1- yl)pyrazolo[1,5-a]pyrimidine-2-carboxamide [0320]
  • Compound 33 was prepared by the same procedure used for Example 19 by reaction of Intermediate F with N-methoxy-N-methylamine.
  • Example 34 N-methoxy-7-morpholino-5-(3-(m-tolyl)-1H-pyrazol-1- yl)pyrazolo[1,5-a]pyrimidine-2-carboxamide [0322]
  • Compound 34 was prepared by the same procedure used for Example 19 by reaction of Intermediate F with N-methoxylamine.
  • Example 35 N-(methylsulfonyl)-7-morpholino-5-(3-(m-tolyl)-1H-pyrazol-1- yl)pyrazolo[1,5-a]pyrimidine-2-carboxamide [0324]
  • Compound 35 was prepared by the same procedure used for Example 19 by reaction of Intermediate F with methylsulfonamide.
  • Example 36 N-cyclopentyl-7-morpholino-5-(3-(m-tolyl)-1H-pyrazol-1- yl)pyrazolo[1,5-a]pyrimidine-2-carboxamide [0326]
  • Compound 36 was prepared by the same procedure used for Example 19 by reaction of Intermediate F with cyclopentylamine.
  • Example 37 N-cyclopentyl-N-methyl-7-morpholino-5-(3-(m-tolyl)-1H-pyrazol-1- yl)pyrazolo[1,5-a]pyrimidine-2-carboxamide
  • N-cyclopentyl-7-morpholino-5-(3-(m-tolyl)-1H-pyrazol-1- yl)pyrazolo[1,5-a]pyrimidine-2-carboxamide Compound 36, 55 mg, 0.11 mmol
  • DMF 5 mL
  • NaH 10 mg, 0.24 mmol
  • Example 38 N-isopropyl-7-morpholino-5-(3-(m-tolyl)-1H-pyrazol-1- yl)pyrazolo[1,5-a]pyrimidine-2-carboxamide [0330] Compound 38 was prepared by the same procedure used for Example 19 by reaction of Intermediate F with isopropylamine. LC-MS (ESI+): m/z 446 (MH + ).
  • Example 39 N-(1,3-dimethoxypropan-2-yl)-7-morpholino-5-(3-(m-tolyl)-1H- pyrazol-1-yl)pyrazolo[1,5-a]pyrimidine-2-carboxamide [0332]
  • Compound 39 was prepared by the same procedure used for Example 19 by reaction of Intermediate F with N-(1,3-dimethoxypropan-2-yl)amine.
  • Example 40 N-(2-(dimethylamino)ethyl)-7-morpholino-5-(3-(m-tolyl)-1H-pyrazol- [0334]
  • Compound 40 was prepared by the same procedure used for Example 19 by reaction of Intermediate F with N-(2-(dimethylamino)ethyl)amine.
  • Example 41 N-(4-(dimethylamino)butyl)-7-morpholino-5-(3-(m-tolyl)-1H- pyrazol-1-yl)pyrazolo[1,5-a]pyrimidine-2-carboxamide
  • Compound 41 was prepared by the same procedure used for Example 19 by reaction of Intermediate F with N-(4-(dimethylamino)butyl)amine.
  • Example 42 7-morpholino-N-(oxetan-3-yl)-5-(3-(m-tolyl)-1H-pyrazol-1- yl)pyrazolo[1,5-a]pyrimidine-2-carboxamide
  • Compound 42 was prepared by the same procedure used for Example 19 by reaction of Intermediate F with N-(oxetan-3-yl)amine.
  • Example 43 7-morpholino-N-(oxetan-3-ylmethyl)-5-(3-(m-tolyl)-1H-pyrazol-1- yl)pyrazolo[1,5-a]pyrimidine-2-carboxamide
  • Compound 43 was prepared by the same procedure used for Example 19 by reaction of Intermediate F with N-(oxetan-3-ylmethyl)amine.
  • Example 44 N-((3-(hydroxymethyl)oxetan-3-yl)methyl)-7-morpholino-5-(3-(m- tolyl)-1H-pyrazol-1-yl)pyrazolo[1,5-a]pyrimidine-2-carboxamide [0342]
  • Compound 44 was prepared by the same procedure used for Example 19 by reaction of Intermediate F with N-((3-(hydroxymethyl)oxetan-3-yl)methyl)amine.
  • Example 45 (7-morpholino-5-(3-(m-tolyl)-1H-pyrazol-1-yl)pyrazolo[1,5- a]pyrimidin-2-yl)(2-oxa-6-azaspiro[3.3]heptan-6-yl)methanone [0344]
  • Compound 45 was prepared by the same procedure used for Example 19 by reaction of Intermediate F with 2-oxa-6-azaspiro[3.3]heptane.
  • Example 46 (7-morpholino-5-(3-(m-tolyl)-1H-pyrazol-1-yl)pyrazolo[1,5- a]pyrimidin-2-yl)(6-oxa-1-azaspiro[3.3]heptan-1-yl)methanone [0346]
  • Compound 46 was prepared by the same procedure used for Example 19 by reaction of Intermediate F with 6-oxa-1-azaspiro[3.3]heptane.
  • Example 47 7-morpholino-N-(oxetan-3-yloxy)-5-(3-(m-tolyl)-1H-pyrazol-1- yl)pyrazolo[1,5-a]pyrimidine-2-carboxamide [0349] To a solution of 2-hydroxyisoindoline-1,3-dione (4.84 g, 27 mmol) in THF (120 mL) at 0°C was added PPh 3 (8.5 g, 32.4 mmol), DEAD (6.56 g, 32.4 mmol) and oxetan-3-ol (2 g, 29.7 mmol). The reaction was heated to 30°C overnight.
  • reaction mixture was concentrated directly and purified by silica gel column chromatography with a gradient elution of 2% MeOH/DCM to 3% MeOH/DCM to provide ethyl 5-chloro-7-morpholinopyrazolo[1,5-a]pyrimidine-2-carboxylate (3.3 g, 10.6 mmol) as a yellow solid.
  • reaction mixture was concentrated directly and purified by silica gel column chromatography with a gradient elution of 20% EtOAc/HeX to 33% EtOAc/HeX to provide 4-(2-bromo-5-(3-(m-tolyl)-1H-pyrazol-1-yl)pyrazolo[1,5-a]pyrimidin-7- yl)morpholine (5.0 g, 11.4 mmol) as a yellow solid.
  • reaction mixture was concentrated directly and purified by silica gel column chromatography with a gradient elution of 20% EtOAc/Hex to 33% EtOAc/Hex to provide 5-methyl-3-(7-morpholino-5-(3-(m- tolyl)-1H-pyrazol-1-yl)pyrazolo[1,5-a]pyrimidin-2-yl)-1H-pyrazole-1-carboxylate (220 mg, 0.41 mmol) as colorless oil.
  • the reaction mixture was quenched with 1 M aqueous HCl solution and then adjusted the PH to 8 using NaHCO 3 solution.
  • the aqueous solution was extracted with DCM/MeOH (10:1, 3 x 20 mL).
  • the combined organic phase was dried over anhydrous Na 2 SO 4 , filtrated and concentrated under reduce pressure.
  • the crude product was purified by silica gel column chromatography with a gradient elution of 2% MeOH/DCM to 5% MeOH/DCM to provide (7-morpholino-5-(3-phenyl-1H-pyrazol-1- yl)pyrazolo[1,5-a]pyrimidin-2-yl)methanamine (110 mg, 0.29 mmol) as white solid.
  • Example 48 N-[(3S)-1-methylpyrrolidin-3-yl]-7-morpholino-5-[3-(m- tolyl)pyrazol-1-yl]pyrazolo[1,5-a]pyrimidine-2-carboxamide [0439]
  • Compound 48 was prepared by General Procedure 1.
  • Example 49 N-[(3R)-1-methylpyrrolidin-3-yl]-7-morpholino-5-[3-(m- tolyl)pyrazol-1-yl]pyrazolo[1,5-a]pyrimidine-2-carboxamide [0441]
  • Compound 49 was prepared by General Procedure 1.
  • Example 50 7-morpholino-5-[3-(m-tolyl)pyrazol-1-yl]-N-[(3S)-tetrahydrofuran-3- yl]pyrazolo[1,5-a]pyrimidine-2-carboxamide [0443]
  • Compound 50 was prepared by General Procedure 1. LC-MS (ESI+): m/z 474 (MH + ).
  • Example 51 7-morpholino-5-[3-(m-tolyl)pyrazol-1-yl]-N-[(3R)-tetrahydrofuran- 3-yl]pyrazolo[1,5-a]pyrimidine-2-carboxamide [0445]
  • Compound 51 was prepared by General Procedure 1.
  • Example 52 N-[(3R)-1-methyl-3-piperidyl]-7-morpholino-5-[3-(m-tolyl)pyrazol-1- yl]pyrazolo[1,5-a]pyrimidine-2-carboxamide [0447]
  • Compound 52 was prepared by General Procedure 1. LC-MS (ESI+): m/z 501 (MH+).
  • Example 54 N-(1-methyl-4-piperidyl)-7-morpholino-5-[3-(m-tolyl)pyrazol-1- yl]pyrazolo[1,5-a]pyrimidine-2-carboxamide [0451]
  • Compound 54 was prepared by General Procedure 1. LC-MS (ESI+): m/z 501 (MH + ).
  • Example 55 N-(1-methyl-4-piperidyl)-7-morpholino-5-[4-(m-tolyl)pyrazol-1- yl]pyrazolo[1,5-a]pyrimidine-2-carboxamide
  • Compound 55 was prepared by General Procedure 1. LC-MS (ESI+): m/z 501 (MH + ).
  • Example 57 4-[5-[4-(m-tolyl)pyrazol-1-yl]-2-pyrimidin-4-yl-pyrazolo[1,5- a]pyrimidin-7-yl]morpholine [0457] Compound 57 was prepared by General Procedures 2 and 6. LC-MS (ESI+): m/z 439 (MH + ).
  • Example 58 4-[5-[4-(m-tolyl)pyrazol-1-yl]-2-pyrimidin-5-yl-pyrazolo[1,5- a]pyrimidin-7-yl]morpholine [0459]
  • Compound 58 was prepared by General Procedures 2 and 7.
  • Example 59 3-[7-morpholino-5-[4-(m-tolyl)pyrazol-1-yl]pyrazolo[1,5- a]pyrimidin-2-yl]pyridin-2-amine [0461]
  • Compound 59 was prepared by General Procedures 2 and 7.
  • Example 60 5-[7-morpholino-5-[4-(m-tolyl)pyrazol-1-yl]pyrazolo[1,5- a]pyrimidin-2-yl]pyrimidin-2-amine [0463] Compound 60 was prepared by General Procedures 2 and 7. LC-MS (ESI+): m/z 454 (MH + ).
  • Example 61 4-[2-(1-methylpyrazol-3-yl)-5-[3-(m-tolyl)pyrazol-1-yl]pyrazolo[1,5- a]pyrimidin-7-yl]morpholine [0465] Compound 61 was prepared by General Procedures 2 and 7. LC-MS (ESI+): m/z 441 (MH + ).
  • Example 62 4-[5-[3-(m-tolyl)pyrazol-1-yl]-2-(1H-pyrazol-3-yl)pyrazolo[1,5- a]pyrimidin-7-yl]morpholine [0467] Compound 62 was prepared by General Procedures 2 and 7.
  • Example 63 4-[5-(3-phenylpyrazol-1-yl)-2-pyrimidin-2-yl-pyrazolo[1,5- a]pyrimidin-7-yl]morpholine [0469] Compound 63 was prepared by General Procedures 2 and 6. LC-MS (ESI+): m/z 425 (MH + ).
  • Example 64 4-[5-(3-phenylpyrazol-1-yl)-2-(3-pyridyl)pyrazolo[1,5-a]pyrimidin-7- yl]morpholine [0471] Compound 64 was prepared by General Procedures 2 and 7.
  • Example 65 4-[2-(5-methyl-1H-pyrazol-3-yl)-5-[3-(m-tolyl)pyrazol-1- yl]pyrazolo[1,5-a]pyrimidin-7-yl]morpholine [0473]
  • Compound 65 was prepared by General Procedures 2 and 7.
  • Example 66 4-[2-(1-methylpyrazol-3-yl)-5-(3-phenylpyrazol-1-yl)pyrazolo[1,5- a]pyrimidin-7-yl]morpholine [0475] Compound 66 was prepared by General Procedures 2 and 7. LC-MS (ESI+): m/z 427 (MH + ).
  • Example 67 4-[5-(3-phenylpyrazol-1-yl)-2-(1H-pyrazol-3-yl)pyrazolo[1,5- a]pyrimidin-7-yl]morpholine [0477]
  • Compound 67 was prepared by General Procedures 2 and 7.
  • Example 68 4-[5-(3-phenylpyrazol-1-yl)-2-pyrimidin-4-yl-pyrazolo[1,5- a]pyrimidin-7-yl]morpholine [0479]
  • Compound 68 was prepared by General Procedures 2 and 6.
  • Example 69 4-[5-(3-phenylpyrazol-1-yl)-2-pyrimidin-5-yl-pyrazolo[1,5- a]pyrimidin-7-yl]morpholine [0481] Compound 69 was prepared by General Procedures 2 and 7. LC-MS (ESI+): m/z 425 (MH + ).
  • Example 70 3-[7-morpholino-5-(3-phenylpyrazol-1-yl)pyrazolo[1,5-a]pyrimidin- 2-yl]pyridin-2-amine [0483] Compound 70 was prepared by General Procedures 2 and 7.
  • Example 71 5-[7-morpholino-5-(3-phenylpyrazol-1-yl)pyrazolo[1,5-a]pyrimidin- 2-yl]pyrimidin-2-amine [0485] Compound 71 was prepared by General Procedures 2 and 7. LC-MS (ESI+): m/z 440 (MH + ).
  • Example 72 4-[5-(3-phenylpyrazol-1-yl)-2-(2-pyridyl)pyrazolo[1,5-a]pyrimidin-7- yl]morpholine [0487]
  • Compound 72 was prepared by General Procedures 2 and 6.
  • Example 73 N-isopropyl-7-morpholino-5-(3-phenylpyrazol-1-yl)pyrazolo[1,5- a]pyrimidine-2-carboxamide [0489] Compound 73 was prepared by General Procedure 1. LC-MS (ESI+): m/z 432 (MH + ).
  • Example 75 N-[(1S)-1-cyclopropylethyl]-7-morpholino-5-(3-phenylpyrazol-1- yl)pyrazolo[1,5-a]pyrimidine-2-carboxamide [0493]
  • Compound 75 was prepared by General Procedure 1.
  • Example 76 7-morpholino-N-(oxetan-3-ylmethyl)-5-(3-phenylpyrazol-1- yl)pyrazolo[1,5-a]pyrimidine-2-carboxamide [0495]
  • Compound 76 was prepared by General Procedure 1.
  • Example 77 N-cyclopentyl-7-morpholino-5-(3-phenylpyrazol-1-yl)pyrazolo[1,5- a]pyrimidine-2-carboxamide [0497]
  • Compound 77 was prepared by General Procedure 1.
  • Example 78 4-[5-(4-phenylpyrazol-1-yl)-2-(2-pyridyl)pyrazolo[1,5-a]pyrimidin-7- yl]morpholine [0499]
  • Compound 78 was prepared by General Procedures 2 and 6.
  • Example 79 4-[5-(4-phenylpyrazol-1-yl)-2-(3-pyridyl)pyrazolo[1,5-a]pyrimidin-7- yl]morpholine [0501]
  • Compound 79 was prepared by General Procedures 2 and 7.
  • Example 80 N-isopropyl-7-morpholino-5-(4-phenylpyrazol-1-yl)pyrazolo[1,5- a]pyrimidine-2-carboxamide [0503]
  • Compound 80 was prepared by General Procedure 1. LC-MS (ESI+): m/z 432 (MH + ).
  • Example 81 4-[2-(5-methyl-1H-pyrazol-3-yl)-5-(3-phenylpyrazol-1- yl)pyrazolo[1,5-a]pyrimidin-7-yl]morpholine [0505]
  • Compound 81 was prepared by General Procedures 2 and 7.
  • Example 82 4-[5-(3-phenylpyrazol-1-yl)-2-pyrazin-2-yl-pyrazolo[1,5-a]pyrimidin- 7-yl]morpholine [0507] Compound 82 was prepared by General Procedures 2 and 6.
  • Example 83 N,N-dimethyl-2-[3-methyl-5-[7-morpholino-5-[3-(m-tolyl)pyrazol-1- yl]pyrazolo[1,5-a]pyrimidin-2-yl]pyrazol-1-yl]ethanamine [0509]
  • Compound 83 was prepared by General Procedure 2.
  • Example 84 4-[2-(2,5-dimethylpyrazol-3-yl)-5-[3-(m-tolyl)pyrazol-1- yl]pyrazolo[1,5-a]pyrimidin-7-yl]morpholine [0511]
  • Compound 84 was prepared by General Procedures 2 and 5.
  • Example 85 4-[2-(2,5-dimethylpyrazol-3-yl)-5-(3-phenylpyrazol-1- yl)pyrazolo[1,5-a]pyrimidin-7-yl]morpholine [0513]
  • Compound 85 was prepared by General Procedures 2 and 5.
  • Example 86 4-[2-(1,5-dimethylpyrazol-3-yl)-5-(3-phenylpyrazol-1- yl)pyrazolo[1,5-a]pyrimidin-7-yl]morpholine [0515]
  • Compound 86 was prepared by General Procedures 2 and 5.
  • Example 87 4-[2-(1,5-dimethylpyrazol-3-yl)-5-[3-(m-tolyl)pyrazol-1- yl]pyrazolo[1,5-a]pyrimidin-7-yl]morpholine [0517]
  • Compound 87 was prepared by General Procedures 2 and 5.
  • Example 88 4-[5-(3-phenylpyrazol-1-yl)-2-pyridazin-3-yl-pyrazolo[1,5- a]pyrimidin-7-yl]morpholine [0519] Compound 88 was prepared by General Procedures 2 and 6. LC-MS (ESI+): m/z 425 (MH + ).
  • Example 89 methyl N-[[7-morpholino-5-(3-phenylpyrazol-1-yl)pyrazolo[1,5- a]pyrimidin-2-yl]methyl]carbamate [0521]
  • Compound 89 was prepared by General Procedure 8.
  • Example 90 N-[[7-morpholino-5-(3-phenylpyrazol-1-yl)pyrazolo[1,5-a]pyrimidin- 2-yl]methyl]propenamide [0523]
  • Compound 90 was prepared by General Procedure 8.
  • Example 91 N,N-dimethyl-2-[5-methyl-3-[7-morpholino-5-[3-(m-tolyl)pyrazol-1- yl]pyrazolo[1,5-a]pyrimidin-2-yl]pyrazol-1-yl]ethanamine [0525]
  • Compound 91 was prepared by General Procedure 2.
  • Example 92 N,N-dimethyl-2-[5-methyl-3-[7-morpholino-5-[3-(2-pyridyl)pyrazol- 1-yl]pyrazolo[1,5-a]pyrimidin-2-yl]pyrazol-1-yl]ethanamine [0527]
  • Compoun d 92 was prepared by General Procedure 2.
  • Example 93 N,N-dimethyl-2-[5-methyl-3-[7-morpholino-5-[3-(4-pyridyl)pyrazol- 1-yl]pyrazolo[1,5-a]pyrimidin-2-yl]pyrazol-1-yl]ethanamine [0529]
  • Compound 93 was prepared by General Procedure 2.
  • Example 94 N,N-dimethyl-2-[5-methyl-3-[5-[3-(6-methyl-2-pyridyl)pyrazol-1-yl]- 7-morpholino-pyrazolo[1,5-a]pyrimidin-2-yl]pyrazol-1-yl]ethanamine [0531]
  • Compound 94 was prepared by General Procedure 2.
  • Example 95 N,N-dimethyl-2-[5-methyl-3-[5-[3-(4-methyl-2-pyridyl)pyrazol-1-yl]- 7-morpholino-pyrazolo[1,5-a]pyrimidin-2-yl]pyrazol-1-yl]ethanamine [0533]
  • Compound 95 was prepared by General Procedures 2 and 4.
  • Example 96 N,N-dimethyl-2-[5-methyl-3-[5-[3-(2-methyl-4-pyridyl)pyrazol-1-yl]- 7-morpholino-pyrazolo[1,5-a]pyrimidin-2-yl]pyrazol-1-yl]ethanamine [0535]
  • Compound 96 was prepared by General Procedures 2 and 4.
  • Example 97 2-[3-[5-[3-(3-chlorophenyl)pyrazol-1-yl]-7-morpholino-pyrazolo[1,5- a]pyrimidin-2-yl]-5-methyl-pyrazol-1-yl]-N,N-dimethyl-ethanamine [0537]
  • Compound 97 was prepared by General Procedures 2 and 3.
  • Example 98 5-[7-morpholino-5-(3-phenylpyrazol-1-yl)pyrazolo[1,5-a]pyrimidin- 2-yl]pyridin-2-amine [0539]
  • Compound 98 was prepared by General Procedures 2 and 7.
  • Example 99 N,N-dimethyl-2-[5-methyl-3-[7-morpholino-5-(3-phenylpyrazol-1- yl)pyrazolo[1,5-a]pyrimidin-2-yl]pyrazol-1-yl]ethanamine [0541]
  • Compound 99 was prepared by General Procedure 2.
  • Example 100 2-[3-[5-[3-(3-methoxyphenyl)pyrazol-1-yl]-7-morpholino- pyrazolo[1,5-a]pyrimidin-2-yl]-5-methyl-pyrazol-1-yl]-N,N-dimethyl-ethanamine [0543]
  • Compound 100 was prepared by General Procedure 2.
  • Example 101 4-[5-[3-(3-chlorophenyl)pyrazol-1-yl]-2-(1-methylpyrazol-3- yl)pyrazolo[1,5-a]pyrimidin-7-yl]morpholine [0545]
  • Compound 101 was prepared by General Procedures 2 and 3.
  • Example 102 4-[5-[3-(5-methyl-3-pyridyl)pyrazol-1-yl]-2-(3-pyridyl)pyrazolo[1,5- a]pyrimidin-7-yl]morpholine [0547] Compound 102 was prepared by General Procedure 2.
  • Example 103 4-[5-[3-(2-methyl-4-pyridyl)pyrazol-1-yl]-2-(3-pyridyl)pyrazolo[1,5- a]pyrimidin-7-yl]morpholine [0549]
  • Compound 1 03 was prepared by General Procedures 2 and 4.
  • Example 104 4-[7-morpholino-5-(3-phenylpyrazol-1-yl)pyrazolo[1,5-a]pyrimidin- 2-yl]pyrimidin-2-amine [0551]
  • Compound 104 was prepared by General Procedures 2 and 7.
  • Example 105 4-[5-[3-(3-chlorophenyl)pyrazol-1-yl]-2-(3-pyridyl)pyrazolo[1,5- a]pyrimidin-7-yl]morpholine [0553] Compound 105 was prepared by General Procedures 2 and 3.
  • Example 106 4-[5-[3-(3-bromophenyl)pyrazol-1-yl]-2-(3-pyridyl)pyrazolo[1,5- a]pyrimidin-7-yl]morpholine [0555] Compound 106 was prepared by General Procedures 2 and 7. LC-MS (ESI+): m/z 502/504 (MH + ).
  • Example 107 4-[5-[3-(3-methoxyphenyl)pyrazol-1-yl]-2-(3-pyridyl)pyrazolo[1,5- a]pyrimidin-7-yl]morpholine [0557]
  • Compound 107 was prepared by General Procedures 2 and 3.
  • Example 108 4-[5-[3-(6-methyl-2-pyridyl)pyrazol-1-yl]-2-(3-pyridyl)pyrazolo[1,5- a]pyrimidin-7-yl]morpholine [0559]
  • Compound 108 was prepared by General Procedure 2.
  • Example 109 6-[7-morpholino-5-(3-phenylpyrazol-1-yl)pyrazolo[1,5-a]pyrimidin- 2-yl]pyridin-2-amine [0561] Compound 109 was prepared by the methods described above. LC-MS (ESI+): m/z 438 (MH + ). [0562] Example 110: 4-[2-(3-methylisoxazol-5-yl)-5-[3-(m-tolyl)pyrazol-1- yl]pyrazolo[1,5-a]pyrimidin-7-yl]morpholine [0563] Compound 110 was prepared by the methods described above.
  • Example 111 4-[5-[3-(m-tolyl)pyrazol-1-yl]-2-thiazol-2-yl-pyrazolo[1,5- a]pyrimidin-7-yl]morpholine [0565] Compound 111 was prepared by the methods described above. LC-MS (ESI+): m/z 443 (MH + ).
  • Example 112 4-[2-(1-methylpyrazol-4-yl)-5-[3-(m-tolyl)pyrazol-1-yl]pyrazolo[1,5- a]pyrimidin-7-yl]morpholine [0567] Compound 112 was prepared by the methods described above. LC-MS (ESI+): m/z 440 (MH + ). [0568] Example 113: 4-[5-[3-(m-tolyl)pyrazol-1-yl]-2-(1H-pyrazol-4-yl)pyrazolo[1,5- a]pyrimidin-7-yl]morpholine [0569] Compound 113 was prepared by the methods described above.
  • Example 114 4-[5-(3-phenylpyrazol-1-yl)-2-thiazol-2-yl-pyrazolo[1,5- a]pyrimidin-7-yl]morpholine [0571] Compound 114 was prepared by the methods described above. LC-MS (ESI+): m/z 429 (MH + ).
  • Example 115 4-[2-(1-methylpyrazol-4-yl)-5-(3-phenylpyrazol-1-yl)pyrazolo[1,5- a]pyrimidin-7-yl]morpholine [0573] Compound 115 was prepared by the methods described above. LC-MS (ESI+): m/z 426 (MH + ). [0574] Example 116: 4-[5-(3-phenylpyrazol-1-yl)-2-(1H-pyrazol-4-yl)pyrazolo[1,5- a]pyrimidin-7-yl]morpholine [0575] Compound 116 was prepared by the methods described above.
  • Example 117 methyl 3-[1-[7-morpholino-2-(3-pyridyl)pyrazolo[1,5-a]pyrimidin- 5-yl]pyrazol-3-yl]benzoate [0577] Compound 117 was prepared by the methods described above. LC-MS (ESI+): m/z 481 (MH + ). [0578] Example 118: 3-[1-[7-morpholino-2-(3-pyridyl)pyrazolo[1,5-a]pyrimidin-5- yl]pyrazol-3-yl]benzamide [0579] Compound 118 was prepared by the methods described above.
  • Example 120 4-[5-[3-(m-tolyl)pyrazol-1-yl]-2-oxazol-2-yl-pyrazolo[1,5- a]pyrimidin-7-yl]morpholine [0583] Compound 120 was prepared by the methods described above. LC-MS (ESI+): m/z 427 (MH + ). [0584] Example 121: 4-[2-(3-methylisoxazol-5-yl)-5-(3-phenylpyrazol-1-yl)pyrazolo[1,5- a]pyrimidin-7-yl]morpholine [0585] Compound 121 was prepared by the methods described above.
  • Example 123 4-[2-(1-methylpyrazol-3-yl)-5-[3-[3- (trideuteriomethyl)phenyl]pyrazol-1-yl]pyrazolo[1,5-a]pyrimidin-7-yl]morpholine [0589]
  • Compound 123 was prepared by the methods described above.
  • Biological Example 1 Inhibition of PIKfyve
  • the kinase substrate was prepared by mixing and sonicating fluorescently-labeled phosphatidylinositol 3-phosphate (PI3P) with phospho-L-serine (PS) at a 1:10 ratio in 50 mM HEPES buffer pH 7.5. [0591] The kinase reactions were assembled in 384-well plates (Greiner) in a total volume of 20 mL as follows.
  • Kinase protein was pre-diluted in an assay buffer comprising 25 mM HEPES, pH 7.5, 1 mM DTT, 2.5 mM MgCl2, and 2.5 mM MnCl2, and 0.005% Triton X-100, and dispensed into a 384-well plate (10 ⁇ L per well).
  • Test compounds were serially pre-diluted in DMSO and added to the protein samples by acoustic dispensing (Labcyte Echo). The concentration of DMSO was equalized to 1% in all samples. All test compounds were tested at 12 concentrations. Apilimod was used as a reference compound and was tested in identical manner in each assay plate.
  • Control samples (0%-inhibition, in the absence of inhibitor, DMSO only) and 100%-inhibition (in the absence of enzyme) were assembled in replicates of four and of enzyme was 2 nM, the final concentration of ATP was 10 mM, and the final concentration of PI3P/PS substrate was 1 ⁇ M (PI3P).
  • the kinase reactions were allowed to proceed for 3 h at room temperature. Following incubation, the reactions were quenched by addition of 50 mL of termination buffer (100 mM HEPES, pH 7.5, 0.01% Triton X-100, 20 mM EDTA).
  • Terminated plates were analyzed on a microfluidic electrophoresis instrument (Caliper LabChip® 3000, Caliper Life Sciences/Perkin Elmer). The change in the relative fluorescence intensity of the PI(3)P substrate and PI(3,5)P product peaks was measured. The activity in each test sample was determined as the product to sum ratio (PSR): P/(S+P), where P is the peak height of the product, and S is the peak height of the substrate.
  • PSR product to sum ratio
  • the IC50 of test compounds (50%-inhibition) the %-inh cdata (P inh versus compound concentration) were fitted by a four-parameter sigmoid dose- response model using XLfit software (IDBS). [0592]
  • IDBS XLfit software
  • VeroE6-EGFP cells (provided by Lab of Virology & Chemotherapy, Rega Institute, KU Leuven, Leuven, Belgium) (sometimes referred to herein as VeroE6, Vero-E6, or Vero-E6-GFP) were propagated in growth medium which was prepared by supplementing DMEM (Gibco 41965- 039) with 10% v/v heat-inactivated FCS and 5 mL sodium bicarbonate 7.5% (Gibco 25080-060). Cells were cultured in T150 bottles and split 1/4 twice a week. Pen-strep was added directly to the T150 bottle at a 1/100 dilution.
  • Assay medium was prepared by supplementing DMEM (Gibco 41965-039) with 2% v/v heat-inactivated FCS and 5 mL sodium bicarbonate 7.5% (Gibco 25080-060). [0597] Serial dilutions of compounds were prepared in 96-well plates to which cells were added (25,000 cells/well) after which plates were incubated overnight (37°C; 5% CO2).
  • virus inoculum SARS-CoV-2 clinical isolate, Belgian strain: BetaCov/Belgium/GHB-03021/2020
  • CPE cytopathic effect
  • GFP signal was determined using high-content imaging.
  • Antiviral activity is expressed as the EC 50 or concentration required to rescue 50% of the GFP signal from the virus-induced cytopathogenicy. The signal is provided as the logarithm of the surface of the well that is covered with fluorescent pixels which correlates with living cells.
  • cytotoxicity was assessed by growing uninfected cells in the presence of the test compound at the concentrations tested. After a 4 day incubation, cell viability was measured using a commercial kit.
  • Antiviral readout was performed using high-content imagers. Using a 5x objective, almost the entire well of a 384-well plate is captured at once (for a 96-well plate, the well is covered by 4 field of views). A GFP marker located in both the cell cytoplasm as well as the nucleus allowed for the calculation of the surface of the well that is (still) covered by cells (SpotTotalAreaCh2).
  • the Safety index is the ratio of CC 50 to EC 50 .
  • Table 3 Compound # EC 50 ( ⁇ M) CC 50 ( ⁇ M) Safety index Biological E xamp e : nt v ra e ects o yve n tors n an - cell assay [0603] Materials and Methods [0604] PIKfyve inhibitors were assessed for anti-viral activity against SARS-COV-2 using A549- ACE2 cells (adenocarcinomic human alveolar basal epithelial cells) transduced to express the human Angiotensin-converting enzyme 2 (ACE2), provided by Institut Pasteur, Paris, France.
  • ACE2 Angiotensin-converting enzyme 2
  • A549-ACE2 cells were grown in 96-well plates, and ten concentrations of each tested PIKfyve inhibitor was tested in triplicate. Concentrations of compounds tested included: 0.001 – 1uM (0, 0.001, 0.003, 0.01, 0.03, 0.1, 0.3, 1, 3, 10). The DMSO concentration in the final assay was ⁇ 1% (dilution of stock made in serial dilutions of media). [0605] Cells were pretreated for 2 hours before infection with SARS-CoV-2 virus (isolate BetaCoV/France/IDF0372/2020 C2). After virus infection, cells were incubated for 1 hour at 37°C. All cells were washed after a 48-hour incubation.
  • Viral replication was measured by quantitative RT-PCR in the presence and absence of the tested compounds.
  • cytotoxicity was assessed by growing uninfected cells in the presence of the three tested compounds at the concentrations tested. After 48 hours of incubation, cell viability was measured using a commercial kit.
  • Three test compounds, Compound 14, Compound 17, and Compound 43 were provided in 10 mM solutions in 100 uL DMSO.
  • Results [0610] Exemplary results are shown in Figures 2A-C. The tested compounds showed potent antiviral effects with minimal toxicity.
  • Viral titer is shown as Log 10 of plaque forming units per mL (PFU/mL) on the left axis (data represented by circles). Percent cell viability is shown on the right axis (data represented by squares).
  • the IC 50 values for other tested compounds are shown in Table 4 below.
  • Compounds 61, 65, 81, and 114 have CC50 of > 10 ⁇ M.
  • test compounds were prepared at four serial log10 concentrations, 0.1, 1.0, 10, and 100 ⁇ g/ml or ⁇ M. Five microwells were used per dilution: three for infected cultures and two for uninfected toxicity cultures. Controls for the experiment consisted of six microwells that were infected and not treated (virus controls) and six that were untreated and uninfected (cell controls) on every plate. A known active drug, the protease inhibitor M128533, was tested in parallel as a positive control drug using the same method as is applied for test compounds. [0613] Growth media was removed from the cells and the test compound was applied in 0.1 ml volume to wells at 2X concentration.
  • the SARS-CoV-2, USA_WA1/2020 strain normally at ⁇ 60 CCID50 (50% cell culture infectious dose) in 0.1 ml volume, was added to the wells designated for virus infection.
  • Medium devoid of virus was placed in toxicity control wells and cell control wells. Plates were incubated at 37°C with 5% CO 2 until marked CPE (>80% CPE for most virus strains) was observed in virus control wells.
  • the plates were then stained with 0.011% neutral red for approximately two hours at 37°C in a 5% CO2 incubator. The neutral red medium was removed by complete aspiration, and the cells were optionally rinsed 1X with phosphate buffered solution (PBS) to remove residual dye.
  • PBS phosphate buffered solution
  • the PBS was completely removed, and the incorporated neutral red was eluted with 50% Sorensen’s citrate buffer/50% ethanol for at least 30 minutes.
  • Neutral red dye penetrates into living cells, thus, the more intense the red color, the larger the number of viable cells present in the wells.
  • the dye content in each well was quantified using a spectrophotometer at 540 nm wavelength.
  • the dye content in each set of wells was converted to a percentage of dye present in untreated control wells using a Microsoft Excel computer-based spreadsheet and normalized based on the virus control.
  • the 50% effective (EC 50 , virus-inhibitory) concentrations and 50% cytotoxic (CC 50 , cell-inhibitory) concentrations were then calculated by regression analysis.
  • cytophathic/cytopathogenic effect CPE
  • a colorimetric mammalian cell viability assay Thiazolyl Blue Tetrazolium Bromide (MTT) assay; Sigma Catalog # M5655-1G; Lot # MKCL1832
  • MTT Thiazolyl Blue Tetrazolium Bromide
  • Percentage viral inhibition was calculated as follows: Inhibition where: A: mean optical density of test, B: mean optical density of virus controls, C: mean optical density of cell controls. [0619] Values above 100% inhibition occur when A>C, due either to natural variation or compound effects.
  • CC 50 values were calculated used non-linear regression as above for EC 50 .
  • the virus strains/serotypes used were human alphacoronavirus 229E (HCoV 229E; ATCC ® CVR-740TM) and betacoronavirus 1 OC-4 (HCoV OC43; ATCC ® VR-1558TM).
  • HCoV 229E was tested with human bronchial epithelial (16BHE) cells.
  • HCoV OC43 was tested with human lung mucoepidermoid (H292) cells. Remdesivir was used as the control compound.
  • Compound 18 increased cell viability to levels that were higher than the uninfected cells, leading to percentages above 100%. The viability in infected cells was reduced to around 0% at 10 ⁇ M. In order to calculate a more representative EC50, we excluded the 10 ⁇ M-data point. The compound had a CC 50 ⁇ M.
  • Biological Example 6 PIKfyve inhibition of SARS CoV2–induced cytopathic effect (CPE) in Vero E6 cells [0630] Materials and Methods [0631] Five PIKfyve inhibitors were assessed for anti-viral activity against SARS-CoV-2, strain USA_WA1/2020, using Vero E6 and Vero 6 cells according to the methods of Severson et al.
  • Solution A was prepared by adding 7.098 g of disodium hydrogen phosphate into 500 mL of pure water, followed by sonication.
  • Solution B was prepared by adding 3.400 g of potassium dihydrogen phosphate to 250 mL of pure water, followed by sonication.
  • Solution A was placed on a stirrer and Solution B was added slowly into Solution A until the pH reached 7.4.
  • 10 mM NADPH solution was prepared, fresh prior to use, by dissolving nicotinamide adenine dinucleotide phosphate (NADPH) at 8.334 mg/mL in phosphate buffer.
  • NADPH nicotinamide adenine dinucleotide phosphate
  • master solution was prepared according to Table 9. The incubation was carried out in 96 deep well plates. The following volumes were dispensed into each well of the incubation plate, ⁇ / ⁇ RI ⁇ WKH ⁇ VXEVWUDWH ⁇ DQG ⁇ human liver microsomes (HLM) PL[WXUH ⁇ LQ ⁇ SKRVSKDWH ⁇ EXIIHU ⁇ / ⁇ RI ⁇ the compound working solution, or vehicle (mixture of DMSO and acetonitrile (1:4)).
  • HLM human liver microsomes
  • the incubation plate was placed into the water bath and pre-warmed at 37°C for 15 minutes before the reactions ZHUH ⁇ VWDUWHG ⁇ E ⁇ WKH ⁇ DGGLWLRQ ⁇ RI ⁇ / ⁇ RI ⁇ P0 ⁇ 1$'3+ ⁇ VROXWLRQ ⁇ LQ ⁇ SKRVSKDWH ⁇ EXIIHU ⁇ After the addition of NADPH, the incubation plate was incubated at 37°C for the corresponding time. The assay was performed in duplicate. Table 9. B uffer Stock Final C oncentration Volume Concentration [0645] DFHWRQLWULOH ⁇ containing 3% formic acid and internal standards (200 nM Labetalol, 200 nM Alprazolam and 200 nM tolbutamide).
  • Example 8 Permeability Study [0650] Preparation of MDCK-MDR1 Cells [0651] 50 ⁇ Land25mL of cell culture medium were added to each well of the Transwell ® insert and reservoir, respectively. The HTS Transwell ® plates were incubated at 37 °C, 5% CO2 for 1 hour before cell seeding. [0652] MDCK-MDR1 cells were diluted to 56x10 6 cells/mL withculturemediumand50 ⁇ Lof cell suspension were dispensed into the filter well of the 96-well HTS Transwell plate.
  • the MDCK-MDRI plate was removed from the incubator and washed twice with prewarmed Hanks * Balanced Salt solution (HBSS) (10 mM HEPES, pH 7.4), and then incubated at 37 °C for 30 minutes.
  • HBSS Balanced Salt solution
  • the stock solutions of control compounds were diluted in DMSO to get 200 mM solutions and then diluted with HBSS (10 rnM HEPES, pH 7.4) to get 1 mM working solutions.
  • the test compounds were diluted in DMSO to get 200 mM solutions and then diluted with HBSS (10 rnM HEPES with 4% BSA, pH 7.4) to get 1 mM working solutions.
  • the final concentration of DMSO in Ore incubation system was 0.5%.
  • Lucifer Yellow leakage after 2-hour transport period stock solution of Lucifer yellow was prepared in DMSO and diluted with BBSS (10 mM HEPES, pH 7.4) to reach the final concentration of 100 pM. 100 pL of the Lucifer yellow solution was added to each Transwell insert (apical compartment), followed by filling the wells in the receiver plate (basolateral compartment) with 300 pL of BBSS (10 mM HEPES, pH 7.4). The plates were Incubated at. 37 °C for 30 mins. 80 pl, samples were removed directly from the apical and basolateral wells (using the basolateral access holes) and transferred to wells of new 96 wells plates. The Lucifer Yellow 7 fluorescence (to monitor monolayer integrity) signal was measured in a fluorescence plate reader at 480 nM excitation and 530 nM emission.
  • test compound and the positive control were diluted to 100 uM by combining 198 pL of 50% acetonitrile / 50% water and 2 pL of 10 mM stock.
  • Incubation medium (William’s E Medium supplemented with GlutaMAX jM ) and hepatocyte thawing medium w-ere placed in a 37 °C water bath, and allowed to warm for at least 15 minutes prior to use.
  • a vial of cryopreserved hepatocytes was transferred from storage, ensuring that vials remain at cryogenic temperatures until thawing process ensued.
  • the cells w-ere thawed by placing the vial in a 37°C water bath and gently shaking the vials for 2 minutes. After thawing was completed, the vial was sprayed with 70% ethanol, and transferred to a biosafety cabinet.
  • fhe hepatocytes were transferred into 50 mL conical tube containing thawing medium.
  • the 50 mL conical tube was placed into a centrifuge and spun at 100 g for 10 minutes. Upon completion of spin, the thawing medium was aspirated and the hepatocytes resuspended in enough incubation medium to yield ⁇ 1.5 x 106 cells/mL.
  • AO/PI Staining was used to count cells and determine the viable cell density, after which, the cells were diluted with incubation medium to a working cell density of 0.5 x ] 06 viable cells/mL.
  • Table 11 shows scaling factors for intrinsic clearance prediction in human, monkey, dog, rat and mouse hepatocytes. Table 11. H epatocellularity Liver weight Scaling factors Liver blood Species flow [0684] The rules for data processing are shown in Table 12. Table 12. Remaining % Processing Rules If Tt t ith ⁇ 005 i bti d t th l ltd Clit l [0685 ] p Table 13.
  • T reatment group hamster RNA Genome TCID50/mg % Weight change I D copies/mg lung lung on d4 ⁇ from d0 sing the following histopathologic markers: congestion (column A), intracellular heme (column B), lymphoid follicles (column C), apoptotic bodies in bronchus wall (column D), necrotizing bronchiolitis (column E), perivascular edema (column F), bronchopneumonia (column G), perivascular inflammation (column H), peribronchial inflammation (column I), and vasculitis (column J).
  • Figure 6 is a plot of the cumulative lung score for each dose group and the control.
  • the individual lung scores shown in Table 16 were used to calculate the cumulative lung score.
  • a blank box in the table indicates a score of 0.
  • a reduction in cumulative lung score was seen in treated hamster lung histopathology.
  • Biological Example 11 Antiviral effects of PIKfyve inhibitors in a Vero-E6 SARS- CoV-2 cytopathic assay
  • Materials and Methods [0699] To determine the half maximal inhibitory concentration (IC50) of the compounds against SARS-CoV-2, anti-viral screening was performed against two (2) SARS-CoV-2 strains, Wildtype (WT) and Delta (D).
  • IC50 half maximal inhibitory concentration
  • WT Wildtype
  • D Delta
  • Adherent Vero-E6 cells were seeded in multi-well plates. Cell cultures were inoculated with a standardized amount of virus, in the absence and presence of serial compound dilutions, followed by 18-24 hours of incubation and an appropriate virus detection method (e.g., Immunostaining).

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Abstract

La présente divulgation concerne des composés qui sont utiles pour le traitement de certaines infections à coronavirus.
PCT/US2022/032465 2021-06-08 2022-06-07 Procédés et traitement d'infection virale avec des pyrazolo-pyrimidines substituées WO2022261069A1 (fr)

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WO2024168016A1 (fr) * 2023-02-10 2024-08-15 Verge Analytics, Inc. Pyrazolo-pyrimidines substituées et leurs utilisations

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WO2011031979A1 (fr) * 2009-09-11 2011-03-17 Cylene Pharmaceuticals Inc. Lactames substitués par hétérocycle pharmaceutiquement utiles

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WO2011031979A1 (fr) * 2009-09-11 2011-03-17 Cylene Pharmaceuticals Inc. Lactames substitués par hétérocycle pharmaceutiquement utiles

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024168016A1 (fr) * 2023-02-10 2024-08-15 Verge Analytics, Inc. Pyrazolo-pyrimidines substituées et leurs utilisations

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