WO2023211850A1 - Kcnt1 inhibitors comprising an isoxazole or oxadiazole core and methods of use - Google Patents

Kcnt1 inhibitors comprising an isoxazole or oxadiazole core and methods of use Download PDF

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WO2023211850A1
WO2023211850A1 PCT/US2023/019648 US2023019648W WO2023211850A1 WO 2023211850 A1 WO2023211850 A1 WO 2023211850A1 US 2023019648 W US2023019648 W US 2023019648W WO 2023211850 A1 WO2023211850 A1 WO 2023211850A1
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compound
mmol
formula
disorder
disorder associated
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PCT/US2023/019648
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French (fr)
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Andrew Mark Griffin
Gabriel Martinez Botella
Ricardo Lira
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Praxis Precision Medicines, Inc.
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Publication of WO2023211850A1 publication Critical patent/WO2023211850A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D413/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms
    • C07D413/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing three or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D413/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms
    • C07D413/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings
    • C07D413/04—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings directly linked by a ring-member-to-ring-member bond

Definitions

  • the present disclosure is generally directed to KCNT1 inhibitors canprising an isoxazole core or an oxadiazole core, as well as pharmaceutical compositions and methods of treatment involving the use of such compounds.
  • KCNT1 Potassium sodium-activated channel subfamily T member 1
  • Slack sodium-activated potassium channels known as Slack (Sequence like a calcium- activated K + channel). These channels are found in neurons throughout the brain and can mediate a sodium-activated potassium current IkNa. This delayed outward current can regulate neuronal excitability and the rate of adaptation in response to maintained stimulation. Abnormal Slack activity has been associated with development of early onset epilepsies and intellectual impairment.
  • pharmaceutical compounds that selectively regulate sodium-activated potassium channels e.g., abnormal KCNT1 or abnormal IkNa
  • KCNT1 or abnormal IkNa are useful in treating a neurological disease or disorder or a disease or condition related to excessive neuronal excitability and/or KCNT1 gain-of-function mutations.
  • KCNT1 inhibitors and their preparation are disclosed, for example, in WO 2021/195066, incorporated herein by reference in its entirety.
  • WO 2021/195066 discloses, for example, compounds having the Formula A: wherein X is CR 7 or N and Y is S; or
  • X is CR 7 and Y is O;
  • ring A is selected from the group consisting of phenyl, 6-membered heteroaryl, and 5- 7 membered heteroaryl;
  • R 1 is selected from the group consisting of phenyl, 5-6 membered heteroaryl, -CH 2- phenyl, 5-8 membered carbocyclyl, and 5-10 membered heterocyclyl; wherein the phenyl 5-6 membered heteroaryl, -CH2-phenyl, 5-8 membered carbocyclyl, and 5-10 membered heterocyclyl is optionally substituted with one or more R 6 ;
  • R 2 is hydrogen or C 1 -ealkyl
  • R 3 is selected from the group consisting of hydrogen, C 1 -ealkyl, C 1 -ehaloalkyl, C 1- ealkoxy, C 1-6 haloalkoxy; and C 3-8 cycloakyl, wherein the C 1 -ealkyl is optionally substituted with C 1- ealkoxy or C 1 -ehaloalkoxy, and R 4 is hydrogen; or
  • R 3 and R 4 can be taken together with the carbon attached to R 3 and R 4 to form a C3- scycloakylene or 3-7 membered heterocycloalkylene;
  • R 5 and Re are each independently selected from the group consisting of halogen, C 1- ealkyl, C 1 -ealkylene-O-C 1 -ealkyl, C 1 -ehaloalkyl, C 1 -ealkoxy, C 1 -ehaloalkoxy, -S(O) 2 R 8 , -S(O) 2 - N(R 9 ) 2 , and C 3-8 cycloalkyl;
  • R7 is selected from the group consisting of hydrogen, C 1 -ealkyl, and C 1 -ehaloalkyl;
  • R 9 is hydrogen or C 1 -ealkyl; each R 9 is independently selected from the group consisting of hydrogen, C 1 -ealkyl, and -(C 1 -ealkylene)-OH, or the two R 9 can be taken together with the nitrogen atom attached to the two R 9 to form a heterocycle optionally substituted with one or more substituents each independently selected from halogen and -OH; and n is selected from the group consisting of 0, 1, 2, and 3; provided that when R 3 is hydrogen and ring A is a 6-membered heterocyclyl or 6-membered heteroaryl, R 1 is not thiophene; and provided that when R 3 is hydrogen and ring A is a 6-membered heteroaryl or 5- membered heterocyclyl, R 1 is not phenyl; or a pharmaceutically acceptable salt thereof.
  • WO 2021/195066 also discloses several subgenera of Formula (A), including, for example, a compound of Formula I-IB: or a pharmaceutically acceptable salt thereof.
  • KCNT1 inhibitors and their preparation are disclosed, for example, in WO 2020/227101, incorporated herein by reference in its entirety.
  • WO 2020/227101 discloses, for example, compounds having the Formula (I): wherein X, Y, Z, Y’, and Z’ are each independently selected from CH and N, wherein the hydrogen of CH may be substituted with Rs, wherein at least 3 selected from X, Y, Z, Y’, and Z’ are CH; R 1 is selected from the group consisting of C 1-6 alkyl, C 3-10 cycloalkyl, 3-10 membered heterocyclyl, 3-10 membered heteroaryl, and phenyl, wherein C 1-6 alkyl, C 3-10 cycloalkyl, 3-10 membered heterocyclyl, 3-10 membered heteroaryl, or phenyl is optionally substituted with one or more substituents each independently selected from the group consisting of halogen, C(O)N(R 9 ) 2 , N
  • R12 is selected from the group consisting of C 1-6 alkyl, C 3-10 cycloalkyl, 3-10 membered heterocyclyl, 3-10 membered heteroaryl, and phenyl, wherein the C 1-6 alkyl, C 3-10 cycloalkyl, 3-10 membered heterocyclyl, 3-10 membered heteroaryl, or phenyl is optionally substituted with one or more substituents each independently selected from the group consisting of halogen, -OH, -CN, C 1-6 alkyl, C 1-6 haloalkyl, and C 1-6 alkoxy; or two R12 on adjacent carbons can be taken together with the two carbons where R 12 are attached to form a carbocyclic ring; x is 0, 1, or 2;
  • R2 is hydrogen or C 1-4 alkyl
  • R 3 is selected from the group consisting of hydrogen, C 1-6 alkyl, C 3-10 cycloalkyl, 3-10 membered heterocyclyl, 3-10 membered heteroaryl, and phenyl
  • R 4 is selected from C 1-6 alkyl and hydrogen; or R 3 and R 4 can be taken together with the carbon attached to R 3 and R 4 to form a C 3-7 cycloalkylene or 3-7 membered heterocyclene; wherein the C 1-6 alkyl, C 3-10 cycloalkyl, 3-10 membered heterocyclyl, 3-10 membered heteroaryl, phenyl, C 3-7 Cycloalkylene, or 3-7 membered heterocyclene may be optionally substituted with one or more R7; each R 5 is independently selected from the group consisting of halogen, C 1-6 alkyl, C 1- 6 haloalkyl, C 1-6 alkylene-N(R 9 ) 2 , C 1-6
  • R7 is each independently selected from the group consisting of phenyl, C 1-6 alkoxy, - OH, -N(R 9 ) 2 , -NR 9 -SO 2 -Cl-6alkyl, -O-(C 1-6 alkylene)-phenyl, C 3-10 cycloalkyl, -C(O)ORs, - C(O)N(R 9 ) 2 ,-NR 10 C(O)-RII, -CN, -S(O) 2 -C 1-6 alkyl, -S(O) 2 - N(R 9 ) 2 , 3-10 membered heterocyclyl, and 3-10 membered heteroaryl, wherein the phenyl, C 3-10 cycloalkyl, 3-10 membered heterocyclyl, or 3-10 membered heteroaryl is optionally substituted with one or more substituents each independently selected from the group consisting of C 1-6 alkyl, halogen, -OH, C 1-6 al
  • R 8 is hydrogen or C 1-6 alkyl; each R 9 is independently selected from the group consisting of hydrogen, C 1-6 alkyl, and -(C 1-6 alkylene )-OH, or the two R 9 can be taken together with the nitrogen atom attached to the two R 9 to form a heterocycle optionally substituted with one or more substituents each independently selected from halogen and -OH; each R 10 is independently hydrogen or C 1-6 alkyl;
  • R11 is selected from the group consisting of C 1-6 alkyl, C 1-6 alkoxy, and -O-(C 1- 6alkylene)-phenyl; and when R 3 and R 4 are both hydrogen, at least one selected from X, Y, Z, Y’, and Z’ is N, or a pharmaceutically acceptable salt thereof, and numerous species thereof.
  • Described herein are compounds and compositions useful for preventing and/or treating a disease, disorder, or condition, e g., a neurological disorder, a disorder associated with excessive neuronal excitability, or disorder associated with a gain-of-function mutation in a gene, for example, KCNT1.
  • a disease, disorder, or condition e g., a neurological disorder, a disorder associated with excessive neuronal excitability, or disorder associated with a gain-of-function mutation in a gene, for example, KCNT1.
  • a compound of Formula (I-IB) as described in WO 2021/195066 having an isoxazole core and chosen from:
  • a compound of general Formula (I) having an oxadiazole core as described in WO 2020/227101, and chosen from: or a pharmaceutically acceptable salt thereof.
  • provided herein is a compound of Formula (II) having an oxadiazole core: or a pharmaceutically acceptable salt thereof.
  • a compound of Formula (III) having an oxadiazole core: or a pharmaceutically acceptable salt thereof is provided herein.
  • a compound of Formula (IV) having an oxadiazole core or a pharmaceutically acceptable salt thereof.
  • a compound of Formula (V) having an oxadiazole core or a pharmaceutically acceptable salt thereof.
  • a compound of Formula (VI) having an oxadiazole core or a pharmaceutically acceptable salt thereof.
  • R 1 is chosen from -H or a C 1-6 alkyl, such a methyl
  • R 2 is chosen from -H or a C 1-6 alkyl, such a methyl
  • R 3 is independently chosen from a halogen, a C 1-6 alkyl, a carbocyclyl, or an alkoxy, wherein the C 1-6 alkyl, carbocyclyl or alkoxy optionally comprises at least one halogen substituent; and n is 0, 1, 2, 3, or 4.
  • a method of treating a neurological disorder, a disorder associated with excessive neuronal excitability, or a disorder associated with a gain-of-function mutation of a gene by administering to a subject in need thereof an effective amount of a compound described herein having an isoxazole core, such as a compound of Formula (I-IB) [e.g., Formulae (I-IBa) - (I-IBt)] or Formula (VII) [e.g., Formula (Vll-a) - (VII-f)], or a pharmaceutically acceptable salt thereof, or pharmaceutical compositions described herein comprising such compounds or a pharmaceutically acceptable salt thereof.
  • a compound described herein having an isoxazole core such as a compound of Formula (I-IB) [e.g., Formulae (I-IBa) - (I-IBt)] or Formula (VII) [e.g., Formula (Vll-a) - (VII-f)] or a pharmaceutically acceptable salt
  • a method of treating a neurological disorder, a disorder associated with excessive neuronal excitability, or a disorder associated with a gain-of-function mutation of a gene by administering to a subject in need thereof an effective amount of a compound described herein having an oxadiazole core, such as a compound of Formula (I-a), Formula (I-b), Formula (I-c), Formula (I-d), Formula (II), Formula (III), Formula (IV), Formula (V), or Formula (VI), or a pharmaceutically acceptable salt thereof, or pharmaceutical compositions described herein comprising such compounds or a pharmaceutically acceptable salt thereof.
  • a compound described herein having an oxadiazole core such as a compound of Formula (I-a), Formula (I-b), Formula (I-c), Formula (I-d), Formula (II), Formula (III), Formula (IV), Formula (V), or Formula (VI), or a pharmaceutically acceptable salt thereof, or pharmaceutical compositions described herein comprising such compounds or a pharmaceutically acceptable salt thereof.
  • the method provided involves treating a disorder associated with a gain-of-function mutation of KCNT1.
  • the neurological disorder, the disorder associated with excessive neuronal excitability, or the disorder associated with a gain-of-function mutation of a gene is epilepsy, an epilepsy syndrome, or an encephalopathy.
  • the neurological disorder, the disorder associated with excessive neuronal excitability, or the disorder associated with a gain-of-function mutation of a gene is a genetic or pediatric epilepsy or a genetic or pediatric epilepsy syndrome.
  • the neurological disorder, the disorder associated with excessive neuronal excitability, or the disorder associated with a gain-of-function mutation of a gene is a cardiac dysfunction.
  • the neurological disorder, the disorder associated with excessive neuronal excitability, or the disorder associated with a gain-of-function mutation of a gene is selected from the group consisting of epilepsy and other encephalopathies (e.g., malignant migrating focal seizures of infancy (MMFSI) or epilepsy of infancy with migrating focal seizures (EIMFS), autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE), West syndrome, infantile spasms, epileptic encephalopathy, focal epilepsy, Ohtahara syndrome, developmental and epileptic encephalopathy, Lennox-Gastaut syndrome, seizures (e.g., Generalized tonic clonic seizures, Asymmetric Tonic Seizures), leukodystrophy, leukoencephalopathy, intellectual disability, Multifocal Epilepsy, Drug resistant epilepsy, Temporal lobe epilepsy, or cerebellar ataxia.
  • epilepsy and other encephalopathies e.g., mal
  • the neurological disorder, the disorder associated with excessive neuronal excitability, or the disorder associated with a gain-of-function mutation of a gene is chosen from cardiac arrhythmia, Brugada syndrome, or myocardial infarction.
  • the neurological disorder, the disorder associated with excessive neuronal excitability, or the disorder associated with a gain-of-function mutation of a gene is selected from pain and related conditions (e.g., neuropathic pain, acute/chronic pain, migraine).
  • the neurological disorder, the disorder associated with excessive neuronal excitability, or the disorder associated with a gain-of-function mutation of a gene is a muscle disorder (e.g., myotonia, neuromyotonia, cramp muscle spasms, spasticity).
  • the neurological disorder, the disorder associated with excessive neuronal excitability, or the disorder associated with a gain-of-function mutation of a gene is selected from itch and pruritis, ataxia, or cerebellar ataxias.
  • the neurological disorder, the disorder associated with excessive neuronal excitability, or the disorder associated with a gain-of-function mutation of a gene is a psychiatric disorder (e.g., major depression, anxiety, bipolar disorder, schizophrenia).
  • the neurological disorder, the disorder associated with excessive neuronal excitability, or the disorder associated with a gain-of-function mutation in a gene is chosen from a learning disorder, Fragile X, neuronal plasticity, or an autism spectrum disorder.
  • the neurological disorder, the disorder associated with excessive neuronal excitability, or the disorder associated with a gain-of-function mutation of a gene is chosen from epileptic encephalopathy with SCN1A, SCN2A, and/or SCN8A mutations, early infantile epileptic encephalopathy, Dravet syndrome, Dravet syndrome with SCN1A mutation, generalized epilepsy with febrile seizures, intractable childhood epilepsy with generalized tonic-clonic seizures, infantile spasms, benign familial neonatal-infantile seizures, SCN2A epileptic encephalopathy, focal epilepsy with SCN3A mutation, cryptogenic pediatric partial epilepsy with SCN3 A mutation, SCN8A epileptic encephalopathy, Rasmussen encephalitis, malignant migrating partial seizures of infancy, autosomal dominant nocturnal frontal lobe epilepsy, KCNQ2 epileptic encephal
  • FIG. 1 is a graph showing the mean response in terms of percent inhibition of K NA 1.1 for varying concentrations of Formula (I) in various species for KCNT1 wild type and gain-of- function variants, as described in Example 3.
  • compositions useful for preventing and/or treating a disease, disorder, or condition described herein e.g., a neurological disorder, a disorder associated with excessive neuronal excitability, or a disorder associated with gain-of-function mutations in a gene (e.g., KCNT1).
  • a disease, disorder, or condition described herein e.g., a neurological disorder, a disorder associated with excessive neuronal excitability, or a disorder associated with gain-of-function mutations in a gene (e.g., KCNT1).
  • Exemplary diseases, disorders, or conditions include epilepsy and other encephalopathies (e.g., MMFSI or EIMFS, ADNFLE, West syndrome, infantile spasms, epileptic encephalopathy, focal epilepsy, Ohtahara syndrome, developmental and epileptic encephalopathy, Lennox-Gastaut syndrome, seizures, leukodystrophy, leukoencephalopathy, Intellectual disability, Multifocal Epilepsy, Generalized tonic clonic seizures, Drug resistant epilepsy, Temporal lobe epilepsy, cerebellar ataxia, Asymmetric Tonic Seizures); cardiac dysfunctions (e.g., cardiac arrhythmia, Brugada syndrome, myocardial infarction); pain and related conditions (e.g., neuropathic pain, acute/chronic pain, migraine, etc.); muscle disorders (e.g., myotonia, neuromyotonia, cramp muscle spasms, spasticity); itch and pruritis; ataxia and cerebell
  • ranges excluding either or both of those included limits are also included in the disclosure.
  • two opposing and open-ended ranges are provided for a feature, and in such description it is envisioned that combinations of those two ranges are provided herein.
  • a feature is greater than about 10 units, and it is described (such as in another sentence) that the feature is less than about 20 units, and thus, the range of about 10 units to about 20 units is described herein.
  • analogue means one analogue or more than one analogue.
  • C 1-6 alkyl is intended to encompass, C 1 , C 2 , C 3 , C 4 , C 5 , C 6 , C 1-6 , C 1-5 , C 1-4 , C 1-3 , C 1-2 , C 2-6 , C 2-5 , C 2-4 , C 2-3 , C 3-6 , C 3-5 , C 3-4 , C 4-6 , C 4-5 , and C 5-6 alkyl.
  • Alkyl refers to a radical of a straight-chain or branched saturated hydrocarbon group, e.g., having 1 to 20 carbon atoms (“C1-20 alkyl”). In some embodiments, an alkyl group has 1 to 10 carbon atoms (“C1-10 alkyl”). In some embodiments, an alkyl group has 1 to. 9 carbon atoms (“C 1-9 alkyl”). In some embodiments, an alkyl group has 1 to 8 carbon atoms (“C 1-8 alkyl”). In some embodiments, an alkyl group has 1 to 7 carbon atoms (“C1-7 alkyl”). In some embodiments, an alkyl group has 1 to 6 carbon atoms (“ C 1-6 alkyl”).
  • an alkyl group has 1 to 5 carbon atoms (“ C 1-5 alkyl”). In some embodiments, an alkyl group has 1 to 4 carbon atoms (“ C 1-4 alkyl”). In some embodiments, an alkyl group has 1 to 3 carbon atoms (“ C 1-3 alkyl”). In some embodiments, an alkyl group has 1 to 2 carbon atoms (“C1-2 alkyl”). In some embodiments, an alkyl group has 1 carbon atom (“ C 1 alkyl”). Examples of C 1-6 alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, pentyl, hexyl, and the like.
  • alkenyl refers to a radical of a straight-chain or branched hydrocarbon group having from 2 to 20 carbon atoms, one or more carbon-carbon double bonds (e.g., 1, 2, 3, or 4 carbon-carbon double bonds), and optionally one or more carbon-carbon triple bonds (e.g., 1, 2, 3, or 4 carbon-carbon triple bonds) (“C 2-20 alkenyl”). In certain embodiments, alkenyl does not contain any triple bonds. In some embodiments, an alkenyl group has 2 to 10 carbon atoms (“C 2- 10 alkenyl”). In some embodiments, an alkenyl group has 2 to 9 carbon atoms (“C 2-9 alkenyl”).
  • an alkenyl group has 2 to 8 carbon atoms (“C 2-8 alkenyl”). In some embodiments, an alkenyl group has 2 to 7 carbon atoms (“C 2-7 alkenyl”). In some embodiments, an alkenyl group has 2 to 6 carbon atoms (“C2-6 alkenyl”). In some embodiments, an alkenyl group has 2 to 5 carbon atoms (“C2-5 alkenyl”). In some embodiments, an alkenyl group has 2 to 4 carbon atoms (“C2-4 alkenyl”). In some embodiments, an alkenyl group has 2 to 3 carbon atoms (“C2-3 alkenyl”). In some embodiments, an alkenyl group has 2 carbon atoms (“C2 alkenyl”).
  • the one or more carbon-carbon double bonds can be internal (such as in 2-butenyl) or terminal (such as in 1- butenyl).
  • Examples of C2-4 alkenyl groups include ethenyl (C2), 1 -propenyl (C3), 2-propenyl (C3), 1-butenyl (C4), 2-butenyl (C4), butadienyl (C4), and the like.
  • Examples of C2-6 alkenyl groups include the aforementioned C24 alkenyl groups as well as pentenyl (C 5 ), pentadienyl (C 5 ), hexenyl (C 6 ), and the like.
  • Additional examples of alkenyl include heptenyl (C 7 ), octenyl (C 8 ), octatrienyl (C 8 ), and the like.
  • Alkynyl refers to a radical of a straight-chain or branched hydrocarbon group having from 2 to 20 carbon atoms, one or more carbon-carbon triple bonds (e.g., 1, 2, 3, or 4 carbon-carbon triple bonds), and optionally one or more carbon-carbon double bonds (e.g., 1, 2, 3, or 4 carbon-carbon double bonds) (“C2-20 alkynyl”). In certain embodiments, alkynyl does not contain any double bonds. In some embodiments, an alkynyl group has 2 to 10 carbon atoms (“C2- 10 alkynyl”). In some embodiments, an alkynyl group has 2 to 9 carbon atoms (“C 2-9 alkynyl”).
  • an alkynyl group has 2 to 8 carbon atoms (“C2-8 alkynyl”). In some embodiments, an alkynyl group has 2 to 7 carbon atoms (“C2-7 alkynyl”). In some embodiments, an alkynyl group has 2 to 6 carbon atoms (“C2-6 alkynyl”). In some embodiments, an alkynyl group has 2 to 5 carbon atoms (“C2-5 alkynyl”). In some embodiments, an alkynyl group has 2 to 4 carbon atoms (“C2-4 alkynyl”). In some embodiments, an alkynyl group has 2 to 3 carbon atoms (“C2-3 alkynyl”).
  • an alkynyl group has 2 carbon atoms (“C2 alkynyl”).
  • the one or more carbon-carbon triple bonds can be internal (such as in 2-butynyl) or terminal (such as in 1- butynyl).
  • Examples of C24 alkynyl groups include, without limitation, ethynyl (C2), 1-propynyl (C3), 2-propynyl (C3), 1-butynyl (C4), 2-butynyl (C4), and the like.
  • Examples of C2-6 alkenyl groups include the aforementioned C24 alkynyl groups as well as pentynyl (C 5 ), hexynyl (C 6 ), and the like. Additional examples of alkynyl include heptynyl (C 7 ), octynyl (C 8 ), and the like.
  • Aryl refers to a radical of a monocyclic or polycyclic (e.g., bicyclic or tricyclic) 4n+2 aromatic ring system (e.g., having 6, 10, or 14 ⁇ electrons shared in a cyclic array) having 6- 14 ring carbon atoms and zero heteroatoms provided in the aromatic ring system (“C 6-14 aryl”).
  • an aryl group has six ring carbon atoms (“C 6 aryl”; e.g., phenyl).
  • an aryl group has ten ring carbon atoms (“C 10 aryl”; e.g., naphthyl such as 1- naphthyl and 2-naphthyl).
  • an aryl group has fourteen ring carbon atoms (“C 14 aryl”; e.g., anthracyl).
  • Aryl also includes ring systems wherein the aryl ring, as defined above, is fused with one or more carbocyclyl or heterocyclyl groups wherein the radical or point of attachment is on the aryl ring, and in such instances, the number of carbon atoms continue to designate the number of carbon atoms in the aryl ring system.
  • Typical aryl groups include, but are not limited to, groups derived from aceanthrylene, acenaphthylene, acephenanthrylene, anthracene, azulene, benzene, chrysene, coronene, fluoranthene, fluorene, hexacene, hexaphene, hexalene, as-indacene, s-indacene, indane, indene, naphthalene, octacene, octaphene, octalene, ovalene, penta-2,4-diene, pentacene, pentalene, pentaphene, perylene, phenalene, phenanthrene, picene, pleiadene, pyrene, pyranthrene, rubicene, triphenylene, and trinaphthalene.
  • Particularly aryl groups include pheny
  • Hetero when used to describe a compound or a group present on a compound means that one or more carbon atoms in the compound or group have been replaced by a nitrogen, oxygen, or sulfur heteroatom. Hetero may be applied to any of the alkyl groups described above such as alkyl, e.g., heteroalkyl; alkenyl, e.g., heteroalkenyl; alkynyl, e.g., heteroalkynyl; carbocyclyl, e.g., heterocyclyl; aryl, e.g., heteroaryl, and the like having from 1 to 5, and particularly from 1 to 3 heteroatoms.
  • alkyl e.g., heteroalkyl
  • alkenyl e.g., heteroalkenyl
  • alkynyl e.g., heteroalkynyl
  • carbocyclyl e.g., heterocyclyl
  • aryl e.g., heteroaryl, and the like having from
  • Heteroaryl refers to a radical of a 5-10 membered monocyclic or bicyclic 4n+2 aromatic ring system (e.g., having 6 or 10 ⁇ electrons shared in a cyclic array) having ring carbon atoms and 1-4 ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen and sulfur (“5-10 membered heteroaryl”).
  • the point of attachment can be a carbon or nitrogen atom, as valency permits.
  • Heteroaryl bicyclic ring systems can include one or more heteroatoms in one or both rings.
  • Heteroaryl includes ring systems wherein the heteroaryl ring, as defined above, is fused with one or more carbocyclyl or heterocyclyl groups wherein the point of attachment is on the heteroaryl ring, and in such instances, the number of ring members continue to designate the number of ring members in the heteroaryl ring system. “Heteroaryl” also includes ring systems wherein the heteroaryl ring, as defined above, is fused with one or more aryl groups wherein the point of attachment is either on the aryl or heteroaryl ring, and in such instances, the number of ring members designates the number of ring members in the fused (aryl/heteroaryl) ring system.
  • Bicyclic heteroaryl groups wherein one ring does not contain a heteroatom e.g., indolyl, quinolinyl, carbazolyl, and the like
  • the point of attachment can be on either ring, i.e., either the ring bearing a heteroatom (e.g., 2-indolyl) or the ring that does not contain a heteroatom (e.g., 5- indolyl).
  • a heteroaryl group is a 5-10 membered aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-10 membered heteroaryl”).
  • a heteroaryl group is a 5-8 membered aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-8 membered heteroaryl”).
  • a heteroaryl group is a 5-6 membered aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-6 membered heteroaryl”).
  • the 5-6 membered heteroaryl has 1-3 ring heteroatoms selected from nitrogen, oxygen, and sulfur.
  • the 5-6 membered heteroaryl has 1-2 ring heteroatoms selected from nitrogen, oxygen, and sulfur.
  • the 5- 6 membered heteroaryl has 1 ring heteroatom selected from nitrogen, oxygen, and sulfur.
  • Carbocyclyl or “carbocyclic” refers to a radical of a non-aromatic cyclic hydrocarbon group having from 3 to 10 ring carbon atoms (“C 3-10 carbocyclyl”) and zero heteroatoms in the non-aromatic ring system.
  • a carbocyclyl group has 3 to 8 ring carbon atoms (“ C 3-8 carbocyclyl”).
  • a carbocyclyl group has 3 to 6 ring carbon atoms (“C 3-6 carbocyclyl”).
  • a carbocyclyl group has 3 to 6 ring carbon atoms (“C 3-6 carbocyclyl”).
  • a carbocyclyl group has 5 to 10 ring carbon atoms (“C5-10 carbocyclyl”).
  • Exemplary C 3-6 carbocyclyl groups include, without limitation, cyclopropyl (C3), cyclopropenyl (C3), cyclobutyl (C4), cyclobutenyl (C 4 ), cyclopentyl (C 5 ), cyclopentenyl (C 5 ), cyclohexyl (C 6 ), cyclohexenyl (C 6 ), cyclohexadienyl (C 6 ), and the like.
  • Exemplary C3-5 carbocyclyl groups include, without limitation, the aforementioned C 3-6 carbocyclyl groups as well as cycloheptyl (C7), cycloheptenyl (C 7 ), cycloheptadienyl (C 7 ), cycloheptatrienyl (C7), cyclooctyl (C 8 ), cyclooctenyl (C 8 ), bicyclo[2.2.1]heptanyl (C 7 ), bicyclo[2.2.2]octanyl (C 8 ), and the like.
  • Exemplary C3-10 carbocyclyl groups include, without limitation, the aforementioned C 3-8 carbocyclyl groups as well as cyclononyl (C9), cyclononenyl (C9), cyclodecyl (C10), cyclodecenyl (C10), octahydro-lH-indenyl (C9), decahydronaphthalenyl (C10), spiro[4.5]decanyl (C10), and the like.
  • the carbocyclyl group is either monocyclic (“monocyclic carbocyclyl”) or contain a fused, bridged or spiro ring system such as a bicyclic system (“bicyclic carbocyclyl”) and can be saturated or can be partially unsaturated.
  • “Carbocyclyl” also includes ring systems wherein the carbocyclyl ring, as defined above, is fused with one or more aryl or heteroaryl groups wherein the point of attachment is on the carbocyclyl ring, and in such instances, the number of carbons continue to designate the number of carbons in the carbocyclic ring system.
  • Heterocyclyl refers to a radical of a 3- to 10-membered non-aromatic ring system having ring carbon atoms and 1 to 4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, sulfur, boron, phosphorus, and silicon (“3-10 membered heterocyclyl”).
  • the point of attachment can be a carbon or nitrogen atom, as valency permits.
  • a heterocyclyl group can either be monocyclic (“monocyclic heterocyclyl”) or a fused, bridged or spire ring system such as a bicyclic system (“bicyclic heterocyclyl”), and can be saturated or can be partially unsaturated.
  • Heterocyclyl bicyclic ring systems can include one or more heteroatoms in one or both rings.
  • Heterocyclyl also includes ring systems wherein the heterocyclyl ring, as defined above, is fused with one or more carbocyclyl groups wherein the point of attachment is either on the carbocyclyl or heterocyclyl ring, or ring systems wherein the heterocyclyl ring, as defined above, is fused with one or more aryl or heteroaryl groups, wherein the point of attachment is on the heterocyclyl ring, and in such instances, the number of ring members continue to designate the number of ring members in the heterocyclyl ring system.
  • a heterocyclyl group is a 5-10 membered non-aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, sulfur, boron, phosphorus, and silicon (“5-10 membered heterocyclyl”).
  • a heterocyclyl group is a 5-8 membered non-aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-8 membered heterocyclyl”).
  • a heterocyclyl group is a 5-6 membered non-aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-6 membered heterocyclyl”).
  • the 5-6 membered heterocyclyl has 1-3 ring heteroatoms selected from nitrogen, oxygen, and sulfur.
  • the 5-6 membered heterocyclyl has 1-2 ring heteroatoms selected from nitrogen, oxygen, and sulfur.
  • the 5-6 membered heterocyclyl has one ring heteroatom selected from nitrogen, oxygen, and sulfur.
  • Exemplary 3 -membered heterocyclyl groups containing one heteroatom include, without limitation, azirdinyl, oxiranyl, thiorenyl.
  • Exemplary 4-membered heterocyclyl groups containing one heteroatom include, without limitation, azetidinyl, oxetanyl and thietanyl.
  • Exemplary 5-membered heterocyclyl groups containing one heteroatom include, without limitation, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothiophenyl, dihydrothiophenyl, pyrrolidinyl, dihydropyrrolyl and pyrrolyl-2, 5-dione.
  • Exemplary 5-membered heterocyclyl groups containing two heteroatoms include, without limitation, dioxolanyl, oxasulfuranyl, disulfuranyl, and oxazolidin-2-one.
  • Exemplary 5-membered heterocyclyl groups containing three heteroatoms include, without limitation, triazolinyl, oxadiazolinyl, and thiadiazolinyl.
  • Exemplary 6-membered heterocyclyl groups containing one heteroatom include, without limitation, piperidinyl, tetrahydropyranyl, dihydropyridinyl, and thianyl.
  • Exemplary 6-membered heterocyclyl groups containing two heteroatoms include, without limitation, piperazinyl, morpholinyl, dithianyl, dioxanyl.
  • Exemplary 6-membered heterocyclyl groups containing three heteroatoms include, without limitation, triazinanyl.
  • Exemplary 7-membered heterocyclyl groups containing one heteroatom include, without limitation, azepanyl, oxepanyl and thiepanyl.
  • Exemplary 8-membered heterocyclyl groups containing one heteroatom include, without limitation, azocanyl, oxecanyl and thiocanyl.
  • Exemplary 5-membered heterocyclyl groups fused to a C 6 aryl ring include, without limitation, indolinyl, isoindolinyl, dihydrobenzofuranyl, dihydrobenzothienyl, benzoxazolinonyl, and the like.
  • Exemplary 6- membered heterocyclyl groups fused to an aryl ring include, without limitation, tetrahydroquinolinyl, tetrahydroisoquinolinyl, and the like.
  • Cyano refers to -CN.
  • Halo or “halogen” refers to a fluorine atom (i.e., fluoro or -F), a chlorine atom (i.e., chloro or -Cl), a bromine atom (i.e., bromo or -Br), and an iodine atom (i.e., iodo or -I).
  • the halo group is fluoro or chloro.
  • Haloalkyl refers to an alkyl group substituted with one or more halogen atoms.
  • Niro refers to -NO 2 .
  • substituted means that at least one hydrogen present on a group (e.g., a carbon or nitrogen atom) is replaced with a permissible substituent, e.g., a substituent which upon substitution results in a stable compound, e.g., a compound which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, or other reaction.
  • a “substituted” group has a substituent at one or more substitutable positions of the group, and when more than one position in any given structure is substituted, the substituent is either the same or different at each position.
  • pharmaceutically acceptable salt refers to those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio.
  • the general concept of pharmaceutically acceptable salts has been discussed in the art, including, for example, Berge et al., which describes pharmaceutically acceptable salts in detail in J Pharmaceutical Sciences (1977) 66: 1-19.
  • Pharmaceutically acceptable salts of the compounds described herein include those derived from suitable inorganic and organic acids and bases.
  • Examples of pharmaceutically acceptable, nontoxic acid addition salts are salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid or by using other methods used in the art such as ion exchange.
  • inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid
  • organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid or by using other methods used in the art such as ion exchange.
  • salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2- naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pect
  • Pharmaceutically acceptable salts derived from appropriate bases include alkali metal, alkaline earth metal, ammonium and N + (C 1-4 alkyl) 4 salts.
  • Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like.
  • Further pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, lower alkyl sulfonate, and aryl sulfonate.
  • modified-release polymer refers to a polymer that is used in a formulation (e.g., tablets and capsules) to modify the release rate of the drug upon administration to a subject.
  • a modified-release polymer is used to dissolve a drug overtime in order to be released slower and steadier into the bloodstream.
  • a modified-release polymer is a controlled- release polymer.
  • a modified-release polymer or a controlled-release polymer is an HPMC polymer.
  • a modified-release polymer may include hydrophilic matrix polymers (e.g., hypromellose, hydroxyl-propyl methylcellulose (HPMC)), hydrophobic matrix polymers (e.g., ethyl cellulose, ethocel), or polyacrylate polymers (e.g., Eudragit® RL100, Eudragit® RS 100).
  • hydrophilic matrix polymers e.g., hypromellose, hydroxyl-propyl methylcellulose (HPMC)
  • hydrophobic matrix polymers e.g., ethyl cellulose, ethocel
  • polyacrylate polymers e.g., Eudragit® RL100, Eudragit® RS 100.
  • diluent refers to an excipient used to increase weight and improve content uniformity.
  • diluents include cellulose derivatives (e.g., microcrystalline cellulose), starches (e.g., hydrolyzed starches, and partially pregelatinized starches), anhydrous lactose, lactose monohydrate, di-calcium phosphate (DCP), sugar alcohols (e.g., sorbitol, xylitol and mannitol)).
  • glidanf refers to an excipient used to promote powder flow by reducing interparticle friction and cohesion.
  • glidants include fumed silica (e.g., colloidal silicon dioxide), talc, and magnesium carbonate.
  • lubricant refers to an excipient used to prevent ingredients from clumping together and from sticking to the tablet punches or capsule filling machine. Lubricants are also used to ensure that tablet formation and ejection can occur with low friction between the solid and die wall.
  • lubricants include magnesium stearate, calcium stearate, stearic acid, talc, silica, and fats (e.g., vegetable stearin).
  • coating refers to an excipient to protect tablet ingredients from deterioration by moisture in the air and make large or unpleasant-tasting tablets easier to swallow.
  • a compound of Formula (I-b) having an oxadiazole core or a pharmaceutically acceptable salt thereof.
  • a compound of Formula (II) having an oxadiazole core or a pharmaceutically acceptable salt thereof.
  • a compound of Formula (III) having an oxadiazole core: or a pharmaceutically acceptable salt thereof is provided herein.
  • a compound of Formula (IV) having an oxadiazole core: or a pharmaceutically acceptable salt thereof is provided herein.
  • a compound of Formula (V) having an oxadiazole core or a pharmaceutically acceptable salt thereof.
  • R 1 is chosen from -H or a C 1-6 alkyl, such a methyl
  • R 2 is chosen from -H or a C 1-6 alkyl, such a methyl
  • R 3 is independently chosen from a halogen, a C 1-6 alkyl, a carbocyclyl, or an alkoxy, wherein the C 1-6 alkyl, carbocyclyl or alkoxy optionally comprises at least one halogen substituent; and n is 0, 1, 2, 3, or 4.
  • R 1 is methyl
  • R 2 is hydrogen
  • R 3 is chosen from a cyclopropyl
  • a halogen such as bromine, or -CF 3
  • n is 1.
  • Compounds described herein can comprise one or more asymmetric centers, and thus can exist in various isomeric forms, e.g., enantiomers and/or diastereomers.
  • the compounds described herein can be in the form of an individual enantiomer, diastereomer or geometric isomer, or can be in the form of a mixture of stereoisomers, including racemic mixtures and mixtures enriched in one or more stereoisomer.
  • Isomers can be isolated from mixtures by methods known to those skilled in the art, including chiral high pressure liquid chromatography (HPLC) and the formation and crystallization of chiral salts; or preferred isomers can be prepared by asymmetric syntheses.
  • HPLC high pressure liquid chromatography
  • Embodiments disclosed herein additionally encompasses compounds described herein as individual isomers substantially free of other isomers, and alternatively, as mixtures of various isomers.
  • a pure enantiomeric compound is substantially free from other enantiomers or stereoisomers of the compound (i.e., in enantiomeric excess).
  • an “S” form of the compound is substantially free from the “R” form of the compound and is, thus, in enantiomeric excess of the “R” form.
  • enantiomerically pure or “pure enantiomer” denotes that the compound comprises more than 75% by weight, such as more than 80% by weight, more than 85% by weight, more than 90% by weight, more than 91% by weight, more than 92% by weight, more than 93% by weight, more than 94% by weight, more than 95% by weight, more than 96% by weight, more than 97% by weight, more than 98% by weight, more than 98.5% by weight, more than 99% by weight, more than 99.2% by weight, more than 99.5% by weight, more than 99.6% by weight, more than 99.7% by weight, more than 99.8% by weight, or more than 99.9% by weight, of the enantiomer.
  • the weights are based upon total weight of all enantiomers or stereoisomers of the compound.
  • compositions comprising the compounds described herein.
  • an enantiomerically pure compound can be present in the compositions with other active or inactive ingredients.
  • a pharmaceutical composition comprising enantiomerically pure R-compound can comprise, for example, about 90% excipient and about 10% enantiomerically pure R-compound.
  • the enantiomerically pure R-compound in such compositions can, for example, comprise at least about 95% by weight R-compound and at most about 5% by weight S-compound, by total weight of the compound.
  • a pharmaceutical composition comprising enantiomerically pure S- compound can comprise, for example, about 90% excipient and about 10% enantiomerically pure S-compound.
  • the enantiomerically pure S-compound in such compositions can, for example, comprise at least about 95% by weight S-compound and at most about 5% by weight R-compound, by total weight of the compound.
  • the active ingredient can be formulated with little or no excipient or carrier.
  • Compounds described herein may also comprise one or more isotopic substitutions.
  • H may be in any isotopic form, including 1 H, 2 H (D or deuterium), and 3 H (T or tritium); C may be in any isotopic form, including 12 C, 13 C, and 14 C.
  • O may be in any isotopic form, including 16 O and 18 O, and F may be in any isotopic form, including 18 F and 19 F.
  • the compounds and compositions described above and herein can be used to treat a neurological disorder, a disorder associated with excessive neuronal excitability, or a disorder associated with a gain-of-function mutation in a gene (e.g., KCNT1).
  • a neurological disorder e.g., KCNT1
  • a disorder associated with excessive neuronal excitability e.g., KCNT1
  • a disorder associated with a gain-of-function mutation in a gene e.g., KCNT1
  • Exemplary diseases, disorders, or conditions include epilepsy and other encephalopathies (e.g., MMFSI or EIMFS, ADNFLE, West syndrome, infantile spasms, epileptic encephalopathy, developmental and epileptic encephalopathy (DEE), early infantile epileptic encephalopathy (EIEE), generalized epilepsy, focal epilepsy, multifocal epilepsy, temporal lobe epilepsy, Ohtahara syndrome, early myoclonic encephalopathy, Lennox-Gastaut syndrome, drug resistant epilepsy, seizures (e.g., frontal lobe seizures, generalized tonic clonic seizures, asymmetric tonic seizures, focal seizures), leukodystrophy, hypomyelinating leukodystrophy, and leukoencephalopathy), cardiac dysfunctions (e.g., cardiac arrhythmia, Brugada syndrome, myocardial infarction), pulmonary vasculopathy/hemorrhage, pain and related conditions (
  • movement disorders e.g., ataxia and cerebellar ataxias
  • psychiatric disorders e.g., major depression, anxiety, bipolar disorder, schizophrenia, attention-deficit hyperactivity disorder
  • neurodevelopmental disorder e.g., learning disorders, intellectual disability, Fragile X, neuronal plasticity, and autism spectrum disorders.
  • the neurological disorder, the disorder associated with excessive neuronal excitability, or the disorder associated with a gain-of-function mutation in a gene is selected from EIMFS, ADNFLE, or West syndrome.
  • the neurological disorder, the disorder associated with excessive neuronal excitability, or the disorder associated with a gain-of-function mutation in a gene is selected from infantile spasms, epileptic encephalopathy, focal epilepsy, Ohtahara syndrome, developmental and epileptic encephalopathy, or Lennox-Gastaut syndrome.
  • the neurological disorder, the disorder associated with excessive neuronal excitability, or the disorder associated with a gain-of-function mutation in a gene is seizure.
  • the neurological disorder, the disorder associated with excessive neuronal excitability, or the disorder associated with a gain-of-function mutation in a gene is selected from cardiac arrhythmia, Brugada syndrome, or myocardial infarction.
  • the neurological disorder, the disorder associated with excessive neuronal excitability, or the disorder associated with a gain-of-function mutation in a gene is selected from a learning disorder, Fragile X, intellectual function, neuronal plasticity, a psychiatric disorder, or an autism spectrum disorder.
  • the compounds, pharmaceutically acceptable salts thereof, and compositions disclosed herein can be administered to a subject with a neurological disorder, a disorder associated with excessive neuronal excitability, or a disorder associated with a gain-of- function mutation in a gene such as KCNT1 (e.g., EIMFS, ADNFLE, West syndrome, infantile spasms, epileptic encephalopathy, focal epilepsy, Ohtahara syndrome, developmental and epileptic encephalopathy, Lennox-Gastaut syndrome, seizures, cardiac arrhythmia, Brugada syndrome, and myocardial infarction).
  • KCNT1 e.g., EIMFS, ADNFLE, West syndrome, infantile spasms, epileptic encephalopathy, focal epilepsy, Ohtahara syndrome, developmental and epileptic encephalopathy, Lennox-Gastaut syndrome, seizures, cardiac arrhythmia, Brugada syndrome, and myocardial infarction.
  • EIMFS is a rare and debilitating genetic condition characterized by an early onset (before 6 months of age) of almost continuous heterogeneous focal seizures, where seizures appear to migrate from one brain region and hemisphere to another.
  • Patients with EIMFS are generally intellectually impaired, non-verbal and non-ambulatory. While several genes have been implicated to date, the gene that is most commonly associated with EIMFS is KCNT1.
  • mutations may be gain-of-function, missense mutations that are dominant (i.e., present on only one allele) and result in change-in-function of the encoded potassium channel that causes a marked increase in whole cell current when tested in Xenopus oocyte or mammalian expression systems (see e.g. Milligan et al. (2015) Ann Neurol. 75(4): 581-590; Barcia et al. (2012) Nat Genet. 44(11): 1255-1259; and Mikati et al. (2015) Ann Neurol. 78(6): 995-999).
  • ADNFLE has a later onset than EIMFS, generally in mid-childhood, and is generally a less severe condition. It is characterized by nocturnal frontal lobe seizures and can result in psychiatric, behavioral and cognitive disabilities in patients with the condition. While ADNFLE is associated with genes encoding several neuronal nicotinic acetylcholine receptor subunits, mutations in the KCNT1 gene have been implicated in more severe cases of the disease (Heron et al. (2012) Nat Genet. 44: 1188-1190).
  • West syndrome is a severe form of epilepsy composed of a triad of infantile spasms, an interictal electroencephalogram (EEG) pattern termed hypsarrhythmia, and mental retardation, although a diagnosis can be made one of these elements is missing.
  • EEG interictal electroencephalogram
  • Mutations in KCNT1, including G652V and R474H, have been associated with West syndrome (Fukuoka et al. (2017) Brain Dev 39:80-83 and Ohba et al. (2015) Epilepsia 56:el21-el28). Treatment targeting the KCNT1 channel suggests that these mutations are gain-of-function mutations (Fukuoka et al. (2017) Brain Dev 39:80-83).
  • KCNT1 for example, epilepsy and other encephalopathies (e.g., MMFSI or EIMFS), ADNFLE, West syndrome, infantile spasms, epileptic encephalopathy, focal epilepsy, Ohtahara syndrome, DEE, Lennox-Gastaut syndrome, seizures, leukodystrophy, leukoencephalopathy, intellectual disability, Multifocal Epilepsy, Generalized tonic clonic seizures, Drug resistant epilepsy, Temporal lobe epilepsy, cerebellar ataxia, Asymmetric Tonic Seizures), cardiac dysfunctions (e.g., cardiac arrhythmia, Brugada syndrome, myocardial infarction), pain and related conditions (e.g., neuropathic pain, acute/chronic pain, migraine, etc.), muscle disorders (
  • epilepsy and other encephalopathies e.g., MMFSI or EIMFS
  • ADNFLE West syndrome
  • infantile spasms epileptic encephalopathy
  • the subject presenting with a disorder that may be associated with a gain-of-function mutation in KCNT1 is genotyped to confirm the presence of a known gain-of- function mutation in KCNT1 prior to administration of the compounds or a pharmaceutically acceptable salt thereof or compositions disclosed herein.
  • whole exome sequencing can be performed on the subject.
  • Gain-of-function mutations associated with EIMFS may include, but are not limited to, V271F, G288S, R428Q, R474Q, R474H, R474C, I760M, A934T, P924L, G243S, H257D, A259D, R262Q, Q270E, L274I, F346L, C377S, R398Q, P409S, A477T, F502V, M516V, Q550del, K629E, K629N, I760F, E893K, M896K, R933G, R950Q, and KI 154Q.
  • Gain- of-function mutations associated with ADNFLE may include, but are not limited to, M896I, R398Q, Y796H, R928C, and G288S.
  • Gain-of-function mutations associated with West syndrome may include, but are not limited to, G652V and R474H.
  • Gain-of-function mutations associated with temporal lobe epilepsy may include, but are not limited to, R133H and R565H.
  • Gain-of- function mutations associated with Lennox-Gastaut may include, but are not limited to, R209C.
  • Gain-of-function mutations associated with seizures may include, but are not limited to, A259D, G288S, R474C, and R474H.
  • Gain-of-function mutations associated with leukodystrophy may include, but are not limited to, G288S and Q906H.
  • Gain-of-function mutations associated with Multifocal Epilepsy may include, but are not limited to, V340M.
  • Gain-of-function mutations associated with early-onset epilepsy may include, but are not limited to, F346L and A934T.
  • Gain-of-function mutations associated with Early-onset epileptic encephalopathies (EOEE) may include, but are not limited to, R428Q.
  • Gain-of-function mutations associated with developmental and epileptic encephalopathies may include, but are not limited to, F346L, R474H, and A934T.
  • Gain-of-function mutations associated with epileptic encephalopathies may include, but are not limited to, L437F, Y796H, P924L, and R961H.
  • Gain-of-function mutations associated with Early Infantile Epileptic Encephalopathy (EIEE) may include, but are not limited to, M896K.
  • Gain-of- function mutations associated with drug-resistant epilepsy and generalized tonic-clonic seizure may include, but are not limited to, F346L.
  • Gain-of-function mutations associated with migrating partial seizures of infancy may include, but are not limited to, R428Q.
  • Gain-of-function mutations associated with Leukoencephalopathy may include, but are not limited to, F932I.
  • Gain-of-function mutations associated with NFLE may include, but are not limited to, A934T and R950Q.
  • Gain- of-function mutations associated with Ohtahara syndrome may include, but are not limited to, A966T.
  • Gain-of-function mutations associated with infantile spasms may include, but are not limited to, P924L.
  • Gain-of-function mutations associated with Brugada Syndrome may include, but are not limited to, R1106Q.
  • Gain-of-function mutations associated with Brugada Syndrome may include, but are not limited to, R474H.
  • the subject is first genotyped to identify the presence of a mutation in KCNT1, and this mutation is then confirmed to be a gain-of-function mutation using standard in vitro assays, such as those described in Milligan et al. (2015) Ann Neurol. 75(4): 581-590.
  • the presence of a gain-of-function mutation is confirmed when the expression of the mutated KCNT1 allele results an increase in whole cell current compared to the whole cell current resulting from expression of wild-type KCNT1, as may be assessed using whole-cell electrophysiology (such as described in Milligan et al. (2015) Ann Neurol. 75(4): 581-590; Barcia et al. (2012) Nat Genet.
  • This increase of whole cell current can be, for example, an increase of at least or about 50%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, or more.
  • the subject can then be confirmed to have a disease or condition associated with a gain-of- function mutation in KCNT1.
  • the subject is confirmed as having a KCNT1 allele containing a gain-of-function mutation (e.g., V271F, G288S, R398Q, R428Q, R474Q, R474H, R474C, G652V, I760M, Y796H, M896I, P924L, R928C, or A934T).
  • a gain-of-function mutation e.g., V271F, G288S, R398Q, R428Q, R474Q, R474H, R474C, G652V, I760M, Y796H, M896I, P924L, R928C, or A934T.
  • the compounds or pharmaceutically acceptable salts thereof disclosed herein or the pharmaceutical composition disclosed herein can also be used therapeutically for conditions associated with excessive neuronal excitability where the excessive neuronal excitability is not necessarily the result of a gain-of-function mutation in KCNT1. Even in instances where the disease is not the result of increased KCNT1 expression and/or activity, inhibition of KCNT1 expression and/or activity can nonetheless result in a reduction in neuronal excitability, thereby providing a therapeutic effect.
  • the compounds or pharmaceutically acceptable salts thereof disclosed herein or the pharmaceutical compositions disclosed herein can be used to treat a subject with conditions associated with excessive neuronal excitability, for example, epilepsy and other encephalopathies (e.g., EIMFS, ADNFLE, West syndrome, infantile spasms, epileptic encephalopathy, focal epilepsy, Ohtahara syndrome, developmental and epileptic encephalopathy, Lennox-Gastaut syndrome, seizures) or cardiac dysfunctions (e.g., cardiac arrhythmia, Brugada syndrome, myocardial infarction), regardless of whether or not the disorder is associated with a gain-of- function mutation in KCNT1.
  • epilepsy and other encephalopathies e.g., EIMFS, ADNFLE, West syndrome, infantile spasms, epileptic encephalopathy, focal epilepsy, Ohtahara syndrome, developmental and epileptic encephalopathy, Lennox-Gastaut syndrome, seizures
  • cardiac dysfunctions e
  • a “subject” to which administration is contemplated includes, but is not limited to, humans (i.e., a male or female of any age group, e.g., a pediatric subject (e.g., an infant, child, adolescent) or an adult subject (e.g., a young adult, middle-aged adult, or senior adult)) and/or a non-human animal, e.g., a mammal such as primates (e.g., cynomolgus monkeys, rhesus monkeys), cattle, pigs, horses, sheep, goats, rodents, cats, and/or dogs.
  • the subject is a human.
  • the subject is a non-human animal.
  • treating contemplate an action that occurs while a subject is suffering from the specified disease, disorder or condition, which reduces the severity of the disease, disorder or condition, or retards or slows the progression of the disease, disorder or condition (also “therapeutic treatment”).
  • treating refers to a method or procedure for obtaining beneficial or desired results — for example, clinical results.
  • Beneficial or desired results may include: (1) alleviating one or more symptoms caused by or associated with a disease, disorder, or condition; (2) reducing the extent of the disease, disorder, or condition; (3) slowing or stopping the development or progression of one or more symptoms caused by or associated with the disease, disorder, or condition (for example, stabilizing the disease, disorder, or condition); and (4) relieving the disease, for example, by causing the regression of one or more clinical symptoms (e.g., ameliorating the disease state, enhancing the effect of another medication, delaying or stopping the progression of the disease, increasing the quality of life, and/or prolonging survival rates).
  • an “effective amount” of a compound or pharmaceutically acceptable salt thereof refers to an amount sufficient to elicit the desired biological response.
  • the effective amount of a compound or pharmaceutically acceptable salt thereof may vary depending on such factors as the desired biological endpoint, the pharmacokinetics of the compound or pharmaceutically acceptable salt thereof, the disease being treated, the mode of administration, and the age, weight, health, and condition of the subject.
  • a therapeutically effective amount of the compound or pharmaceutically acceptable salt thereof disclosed herein is administered to the subject (e.g., a human).
  • a “therapeutically effective amount” of a compound or pharmaceutically acceptable salt thereof is an amount sufficient to provide a therapeutic benefit in the treatment of a disease, disorder or condition, or to delay or minimize one or more symptoms associated with the disease, disorder or condition.
  • a therapeutically effective amount of a compound or pharmaceutically acceptable salt thereof means an amount of therapeutic agent, alone or in combination with other therapies, which provides a therapeutic benefit in the treatment of the disease, disorder or condition.
  • the term “therapeutically effective amount” can encompass an amount that improves overall therapy, reduces or avoids symptoms or causes of the disease or condition, or enhances the therapeutic efficacy of another therapeutic agent.
  • the method provided involves treating a disorder associated with a gain-of-function mutation of KCNT1.
  • a “disorder associated with a gain-of- function mutation in KCNT1” refers to a disorder that is associated with, is partially or completely caused by, or has one or more symptoms that are partially or completely caused by, a mutation in KCNT1 that results in a gain-of-function phenotype, i.e., an increase in activity of the potassium channel encoded by KCNT1 resulting in an increase in whole cell current.
  • a “gain-of-function mutation of KCNT1” is a mutation in KCNT1 that results in an increase in activity of the potassium channel encoded by KCNT1.
  • Activity can be assessed by, for example, ion flux assay or electrophysiology (e.g., using the whole cell patch clamp technique).
  • a gain-of-function mutation results in an increase of at least or about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 175%, 200%, 225%, 250%, 275%, 300%, 325%, 350%, 375%, 400%, or more compared to the activity of a potassium channel encoded by a wild-type KCNT1.
  • compositions that contain, as the active ingredient, one or more of the compounds described, or a pharmaceutically acceptable salt or ester thereof, and one or more pharmaceutically acceptable excipients, carriers, including inert solid diluents and fillers, diluents, including sterile aqueous solution and various organic solvents, permeation enhancers, solubilizers and adjuvants.
  • the pharmaceutical compositions may be administered alone or in combination with other therapeutic agents.
  • compositions may be prepared in a manner disclosed in the pharmaceutical art, including, for example, in Remington’s Pharmaceutical Sciences, Mace Publishing Co., Philadelphia, Pa. 17th Ed. (1985) and Modern Pharmaceutics, Marcel Dekker, Inc. 3rd Ed. (G. S. Banker & C. T. Rhodes, Eds.).
  • compositions may be administered in either single or multiple doses by any of the accepted modes of administration of agents having similar utilities, for example as described in those patents and patent applications incorporated by reference, including rectal, buccal, intranasal and transdermal routes, by intra-arterial injection, intravenously, intraperitoneally, parenterally, intramuscularly, subcutaneously, orally, topically, as an inhalant, or via an impregnated or coated device such as a stent, for example, or an artery-inserted cylindrical polymer.
  • agents having similar utilities for example as described in those patents and patent applications incorporated by reference, including rectal, buccal, intranasal and transdermal routes, by intra-arterial injection, intravenously, intraperitoneally, parenterally, intramuscularly, subcutaneously, orally, topically, as an inhalant, or via an impregnated or coated device such as a stent, for example, or an artery-inserted cylindrical polymer.
  • One mode for administration is parenteral, particularly by injection.
  • the forms in which the novel compositions disclosed herein may be incorporated for administration by injection include aqueous or oil suspensions, or emulsions, with sesame oil, corn oil, cottonseed oil, or peanut oil, as well as elixirs, mannitol, dextrose, or a sterile aqueous solution, and similar pharmaceutical vehicles.
  • Aqueous solutions in saline are also conventionally used for injection.
  • Ethanol, glycerol, propylene glycol, liquid polyethylene glycol, and the like (and suitable mixtures thereof), cyclodextrin derivatives, and vegetable oils may also be employed.
  • the proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.
  • a coating such as lecithin
  • surfactants for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.
  • Sterile injectable solutions are prepared by incorporating a compound or pharmaceutically acceptable salt thereof as disclosed herein in the required amount in the appropriate solvent with various other ingredients as enumerated above, as desired, followed by filtered sterilization.
  • dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the desired other ingredients from those enumerated above.
  • exemplary methods of preparation include vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-fdtered solution thereof.
  • Oral administration is another route for administration of the compounds or pharmaceutically acceptable salts thereof as disclosed herein. Administration may be via capsule or enteric coated tablets, or the like.
  • the active ingredient may be diluted by an excipient and/or enclosed within such a carrier that can be in the form of a capsule, sachet, paper or other container.
  • the excipient serves as a diluent, it can be in the form of a solid, semi-solid, or liquid material (as above), which acts as a vehicle, carrier or medium for the active ingredient.
  • compositions can be in the form of tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as a solid or in a liquid medium), ointments containing, for example, up to 10% by weight of the active compound, soft and hard gelatin capsules, sterile injectable solutions, and sterile packaged powders.
  • compositions disclosed herein can additionally include lubricating agents such as talc, magnesium stearate, and mineral oil; wetting agents; emulsifying and suspending agents; preserving agents such as methyl and propylhydroxy-benzoates; sweetening agents; glidants; and flavoring agents.
  • compositions disclosed herein can be formulated so as to provide quick, sustained or delayed release of the active ingredient after administration to the patient by employing procedures known in the art.
  • Controlled release drug delivery systems for oral administration include osmotic pump systems and dissolutional systems containing polymer-coated reservoirs or drug-polymer matrix formulations. Examples of controlled release systems are given in U.S. Pat. Nos. 3,845,770; 4,326,525; 4,902,514; and 5,616,345.
  • Another embodiment for use in the methods disclosed herein may employ transdermal delivery devices (“patches”). Such transdermal patches may be used to provide continuous or discontinuous infusion of the compounds or pharmaceutically acceptable salts thereof as disclosed herein in controlled amounts.
  • transdermal patches for the delivery of pharmaceutical agents is described, for example, in U.S. Pat. Nos. 5,023,252, 4,992,445 and 5,001,139. Such patches may be constructed for continuous, pulsatile, or on-demand delivery of pharmaceutical agents.
  • compositions disclosed herein may be formulated in a unit dosage form.
  • unit dosage forms refers to physically discrete units suitable as unitary dosages for human subjects and other mammals, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical excipient (e.g., a tablet, capsule, ampoule).
  • the compounds are generally administered in a pharmaceutically effective amount.
  • each dosage unit contains from about 1 mg to about 2 g of a compound or pharmaceutically acceptable salt thereof as described herein, and for parenteral administration, preferably from about 0.1 to about 700 mg of a compound or pharmaceutically acceptable salt thereof as described herein.
  • the amount of the compound or pharmaceutically acceptable salt thereof actually administered usually will be determined by a physician, in the light of the relevant circumstances, including the condition to be treated, the chosen route of administration, the actual compound or pharmaceutically acceptable salt thereof administered and its relative activity, the age, weight, and response of the individual patient, the severity of the patient’s symptoms, and the like.
  • the principal active ingredient may be mixed with a pharmaceutical excipient to form a solid preformulation composition containing a homogeneous mixture of a compound or pharmaceutically acceptable salt thereof as disclosed herein.
  • a pharmaceutical excipient to form a solid preformulation composition containing a homogeneous mixture of a compound or pharmaceutically acceptable salt thereof as disclosed herein.
  • these preformulation compositions as homogeneous, it is meant that the active ingredient is dispersed evenly throughout the composition so that the composition may be readily subdivided into equally effective unit dosage forms such as tablets, pills and capsules.
  • the tablets or pills disclosed herein may be coated or otherwise compounded to provide a dosage form affording the advantage of prolonged action, or to protect from the acid conditions of the stomach.
  • the tablet or pill can comprise an inner dosage and an outer dosage component, the latter being in the form of an envelope over the former.
  • the two components can be separated by an enteric layer that serves to resist disintegration in the stomach and permit the inner component to pass intact into the duodenum or to be delayed in release.
  • enteric layers or coatings such materials including a number of polymeric acids and mixtures of polymeric acids with such materials as shellac, cetyl alcohol, and cellulose acetate.
  • compositions for inhalation or insufflation include solutions and suspensions in pharmaceutically acceptable, aqueous or organic solvents, or mixtures thereof, and powders.
  • the liquid or solid compositions may contain suitable pharmaceutically acceptable excipients as described herein.
  • the compositions are administered by the oral or nasal respiratory route for local or systemic effect.
  • Compositions in pharmaceutically acceptable solvents may be nebulized by use of inert gases. Nebulized solutions may be inhaled directly from the nebulizing device or the nebulizing device may be attached to a facemask tent or intermittent positive pressure breathing machine. Solution, suspension, or powder compositions may be administered, such as orally or nasally, from devices that deliver the formulation in an appropriate manner.
  • a pharmaceutical composition comprising a compound, or pharmaceutically acceptable salt thereof, as disclosed herein and at least one pharmaceutically acceptable excipient and/or carrier.
  • the compounds provided herein may be isolated and purified by known standard procedures. Such procedures include recrystallization, filtration, flash chromatography, trituration, high performance liquid chromatography (HPLC), or supercritical fluid chromatography (SFC). Note that flash chromatography may either be performed manually or via an automated system.
  • the compounds provided herein may be characterized by known standard procedures, such as nuclear magnetic resonance spectroscopy (NMR) or liquid chromatography mass spectrometry (LCMS). NMR chemical shifts are reported in part per million (ppm) and are generated using methods described in the art.
  • Step 1 Synthesis of (Z)-2-bromoisonicotinaldehyde oxime (2)
  • Step 3 Synthesis of (S)-2-(l-(3-(2-bromopyridin-4-yl)isoxazol-5- yl)ethyl)isoindoline-l, 3-dione (5)
  • Step 4 Synthesis of (S)-l-(3-(2-bromopyridin-4-yl)isoxazol-5-yl)ethan-l-amine (6)
  • Step 5 Synthesis of (S)-N-(l-(3-(2-bromopyridin-4-yl)isoxazol-5-yl)ethyl)-l- methyl-3-(trifluoromethyl)-lH-pyrazole-5-carboxamide (8)
  • Step 6 Synthesis of Synthesis of (S)-l-methyl-3-(trifluoromethyl)-N-(l-(3-(2- vinylpyridin-4-yl)isoxazol-5-yl)ethyl)-lH-pyrazole-5-carboxamide (10)
  • Step 7 Synthesis of (S)-N-(l-(3-(2-ethylpyridin-4-yl)isoxzol-5-yl)ethyl)-l-methyl- 3-(trifluoromethyl)-lH-pyrazole-5-carboxamide hydrochloride (Formula (I-IBa) HC1)
  • HPLC Rt 7.101 min, 99.26%.
  • Method HPLC-AMM BICARB-X Bridge- 10-90- 100- NEW.lcm; Column: X-Bridge C18 (4.6*150) mm 5u; Mobile Phase: A - 5mM Ammonium Bicarbonate in water B - Acetonitrile; Flow Rate: 1.0. mL/minute; Gradient program: Time(min)/ B Cone.: 0.01 B Cone. 10, 6.00 B Cone. 90, 10.00 B Cone. 100, 12.00 B Cone. 100, 14.00 B Cone. 10, 18.00 B Cone. 10, 18.00 Controller Stop.
  • LCMS 393.95 (M+H), Rt 1.608 min, 99.68%.
  • Step A Synthesis of (S)-2-(but-3-yn-2-yl)isoindoline- 1,3-dione (4)
  • Step 3 Synthesis of ethyl 3-(2-bromo-3-fluoropyridin-4-yl)isoxazole-5-carboxylate (5)
  • Step 4 Synthesis of ethyl 3-(3-fluoro-2-methylpyridin-4-yl)isoxazole-5-carboxylate (7)
  • Step 5 Synthesis of (3-(3-fluoro-2-methylpyridin-4-yl)isoxazol-5-yl)methanol (8)
  • Step 8 Synthesis of l-(3-(3-fluoro-2-methylpyridin-4-yl)isoxazol-5-yl)cyclopropane-l- carbonitrile (11)
  • Step 9 Synthesis of l-(3-(3-fluoro-2-methylpyridin-4-yl)isoxazol-5-yl)cyclopropane-l- carboxamide (12)
  • Step 10 Synthesis of l-(3-(3-fluoro-2-methylpyridin-4-yl)isoxazol-5- yl)cyclopropane-l-carboxamide (13) [0152] To a stirred solution of compound 12 (0.2 g, 0.765 mmol) in 1,4 dioxane:water (1 :1.5 mL) were added NaOH (0.1 g, 2.684 mmol) and NaOCI (0.10 g, 2.056 mmol) at 0 °C, and the reaction mixture was stirred at 80 °C for 1 hour. After completion of the reaction (monitored by TLC), the reaction mixture was diluted with water and extracted with EtOAc.
  • Step 11 Synthesis of /V-(l-(3-(3-fluoro-2-methylpyridin-4-yl)isoxazol-5-yl)cyclopropyl)-l- methyl-3-(trifluoromethyl)-lH-pyrazole-5-carboxamide (Formula (I-IBh))
  • Example 3 Efficacy of Various Formulae in the inhibition of KCNT1 (KCNT1 - Patch Clamp Assay)
  • Inhibition of KCNT1 was evaluated using a tetracycline inducible cell line (HEK-TREX). Currents were recorded using the SyncroPatch 384PE automated, patch clamp system. Pulse generation and data collection were performed with PatchController384 VI .3.0 and DataController384 VI.2.1 (Nanion Technologies). The access resistance and apparent membrane capacitance were estimated using built-in protocols. Current was recorded in perforated patch mode (10 ⁇ M escin) from a population of cells.
  • the cells were lifted, triturated, and resuspended at 800,000 cells/ml. The cells were allowed to recover in the cell hotel prior to experimentation. Currents were recorded at room temperature.
  • the extracellular solution was used as the wash, reference, and compound delivery solution.
  • Escin is made at a 5mM stock in water, aliquoted, and stored at -20 °C.
  • the compound plate was created at 2x concentrated in the extracellular solution.
  • the compound was diluted to 1:2 when added to the recording well.
  • the amount of DMSO in the extracellular solution was held constant at the level used for the highest tested concentration.
  • a holding potential of -80 mV with a 100ms step to OmV was used.
  • Mean current was measured during the step to 0 mV.
  • 100 ⁇ M Bepridil was used to completely inhibit KCNT1 current to allow for offline subtraction of non-KCNTl current.
  • the average mean current from 3 sweeps was calculated and the percent inhibition of each compound was calculated.
  • the percent inhibition as a function of the compound concentration was fit with a Hill equation to derive IC 50 , slope, minimum parameters, and maximum parameters. If KCNT1 inhibition was less than 50% at the highest tested concentration or if an IC 50 could not be calculated, then a percent inhibition was reported in place of the IC 50 .
  • Results from this example are summarized in Table 1 below.
  • “A” indicates IC 50 of less than or equal to 1 ⁇ M
  • “B” indicates inhibition of between 1 ⁇ M to 20 ⁇ M
  • “C” indicates inhibition of greater than or equal to 20 ⁇ M.
  • Example 4 Formula (I-IBa) selectively inhibits KCNT1 gain-of-function variants across species
  • Formula (I-IBa) was therefore tested in the automated SyncroPatch patch claim assay described above in Example 2 to assess activity directly on K NA 1.1 current at physiological membrane potentials for various species, including human, mouse, rat, and dog.
  • KNAI . I-WT wildtype K NA 1.1
  • I-WT wildtype K NA 1.1
  • IC 50 values were generated using concentrations ranging from 0.001 to 30 gm in half log steps and a minimum of three cells (replicates) per concentration. The data are shown in Table 2, below.
  • A indicates IC 50 of less than or equal to 1 ⁇ M
  • B indicates inhibition of between 1 ⁇ M to 20 ⁇ M
  • C indicates inhibition of greater than or equal to 20 ⁇ M.
  • NA indicates that the IC 50 was not calculated, as the percent inhibitions at the top dose was not significant.
  • FIG. 1 shows the percent inhibition of KCNT1 for increasing concentrations of a compound of Formula (I-IBa).
  • Kinetic Solubility Assay employed the shake flask method followed by HPLC-UV analysis. The following step-wise procedure was used:
  • test compounds and controls (10 mM in DMSO, 10 ⁇ L/vial) into the 50 mM pH 7.4 phosphate buffer (490 ⁇ L/well) placed in a Mini-Uniprep filter.
  • Log D The Log D assay is a miniaturized 1-octanol/buffer shake flask method followed by LC/MS/MS analysis. It is typically measured by determining the partition of a compound between an organic solvent (1 -octanol) and an aqueous buffer (0.1 M phosphate buffer, pH 7.4; Varied buffer pH can be set). Since logD is pH dependent, the pH of the aqueous phase is always specified and is commonly measured at pH 7.4, the physiological pH of body fluids. The following Log D method was used to calculate the Log D values in Table 3 below:
  • test compounds (10 mM in DMSO; 2 ⁇ L/well) and QC samples (10 mM in DMSO; 2 ⁇ L/well) from storage tubes to the 96-well polypropylene cluster tubes.
  • NADPH Liver Microsome Metabolic Stability Assay
  • Test compounds were incubated at 37°C with liver microsomes (pooled from multiple donors) at 1.0 ⁇ M in the presence of NADPH ( ⁇ 1.0 mM) at 0.5 mg/ml microsomal protein.
  • Positive controls include testosterone (3A4 substrate), propafenone (2D6) and diclofenac (2C9). They are also incubated with microsomes in the presence of NADPH.
  • the mg microsomal protein / g liver weight is 45 for 5 species.
  • the liver weight values will use 40 g/kg, 30 g/kg, 32 g/kg, 20 g/kg and 88 g/kg for rat, monkey, dog, human and mouse, respectively.
  • the liver clearance will be calculated using CL int(mic) with the following equation:
  • reaction solution was concentrated under reduced pressure.
  • the residue was acidified to pH 2-3 with a solution of IM aqueous potassium hydrogen sulfate.
  • the solution was extracted with ethyl acetate (x2).
  • the organic extracts were combined and washed with a solution of IN aqueous sodium bicarbonate, water, and brine, and then dried (Na 2 SO 4 ).
  • the solvent was removed under reduced pressure to afford the title compound (2.8 g, 14.88 mmol, 56% yield).
  • Step 2 Synthesis of tert-butyl (S)-(l-cyanoethyl)carbamate
  • Step 3 Synthesis of tert-butyl (S,E)-(l-amino-l-(hydroxyimino)propan-2-yl)carbamate
  • Step 4 Synthesis of tert-butyl (S)-(l-(5-(2-cyclopropylpyridin-4-yl)-l,2,4-oxadiazol-3-yl)ethyl) ⁇ carbamate
  • Step 5 Synthesis of (S)-l-(5-(2-cyclopropylpyridin-4-yl)-l,2 > 4-oxadiazol-3-yl)ethan-l-amine
  • Step 6 Synthesis of (S)-N-(l-(5-(2-cyclopropylpyridin-4-yl)-l,2,4-oxadiazol-3-yl)ethyl)-l- meth-yl-3-(trifluoromethyl)-lH-pyrazole-5-carboxamide [Formula (V)]
  • LCMS 407.15 (M+H), Rt 1.972 min, 95.178%.
  • Method File LCMS X- Select(Ammonium Bicarbonate) Column : X-Bridge C18 (3.0*50)mm 2.5u; Mobile Phase: A: 2.5 mM Ammonium Bicarbonate in water B: Acetonitrile Flow Rate: 1.2 mL/minute; Column oven temp. 50°C; Gradient program: 0% B to 98 % B in 2.0 minute, hold till 3.0 min, at 3.2 min B cone is 0 % up to 4.0 min.
  • Step 4 Synthesis of (E)-N-hydroxy-2-(trifluoromethyl)isonicotinimidoyl chloride (5) [0185] To a stirred solution of compound 4 (3000 mg, 15.78 mmol) in DMF (10 mL) was added N-chlorosuccinimide (4214.08 mg, 31.56 mmol) at 65 °C, and then stirring was continued further for 30 minutes at 80 °C. After completion of the reaction (monitored by TLC), the reaction mixture was allowed to cool to room temperature, quenched by adding a minimum quantity of crushed ice, and then diluted with Et2O (40 mL). The organic layer was separated and dried over anhydrous Na 2 SO 4 and concentrated under reduced pressure, giving crude compound 5 (3 g). The obtained crude was then used directly for further reaction transformations.
  • Step 5 Synthesis of 2-((3-(2-(trifluoromethyl)pyridin-4-yl)isoxazol-5-yl)methyl)isoindoline- 1, 3-dione (7)
  • Step 6 Synthesis of (3-(2-(trifluoromethyl)pyridin-4-yl)isoxazol-5-yl)methanamine (8)
  • Step 7 Synthesis of l-methyl-3-(trifluoromethyl)-N-((3-(2-(trifluoromethyl)pyridin-4- yl)isoxazol-5-yl)methyl)-lH-pyrazole-5-carboxamide (Formula (I-IBi)):
  • LCMS 420.50 (M+H), Rt 2.021 min, 99.49%.
  • Step A Synthesis of l-methyl-3-(trifluoromethyl)-N-((3-(2-(trifluoromethyl)pyridin-4- yl)isoxazol-5-yl)methyl)-lH-pyrazole-5-carboxamide (6):
  • Example 8 Synthesis of (S)-3-(difluoromethyl)-N-(l-(3-(2-ethylpyridin-4-yl)isoxazol-5- yl)ethyl)-l-methyl-lH-pyrazole-5-carboxamide hydrochloride [Formula I-IBj)] and (R)-3- (difluoromethyl)-N-(l-(3-(2-ethylpyridin-4-yl)isoxazol-5-yl)ethyl)-l-methyl-lH-pyrazole-5- carboxamide hydrochloride [Formula I-IBk)]: Step-1: Synthesis of (Z)-2-bromoisonicotinaldehyde oxime (2)
  • Step-3 Synthesis of 2-(l-(3-(2-bromopyridin-4-yl)isoxazol-5-yl)ethyl)isoindoline-1, 3-dione (5)
  • Step-4 Synthesis of 2-(l-(3-(2-vinylpyridin-4-yl)isoxazol-5-yl)ethyl)isoindoline-l, 3-dione (7)
  • Step-5 Synthesis of l-(3-(2-ethylpyridin-4-yl)isoxazol-5-yl)ethan-l-amine (8)
  • Step-7 (S)-3-(difluoromethyl)-N-(l-(3-(2-ethylpyridin-4-yl)isoxazol-5-yl)ethyl)-l-methyl- lH-pyrazole-5-carboxamide hydrochloride [Formula I-IBj)] and (R)-3-(difluoromethyl)-N- (l-(3-(2-ethylpyridin-4-yl)isoxazol-5-yl)ethyl)-l-methyl-lH-pyrazole-5-carboxamide hydrochloride [Formula I-IBk)] :
  • LCMS 376.0 (M+H), Rt 1.395 min, 99.69%.
  • Column X-Select CSH C18 (3.0*50) mm 2.5um; Mobile Phase: A: 0.05% Formic acid in water : ACN (95:5); B: ACN; Inj Volume: 2.0 ⁇ L; Flow Rate : 1.2. mL/minute; Column oven temperature: 50 °C; Gradient program: 0% B to 98 % B in 2.0 min, hold till 3.0 min, at 3.2 min B cone is 0 % up to 4.0 min.
  • Step A Synthesis of 2-(but-3-yn-2-yl)isoindoline- 1,3-dione (4)
  • Step-1 Synthesis of (Z)-2-bromoisonicotinaldehyde oxime (2)
  • Step-3 Synthesis of 2-(l-(3-(2-bromopyridin-4-yl)isoxazol-5-yl)ethyl)isoindoline-l, 3-dione (5)
  • Step-4 Synthesis of 2-(l-(3-(2-vinylpyridin-4-yl)isoxazol-5-yl)ethyl)isoindoline-l, 3-dione (7)
  • Step-5 Synthesis of l-(3-(2-ethylpyridin-4-yl)isoxazol-5-yl)ethan-l-amine (8)
  • Step-7 (S)-N-(l-(3-(2-ethylpyridin-4-yl)isoxazol-5-yl)ethyl)benzamide hydrochloride [Formula (I-IBn) HC1] and (R)-N-(l-(3-(2-ethylpyridin-4-yl)isoxazol-5-yl)ethyl)benzamide hydro-chloride [Formula (I-IBo) HCI]:
  • LCMS 322.2 (M+H), Rt 1.511 min, 98.118 %.
  • Column X-Select CSH (3.0*50) mm 2.5u; Mobile Phase: A: 0.025% Formic acid in water; B: ACN; Inj Volume: 2.0 ⁇ L, Flow Rate : 1.2. mL/minute; Column oven temperature: 50 °C; Gradient program: 0% B to 98 % B in 2.0 min, hold till 3.0 min, at 3.2 min B cone is 0 % up to 4.0 min.
  • LCMS 322.10 (M+H), Rt 1.516 min, 98.934%.
  • Column X-Select CSH (3.0*50) mm 2.5u; Mobile Phase: A: 0.025% Formic acid in water; B: ACN; Inj Volume: 2.0 ⁇ L; Flow Rate : 1.2. mL/minute; Column oven temperature: 50 °C; Gradient program: 0% B to 98 % B in 2.0 min, hold till 3.0 min, at 3.2 min B cone is 0 % up to 4.0 min.
  • Step A Synthesis of (S)-2-(but-3-yn-2-yl)isoindoline-l, 3-dione (4)
  • Step-1 Synthesis of (Z)-2-bromoisonicotinaldehyde oxime (2)
  • Step-2 Synthesis of (E)-2-bromo-N-hydroxyisonicotinimidoyl chloride (3) [0225] To a stirred solution of compound 2 (20 g, 99.49 mmol) in DMF (200 mL) was added NCS (26.57 g, 198.99 mmol), and the reaction mixture was stirred at room temperature for 3 days. Progress of the reaction was monitored by TLC. After completion, the reaction mixture was concentrated under vacuo, the obtained residue was diluted with EtOAc (200 mL) and washed with water (2x100 mL) followed by saturated brine solution (1x100 mL). The combined organic fractions were then separated, dried over MgSO 4 and concentrated under reduced pressure to afford compound 3 (23 g, 97.67 mmol, 98% yield).
  • Step-3 Synthesis of 2-(l-(3-(2-bromopyridin-4-yl)isoxazol-5-yl)ethyl)isoindoline-l, 3-dione (5)
  • Step-4 Synthesis of 2-(l-(3-(2-vinylpyridin-4-yl)isoxazol-5-yl)ethyl)isoindoline-1, 3-dione (7)
  • Step-5 Synthesis of l-(3-(2-ethylpyridin-4-yl)isoxazol-5-yl)ethan-l-amine (8)
  • Step-7 Synthesis of (R)-3-chloro-N-(l-(3-(2-ethylpyridin-4-yl)isoxazol-5-yl)ethyl)benzamide hydrochloride [Formula (I-IBm) HC1] and ((S)-3-chloro-N-(l-(3-(2-ethylpyridin-4- yl)isoxazol-5-yl)ethyl)benz-amide hydrochloride [Formula (I-IBp) HCI]:
  • LCMS 355.95 (M+H), Rt 1.797 min, 97.84%.
  • Column X-Select CSH (3.0*50) mm 2.5u; Mobile Phase: A: 0.05% Formic acid in water; B: ACN Inj Volume: 2.0 ⁇ L; Flow Rate : 1.2. mL/minute; Gradient program: 0% B to 98 % B in 2.0 minute, Hold till 3.0 min, At 3.2 min B cone is 0 % up to 4.0 min.
  • Step A Synthesis of (S)-2-(but-3-yn-2-yl)isoindoline-l, 3-dione (4) [0238] To a stirred solution of compound 4A (31.49 g, 214.01 mmol) and compound 4B (15 g, 214.01 mmol) in THF (150 mL) was added and DEAD (55.9 g, 321.02 mmol) followed by triphenylphosphine (84.2 g, 321.02 mmol) at 0 °C, and the mixture was stirred at room temperature for 16 hours. Progress of the reaction was monitored by TLC.
  • Step-1 Synthesis of (Z)-2-bromoisonicotinaldehyde oxime (2)
  • Step-3 Synthesis of 2-(l-(3-(2-bromopyridin-4-yl)isoxazol-5-yl)ethyl)isoindoline-l, 3-dione (5)
  • Step-4 Synthesis of l-(3-(2-bromopyridin-4-yl)isoxazol-5-yl)ethan-l-amine (6)
  • Step-5 Synthesis of N-(l-(3-(2-bromopyridin-4-yl)isoxazol-5-yl)ethyl)benzamide (8)
  • Step-6 Synthesis of (S)-N-(l-(3-(2-(ethylamino)pyridin-4-yl)isoxazol-5-yl)ethyl)benzamide [Formula (I-IBq)] and (R)-N-(l-(3-(2-(ethylamino)pyridin-4-yl)isoxazol-5- yl)ethyl)benzamide [Formula (I-IBr)]:
  • Step A Synthesis of 2-(but-3-yn-2-yl)isoindoline- 1,3-dione (4)
  • Step-1 Synthesis of (E)-2-bromoisonicotinaldehyde oxime (2)
  • Step-2 Synthesis of (E)-2-bromo-N-hydroxyisonicotinimidoyl chloride (3) [0257] To a stirred solution of compound 2 (20 g, 99.49 mmol) in DMF (200 mL) was added NCS (26.57 g, 198.99 mmol), and the reaction mixture was stirred at room temperature for 3 days. Progress of the reaction was monitored by TLC. After completion, the reaction mixture was concentrated under vacuo, and the obtained residue was diluted with EtOAc (200 mL) and washed with water (2x100 mL) followed by saturated brine solution (1x100 mL). The combined organic fractions were then separated, dried over MgSO 4 and concentrated under reduced pressure to afford compound 3 (23 g, 97.67 mmol, 98% yield).
  • Step-3 Synthesis of 2-(l-(3-(2-bromopyridin-4-yl)isoxazol-5-yl)ethyl)isoindoline-l, 3-dione (5)
  • Step-4 Synthesis of l-(3-(2-bromopyridin-4-yl)isoxazol-5-yl)ethan-l-amine (6)
  • Step-5 Synthesis of N-(l-(3-(2-bromopyridin-4-yl)isoxazol-5-yl)ethyl)-3-isopropyl-l- methyl-lH-pyrazole-5-carboxamide (8)
  • Step-6 Synthesis of (S)-N-(l-(3-(2-(ethylamino)pyridin-4-yl)isoxazol-5-yl)ethyl)-3- isopropyl-l-methyl-lH-pyrazole-5-carboxamide [Formula (I-IBs)] and (R)-N-(l-(3-(2- (ethylamino)pyridin-4-yl)isoxazol-5-yl)ethyl)-3-isopropyl-l-methyl-lH-pyrazole-5- carboxamide [Formula (I-IBt)] :
  • LCMS 383.10 (M+H), Rt 1.475 min, 99.65%.
  • LCMS 383.10 (M+H), Rt 1.475 min, 99.73%.
  • Column X-Select CSH C18 (3.0*50) mm 2.5um; Mobile Phase: A: 0.05% Formic acid in water:ACN(95:05); B: ACN; Inj Volume: 2.0 ⁇ L; Flow Rate : 1.2. mL/minute; Column oven Temp: 50°C; Gradient program: 0% B to 98 % B in 2.0 minute, Hold till 3.0 min, At 3.2 min B cone is 0 % up to 4.0 min.
  • Step A Synthesis of 2-(but-3-yn-2-yl)isoindoline- 1,3-dione (4)

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Abstract

Disclosed herein are compounds having an isoxazole core or an oxadiazole core or a pharmaceutically acceptable salt thereof and compositions useful for preventing and/or treating a neurological disorder, a disorder associated with excessive neuronal excitability, or disorder associated with a gain-of-function mutation in a gene (e.g., KCNT1). Methods of treating a neurological disorder, a disorder associated with excessive neuronal excitability, or disorder associated with a gain-of-function mutation in a gene, such as KCNT1, are also provided herein.

Description

KCNTl INHIBITORS COMPRISING AN ISOXAZOLE OR OXADIAZOLE CORE AND
METHODS OF USE
Cross-Reference to Related Applications
[0001] This application claims priority to U.S. Provisional Patent Application No. 63/334,359, filed April 25, 2022, and U.S. Provisional Application No. 63/386,022, filed December 5, 2022, which are incorporated herein by reference in their entireties.
Field of the Disclosure
[0002] The present disclosure is generally directed to KCNT1 inhibitors canprising an isoxazole core or an oxadiazole core, as well as pharmaceutical compositions and methods of treatment involving the use of such compounds.
Background of the disclosure
[0003] Potassium sodium-activated channel subfamily T member 1 (“KCNT1”) is one of the genes in a family of genes responsible for providing the instructions to make potassium channels. KCNT1 encodes sodium-activated potassium channels known as Slack (Sequence like a calcium- activated K+ channel). These channels are found in neurons throughout the brain and can mediate a sodium-activated potassium current IkNa. This delayed outward current can regulate neuronal excitability and the rate of adaptation in response to maintained stimulation. Abnormal Slack activity has been associated with development of early onset epilepsies and intellectual impairment. Accordingly, pharmaceutical compounds that selectively regulate sodium-activated potassium channels, e.g., abnormal KCNT1 or abnormal IkNa, are useful in treating a neurological disease or disorder or a disease or condition related to excessive neuronal excitability and/or KCNT1 gain-of-function mutations.
[0004] KCNT1 inhibitors and their preparation are disclosed, for example, in WO 2021/195066, incorporated herein by reference in its entirety. WO 2021/195066 discloses, for example, compounds having the Formula A:
Figure imgf000003_0001
wherein X is CR7 or N and Y is S; or
X is CR7 and Y is O; ring A is selected from the group consisting of phenyl, 6-membered heteroaryl, and 5- 7 membered heteroaryl; R1 is selected from the group consisting of phenyl, 5-6 membered heteroaryl, -CH2- phenyl, 5-8 membered carbocyclyl, and 5-10 membered heterocyclyl; wherein the phenyl 5-6 membered heteroaryl, -CH2-phenyl, 5-8 membered carbocyclyl, and 5-10 membered heterocyclyl is optionally substituted with one or more R6;
R2 is hydrogen or C1-ealkyl;
R3 is selected from the group consisting of hydrogen, C1-ealkyl, C1-ehaloalkyl, C1- ealkoxy, C1-6haloalkoxy; and C3-8cycloakyl, wherein the C1-ealkyl is optionally substituted with C1- ealkoxy or C1-ehaloalkoxy, and R4 is hydrogen; or
R3 and R4 can be taken together with the carbon attached to R3 and R4 to form a C3- scycloakylene or 3-7 membered heterocycloalkylene;
R5 and Re are each independently selected from the group consisting of halogen, C1- ealkyl, C1-ealkylene-O-C1-ealkyl, C1-ehaloalkyl, C1-ealkoxy, C1-ehaloalkoxy, -S(O)2R8, -S(O)2- N(R9)2, and C3-8cycloalkyl;
R7 is selected from the group consisting of hydrogen, C1-ealkyl, and C1-ehaloalkyl;
Rs is hydrogen or C1-ealkyl; each R9 is independently selected from the group consisting of hydrogen, C1-ealkyl, and -(C1-ealkylene)-OH, or the two R9 can be taken together with the nitrogen atom attached to the two R9 to form a heterocycle optionally substituted with one or more substituents each independently selected from halogen and -OH; and n is selected from the group consisting of 0, 1, 2, and 3; provided that when R3 is hydrogen and ring A is a 6-membered heterocyclyl or 6-membered heteroaryl, R1 is not thiophene; and provided that when R3 is hydrogen and ring A is a 6-membered heteroaryl or 5- membered heterocyclyl, R1 is not phenyl; or a pharmaceutically acceptable salt thereof.
[0005] WO 2021/195066 also discloses several subgenera of Formula (A), including, for example, a compound of Formula I-IB:
Figure imgf000005_0001
or a pharmaceutically acceptable salt thereof.
[0006] KCNT1 inhibitors and their preparation are disclosed, for example, in WO 2020/227101, incorporated herein by reference in its entirety. WO 2020/227101 discloses, for example, compounds having the Formula (I):
Figure imgf000005_0002
wherein X, Y, Z, Y’, and Z’ are each independently selected from CH and N, wherein the hydrogen of CH may be substituted with Rs, wherein at least 3 selected from X, Y, Z, Y’, and Z’ are CH; R1 is selected from the group consisting of C1-6alkyl, C3-10cycloalkyl, 3-10 membered heterocyclyl, 3-10 membered heteroaryl, and phenyl, wherein C1-6alkyl, C3-10cycloalkyl, 3-10 membered heterocyclyl, 3-10 membered heteroaryl, or phenyl is optionally substituted with one or more substituents each independently selected from the group consisting of halogen, C(O)N(R9)2, N(R9)2, C3-7cycloalkyl, phenyl, 3-10 membered heteroaryl, and C1-6alkoxy;
R12 is selected from the group consisting of C1-6alkyl, C3-10cycloalkyl, 3-10 membered heterocyclyl, 3-10 membered heteroaryl, and phenyl, wherein the C1-6alkyl, C3-10cycloalkyl, 3-10 membered heterocyclyl, 3-10 membered heteroaryl, or phenyl is optionally substituted with one or more substituents each independently selected from the group consisting of halogen, -OH, -CN, C1-6alkyl, C1-6haloalkyl, and C1-6alkoxy; or two R12 on adjacent carbons can be taken together with the two carbons where R12 are attached to form a carbocyclic ring; x is 0, 1, or 2;
R2 is hydrogen or C1-4alkyl; R3 is selected from the group consisting of hydrogen, C1-6alkyl, C3-10cycloalkyl, 3-10 membered heterocyclyl, 3-10 membered heteroaryl, and phenyl; and R4 is selected from C1-6alkyl and hydrogen; or R3 and R4 can be taken together with the carbon attached to R3 and R4 to form a C3-7cycloalkylene or 3-7 membered heterocyclene; wherein the C1-6alkyl, C3-10cycloalkyl, 3-10 membered heterocyclyl, 3-10 membered heteroaryl, phenyl, C3-7Cycloalkylene, or 3-7 membered heterocyclene may be optionally substituted with one or more R7; each R5 is independently selected from the group consisting of halogen, C1-6alkyl, C1- 6haloalkyl, C1-6alkylene-N(R9)2, C1-6alkylene-0-C3-10cycloalkyl, C1-6alkoxy, C1-6alkoxy substituted with C3-10cycloalkyl optionally substituted with one or more halogensh C1-6haloalkoxy, 3-10 membered heterocyclyl optionally substituted with one or more halogens or C1-6alkoxy, 3-10 membered heteroaryl, C1-6alkylene-OH, C1-6alkylene-C1-6alkoxy, OH, N(R9)2, -C(O)ORs, C(O)N(R9)2, C1-6alkylene-CN, -CN, -S(O)2- C1-6alkyl, C1-6alkylene-S(O)2-C1-6alkyl, -S(O)2- N(R9)2, -OC(O)C1-6alkyl, -0-C3-10cycloalkyl optionally substituted with one or more halogen or C1-oalkyl, and C3-10cycloalkyl optionally substituted with one or more substituents selected from halogen, C1-6alkyl, and C1-6alkoxy; n is selected from the group consisting of 0, 1, 2, and 3;
R7 is each independently selected from the group consisting of phenyl, C1-6alkoxy, - OH, -N(R9)2, -NR9-SO2-Cl-6alkyl, -O-(C1-6alkylene)-phenyl, C3-10cycloalkyl, -C(O)ORs, - C(O)N(R9)2,-NR10C(O)-RII, -CN, -S(O)2-C1-6alkyl, -S(O)2- N(R9)2, 3-10 membered heterocyclyl, and 3-10 membered heteroaryl, wherein the phenyl, C3-10cycloalkyl, 3-10 membered heterocyclyl, or 3-10 membered heteroaryl is optionally substituted with one or more substituents each independently selected from the group consisting of C1-6alkyl, halogen, -OH, C1-6alkoxy, and - N(R9)2;
R8 is hydrogen or C1-6alkyl; each R9 is independently selected from the group consisting of hydrogen, C1-6alkyl, and -(C1-6alkylene )-OH, or the two R9 can be taken together with the nitrogen atom attached to the two R9 to form a heterocycle optionally substituted with one or more substituents each independently selected from halogen and -OH; each R10 is independently hydrogen or C1-6alkyl;
R11 is selected from the group consisting of C1-6alkyl, C1-6alkoxy, and -O-(C1- 6alkylene)-phenyl; and when R3 and R4 are both hydrogen, at least one selected from X, Y, Z, Y’, and Z’ is N, or a pharmaceutically acceptable salt thereof, and numerous species thereof.
Summary of the Disclosure
[0007] Described herein are compounds and compositions useful for preventing and/or treating a disease, disorder, or condition, e g., a neurological disorder, a disorder associated with excessive neuronal excitability, or disorder associated with a gain-of-function mutation in a gene, for example, KCNT1.
[0008] In some aspects, provided is a compound of Formula (I-IB) as described in WO 2021/195066 having an isoxazole core and chosen from:
Figure imgf000007_0001
Figure imgf000008_0001
Figure imgf000009_0001
Figure imgf000010_0001
Figure imgf000011_0001
or a pharmaceutically acceptable salt thereof.
[0009] In some aspects, provided herein is a compound of general Formula (I) having an oxadiazole core, as described in WO 2020/227101, and chosen from:
Figure imgf000011_0002
Figure imgf000012_0001
or a pharmaceutically acceptable salt thereof.
[0010] In some aspects, provided herein is a compound of Formula (II) having an oxadiazole core:
Figure imgf000012_0002
or a pharmaceutically acceptable salt thereof. [0011] In some aspects, provided herein is a compound of Formula (III) having an oxadiazole core:
Figure imgf000013_0003
or a pharmaceutically acceptable salt thereof.
[0012] In some aspects, provided herein is a compound of Formula (IV) having an oxadiazole core:
Figure imgf000013_0001
or a pharmaceutically acceptable salt thereof.
[0013] In some aspects, provided herein is a compound of Formula (V) having an oxadiazole core:
Figure imgf000013_0002
or a pharmaceutically acceptable salt thereof.
[0014] In some aspects, provided herein is a compound of Formula (VI) having an oxadiazole core:
Figure imgf000014_0001
or a pharmaceutically acceptable salt thereof.
[0015] In some aspects, provided herein is a compound of Formula (VII) having an isoxazole core:
Figure imgf000014_0002
or a pharmaceutically acceptable salt thereof, wherein:
R1 is chosen from -H or a C1-6alkyl, such a methyl;
R2 is chosen from -H or a C1-6alkyl, such a methyl;
R3 is independently chosen from a halogen, a C1-6alkyl, a carbocyclyl, or an alkoxy, wherein the C1-6alkyl, carbocyclyl or alkoxy optionally comprises at least one halogen substituent; and n is 0, 1, 2, 3, or 4.
[0016] In some aspects, provided herein is a compound of Formula (VII) as described having an isoxazole core and chosen from:
Figure imgf000015_0001
Figure imgf000016_0001
or a pharmaceutically acceptable salt thereof.
[0017] In other aspects, provided is a method of treating a neurological disorder, a disorder associated with excessive neuronal excitability, or a disorder associated with a gain-of-function mutation of a gene, by administering to a subject in need thereof an effective amount of a compound described herein having an isoxazole core, such as a compound of Formula (I-IB) [e.g., Formulae (I-IBa) - (I-IBt)] or Formula (VII) [e.g., Formula (Vll-a) - (VII-f)], or a pharmaceutically acceptable salt thereof, or pharmaceutical compositions described herein comprising such compounds or a pharmaceutically acceptable salt thereof.
[0018] In other aspects, provided is a method of treating a neurological disorder, a disorder associated with excessive neuronal excitability, or a disorder associated with a gain-of-function mutation of a gene, by administering to a subject in need thereof an effective amount of a compound described herein having an oxadiazole core, such as a compound of Formula (I-a), Formula (I-b), Formula (I-c), Formula (I-d), Formula (II), Formula (III), Formula (IV), Formula (V), or Formula (VI), or a pharmaceutically acceptable salt thereof, or pharmaceutical compositions described herein comprising such compounds or a pharmaceutically acceptable salt thereof.
[0019] In some embodiments, the method provided involves treating a disorder associated with a gain-of-function mutation of KCNT1. [0020] In some variations, the neurological disorder, the disorder associated with excessive neuronal excitability, or the disorder associated with a gain-of-function mutation of a gene (e.g., KCNT1) is epilepsy, an epilepsy syndrome, or an encephalopathy.
[0021] In some variations, the neurological disorder, the disorder associated with excessive neuronal excitability, or the disorder associated with a gain-of-function mutation of a gene (e.g., KCNT1) is a genetic or pediatric epilepsy or a genetic or pediatric epilepsy syndrome.
[0022] In some variations, the neurological disorder, the disorder associated with excessive neuronal excitability, or the disorder associated with a gain-of-function mutation of a gene (e.g., KCNT1) is a cardiac dysfunction.
[0023] In some variations, the neurological disorder, the disorder associated with excessive neuronal excitability, or the disorder associated with a gain-of-function mutation of a gene (e.g., KCNT1) is selected from the group consisting of epilepsy and other encephalopathies (e.g., malignant migrating focal seizures of infancy (MMFSI) or epilepsy of infancy with migrating focal seizures (EIMFS), autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE), West syndrome, infantile spasms, epileptic encephalopathy, focal epilepsy, Ohtahara syndrome, developmental and epileptic encephalopathy, Lennox-Gastaut syndrome, seizures (e.g., Generalized tonic clonic seizures, Asymmetric Tonic Seizures), leukodystrophy, leukoencephalopathy, intellectual disability, Multifocal Epilepsy, Drug resistant epilepsy, Temporal lobe epilepsy, or cerebellar ataxia.
[0024] In some variations, the neurological disorder, the disorder associated with excessive neuronal excitability, or the disorder associated with a gain-of-function mutation of a gene (e.g., KCNT1) is chosen from cardiac arrhythmia, Brugada syndrome, or myocardial infarction.
[0025] In some variations, the neurological disorder, the disorder associated with excessive neuronal excitability, or the disorder associated with a gain-of-function mutation of a gene (e.g., KCNT1) is selected from pain and related conditions (e.g., neuropathic pain, acute/chronic pain, migraine).
[0026] In some variations, the neurological disorder, the disorder associated with excessive neuronal excitability, or the disorder associated with a gain-of-function mutation of a gene (e.g., KCNT1) is a muscle disorder (e.g., myotonia, neuromyotonia, cramp muscle spasms, spasticity). [0027] In some variations, the neurological disorder, the disorder associated with excessive neuronal excitability, or the disorder associated with a gain-of-function mutation of a gene (e.g., KCNT1) is selected from itch and pruritis, ataxia, or cerebellar ataxias.
[0028] In some variations, the neurological disorder, the disorder associated with excessive neuronal excitability, or the disorder associated with a gain-of-function mutation of a gene (e.g., KCNT1) is a psychiatric disorder (e.g., major depression, anxiety, bipolar disorder, schizophrenia). [0029] In other variations, the neurological disorder, the disorder associated with excessive neuronal excitability, or the disorder associated with a gain-of-function mutation in a gene (e.g., KCNT1) is chosen from a learning disorder, Fragile X, neuronal plasticity, or an autism spectrum disorder.
[0030] In yet other variations, the neurological disorder, the disorder associated with excessive neuronal excitability, or the disorder associated with a gain-of-function mutation of a gene (e.g., KCNT1) is chosen from epileptic encephalopathy with SCN1A, SCN2A, and/or SCN8A mutations, early infantile epileptic encephalopathy, Dravet syndrome, Dravet syndrome with SCN1A mutation, generalized epilepsy with febrile seizures, intractable childhood epilepsy with generalized tonic-clonic seizures, infantile spasms, benign familial neonatal-infantile seizures, SCN2A epileptic encephalopathy, focal epilepsy with SCN3A mutation, cryptogenic pediatric partial epilepsy with SCN3 A mutation, SCN8A epileptic encephalopathy, Rasmussen encephalitis, malignant migrating partial seizures of infancy, autosomal dominant nocturnal frontal lobe epilepsy, KCNQ2 epileptic encephalopathy, or KCNT1 epileptic encephalopathy.
[0031] Other objects and advantages will become apparent to those skilled in the art from consideration of the ensuing description.
Brief Description of the Figures
[0032] FIG. 1 is a graph showing the mean response in terms of percent inhibition of KNA1.1 for varying concentrations of Formula (I) in various species for KCNT1 wild type and gain-of- function variants, as described in Example 3.
Description of the Disclosure
[0033] Provided herein, in certain aspects, are compounds and compositions useful for preventing and/or treating a disease, disorder, or condition described herein, e.g., a neurological disorder, a disorder associated with excessive neuronal excitability, or a disorder associated with gain-of-function mutations in a gene (e.g., KCNT1). Exemplary diseases, disorders, or conditions include epilepsy and other encephalopathies (e.g., MMFSI or EIMFS, ADNFLE, West syndrome, infantile spasms, epileptic encephalopathy, focal epilepsy, Ohtahara syndrome, developmental and epileptic encephalopathy, Lennox-Gastaut syndrome, seizures, leukodystrophy, leukoencephalopathy, Intellectual disability, Multifocal Epilepsy, Generalized tonic clonic seizures, Drug resistant epilepsy, Temporal lobe epilepsy, cerebellar ataxia, Asymmetric Tonic Seizures); cardiac dysfunctions (e.g., cardiac arrhythmia, Brugada syndrome, myocardial infarction); pain and related conditions (e.g., neuropathic pain, acute/chronic pain, migraine, etc.); muscle disorders (e.g., myotonia, neuromyotonia, cramp muscle spasms, spasticity); itch and pruritis; ataxia and cerebellar ataxias; and psychiatric disorders (e.g., major depression, anxiety, bipolar disorder, schizophrenia).
I. Definitions
[0034] Unless defined otherwise, all terms of art, notations and other technical and scientific terms or terminology used herein are intended to have the same meaning as is commonly understood by one of ordinary skill in the art to which the claimed subject matter pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art.
[0035] Throughout this disclosure, various aspects of the claimed subject matter are presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the claimed subject matter. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For instance, where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit, unless the context clearly dictate otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure. In some embodiments, two opposing and open-ended ranges are provided for a feature, and in such description it is envisioned that combinations of those two ranges are provided herein. For example, in some embodiments, it is described that a feature is greater than about 10 units, and it is described (such as in another sentence) that the feature is less than about 20 units, and thus, the range of about 10 units to about 20 units is described herein.
[0036] The term “about” as used herein refers to the usual error range for the respective value readily known in this technical field. Reference to “about” a value or parameter herein includes (and describes) variations that are directed to that value or parameter per se. For example, description referring to “about X” includes description of “X.”
[0037] As used herein, including in the appended claims, the singular forms “a,” “or,” and “the” include plural referents unless the context clearly dictates otherwise. For example, “a” or “an” means “at least one” or “one or more.” It is understood that aspects and variations described herein include embodiments “consisting” and/or “consisting essentially of’ such aspects and variations.
[0038] The terms “disease,” “disorder,” and “condition” are used interchangeably herein.
[0039] As used herein, the term “in some embodiments,” “in other embodiments,” or the like, refers to embodiments of all aspects of the disclosure, unless the context clearly indicates otherwise.
[0040] Definitions of specific functional groups and chemical terms are described in more detail below. The chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75th Ed., inside cover, and specific functional groups are generally defined as described therein. Additionally, general principles of organic chemistry, as well as specific functional moieties and reactivity, are described, for example, in Thomas Sorrell, Organic Chemistry, University Science Books, Sausalito, 1999; Smith and March, March ’s Advanced Organic Chemistry, 5th Edition, John Wiley & Sons, Inc., New York, 2001; Larock, Comprehensive Organic Transformations, VCH Publishers, Inc., New York, 1989; and Carruthers, Some Modern Methods of Organic Synthesis, 3rd Edition, Cambridge University Press, Cambridge, 1987.
[0041] The following terms are intended to have the meanings presented therewith below and are useful in understanding the description and intended scope of the present disclosure. When describing certain aspects of the disclosure, which may include compounds, pharmaceutical compositions containing such compounds, and methods of using such compounds and compositions, the following terms, if present, have the following meanings unless otherwise indicated. It should also be understood that when described herein any of the moieties defined forth below may be substituted with a variety of substituents, and that the respective definitions are intended to include such substituted moieties within their scope as set out below. Unless otherwise stated, the term “substituted” is to be defined as set out below. It should be further understood that the terms “groups” and “radicals” can be considered interchangeable when used herein. The articles “a” and “an” may be used herein to refer to one or to more than one (i.e., at least one) of the grammatical objects of the article. By way of example “an analogue” means one analogue or more than one analogue.
[0042] When a range of values is listed, it is intended to encompass each value and sub-range within the range. For example, “C1-6 alkyl” is intended to encompass, C1, C2, C3, C4, C5, C6, C1-6, C1-5, C1-4, C1-3, C1-2, C2-6, C2-5, C2-4, C2-3, C3-6, C3-5, C3-4, C4-6, C4-5, and C5-6 alkyl.
[0043] “Alkyl” refers to a radical of a straight-chain or branched saturated hydrocarbon group, e.g., having 1 to 20 carbon atoms (“C1-20 alkyl”). In some embodiments, an alkyl group has 1 to 10 carbon atoms (“C1-10 alkyl”). In some embodiments, an alkyl group has 1 to. 9 carbon atoms (“C1-9 alkyl”). In some embodiments, an alkyl group has 1 to 8 carbon atoms (“C1-8 alkyl”). In some embodiments, an alkyl group has 1 to 7 carbon atoms (“C1-7 alkyl”). In some embodiments, an alkyl group has 1 to 6 carbon atoms (“ C1-6 alkyl”). In some embodiments, an alkyl group has 1 to 5 carbon atoms (“ C1-5 alkyl”). In some embodiments, an alkyl group has 1 to 4 carbon atoms (“ C1-4 alkyl”). In some embodiments, an alkyl group has 1 to 3 carbon atoms (“ C1-3 alkyl”). In some embodiments, an alkyl group has 1 to 2 carbon atoms (“C1-2 alkyl”). In some embodiments, an alkyl group has 1 carbon atom (“ C1 alkyl”). Examples of C1-6 alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, pentyl, hexyl, and the like.
[0044] “Alkenyl” refers to a radical of a straight-chain or branched hydrocarbon group having from 2 to 20 carbon atoms, one or more carbon-carbon double bonds (e.g., 1, 2, 3, or 4 carbon-carbon double bonds), and optionally one or more carbon-carbon triple bonds (e.g., 1, 2, 3, or 4 carbon-carbon triple bonds) (“C2-20 alkenyl”). In certain embodiments, alkenyl does not contain any triple bonds. In some embodiments, an alkenyl group has 2 to 10 carbon atoms (“C2- 10 alkenyl”). In some embodiments, an alkenyl group has 2 to 9 carbon atoms (“C2-9 alkenyl”). In some embodiments, an alkenyl group has 2 to 8 carbon atoms (“C2-8 alkenyl”). In some embodiments, an alkenyl group has 2 to 7 carbon atoms (“C2-7 alkenyl”). In some embodiments, an alkenyl group has 2 to 6 carbon atoms (“C2-6 alkenyl”). In some embodiments, an alkenyl group has 2 to 5 carbon atoms (“C2-5 alkenyl”). In some embodiments, an alkenyl group has 2 to 4 carbon atoms (“C2-4 alkenyl”). In some embodiments, an alkenyl group has 2 to 3 carbon atoms (“C2-3 alkenyl”). In some embodiments, an alkenyl group has 2 carbon atoms (“C2 alkenyl”). The one or more carbon-carbon double bonds can be internal (such as in 2-butenyl) or terminal (such as in 1- butenyl). Examples of C2-4 alkenyl groups include ethenyl (C2), 1 -propenyl (C3), 2-propenyl (C3), 1-butenyl (C4), 2-butenyl (C4), butadienyl (C4), and the like. Examples of C2-6 alkenyl groups include the aforementioned C24 alkenyl groups as well as pentenyl (C5), pentadienyl (C5), hexenyl (C6), and the like. Additional examples of alkenyl include heptenyl (C7), octenyl (C8), octatrienyl (C8), and the like.
[0045] “Alkynyl” refers to a radical of a straight-chain or branched hydrocarbon group having from 2 to 20 carbon atoms, one or more carbon-carbon triple bonds (e.g., 1, 2, 3, or 4 carbon-carbon triple bonds), and optionally one or more carbon-carbon double bonds (e.g., 1, 2, 3, or 4 carbon-carbon double bonds) (“C2-20 alkynyl”). In certain embodiments, alkynyl does not contain any double bonds. In some embodiments, an alkynyl group has 2 to 10 carbon atoms (“C2- 10 alkynyl”). In some embodiments, an alkynyl group has 2 to 9 carbon atoms (“C2-9 alkynyl”). In some embodiments, an alkynyl group has 2 to 8 carbon atoms (“C2-8 alkynyl”). In some embodiments, an alkynyl group has 2 to 7 carbon atoms (“C2-7 alkynyl”). In some embodiments, an alkynyl group has 2 to 6 carbon atoms (“C2-6 alkynyl”). In some embodiments, an alkynyl group has 2 to 5 carbon atoms (“C2-5 alkynyl”). In some embodiments, an alkynyl group has 2 to 4 carbon atoms (“C2-4 alkynyl”). In some embodiments, an alkynyl group has 2 to 3 carbon atoms (“C2-3 alkynyl”). In some embodiments, an alkynyl group has 2 carbon atoms (“C2 alkynyl”). The one or more carbon-carbon triple bonds can be internal (such as in 2-butynyl) or terminal (such as in 1- butynyl). Examples of C24 alkynyl groups include, without limitation, ethynyl (C2), 1-propynyl (C3), 2-propynyl (C3), 1-butynyl (C4), 2-butynyl (C4), and the like. Examples of C2-6 alkenyl groups include the aforementioned C24 alkynyl groups as well as pentynyl (C5), hexynyl (C6), and the like. Additional examples of alkynyl include heptynyl (C7), octynyl (C8), and the like.
[0046] “Aryl” refers to a radical of a monocyclic or polycyclic (e.g., bicyclic or tricyclic) 4n+2 aromatic ring system (e.g., having 6, 10, or 14 π electrons shared in a cyclic array) having 6- 14 ring carbon atoms and zero heteroatoms provided in the aromatic ring system (“C6-14 aryl”). In some embodiments, an aryl group has six ring carbon atoms (“C6 aryl”; e.g., phenyl). In some embodiments, an aryl group has ten ring carbon atoms (“C10 aryl”; e.g., naphthyl such as 1- naphthyl and 2-naphthyl). In some embodiments, an aryl group has fourteen ring carbon atoms (“C14 aryl”; e.g., anthracyl). “Aryl” also includes ring systems wherein the aryl ring, as defined above, is fused with one or more carbocyclyl or heterocyclyl groups wherein the radical or point of attachment is on the aryl ring, and in such instances, the number of carbon atoms continue to designate the number of carbon atoms in the aryl ring system. Typical aryl groups include, but are not limited to, groups derived from aceanthrylene, acenaphthylene, acephenanthrylene, anthracene, azulene, benzene, chrysene, coronene, fluoranthene, fluorene, hexacene, hexaphene, hexalene, as-indacene, s-indacene, indane, indene, naphthalene, octacene, octaphene, octalene, ovalene, penta-2,4-diene, pentacene, pentalene, pentaphene, perylene, phenalene, phenanthrene, picene, pleiadene, pyrene, pyranthrene, rubicene, triphenylene, and trinaphthalene. Particularly aryl groups include phenyl, naphthyl, indenyl, and tetrahydronaphthyl.
[0047] “Hetero” when used to describe a compound or a group present on a compound means that one or more carbon atoms in the compound or group have been replaced by a nitrogen, oxygen, or sulfur heteroatom. Hetero may be applied to any of the alkyl groups described above such as alkyl, e.g., heteroalkyl; alkenyl, e.g., heteroalkenyl; alkynyl, e.g., heteroalkynyl; carbocyclyl, e.g., heterocyclyl; aryl, e.g., heteroaryl, and the like having from 1 to 5, and particularly from 1 to 3 heteroatoms.
[0048] “Heteroaryl” refers to a radical of a 5-10 membered monocyclic or bicyclic 4n+2 aromatic ring system (e.g., having 6 or 10 π electrons shared in a cyclic array) having ring carbon atoms and 1-4 ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen and sulfur (“5-10 membered heteroaryl”). In heteroaryl groups that contain one or more nitrogen atoms, the point of attachment can be a carbon or nitrogen atom, as valency permits. Heteroaryl bicyclic ring systems can include one or more heteroatoms in one or both rings. “Heteroaryl” includes ring systems wherein the heteroaryl ring, as defined above, is fused with one or more carbocyclyl or heterocyclyl groups wherein the point of attachment is on the heteroaryl ring, and in such instances, the number of ring members continue to designate the number of ring members in the heteroaryl ring system. “Heteroaryl” also includes ring systems wherein the heteroaryl ring, as defined above, is fused with one or more aryl groups wherein the point of attachment is either on the aryl or heteroaryl ring, and in such instances, the number of ring members designates the number of ring members in the fused (aryl/heteroaryl) ring system. Bicyclic heteroaryl groups wherein one ring does not contain a heteroatom (e.g., indolyl, quinolinyl, carbazolyl, and the like) the point of attachment can be on either ring, i.e., either the ring bearing a heteroatom (e.g., 2-indolyl) or the ring that does not contain a heteroatom (e.g., 5- indolyl).
[0049] In some embodiments, a heteroaryl group is a 5-10 membered aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-10 membered heteroaryl”). In some embodiments, a heteroaryl group is a 5-8 membered aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-8 membered heteroaryl”). In some embodiments, a heteroaryl group is a 5-6 membered aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-6 membered heteroaryl”). In some embodiments, the 5-6 membered heteroaryl has 1-3 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6 membered heteroaryl has 1-2 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5- 6 membered heteroaryl has 1 ring heteroatom selected from nitrogen, oxygen, and sulfur.
[0050] “Carbocyclyl” or “carbocyclic” refers to a radical of a non-aromatic cyclic hydrocarbon group having from 3 to 10 ring carbon atoms (“C3-10 carbocyclyl”) and zero heteroatoms in the non-aromatic ring system. In some embodiments, a carbocyclyl group has 3 to 8 ring carbon atoms (“ C3-8 carbocyclyl”). In some embodiments, a carbocyclyl group has 3 to 6 ring carbon atoms (“C3-6 carbocyclyl”). In some embodiments, a carbocyclyl group has 3 to 6 ring carbon atoms (“C3-6 carbocyclyl”). In some embodiments, a carbocyclyl group has 5 to 10 ring carbon atoms (“C5-10 carbocyclyl”). Exemplary C3-6 carbocyclyl groups include, without limitation, cyclopropyl (C3), cyclopropenyl (C3), cyclobutyl (C4), cyclobutenyl (C4), cyclopentyl (C5), cyclopentenyl (C5), cyclohexyl (C6), cyclohexenyl (C6), cyclohexadienyl (C6), and the like. Exemplary C3-5 carbocyclyl groups include, without limitation, the aforementioned C3-6 carbocyclyl groups as well as cycloheptyl (C7), cycloheptenyl (C7), cycloheptadienyl (C7), cycloheptatrienyl (C7), cyclooctyl (C8), cyclooctenyl (C8), bicyclo[2.2.1]heptanyl (C7), bicyclo[2.2.2]octanyl (C8), and the like. Exemplary C3-10 carbocyclyl groups include, without limitation, the aforementioned C3-8 carbocyclyl groups as well as cyclononyl (C9), cyclononenyl (C9), cyclodecyl (C10), cyclodecenyl (C10), octahydro-lH-indenyl (C9), decahydronaphthalenyl (C10), spiro[4.5]decanyl (C10), and the like. As the foregoing examples illustrate, in certain embodiments, the carbocyclyl group is either monocyclic (“monocyclic carbocyclyl”) or contain a fused, bridged or spiro ring system such as a bicyclic system (“bicyclic carbocyclyl”) and can be saturated or can be partially unsaturated. “Carbocyclyl” also includes ring systems wherein the carbocyclyl ring, as defined above, is fused with one or more aryl or heteroaryl groups wherein the point of attachment is on the carbocyclyl ring, and in such instances, the number of carbons continue to designate the number of carbons in the carbocyclic ring system.
[0051] “Heterocyclyl” or “heterocyclic” refers to a radical of a 3- to 10-membered non-aromatic ring system having ring carbon atoms and 1 to 4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, sulfur, boron, phosphorus, and silicon (“3-10 membered heterocyclyl”). In heterocyclyl groups that contain one or more nitrogen atoms, the point of attachment can be a carbon or nitrogen atom, as valency permits. A heterocyclyl group can either be monocyclic (“monocyclic heterocyclyl”) or a fused, bridged or spire ring system such as a bicyclic system (“bicyclic heterocyclyl”), and can be saturated or can be partially unsaturated. Heterocyclyl bicyclic ring systems can include one or more heteroatoms in one or both rings. “Heterocyclyl” also includes ring systems wherein the heterocyclyl ring, as defined above, is fused with one or more carbocyclyl groups wherein the point of attachment is either on the carbocyclyl or heterocyclyl ring, or ring systems wherein the heterocyclyl ring, as defined above, is fused with one or more aryl or heteroaryl groups, wherein the point of attachment is on the heterocyclyl ring, and in such instances, the number of ring members continue to designate the number of ring members in the heterocyclyl ring system.
[0052] In some embodiments, a heterocyclyl group is a 5-10 membered non-aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, sulfur, boron, phosphorus, and silicon (“5-10 membered heterocyclyl”). In some embodiments, a heterocyclyl group is a 5-8 membered non-aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-8 membered heterocyclyl”). In some embodiments, a heterocyclyl group is a 5-6 membered non-aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-6 membered heterocyclyl”). In some embodiments, the 5-6 membered heterocyclyl has 1-3 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6 membered heterocyclyl has 1-2 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6 membered heterocyclyl has one ring heteroatom selected from nitrogen, oxygen, and sulfur.
[0053] Exemplary 3 -membered heterocyclyl groups containing one heteroatom include, without limitation, azirdinyl, oxiranyl, thiorenyl. Exemplary 4-membered heterocyclyl groups containing one heteroatom include, without limitation, azetidinyl, oxetanyl and thietanyl. Exemplary 5-membered heterocyclyl groups containing one heteroatom include, without limitation, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothiophenyl, dihydrothiophenyl, pyrrolidinyl, dihydropyrrolyl and pyrrolyl-2, 5-dione. Exemplary 5-membered heterocyclyl groups containing two heteroatoms include, without limitation, dioxolanyl, oxasulfuranyl, disulfuranyl, and oxazolidin-2-one. Exemplary 5-membered heterocyclyl groups containing three heteroatoms include, without limitation, triazolinyl, oxadiazolinyl, and thiadiazolinyl. Exemplary 6-membered heterocyclyl groups containing one heteroatom include, without limitation, piperidinyl, tetrahydropyranyl, dihydropyridinyl, and thianyl. Exemplary 6-membered heterocyclyl groups containing two heteroatoms include, without limitation, piperazinyl, morpholinyl, dithianyl, dioxanyl. Exemplary 6-membered heterocyclyl groups containing three heteroatoms include, without limitation, triazinanyl. Exemplary 7-membered heterocyclyl groups containing one heteroatom include, without limitation, azepanyl, oxepanyl and thiepanyl. Exemplary 8-membered heterocyclyl groups containing one heteroatom include, without limitation, azocanyl, oxecanyl and thiocanyl. Exemplary 5-membered heterocyclyl groups fused to a C6 aryl ring (also referred to herein as a 5,6-bicyclic heterocyclic ring) include, without limitation, indolinyl, isoindolinyl, dihydrobenzofuranyl, dihydrobenzothienyl, benzoxazolinonyl, and the like. Exemplary 6- membered heterocyclyl groups fused to an aryl ring (also referred to herein as a 6,6-bicyclic heterocyclic ring) include, without limitation, tetrahydroquinolinyl, tetrahydroisoquinolinyl, and the like.
[0054] “Cyano” refers to -CN.
[0055] “Halo” or “halogen” refers to a fluorine atom (i.e., fluoro or -F), a chlorine atom (i.e., chloro or -Cl), a bromine atom (i.e., bromo or -Br), and an iodine atom (i.e., iodo or -I). In certain embodiments, the halo group is fluoro or chloro.
[0056] “Haloalkyl” refers to an alkyl group substituted with one or more halogen atoms. [0057] “Nitro” refers to -NO2.
[0058] In general, the term “substituted,” whether preceded by the term “optionally” or not, means that at least one hydrogen present on a group (e.g., a carbon or nitrogen atom) is replaced with a permissible substituent, e.g., a substituent which upon substitution results in a stable compound, e.g., a compound which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, or other reaction. Unless otherwise indicated, a “substituted” group has a substituent at one or more substitutable positions of the group, and when more than one position in any given structure is substituted, the substituent is either the same or different at each position.
[0059] The term “pharmaceutically acceptable salt” refers to those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio. The general concept of pharmaceutically acceptable salts has been discussed in the art, including, for example, Berge et al., which describes pharmaceutically acceptable salts in detail in J Pharmaceutical Sciences (1977) 66: 1-19. Pharmaceutically acceptable salts of the compounds described herein include those derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable, nontoxic acid addition salts are salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid or by using other methods used in the art such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2- naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3 -phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and the like. Pharmaceutically acceptable salts derived from appropriate bases include alkali metal, alkaline earth metal, ammonium and N+(C1-4alkyl)4 salts. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Further pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, lower alkyl sulfonate, and aryl sulfonate.
[0060] The term “modified-release polymer” refers to a polymer that is used in a formulation (e.g., tablets and capsules) to modify the release rate of the drug upon administration to a subject. For example, a modified-release polymer is used to dissolve a drug overtime in order to be released slower and steadier into the bloodstream. For example, a modified-release polymer is a controlled- release polymer. For example, a modified-release polymer or a controlled-release polymer is an HPMC polymer. In some embodiments, a modified-release polymer may include hydrophilic matrix polymers (e.g., hypromellose, hydroxyl-propyl methylcellulose (HPMC)), hydrophobic matrix polymers (e.g., ethyl cellulose, ethocel), or polyacrylate polymers (e.g., Eudragit® RL100, Eudragit® RS 100).
[0061] The term “diluent” as used herein refers to an excipient used to increase weight and improve content uniformity. For example, diluents include cellulose derivatives (e.g., microcrystalline cellulose), starches (e.g., hydrolyzed starches, and partially pregelatinized starches), anhydrous lactose, lactose monohydrate, di-calcium phosphate (DCP), sugar alcohols (e.g., sorbitol, xylitol and mannitol)).
[0062] The term “glidanf ’ as used herein refers to an excipient used to promote powder flow by reducing interparticle friction and cohesion. For example, glidants include fumed silica (e.g., colloidal silicon dioxide), talc, and magnesium carbonate.
[0063] The term “lubricant” as used herein refers to an excipient used to prevent ingredients from clumping together and from sticking to the tablet punches or capsule filling machine. Lubricants are also used to ensure that tablet formation and ejection can occur with low friction between the solid and die wall. For example, lubricants include magnesium stearate, calcium stearate, stearic acid, talc, silica, and fats (e.g., vegetable stearin).
[0064] The term “coating” as used herein refers to an excipient to protect tablet ingredients from deterioration by moisture in the air and make large or unpleasant-tasting tablets easier to swallow.
[0065] The embodiments disclosed herein are not intended to be limited in any manner by the above exemplary listing of chemical groups and substituents. Those skilled in the art will recognize that several embodiments are possible within the scope and spirit of the present disclosure. The following description illustrates the disclosure and, of course, should not be construed in any way as limiting the scope of the inventions described herein.
II. Compounds and Compositions
[0066] In one aspect, provided is a compound of Formula (I-IB) having an isoxazole core and chosen from:
Figure imgf000029_0001
Figure imgf000030_0001
Figure imgf000031_0001
Figure imgf000032_0001
or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof
[0067] In one aspect, provided herein is a compound of Formula (I-a) having an oxadiazole core:
Figure imgf000033_0001
or a pharmaceutically acceptable salt thereof.
[0068] In one aspect, provided herein is a compound of Formula (I-b) having an oxadiazole core:
Figure imgf000033_0002
or a pharmaceutically acceptable salt thereof.
[0069] In one aspect, provided herein is a compound of Formula (I-c) having an oxadiazole core:
Figure imgf000033_0003
or a pharmaceutically acceptable salt thereof.
[0070] In one aspect, provided herein is a compound of Formula (I-d) having an oxadiazole core:
Figure imgf000034_0001
or a pharmaceutically acceptable salt thereof.
[0071] In one aspect, provided herein is a compound of Formula (II) having an oxadiazole core:
Figure imgf000034_0002
or a pharmaceutically acceptable salt thereof.
[0072] In one aspect, provided herein is a compound of Formula (III) having an oxadiazole core:
Figure imgf000034_0003
or a pharmaceutically acceptable salt thereof. [0073] In one aspect, provided herein is a compound of Formula (IV) having an oxadiazole core:
Figure imgf000035_0001
or a pharmaceutically acceptable salt thereof.
[0074] In one aspect, provided herein is a compound of Formula (V) having an oxadiazole core:
Figure imgf000035_0002
or a pharmaceutically acceptable salt thereof.
[0075] In one aspect, provided herein is a compound of Formula (VI) having an oxadiazole core:
Figure imgf000036_0001
[0076] or a pharmaceutically acceptable salt thereof.
[0077] In one aspect, provided herein is a compound of Formula (VII) having an isoxazole core:
Figure imgf000036_0002
or a pharmaceutically acceptable salt thereof, wherein:
R1 is chosen from -H or a C1-6alkyl, such a methyl;
R2 is chosen from -H or a C1-6alkyl, such a methyl;
R3 is independently chosen from a halogen, a C1-6alkyl, a carbocyclyl, or an alkoxy, wherein the C1-6alkyl, carbocyclyl or alkoxy optionally comprises at least one halogen substituent; and n is 0, 1, 2, 3, or 4.
[0078] In one aspect of the compound of Formula (VII), R1 is methyl, R2 is hydrogen, R3 is chosen from a cyclopropyl, a halogen such as bromine, or -CF3, and n is 1.
[0079] In one aspect, provided herein is a compound of Formula (VII) having an isoxazole core and chosen from:
Figure imgf000037_0001
Figure imgf000038_0001
or a pharmaceutically acceptable salt thereof.
[0080] Compounds described herein can comprise one or more asymmetric centers, and thus can exist in various isomeric forms, e.g., enantiomers and/or diastereomers. For example, the compounds described herein can be in the form of an individual enantiomer, diastereomer or geometric isomer, or can be in the form of a mixture of stereoisomers, including racemic mixtures and mixtures enriched in one or more stereoisomer. Isomers can be isolated from mixtures by methods known to those skilled in the art, including chiral high pressure liquid chromatography (HPLC) and the formation and crystallization of chiral salts; or preferred isomers can be prepared by asymmetric syntheses. See, for example, Jacques et al., Enantiomers, Racemates and Resolutions (Wiley Interscience, New York, 1981); Wilen et al., Tetrahedron 33:2725 (1977); Eliel, Stereochemistry of Carbon Compounds (McGraw-Hill, NY, 1962); and Wilen, Tables of Resolving Agents and Optical Resolutions p. 268 (E.L. Eliel, Ed., Univ, of Notre Dame Press, Notre Dame, IN 1972). Embodiments disclosed herein additionally encompasses compounds described herein as individual isomers substantially free of other isomers, and alternatively, as mixtures of various isomers.
[0081] As used herein a pure enantiomeric compound is substantially free from other enantiomers or stereoisomers of the compound (i.e., in enantiomeric excess). In other words, an “S” form of the compound is substantially free from the “R” form of the compound and is, thus, in enantiomeric excess of the “R” form. The term “enantiomerically pure” or “pure enantiomer” denotes that the compound comprises more than 75% by weight, such as more than 80% by weight, more than 85% by weight, more than 90% by weight, more than 91% by weight, more than 92% by weight, more than 93% by weight, more than 94% by weight, more than 95% by weight, more than 96% by weight, more than 97% by weight, more than 98% by weight, more than 98.5% by weight, more than 99% by weight, more than 99.2% by weight, more than 99.5% by weight, more than 99.6% by weight, more than 99.7% by weight, more than 99.8% by weight, or more than 99.9% by weight, of the enantiomer. In certain embodiments, the weights are based upon total weight of all enantiomers or stereoisomers of the compound.
[0082] In certain aspects, provided are compositions comprising the compounds described herein. In some embodiments, an enantiomerically pure compound can be present in the compositions with other active or inactive ingredients. For example, a pharmaceutical composition comprising enantiomerically pure R-compound can comprise, for example, about 90% excipient and about 10% enantiomerically pure R-compound. In certain embodiments, the enantiomerically pure R-compound in such compositions can, for example, comprise at least about 95% by weight R-compound and at most about 5% by weight S-compound, by total weight of the compound. For example, a pharmaceutical composition comprising enantiomerically pure S- compound can comprise, for example, about 90% excipient and about 10% enantiomerically pure S-compound. In certain embodiments, the enantiomerically pure S-compound in such compositions can, for example, comprise at least about 95% by weight S-compound and at most about 5% by weight R-compound, by total weight of the compound. In certain embodiments, the active ingredient can be formulated with little or no excipient or carrier.
[0083] Compounds described herein may also comprise one or more isotopic substitutions. For example, H may be in any isotopic form, including 1H, 2H (D or deuterium), and 3H (T or tritium); C may be in any isotopic form, including 12C, 13C, and 14C. O may be in any isotopic form, including 16O and 18O, and F may be in any isotopic form, including 18F and 19F.
III. Methods of Treatment
[0084] The compounds and compositions described above and herein can be used to treat a neurological disorder, a disorder associated with excessive neuronal excitability, or a disorder associated with a gain-of-function mutation in a gene (e.g., KCNT1).
[0085] In some aspects, provided are methods of treating a neurological disorder, a disorder associated with excessive neuronal excitability, or a disorder associated with a gain-of-function mutation of a gene, by administering to a subject in need thereof an effective amount of any of the compounds described herein or a pharmaceutically acceptable salt thereof, or pharmaceutical compositions comprising such compounds or a pharmaceutically acceptable salt thereof.
[0086] Exemplary diseases, disorders, or conditions include epilepsy and other encephalopathies (e.g., MMFSI or EIMFS, ADNFLE, West syndrome, infantile spasms, epileptic encephalopathy, developmental and epileptic encephalopathy (DEE), early infantile epileptic encephalopathy (EIEE), generalized epilepsy, focal epilepsy, multifocal epilepsy, temporal lobe epilepsy, Ohtahara syndrome, early myoclonic encephalopathy, Lennox-Gastaut syndrome, drug resistant epilepsy, seizures (e.g., frontal lobe seizures, generalized tonic clonic seizures, asymmetric tonic seizures, focal seizures), leukodystrophy, hypomyelinating leukodystrophy, and leukoencephalopathy), cardiac dysfunctions (e.g., cardiac arrhythmia, Brugada syndrome, myocardial infarction), pulmonary vasculopathy/hemorrhage, pain and related conditions (e.g., neuropathic pain, acute/chronic pain, migraine, etc.), muscle disorders (e.g.. myotonia, neuromyotonia, cramp muscle spasms, spasticity), itch and pruritis, movement disorders (e.g., ataxia and cerebellar ataxias), psychiatric disorders (e.g., major depression, anxiety, bipolar disorder, schizophrenia, attention-deficit hyperactivity disorder), neurodevelopmental disorder, learning disorders, intellectual disability, Fragile X, neuronal plasticity, and autism spectrum disorders.
[0087] In some embodiments, the neurological disorder, the disorder associated with excessive neuronal excitability, or the disorder associated with a gain-of-function mutation in a gene (e.g., KCNT1) is selected from EIMFS, ADNFLE, or West syndrome. In some embodiments, the neurological disorder, the disorder associated with excessive neuronal excitability, or the disorder associated with a gain-of-function mutation in a gene (e.g., KCNT1) is selected from infantile spasms, epileptic encephalopathy, focal epilepsy, Ohtahara syndrome, developmental and epileptic encephalopathy, or Lennox-Gastaut syndrome. In some embodiments, the neurological disorder, the disorder associated with excessive neuronal excitability, or the disorder associated with a gain-of-function mutation in a gene (e.g., KCNT1) is seizure. In some embodiments, the neurological disorder, the disorder associated with excessive neuronal excitability, or the disorder associated with a gain-of-function mutation in a gene (e.g., KCNT1) is selected from cardiac arrhythmia, Brugada syndrome, or myocardial infarction. [0088] In some embodiments, the neurological disorder, the disorder associated with excessive neuronal excitability, or the disorder associated with a gain-of-function mutation in a gene (e.g., KCNT1) is selected from a learning disorder, Fragile X, intellectual function, neuronal plasticity, a psychiatric disorder, or an autism spectrum disorder.
[0089] Accordingly, the compounds, pharmaceutically acceptable salts thereof, and compositions disclosed herein can be administered to a subject with a neurological disorder, a disorder associated with excessive neuronal excitability, or a disorder associated with a gain-of- function mutation in a gene such as KCNT1 (e.g., EIMFS, ADNFLE, West syndrome, infantile spasms, epileptic encephalopathy, focal epilepsy, Ohtahara syndrome, developmental and epileptic encephalopathy, Lennox-Gastaut syndrome, seizures, cardiac arrhythmia, Brugada syndrome, and myocardial infarction).
[0090] EIMFS is a rare and debilitating genetic condition characterized by an early onset (before 6 months of age) of almost continuous heterogeneous focal seizures, where seizures appear to migrate from one brain region and hemisphere to another. Patients with EIMFS are generally intellectually impaired, non-verbal and non-ambulatory. While several genes have been implicated to date, the gene that is most commonly associated with EIMFS is KCNT1. Several de novo mutations in KCNT1 have been identified in patients with EIMFS, including V271F, G288S, R428Q, R474Q, R474H, R474C, I760M, A934T, P924L, G243S, H257D, A259D, R262Q, Q270E, L274I, F346L, C377S, R398Q, P409S, A477T, F502V, M516V, Q550del, K629E, K629N, I760F, E893K, M896K, R933G, R950Q, and KI 154Q. Barcia et al. (2012) Nat Genet. 44: 1255-1260; Ishii et al. (2013) Gene 531 :467-471; McTague et al. (2013) Brain. 136: 1578-1591; Epi4K Consortium & Epilepsy Phenome/Genome Project. (2013) Nature 501 :217-221; Lim et al. (2016) Neurogenetics; Ohba et al. (2015) Epilepsia 56:el21-el28; Zhou et al. (2018) Genes Brain Behav. el2456; Moller et al. (2015) Epilepsia, el 14-20; Numis et al. (2018) Epilepsia. 1889-1898; Madaan et al. Brain Dev. 40(3):229-232; McTague et al. (2018) Neurology. 90(l):e55-e66; Kawasaki et al. (2017) J Pediatr. 191 :270-274; Kim et al. (2014) Cell Rep. 9(5): 1661-1672; Ohba et al. (2015) Epilepsia. 56(9):el21-8; Rizzo et al. (2016) Mol Cell Neurosci. 72:54-63; Zhang et al. (2017) Clin Genet. 91 (5):717-724; Mikati et al. (2015) Ann Neurol. 78(6) :995-9; Baumer et al. (2017) Neurology. 89(21):2212; Dilena et al. (2018) Neurotherapeutics. 15(4): 1112-1126. These mutations may be gain-of-function, missense mutations that are dominant (i.e., present on only one allele) and result in change-in-function of the encoded potassium channel that causes a marked increase in whole cell current when tested in Xenopus oocyte or mammalian expression systems (see e.g. Milligan et al. (2015) Ann Neurol. 75(4): 581-590; Barcia et al. (2012) Nat Genet. 44(11): 1255-1259; and Mikati et al. (2015) Ann Neurol. 78(6): 995-999).
[0091] ADNFLE has a later onset than EIMFS, generally in mid-childhood, and is generally a less severe condition. It is characterized by nocturnal frontal lobe seizures and can result in psychiatric, behavioral and cognitive disabilities in patients with the condition. While ADNFLE is associated with genes encoding several neuronal nicotinic acetylcholine receptor subunits, mutations in the KCNT1 gene have been implicated in more severe cases of the disease (Heron et al. (2012) Nat Genet. 44: 1188-1190). Functional studies of the mutated KCNT1 genes associated with ADNFLE indicated that the underlying mutations (M896I, R398Q, Y796H, and R928C) were dominant, gain-of-function mutations (Milligan et al. (2015) Ann Neurol. 75(4): 581-590; Mikati et al. (2015) Ann Neurol. 78(6): 995-999).
[0092] West syndrome is a severe form of epilepsy composed of a triad of infantile spasms, an interictal electroencephalogram (EEG) pattern termed hypsarrhythmia, and mental retardation, although a diagnosis can be made one of these elements is missing. Mutations in KCNT1, including G652V and R474H, have been associated with West syndrome (Fukuoka et al. (2017) Brain Dev 39:80-83 and Ohba et al. (2015) Epilepsia 56:el21-el28). Treatment targeting the KCNT1 channel suggests that these mutations are gain-of-function mutations (Fukuoka et al. (2017) Brain Dev 39:80-83).
[0093] In one aspect, disclosed herein is a method of treating treat a disorder associated with excessive neuronal excitability or a disorder associated with a gain-of-function mutation in a gene such as KCNT1 (for example, epilepsy and other encephalopathies (e.g., MMFSI or EIMFS), ADNFLE, West syndrome, infantile spasms, epileptic encephalopathy, focal epilepsy, Ohtahara syndrome, DEE, Lennox-Gastaut syndrome, seizures, leukodystrophy, leukoencephalopathy, intellectual disability, Multifocal Epilepsy, Generalized tonic clonic seizures, Drug resistant epilepsy, Temporal lobe epilepsy, cerebellar ataxia, Asymmetric Tonic Seizures), cardiac dysfunctions (e.g., cardiac arrhythmia, Brugada syndrome, myocardial infarction), pain and related conditions (e.g., neuropathic pain, acute/chronic pain, migraine, etc.), muscle disorders (e.g., myotonia, neuromyotonia, cramp muscle spasms, spasticity), itch and pruritis, ataxia and cerebellar ataxias, psychiatric disorders (e.g., major depression, anxiety, bipolar disorder, schizophrenia), learning disorders, Fragile X, neuronal plasticity, and autism spectrum disorders), comprising administering to a subject in need thereof a compound disclosed herein or a pharmaceutically acceptable salt thereof or a pharmaceutical composition disclosed herein.
[0094] In some examples, the subject presenting with a disorder that may be associated with a gain-of-function mutation in KCNT1 is genotyped to confirm the presence of a known gain-of- function mutation in KCNT1 prior to administration of the compounds or a pharmaceutically acceptable salt thereof or compositions disclosed herein. For example, whole exome sequencing can be performed on the subject. Gain-of-function mutations associated with EIMFS may include, but are not limited to, V271F, G288S, R428Q, R474Q, R474H, R474C, I760M, A934T, P924L, G243S, H257D, A259D, R262Q, Q270E, L274I, F346L, C377S, R398Q, P409S, A477T, F502V, M516V, Q550del, K629E, K629N, I760F, E893K, M896K, R933G, R950Q, and KI 154Q. Gain- of-function mutations associated with ADNFLE may include, but are not limited to, M896I, R398Q, Y796H, R928C, and G288S. Gain-of-function mutations associated with West syndrome may include, but are not limited to, G652V and R474H. Gain-of-function mutations associated with temporal lobe epilepsy may include, but are not limited to, R133H and R565H. Gain-of- function mutations associated with Lennox-Gastaut may include, but are not limited to, R209C. Gain-of-function mutations associated with seizures may include, but are not limited to, A259D, G288S, R474C, and R474H. Gain-of-function mutations associated with leukodystrophy may include, but are not limited to, G288S and Q906H. Gain-of-function mutations associated with Multifocal Epilepsy may include, but are not limited to, V340M. Gain-of-function mutations associated with early-onset epilepsy (EOE) may include, but are not limited to, F346L and A934T. Gain-of-function mutations associated with Early-onset epileptic encephalopathies (EOEE) may include, but are not limited to, R428Q. Gain-of-function mutations associated with developmental and epileptic encephalopathies may include, but are not limited to, F346L, R474H, and A934T. Gain-of-function mutations associated with epileptic encephalopathies may include, but are not limited to, L437F, Y796H, P924L, and R961H. Gain-of-function mutations associated with Early Infantile Epileptic Encephalopathy (EIEE) may include, but are not limited to, M896K. Gain-of- function mutations associated with drug-resistant epilepsy and generalized tonic-clonic seizure may include, but are not limited to, F346L. Gain-of-function mutations associated with migrating partial seizures of infancy may include, but are not limited to, R428Q. Gain-of-function mutations associated with Leukoencephalopathy may include, but are not limited to, F932I. Gain-of-function mutations associated with NFLE may include, but are not limited to, A934T and R950Q. Gain- of-function mutations associated with Ohtahara syndrome may include, but are not limited to, A966T. Gain-of-function mutations associated with infantile spasms may include, but are not limited to, P924L. Gain-of-function mutations associated with Brugada Syndrome may include, but are not limited to, R1106Q. Gain-of-function mutations associated with Brugada Syndrome may include, but are not limited to, R474H.
[0095] In other examples, the subject is first genotyped to identify the presence of a mutation in KCNT1, and this mutation is then confirmed to be a gain-of-function mutation using standard in vitro assays, such as those described in Milligan et al. (2015) Ann Neurol. 75(4): 581-590. Typically, the presence of a gain-of-function mutation is confirmed when the expression of the mutated KCNT1 allele results an increase in whole cell current compared to the whole cell current resulting from expression of wild-type KCNT1, as may be assessed using whole-cell electrophysiology (such as described in Milligan et al. (2015) Ann Neurol. 75(4): 581-590; Barcia et al. (2012) Nat Genet. 44(11): 1255-1259; Mikati et al. (2015) Ann Neurol. 78(6): 995-999; or Rizzo et al. Mol Cell Neurosci. (2016) 72:54-63). This increase of whole cell current can be, for example, an increase of at least or about 50%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, or more. The subject can then be confirmed to have a disease or condition associated with a gain-of- function mutation in KCNT1.
[0096] In particular examples, the subject is confirmed as having a KCNT1 allele containing a gain-of-function mutation (e.g., V271F, G288S, R398Q, R428Q, R474Q, R474H, R474C, G652V, I760M, Y796H, M896I, P924L, R928C, or A934T).
[0097] The compounds or pharmaceutically acceptable salts thereof disclosed herein or the pharmaceutical composition disclosed herein (e.g., a pharmaceutical composition comprising a compound or pharmaceutically acceptable salt thereof disclosed herein, and a pharmaceutically acceptable excipient) can also be used therapeutically for conditions associated with excessive neuronal excitability where the excessive neuronal excitability is not necessarily the result of a gain-of-function mutation in KCNT1. Even in instances where the disease is not the result of increased KCNT1 expression and/or activity, inhibition of KCNT1 expression and/or activity can nonetheless result in a reduction in neuronal excitability, thereby providing a therapeutic effect. Thus, the compounds or pharmaceutically acceptable salts thereof disclosed herein or the pharmaceutical compositions disclosed herein can be used to treat a subject with conditions associated with excessive neuronal excitability, for example, epilepsy and other encephalopathies (e.g., EIMFS, ADNFLE, West syndrome, infantile spasms, epileptic encephalopathy, focal epilepsy, Ohtahara syndrome, developmental and epileptic encephalopathy, Lennox-Gastaut syndrome, seizures) or cardiac dysfunctions (e.g., cardiac arrhythmia, Brugada syndrome, myocardial infarction), regardless of whether or not the disorder is associated with a gain-of- function mutation in KCNT1.
[0098] In some variations of the foregoing, a “subject” to which administration is contemplated includes, but is not limited to, humans (i.e., a male or female of any age group, e.g., a pediatric subject (e.g., an infant, child, adolescent) or an adult subject (e.g., a young adult, middle-aged adult, or senior adult)) and/or a non-human animal, e.g., a mammal such as primates (e.g., cynomolgus monkeys, rhesus monkeys), cattle, pigs, horses, sheep, goats, rodents, cats, and/or dogs. In certain embodiments, the subject is a human. In certain embodiments, the subject is a non-human animal.
[0099] Some variations of the foregoing, “treating” or “treatment”, as used herein, contemplate an action that occurs while a subject is suffering from the specified disease, disorder or condition, which reduces the severity of the disease, disorder or condition, or retards or slows the progression of the disease, disorder or condition (also “therapeutic treatment”). In some variations, “treating” or “treatment” refers to a method or procedure for obtaining beneficial or desired results — for example, clinical results. Beneficial or desired results may include: (1) alleviating one or more symptoms caused by or associated with a disease, disorder, or condition; (2) reducing the extent of the disease, disorder, or condition; (3) slowing or stopping the development or progression of one or more symptoms caused by or associated with the disease, disorder, or condition (for example, stabilizing the disease, disorder, or condition); and (4) relieving the disease, for example, by causing the regression of one or more clinical symptoms (e.g., ameliorating the disease state, enhancing the effect of another medication, delaying or stopping the progression of the disease, increasing the quality of life, and/or prolonging survival rates).
[0100] In some variations of the foregoing, an “effective amount” of a compound or pharmaceutically acceptable salt thereof refers to an amount sufficient to elicit the desired biological response. As will be appreciated by those of ordinary skill in this art, the effective amount of a compound or pharmaceutically acceptable salt thereof may vary depending on such factors as the desired biological endpoint, the pharmacokinetics of the compound or pharmaceutically acceptable salt thereof, the disease being treated, the mode of administration, and the age, weight, health, and condition of the subject.
[0101] In some embodiments, a therapeutically effective amount of the compound or pharmaceutically acceptable salt thereof disclosed herein is administered to the subject (e.g., a human). In some variations of the foregoing, a “therapeutically effective amount” of a compound or pharmaceutically acceptable salt thereof is an amount sufficient to provide a therapeutic benefit in the treatment of a disease, disorder or condition, or to delay or minimize one or more symptoms associated with the disease, disorder or condition. A therapeutically effective amount of a compound or pharmaceutically acceptable salt thereof means an amount of therapeutic agent, alone or in combination with other therapies, which provides a therapeutic benefit in the treatment of the disease, disorder or condition. The term “therapeutically effective amount” can encompass an amount that improves overall therapy, reduces or avoids symptoms or causes of the disease or condition, or enhances the therapeutic efficacy of another therapeutic agent.
[0102] In some embodiments, the method provided involves treating a disorder associated with a gain-of-function mutation of KCNT1. In some variations, a “disorder associated with a gain-of- function mutation in KCNT1” refers to a disorder that is associated with, is partially or completely caused by, or has one or more symptoms that are partially or completely caused by, a mutation in KCNT1 that results in a gain-of-function phenotype, i.e., an increase in activity of the potassium channel encoded by KCNT1 resulting in an increase in whole cell current. In some variations, a “gain-of-function mutation of KCNT1” is a mutation in KCNT1 that results in an increase in activity of the potassium channel encoded by KCNT1. Activity can be assessed by, for example, ion flux assay or electrophysiology (e.g., using the whole cell patch clamp technique). Typically, a gain-of-function mutation results in an increase of at least or about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 175%, 200%, 225%, 250%, 275%, 300%, 325%, 350%, 375%, 400%, or more compared to the activity of a potassium channel encoded by a wild-type KCNT1.
IV. Pharmaceutical Compositions and Routes of Administration
[0103] Compounds or pharmaceutically acceptable salts thereof provided in accordance with the present disclosure are usually administered in the form of pharmaceutical compositions. Therefore, disclosed herein are pharmaceutical compositions that contain, as the active ingredient, one or more of the compounds described, or a pharmaceutically acceptable salt or ester thereof, and one or more pharmaceutically acceptable excipients, carriers, including inert solid diluents and fillers, diluents, including sterile aqueous solution and various organic solvents, permeation enhancers, solubilizers and adjuvants. The pharmaceutical compositions may be administered alone or in combination with other therapeutic agents. Such compositions may be prepared in a manner disclosed in the pharmaceutical art, including, for example, in Remington’s Pharmaceutical Sciences, Mace Publishing Co., Philadelphia, Pa. 17th Ed. (1985) and Modern Pharmaceutics, Marcel Dekker, Inc. 3rd Ed. (G. S. Banker & C. T. Rhodes, Eds.).
[0104] The pharmaceutical compositions may be administered in either single or multiple doses by any of the accepted modes of administration of agents having similar utilities, for example as described in those patents and patent applications incorporated by reference, including rectal, buccal, intranasal and transdermal routes, by intra-arterial injection, intravenously, intraperitoneally, parenterally, intramuscularly, subcutaneously, orally, topically, as an inhalant, or via an impregnated or coated device such as a stent, for example, or an artery-inserted cylindrical polymer.
[0105] One mode for administration is parenteral, particularly by injection. The forms in which the novel compositions disclosed herein may be incorporated for administration by injection include aqueous or oil suspensions, or emulsions, with sesame oil, corn oil, cottonseed oil, or peanut oil, as well as elixirs, mannitol, dextrose, or a sterile aqueous solution, and similar pharmaceutical vehicles. Aqueous solutions in saline are also conventionally used for injection. Ethanol, glycerol, propylene glycol, liquid polyethylene glycol, and the like (and suitable mixtures thereof), cyclodextrin derivatives, and vegetable oils may also be employed. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.
[0106] Sterile injectable solutions are prepared by incorporating a compound or pharmaceutically acceptable salt thereof as disclosed herein in the required amount in the appropriate solvent with various other ingredients as enumerated above, as desired, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the desired other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, exemplary methods of preparation include vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-fdtered solution thereof.
[0107] Oral administration is another route for administration of the compounds or pharmaceutically acceptable salts thereof as disclosed herein. Administration may be via capsule or enteric coated tablets, or the like. In making the pharmaceutical compositions that include at least one compound or pharmaceutically acceptable salt thereof described herein, the active ingredient may be diluted by an excipient and/or enclosed within such a carrier that can be in the form of a capsule, sachet, paper or other container. When the excipient serves as a diluent, it can be in the form of a solid, semi-solid, or liquid material (as above), which acts as a vehicle, carrier or medium for the active ingredient. Thus, the compositions can be in the form of tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as a solid or in a liquid medium), ointments containing, for example, up to 10% by weight of the active compound, soft and hard gelatin capsules, sterile injectable solutions, and sterile packaged powders.
[0108] Some examples of suitable excipients include lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, sterile water, syrup, and methyl cellulose. In certain embodiments, the compositions disclosed herein can additionally include lubricating agents such as talc, magnesium stearate, and mineral oil; wetting agents; emulsifying and suspending agents; preserving agents such as methyl and propylhydroxy-benzoates; sweetening agents; glidants; and flavoring agents.
[0109] The compositions disclosed herein can be formulated so as to provide quick, sustained or delayed release of the active ingredient after administration to the patient by employing procedures known in the art. Controlled release drug delivery systems for oral administration include osmotic pump systems and dissolutional systems containing polymer-coated reservoirs or drug-polymer matrix formulations. Examples of controlled release systems are given in U.S. Pat. Nos. 3,845,770; 4,326,525; 4,902,514; and 5,616,345. Another embodiment for use in the methods disclosed herein may employ transdermal delivery devices (“patches”). Such transdermal patches may be used to provide continuous or discontinuous infusion of the compounds or pharmaceutically acceptable salts thereof as disclosed herein in controlled amounts. The construction and use of transdermal patches for the delivery of pharmaceutical agents is described, for example, in U.S. Pat. Nos. 5,023,252, 4,992,445 and 5,001,139. Such patches may be constructed for continuous, pulsatile, or on-demand delivery of pharmaceutical agents.
[0110] The compositions disclosed herein may be formulated in a unit dosage form. The term “unit dosage forms” refers to physically discrete units suitable as unitary dosages for human subjects and other mammals, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical excipient (e.g., a tablet, capsule, ampoule). The compounds are generally administered in a pharmaceutically effective amount. Preferably, for oral administration, each dosage unit contains from about 1 mg to about 2 g of a compound or pharmaceutically acceptable salt thereof as described herein, and for parenteral administration, preferably from about 0.1 to about 700 mg of a compound or pharmaceutically acceptable salt thereof as described herein. It will be understood, however, that the amount of the compound or pharmaceutically acceptable salt thereof actually administered usually will be determined by a physician, in the light of the relevant circumstances, including the condition to be treated, the chosen route of administration, the actual compound or pharmaceutically acceptable salt thereof administered and its relative activity, the age, weight, and response of the individual patient, the severity of the patient’s symptoms, and the like.
[0111] For preparing solid compositions such as tablets, the principal active ingredient may be mixed with a pharmaceutical excipient to form a solid preformulation composition containing a homogeneous mixture of a compound or pharmaceutically acceptable salt thereof as disclosed herein. When referring to these preformulation compositions as homogeneous, it is meant that the active ingredient is dispersed evenly throughout the composition so that the composition may be readily subdivided into equally effective unit dosage forms such as tablets, pills and capsules.
[0112] The tablets or pills disclosed herein may be coated or otherwise compounded to provide a dosage form affording the advantage of prolonged action, or to protect from the acid conditions of the stomach. For example, the tablet or pill can comprise an inner dosage and an outer dosage component, the latter being in the form of an envelope over the former. The two components can be separated by an enteric layer that serves to resist disintegration in the stomach and permit the inner component to pass intact into the duodenum or to be delayed in release. A variety of materials can be used for such enteric layers or coatings, such materials including a number of polymeric acids and mixtures of polymeric acids with such materials as shellac, cetyl alcohol, and cellulose acetate.
[0113] Compositions for inhalation or insufflation include solutions and suspensions in pharmaceutically acceptable, aqueous or organic solvents, or mixtures thereof, and powders. The liquid or solid compositions may contain suitable pharmaceutically acceptable excipients as described herein. In certain embodiments, the compositions are administered by the oral or nasal respiratory route for local or systemic effect. Compositions in pharmaceutically acceptable solvents may be nebulized by use of inert gases. Nebulized solutions may be inhaled directly from the nebulizing device or the nebulizing device may be attached to a facemask tent or intermittent positive pressure breathing machine. Solution, suspension, or powder compositions may be administered, such as orally or nasally, from devices that deliver the formulation in an appropriate manner.
[0114] In some embodiments, there is provided a pharmaceutical composition comprising a compound, or pharmaceutically acceptable salt thereof, as disclosed herein and at least one pharmaceutically acceptable excipient and/or carrier.
[0115] Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation many equivalents to the specific embodiments described herein. The scope of the present embodiments described herein is not intended to be limited to the above Description or the Examples that follow, but rather is as set forth in the appended claims. Those of ordinary skill in the art will appreciate that various changes and modifications to this description may be made without departing from the spirit or scope of the embodiments disclosed herein, as defined in the claims.
EXAMPLES
[0116] In order that the embodiments described herein may be more fully understood, the following examples are set forth. The synthetic and biological examples described in this application are offered to illustrate the compounds, pharmaceutical compositions, and methods provided herein and are not to be construed in any way as limiting their scope.
[0117] The compounds provided herein can be prepared from readily available starting materials using the following general methods and procedures. It will be appreciated that where typical or preferred process conditions (i.e., reaction temperatures, times, mole ratios of reactants, solvents, pressures, etc.) are given, other process conditions can also be used unless otherwise stated. Optimal reaction conditions may vary with the particular reactants or solvent used, but such conditions can be determined by one skilled in the art by routine optimization.
[0118] Additionally, as will be apparent to those skilled in the art, conventional protecting groups may be necessary to prevent certain functional groups from undergoing undesired reactions. The choice of a suitable protecting group for a particular functional group as well as suitable conditions for protection and deprotection are described in the art. For example, numerous protecting groups, and their introduction and removal, are described in T. W. Greene and P. G. M. Wuts, Protecting Groups in Organic Synthesis, Second Edition, Wiley, New York, 1991, and references cited therein.
[0119] The compounds provided herein may be isolated and purified by known standard procedures. Such procedures include recrystallization, filtration, flash chromatography, trituration, high performance liquid chromatography (HPLC), or supercritical fluid chromatography (SFC). Note that flash chromatography may either be performed manually or via an automated system. The compounds provided herein may be characterized by known standard procedures, such as nuclear magnetic resonance spectroscopy (NMR) or liquid chromatography mass spectrometry (LCMS). NMR chemical shifts are reported in part per million (ppm) and are generated using methods described in the art.
[0120] Abbreviations
DCM Dichloromethane
DEAD Diethyl azodicarboxylate
DIPEA N,N-Diisopropylethylamine
DMF Dimethylformamide
DMSO Dimethylsulfoxide
DMSO-d6 Deuterated dimethylsulfoxide-d6
EtOAc Ethyl acetate
HATU 2-(7-Azabenzotriazol-l-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate
MeOH Methanol THF Tetrahydrofuran
Synthesis and Characterization of Exemplary Compounds
[0121] Exemplary methods for preparing compounds described herein are illustrated in the following synthetic schemes. These schemes are given for the purpose of illustration, and should not be regarded in any manner as limiting the scope or the spirit of the embodiments disclosed herein.
Example 1 - Synthesis of (S)-N-(l-(3-(2-ethylpyridin-4-yl)isoxzol-5-yl)ethyl)-l-methyl-3-
(trifluoromethyl)-lH-pyrazole-5-carboxamide hydrochloride (Formula I-IBa HC1)
Figure imgf000052_0001
[0122] Step 1: Synthesis of (Z)-2-bromoisonicotinaldehyde oxime (2)
Figure imgf000052_0002
[0123] To a stirred solution of compound 1 (20 g, 107.52 mmol) in ethanol (30 mL) and water (150 mL) was added NH2OH.HCI (8.97 g, 129.03 mmol). The reaction mixture was stirred at room temperature for 16 hours. After completion of the reaction, the reaction mixture was concentrated under reduced pressure, diluted in water (50 mL), extracted in ethyl acetate (3 x 50 mL), dried over Na2SO4, and concentrated under reduced pressure. The crude product was purified by column chromatography (100-200 silica gel), eluting with 30-50% EtOAc:Hexane to afford compound 2 (18 g, 89.54 mmol, 83.2% yield).
[0124] Step 2: Synthesis of (E)-2-bromo-N-hydroxyisonicotinimidoyl chloride (3)
Figure imgf000053_0001
3
[0125] To a stirred solution of compound 2 (18 g, 89.54 mmol) in DMF (90 mL) was added NCS (23.91 g, 179.09 mmol). The reaction mixture was stirred at room temperature for 72 hours. After completion of the reaction, the reaction mixture was diluted with water (100 mL), extracted with ethyl acetate (3 x 50 mL), dried over anhydrous Na2SO4, and concentrated under reduced pressure. The crude was purified by column chromatography (100-200 silica), eluting with 20- 30% EtOAc:Hexane to afford compound 3 (15 g, 63.70 mmol, 71.1% yield) as an off-white solid.
[0126] Step 3: Synthesis of (S)-2-(l-(3-(2-bromopyridin-4-yl)isoxazol-5- yl)ethyl)isoindoline-l, 3-dione (5)
Figure imgf000053_0002
[0127] To a stirred solution of compound 4 (9 g, 45.18 mmol) in toluene (90 mL) was added K2CO3 (20.6 g, 149.09 mmol) and compound 3 (10.59 g, 45.18 mmol). The reaction mixture was stirred at 120 °C for 3 hours, and the mixture was poured into water (10 mL) and extracted with EtOAc (3 x 10 mL). The combined organic phase was washed with brine (2 x 10 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The crude was purified by silica gel chromatography (Hexane/EtOAc= 5/1 to 3/1) to afford compound 5 (8 g, 20.15 mmol, 44.47% yield) as an off-white solid.
[0128] Step 4: Synthesis of (S)-l-(3-(2-bromopyridin-4-yl)isoxazol-5-yl)ethan-l-amine (6)
Figure imgf000053_0003
6 [0129] To a solution of compound 5 (8 g, 20.15 mmol) in ethanol (80 mL) was added hydrazine hydrate (5.92 mL, 120.54 mmol). The reaction mixture was stirred at room temperature for 14 hours. The progress of the reaction was monitored by TLC. After completion of the reaction, the reaction mixture was concentrated under reduced pressure. The crude was diluted with DCM (20 mL) and triturated to afford compound 6 (5 g, 18.65 mmol, 92.83% yield).
[0130] Step 5: Synthesis of (S)-N-(l-(3-(2-bromopyridin-4-yl)isoxazol-5-yl)ethyl)-l- methyl-3-(trifluoromethyl)-lH-pyrazole-5-carboxamide (8)
Figure imgf000054_0001
[0131] To a stirred solution of compound 6 (4.97 g, 18.55 mmol) and compound 7 (3 g, 15.46 mmol) in DCM (50 mL) were added DIPEA (5.38 mL, 30.91 mmol) and HATU (8.81 g, 23.18 mmol) at 0 °C. The reaction mixture was stirred at 0 °C for 1 hour. The progress of the reaction was monitored by TLC. After completion of the reaction, the reaction mixture was diluted with water (5 mL) and extracted with DCM (2 x 30 mL). The organics were dried over MgSO4 and concentrated in vacuo. The crude was then purified by flash column chromatography, eluting with 40%EtOAc in hexane followed by Prep HPLC to obtain a sticky liquid, which was stirred with 4 N HC1 in Dioxane (1 mL) for 1 hour and then concentrated under reduced pressure to afford compound 8 (5 g, 11.26 mmol, 60.70% yield).
[0132] Step 6: Synthesis of Synthesis of (S)-l-methyl-3-(trifluoromethyl)-N-(l-(3-(2- vinylpyridin-4-yl)isoxazol-5-yl)ethyl)-lH-pyrazole-5-carboxamide (10)
Figure imgf000054_0002
[0133] To a solution of compound 8 (5 g, 11.26 mmol) and compound 9 (5.2 g, 33.77 mmol) in 1,4-Dioxane (48 mL) and water (12 mL) was added CsF (5.13 g, 33.77 mmol), and the mixture was purged with nitrogen for 15 minutes, followed by the addition of Pd(PPh3)2Cl2 (0.79 g, 1.13 mmol). The reaction mixture was stirred at 100 °C for 14 hours. The progress of the reaction was monitored by TLC. After completion of reaction, the reaction mixture was concentrated under reduced pressure and diluted with EtOAc (30 mL). The organic layer was washed with water (2 x 30 mL) followed by saturated brine solution (1 x 30 mL). The organic layer was dried over MgSO4 and concentrated under reduced pressure. The crude was then purified by flash column chromatography, eluting with 30% EtOAc in hexane to afford compound 10 (3 g, 7.67 mmol, 68.10% yield).
[0134] Step 7: Synthesis of (S)-N-(l-(3-(2-ethylpyridin-4-yl)isoxzol-5-yl)ethyl)-l-methyl- 3-(trifluoromethyl)-lH-pyrazole-5-carboxamide hydrochloride (Formula (I-IBa) HC1)
Figure imgf000055_0001
[0135] To a solution of compound 10 (6 g, 15.33 mmol) in ethyl acetate (60 mL) was added PtO2 (0.6 g, 2.6422 mmol). The reaction mass was hydrogenated (100 psi) at room temperature for 1 hour. The progress of the reaction was monitored by TLC. After completion of the reaction, the reaction mass was filtered through celite and wash with ethyl acetate (50 mL). The filtrate was concentrated under reduced pressure. The crude was purified by flash column chromatography, eluting with 35% EtOAc in heptane. The product fractions were concentrated under reduced pressure and stirred with 4 N HC1 in Dioxane (20 mL) for 1 hour. The resultant mixture was concentrated under reduced pressure and wash with diethyl ether (2 x 20 mL) to afford HC1 salt of Formula (I-IBa) (5.7 g, 14.383 mmol, 93.82% yield) as an off-white solid.
IL Data for Formula (I-IBa)
[0136] HPLC: Rt 7.101 min, 99.26%. Method: HPLC-AMM BICARB-X Bridge- 10-90- 100- NEW.lcm; Column: X-Bridge C18 (4.6*150) mm 5u; Mobile Phase: A - 5mM Ammonium Bicarbonate in water B - Acetonitrile; Flow Rate: 1.0. mL/minute; Gradient program: Time(min)/ B Cone.: 0.01 B Cone. 10, 6.00 B Cone. 90, 10.00 B Cone. 100, 12.00 B Cone. 100, 14.00 B Cone. 10, 18.00 B Cone. 10, 18.00 Controller Stop.
[0137] LCMS: 393.95 (M+H), Rt 1.608 min, 99.68%. Column: X-Select CSH C18 (3.0*50) mm 2.5um; Mobile Phase: A: 0.05% Formic acid in water:MeCN(95:05) B: 0.05% Formic acid in CAN; Inj Volume: 2.0μL; Flow Rate: 1.2. mL/minute; Column oven Temp: 50°C; Gradient program: 2% B to 98 % B in 2.0 minute, hold until 3.0 min; At 3.2 min B cone is 2% up to 4.0 min.
[0138] 1HNMR (400 MHz, DMSO-d6): δ 9.43 (br d, J= 7.3 Hz, 1H), 8.86 (d, J= 5.9 Hz, 1H), 8.31 (br s, 1H), 8.20 (br d, .7= 5.4 Hz, 1H), 7.53 (s, 1H), 7.38 (s, 1H), 5.41 (quin, J = 7.0 Hz, 1H), 4.15 (s, 3H), 3.06 (q, J= 7.7 Hz, 2H), 1.62 (br d, J= 6.8 Hz, 3H), 1.35 (t, J= 7.6 Hz, 3H).
[0139] Chiral method: Rt 22.445 min, 100%. Method: Chiral-Met-B 10%_1.0 ml.1cm; Description: Column: CHIRAL PAK IG (250*4.6mm, 5um); Mobile phase:: A:0.1% DEA in n-
Hexane Mobile phase ::B:DCM:MeOH(50:50) A:B::90: 10 Flow : 1.0ml/min.
[0140] Step A: Synthesis of (S)-2-(but-3-yn-2-yl)isoindoline- 1,3-dione (4)
Figure imgf000056_0001
[0141] To a mixture of compound 4B (10 g, 142.67 mmol), compound 4A (20.99 g, 142.67 mmol), and PPI13 (56.1 g, 214.01 mmol) in THF (100 mL) was added DEAD (33.58 mL, 214.01 mmol) at 20 °C. The reaction mixture was stirred at 20 °C for 16 hours. After completion of the reaction, the reaction mixture was poured into water (100 mL) and extracted with EtOAc (2 x 100 mL). The combined organic layer was washed with brine (2 x 50 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The crude was purified by flash column chromatography (20-30% of EtOAc in heptane) to afford Compound 4 (12 g, 60.23 mmol, 42.22% yield, 94% purity) as an off-white solid.
Example 2: Synthesis of A-(l-(3-(3-fluoro-2-methylpyridin-4-yl)isoxazol-5-yl)cyclopropyl)- l-methyl-3-(trifluoromethyl)-lH-pyrazole-5-carboxamide (Formula I-IBh)
Figure imgf000057_0001
Step 1: Synthesis of (Z)-2-bromo-3-fluoroisonicotinaldehyde oxime (2)
[0142] To a stirred solution of compound 1 (5.0 g, 24.51 mmol) in ethanol:water (30: 15 mL) was added NH2OH.HCI (2.04 g, 29.412 mmol), and the reaction mixture was stirred at room temperature for 16 hours. After completion of the reaction (monitored by TLC), the reaction mixture was concentrated over reduced pressure. The crude compound was diluted with EtOAc and washed with water. The combined organic layers were dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The crude compound was purified by Combi-Flash chromatography (eluting with 30-40% EtOAc in heptane) to afford the title compound 2 (4.5 g, 20.136 mmol, 82.15% yield) as a pale-yellow solid.
Step 2: Synthesis of (£)-2-bromo-3-fluoro-A-hydroxyisonicotinimidoyl chloride (3)
[0143] To a stirred solution of compound 2 (4.5 g, 20.547 mmol) in DMF (40 mL), was added NCS (8.13 g, 58.861 mmol). The reaction mixture was stirred at room temperature for 24 hours. After completion of reaction (monitored by TLC), the reaction mixture was diluted with ice cold water, solid was precipitated. The obtained solid was filtered off and dried in vacuo to afford the title compound 3 (4.0 g, crude) as an off-white solid. This compound was used as such for the next step without further purification.
Step 3: Synthesis of ethyl 3-(2-bromo-3-fluoropyridin-4-yl)isoxazole-5-carboxylate (5)
[0144] To a stirred solution of compound 3 (4 g, 15.782 mmol) in toluene (40 mL) were added K2CO3 (7.2 g, 52.079 mmol) and compound 4 (3.7 g, 15.782 mmol). The reaction mixture was stirred at 120 °C for 16 hours. After completion of the reaction (monitored by TLC), the reaction mixture was diluted with water and extracted with EtOAc. The combined organic layers were washed with water followed by brine, dried over anhydrous Na2SO4, and concentrated under reduced pressure. The crude compound was purified by Combi-Flash chromatography (eluting with 30-40% EtOAc in heptane) to afford the title compound 5 (4.2 g, 11.996 mmol, 76.01% yield) as a white solid.
Step 4: Synthesis of ethyl 3-(3-fluoro-2-methylpyridin-4-yl)isoxazole-5-carboxylate (7)
[0145] To a stirred solution of compound 5 (3.4 g, 10.79 mmol) in 1,4 dioxane (40 mL) were added compound 6 (2.71 g, 21.58 mmol) and K2CO3 (4.7 g, 32.371 mmol), and the reaction mixture was purged under nitrogen for 10 minutes. PdCl2(PPh3)2 (0.38 g, 0.539 mmol) was added to the reaction mixture under nitrogen atmosphere, and the reaction mixture was stirred at 100 °C for 14 hours. After completion of the reaction (monitored by TLC), the reaction mixture was cooled to room temperature, filtered through a pad of Celite, and washed with ethyl acetate. The filtrate was diluted with water and extracted with EtOAc followed by brine. The combined organic layers were dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The crude compound was purified by Combi-Flash chromatography (eluting with 30-40% ethyl acetate in heptane) to afford the title compound 7 (2.0 g, 7.273 mmol, 67.4% yield) as white solid.
Step 5: Synthesis of (3-(3-fluoro-2-methylpyridin-4-yl)isoxazol-5-yl)methanol (8)
[0146] To a stirred solution of compound 7 (2.0 g, 7.992 mmol) in methanol (20 mL) was added NaBH4 (0.46 g, 12.121 mmol) at 0 °C, and the reaction mixture was stirred at room temperature for 1 hour. After completion of the reaction (monitored by TLC), the reaction mixture was quenched with water and extracted EtOAc. The combined organic layers were washed with water followed by brine, dried over anhydrous Na2SO4, and concentrated under reduced pressure. The crude compound was purified by Combi-Flash chromatography (eluting with 40-50% EtOAc in heptane) to afford the title compound 8 (1.4 g, 6.052 mmol, 60% yield) as a colourless liquid.
Step 6: Synthesis of 5-(bromomethyl)-3-(3-fluoro-2-methylpyridin-4-yl)isoxazole (9)
[0147] To a stirred solution of compound 8 (1.4 g, 6.724 mmol) in DCM (20 mL) were added triphenyl phosphine (2.64 g, 10.087 mmol) and CBri (3.3 g, 10.087 mmol), and the reaction mixture was stirred at room temperature for 2 hours. After completion of the reaction (monitored by TLC), the reaction mixture was diluted with water and extracted EtOAc. The combined organic layers were washed with water followed by brine, dried over anhydrous Na2SO4, and concentrated under reduced pressure. The crude compound was purified by Combi-Flash chromatography (eluting with 10-20% EtOAc in heptane) to afford the title compound 9 (1.0 g, 3.246 mmol, 48.2% yield) as a pale yellow solid. Step 7: Synthesis of 2-(3-(3-fluoro-2-methylpyridin-4-yl)isoxazol-5-yl)acetonitrile (10)
[0148] To a stirred solution of compound 9 (1.0 g, 3.688 mmol) in ACN (10 mL) were added TBAF (1.57 mL, 5.533 mmol) and TMSCN (0.69 mL, 5.533 mmol) at 0 °C. The reaction mixture was stirred at room temperature for 2 hours. After completion of the reaction (monitored by TLC), the reaction mixture was diluted with water and extracted EtOAc. The combined organic layers were washed with water followed by brine, dried over anhydrous Na2SO4, and concentrated under reduced pressure. The crude compound was purified by Combi-Flash chromatography (eluting with 10-30% EtOAc in heptane) to afford the title compound 10 (0.5 g, 1.956 mmol, 53.0% yield) as an off-white solid.
Step 8: Synthesis of l-(3-(3-fluoro-2-methylpyridin-4-yl)isoxazol-5-yl)cyclopropane-l- carbonitrile (11)
[0149] To a stirred solution of compound 10 (0.5 g, 2.302 mmol) in DMF (1 mL) was added NaH (0.13 g, 5.755 mmol) at 0 °C. To this solution was subsequently added 1,2 dibromoethane (0.3 mL, 3.453 mmol), and the reaction mixture was stirred at room temperature for 2 hours. After completion of the reaction (monitored by TLC), the reaction mixture was diluted with water and extracted EtOAc. The combined organic layers were washed with water followed by brine, dried over anhydrous NarSO-i, and concentrated under reduced pressure. The crude compound was purified by Combi-Flash chromatography (eluting with 10-30% EtOAc in heptane) to afford the title compound 11 (0.35 g, 1.316 mmol, 57.2% yield) as an off-white solid.
Step 9: Synthesis of l-(3-(3-fluoro-2-methylpyridin-4-yl)isoxazol-5-yl)cyclopropane-l- carboxamide (12)
[0150] To a stirred solution of compound 11 (0.35 g, 1.438 mmol) in DMSO (3 mL) were added K2CO3 (0.19 g, 1.438 mmol) and H2O2 (0.12 g, 3.597 mmol) at 0 °C. The reaction mixture was stirred at room temperature for 16 hours. After completion of the reaction (monitored by TLC), the reaction mixture was diluted with water and extracted with EtOAc. The combined organic layers were washed with water followed by brine, dried over anhydrous Na2SO4, and concentrated under reduced pressure. The crude compound was triturated with diethyl ether and the precipitated solid was filtered off and dried over vacuo to afford the title compound 12 (0.2 g, 0.574 mmol, 39.9% yield) as an off-white solid.
[0151] Step 10: Synthesis of l-(3-(3-fluoro-2-methylpyridin-4-yl)isoxazol-5- yl)cyclopropane-l-carboxamide (13) [0152] To a stirred solution of compound 12 (0.2 g, 0.765 mmol) in 1,4 dioxane:water (1 :1.5 mL) were added NaOH (0.1 g, 2.684 mmol) and NaOCI (0.10 g, 2.056 mmol) at 0 °C, and the reaction mixture was stirred at 80 °C for 1 hour. After completion of the reaction (monitored by TLC), the reaction mixture was diluted with water and extracted with EtOAc. The combined organic layers were washed with water followed by brine, dried over anhydrous Na2SO4, and concentrated under reduced pressure. The crude compound was triturated with diethyl ether and the precipitated solid was filtered off and dried over vacuo to afford the title compound 13 (0.1 g, 0.343 mmol, 44.8% yield) as an off-white solid.
Step 11: Synthesis of /V-(l-(3-(3-fluoro-2-methylpyridin-4-yl)isoxazol-5-yl)cyclopropyl)-l- methyl-3-(trifluoromethyl)-lH-pyrazole-5-carboxamide (Formula (I-IBh))
[0153] To a stirred solution of compound 14 (0.083 g, 0.428 mmol) in DCM (5 mL) were added DIPEA (0.22 mL, 1.286 mmol) and HATU (0.24 g, 0.643 mmol) at 0 °C. After 10 minutes, compound 13 (0.1 g, 0.428 mmol) was added to the solution, and the reaction mixture was stirred at room temperature for 2 hours. After completion of the reaction (monitored by TLC), the reaction mixture was diluted with water and extracted with DCM. The combined organic layers were washed with water followed by brine, dried over anhydrous Na2SO4, and concentrated under reduced pressure. The crude compound was purified by Combi-Flash chromatography (eluting with 70-80% EtOAc in heptane) to afford the title Compound (XI) (21 mg, 0.505 mmol, 11.7% yield) as an off-white solid.
[0154] Data: HPLC: Rt 8.245 min, 97.42%. Column: X-Select CSH C18 (4.6*150) mm 5u Mobile Phase: A - 0.1% Formic acid in water: Acetonitrile (95:5) B - Acetonitrile Inj Volume; 5. OμL, Flow Rate: 1.0. mL/minute Gradient program: Time(min)/ B Cone. : 0.01/5, 1.0/5, 8.0/100, 12.0/100, 14.0/5, 18.0/5. LCMS: 410.1 (M+H), Rt 1.805 min, 98.95%. Column: X-Select CSH C18 (3.0*50) mm 2.5u Mobile Phase: A: 0.05% Formic acid in water: ACN (95:5) B: 0.05% Formic acid in ACN Inj Volume: 2.0μL, Column oven temperature: 50 C Flow Rate: 1.2. mL/minute Gradient program: 0% B to 98 % B in 2.0 minute, hold till 3.0 min, at 3.2 min B cone is 0 % up to 4.0 min. 1H NMR (400 MHz, DMSO-d6) δ 9.59 (s, 1H), 8.41 (d, J = 5.0 Hz, 1H), 7.69 (t, ./ = 5.1 Hz, 1H), 7.41 (s, 1H), 6.89 (d, ./ = 2.3 Hz, 1H), 4.13 (s, 3H), 2.56 - 2.53 (m, 3H), 1.61 - 1.58 (m, 2H), 1.46 - 1.43 (m, 2H).
Example 3 - Efficacy of Various Formulae in the inhibition of KCNT1 (KCNT1 - Patch Clamp Assay) [0155] Inhibition of KCNT1 (KNal .1, Slack) was evaluated using a tetracycline inducible cell line (HEK-TREX). Currents were recorded using the SyncroPatch 384PE automated, patch clamp system. Pulse generation and data collection were performed with PatchController384 VI .3.0 and DataController384 VI.2.1 (Nanion Technologies). The access resistance and apparent membrane capacitance were estimated using built-in protocols. Current was recorded in perforated patch mode (10 μM escin) from a population of cells. The cells were lifted, triturated, and resuspended at 800,000 cells/ml. The cells were allowed to recover in the cell hotel prior to experimentation. Currents were recorded at room temperature. The external solution contained the following (in mM): NaCl 105, NMDG 40, KC1 4, MgCl2 1, CaCl25, and HEPES 10 (pH = 7.4, Osmolarity -300 mOsm). The extracellular solution was used as the wash, reference, and compound delivery solution. The internal solution contained the following (in mM): NaCl 70, KF 70, KC1 10, EGTA 5, HEPES 5, and Escin 0.01 (pH = 7.2, Osmolarity -295 mOsm). Escin is made at a 5mM stock in water, aliquoted, and stored at -20 °C. The compound plate was created at 2x concentrated in the extracellular solution. The compound was diluted to 1:2 when added to the recording well. The amount of DMSO in the extracellular solution was held constant at the level used for the highest tested concentration. A holding potential of -80 mV with a 100ms step to OmV was used. Mean current was measured during the step to 0 mV. 100 μM Bepridil was used to completely inhibit KCNT1 current to allow for offline subtraction of non-KCNTl current. The average mean current from 3 sweeps was calculated and the percent inhibition of each compound was calculated. The percent inhibition as a function of the compound concentration was fit with a Hill equation to derive IC50, slope, minimum parameters, and maximum parameters. If KCNT1 inhibition was less than 50% at the highest tested concentration or if an IC50 could not be calculated, then a percent inhibition was reported in place of the IC50.
[0156] Results from this example are summarized in Table 1 below. In this table, “A” indicates IC50 of less than or equal to 1 μM; “B” indicates inhibition of between 1 μM to 20 μM; and “C” indicates inhibition of greater than or equal to 20 μM.
[0157] Table 1
Figure imgf000061_0001
Figure imgf000062_0001
Example 4: Formula (I-IBa) selectively inhibits KCNT1 gain-of-function variants across species
[0158] It has previously been disclosed that, for certain KNA1.1 inhibitor compounds, there may be a significant loss of activity at the mouse wildtype KNA1.1 (m KNA1.1-WT) channel compared to the human wildtype KNA1.1 (hkNA1.1) channel. Griffin, A.M., et al., Discovery of the First Orally Available, Selective KNAI.1 Inhibitor: In Vitro and In Vivo Activity of an Oxadiazole Series, ACS Med. Chem. Ltrs. 2021, 12(4):593-602 at 598. As preclinical in vivo testing may typically be performed in a mouse model of KCNT1 gain-of-function mutations, an in vivo assessment of KNA1.1 inhibitor compounds that exhibit lost activity amongst certain species may present technical challenges due to a drop-off of activity in those species.
[0159] Formula (I-IBa) was therefore tested in the automated SyncroPatch patch claim assay described above in Example 2 to assess activity directly on KNA1.1 current at physiological membrane potentials for various species, including human, mouse, rat, and dog.
[0160] Cells stably expressing wildtype KNA1.1 (KNAI . I-WT) cells of each of the aforementioned species, as well as the indicated variants, were voltage clamped at -80 mV, and inhibition was measured using a voltage step to 0 mV. In this assay, KNA1.1 was activated by increasing intracellular Na+ to 70 mM. IC50 values were generated using concentrations ranging from 0.001 to 30 gm in half log steps and a minimum of three cells (replicates) per concentration. The data are shown in Table 2, below.
[0161] In this table, “A” indicates IC50 of less than or equal to 1 μM; “B” indicates inhibition of between 1 μM to 20 μM; and “C” indicates inhibition of greater than or equal to 20 μM. NA indicates that the IC50 was not calculated, as the percent inhibitions at the top dose was not significant.
[0162] Table 2 - KNAI.1 (IC50 in μm) for various species and variants
Figure imgf000063_0001
[0163] As shown in Table 2, strong KNA1.1 inhibition activity was observed across multiple species and variants. FIG. 1 shows the percent inhibition of KCNT1 for increasing concentrations of a compound of Formula (I-IBa).
Example 5 - Pharmacokinetics
[0164] Pharmacokinetic data was obtained for compounds of Formulae (I-IBh), (I-d), and (II) as detailed herein.
[0165] Kinetic Solubility Assay : The Kinetic solubility assay employed the shake flask method followed by HPLC-UV analysis. The following step-wise procedure was used:
1) Weigh and dissolve the samples in 100% DMSO as the stock solution of 10 mM. About 10 μL (compound/Media) of stock solution is needed in this assay.
2) Add the test compounds and controls (10 mM in DMSO, 10 μL/vial) into the 50 mM pH 7.4 phosphate buffer (490 μL/well) placed in a Mini-Uniprep filter.
3) Vortex the samples of kinetic solubility for 2 minutes.
4) Incubate and shake the solubility solutions on an orbital shaker with 800 rpm at room temperature for 24 hours. 5) Centrifuge at 4000 rpm, 20°C for 10 minutes.
6) Transfer 400 μL (with or without dilution) of each solubility supernatant into 96- deep well for analysis after the samples will be directly filtered by the syringeless filter device.
7) Determine the test compound concentration of the filtrate using HPLC-UV.
8) Inject at least 5 UV standard solutions into HPLC from low to high concentration subsequently and then test the Kinetic solubility supernatant in duplicate.
9) Use the QC samples to monitor Kinetic solubility determination process.
[0166] Log D The Log D assay is a miniaturized 1-octanol/buffer shake flask method followed by LC/MS/MS analysis. It is typically measured by determining the partition of a compound between an organic solvent (1 -octanol) and an aqueous buffer (0.1 M phosphate buffer, pH 7.4; Varied buffer pH can be set). Since logD is pH dependent, the pH of the aqueous phase is always specified and is commonly measured at pH 7.4, the physiological pH of body fluids. The following Log D method was used to calculate the Log D values in Table 3 below:
1) Dissolve appropriate test compounds in 100% DMSO to 10 mM solutions.
2) Transfer the test compounds (10 mM in DMSO; 2 μL/well) and QC samples (10 mM in DMSO; 2 μL/well) from storage tubes to the 96-well polypropylene cluster tubes.
3) Add Buffer-saturated 1 -octanol (149 μL/well) and 1 -octanol saturated buffer (149 μL/well) to the well, respectively.
4) Vigorously mix each of the tubes on their sides for 3 minutes and then shake at a speed of 880 rpm at room temperature for 1 hour.
5) Centrifuge the tubes at 4000 rpm for 5 minutes.
6) Dilute the sample of buffer layer by a factor of 20 fold and the sample of 1 -octanol layer by a factor of 200 fold with internal standard (IS) solution. Note: the dilution factor is mainly based on the properties of the compound.
7) Analyze the sample using a triple quadrupole mass spectrometer. Correct the peak areas by dilution factors and embedded internal standard, and the ratio of the corrected peak areas will be used to calculate the results (Log D value). 8) Use the QC samples to monitor the process Log D determination.
9) Data Analysis: The Log D value for each compound is calculated by the following equation:
Figure imgf000065_0001
[0167] Different dilution value in the equation will be performed with different dilution factor for sample handling.
[0168] MW, XLogP and TPSA. These data points were all calculated using Dotmatics.
[0169] Liver Microsome Metabolic Stability Assay (NADPH) :
1) Test compounds were incubated at 37°C with liver microsomes (pooled from multiple donors) at 1.0 μM in the presence of NADPH (~1.0 mM) at 0.5 mg/ml microsomal protein.
2) Positive controls include testosterone (3A4 substrate), propafenone (2D6) and diclofenac (2C9). They are also incubated with microsomes in the presence of NADPH.
3) Time samples (0, 5, 15, 30, 45 and 60 minutes) are removed and immediately mixed with cold acetonitrile containing internal standard (IS). Test compound incubated with microsomes without NADPH for 60 min are also included.
4) Duplicate point for each test condition (n=2).
5) Samples are analyzed by LC/MS/MS; disappearance of test compound is assessed base on peak area ratios of analyte/IS (no standard curve).
6) An excel data summary, calculated intrinsic clearance and Tl/2 values are provided.
7) The following equation is used to calculate the microsome clearance:
Figure imgf000065_0002
Figure imgf000066_0001
The mg microsomal protein / g liver weight is 45 for 5 species. The liver weight values will use 40 g/kg, 30 g/kg, 32 g/kg, 20 g/kg and 88 g/kg for rat, monkey, dog, human and mouse, respectively. The liver clearance will be calculated using CLint(mic) with the following equation:
Figure imgf000066_0002
[0170] Results from this example are summarized in Table 3 below.
Table 3
Figure imgf000066_0004
Example 6 - Synthesis of (S)-N-(l-(5-(2-cyclopropylpyridin-4-yl)-l,2,4-oxadiazol-3- yl)ethyl)-l-methyl-3-(trifluoromethyl)-lH-pyrazole-5-carboxamide [Formula (V)]
Figure imgf000066_0003
Step 1: Synthesis of tert-butyl (S)-(l-amino-l-oxopropan-2-yl)carbamate
Figure imgf000067_0001
[0171] A solution of ethyl chloroformate (2.79 mL, 29.07 mmol) in THF (3 mL) at -15 °C was added drop-wise to a. stirred solution of (terLbutoxycarbonyl)-L-alanine (5 g, 26.43 mmol) and tri ethylamine (4.05 mL, 29.07 mmol) in THF (20 mL) at -15 °C under a nitrogen atmosphere. The solution was stirred at -15 °C for 25 minutes, and then a solution of aqueous ammonia (25%, 9.91 mL, 247.74 mmol) was added. The reaction solution was stirred at 0 °C for 3 hours. After completion of reaction, the reaction solution was concentrated under reduced pressure. The residue was acidified to pH 2-3 with a solution of IM aqueous potassium hydrogen sulfate. The solution was extracted with ethyl acetate (x2). The organic extracts were combined and washed with a solution of IN aqueous sodium bicarbonate, water, and brine, and then dried (Na2SO4). The solvent was removed under reduced pressure to afford the title compound (2.8 g, 14.88 mmol, 56% yield).
[0172] Step 2: Synthesis of tert-butyl (S)-(l-cyanoethyl)carbamate
Figure imgf000067_0002
[0173] To a stirred solution ofte t-butyl ($)-( 1 -amino- l-oxopropan-2-yl)carbamate (2.8 g,
14.88 mmol) in pyridine (25 mL) at 0 °C was added trifluoroacetic anhydride (4.46 mL, 31.66 mmol) dropwise. The reaction solution was continued to stir at room temperature for 3 hours. After completion of the reaction, the solvent was removed under reduced pressure. The crude residue was dissolved in ethyl acetate and washed with a solution of IM aqueous potassium hydrogen sulfate, water and brine, then dried (Na2SO4). The crude material was purified by column chromatography to afford the title compound (2.4 g, 14.1 mmol, 94% yield) as a liquid.
Step 3: Synthesis of tert-butyl (S,E)-(l-amino-l-(hydroxyimino)propan-2-yl)carbamate
Figure imgf000067_0003
[0174] To a stirred solution of tert-butyl (S)-(l-cyanoethyl)carbamate (2.4 g, 14.1 mmol) in ethanol (20 mL) was added hydroxylamine hydrochloride (1.46 g, 21.15 mmol) and triethylamine (3.94 mL, 28.2 mmol) at room temperature. The solution was stirred at 70 °C for 3 hours. After completion of the reaction, the reaction solution was concentrated under reduced pressure. The crude residue was diluted with water and extracted with ethyl acetate (x2). The organic extracts were combined and dried (Na2SO4). The solvent was removed under reduced pressure to afford the title compound (2 g, 9.8 mmol, 69% yield), which was of sufficient purity to use in the next step without further purification.
Step 4: Synthesis of tert-butyl (S)-(l-(5-(2-cyclopropylpyridin-4-yl)-l,2,4-oxadiazol-3-yl)ethyl)~ carbamate
Figure imgf000068_0001
[0175] To a stirred solution of tert-butyl (S,E)-(1 -amino- l-(hydroxyimino)propan-2- yl)carbamate (2 g, 9.84 mmol) in 1,4-dioxane (20 mL) was added 2-cyclopropylisonicotinic acid (1.93 g, 11.81 mmol) and TV, A-di cyclohexyl carbodi imide (8.88 mL, 11.81 mmol) at room temperature. The reaction solution was stirred at 100 °C for 16 hours. After completion of the reaction, the reaction mixture was concentrated under reduced pressure. The obtained crude residue was then purified by column chromatography, eluting with 0-70% EtOAc/heptane, to afford the title compound (1.2 g, 3.64 mmol, 37% yield).
Step 5: Synthesis of (S)-l-(5-(2-cyclopropylpyridin-4-yl)-l,2>4-oxadiazol-3-yl)ethan-l-amine
Figure imgf000068_0002
[0176] To a stirred the solution of tert-butyl (S)-(l-(5-(2-cyclopropylpyridin-4-yl)-l,2,4- oxadiazol-3-yl)ethyl)-carbamate (1 g, 3.03 mmol) in dichloromethane (2 mL) was added a solution of 4M hydrochloric acid in dioxane (10 mL, 40 mmol) at 0 °C. The solution was stirred at room temperature for 2 hours. After completion of the reaction, the reaction mixture was concentrated under reduced pressure to afford crude the title compound (800 mg, 2.99 mmol, 99% yield) as an off-white solid.
Step 6: Synthesis of (S)-N-(l-(5-(2-cyclopropylpyridin-4-yl)-l,2,4-oxadiazol-3-yl)ethyl)-l- meth-yl-3-(trifluoromethyl)-lH-pyrazole-5-carboxamide [Formula (V)]
Figure imgf000069_0001
[0177] To a stirred solution of (S)-l-(5-(2-cyclopropylpyridin-4-yl)-l,2,4-oxadiazol-3- yl)ethan-l -amine (354.9 mg, 1.33 mmol) and l-methyl-3-(trifluoromethyl)-lH-pyrazole-5- carboxylic acid (200 mg, 1.03 mmol) in di chloromethane (10 mL) was added HATU (587.65 mg, 1.55 mmol) and DIPEA (0.54 mL, 3.09 mmol) at 0 °C. The reaction solution was stirred at room temperature for 1 hour. After completion of the reaction, the reaction mixture was quenched with water and extracted with dichloromethane (x2). The organic extracts were combined and washed with water and brine, and then dried (Na2SO4). The crude material was purified by flash chromatography on silica gel, eluting with 0-90% EtOAc/n-heptane, followed by preparative HPLC to afford the title compound (98.55 mg, 0.24 mmol, 23% yield) as an off-white solid.
[0178] Data for Formula (V): HPLC: Rt 10.35 min, 98.76%. Method Name : D:\Methods\X-SELECT-10-90_12MIN TFA - REG.met; Column:X-SELECT CSH C18 (150 X 4.6mm, 3.5μm) Mobile Phase-A: 5mM AMM.ACETATE Mobile Phase-B: ACN Programme:T/B% : 0.01/20, 12/90,16/90,16.1/20,20/20; Flow : 1.0 mL/min. Diluent :ACN:WATER (80:20).
[0179] LCMS : 407.15 (M+H), Rt 1.972 min, 95.178%. Method File : LCMS X- Select(Ammonium Bicarbonate) Column : X-Bridge C18 (3.0*50)mm 2.5u; Mobile Phase: A: 2.5 mM Ammonium Bicarbonate in water B: Acetonitrile Flow Rate: 1.2 mL/minute; Column oven temp. 50°C; Gradient program: 0% B to 98 % B in 2.0 minute, hold till 3.0 min, at 3.2 min B cone is 0 % up to 4.0 min.
[0180] 1H NMR (400 MHz, DMSO-d6) δ 9.29 (d, J = 7.5 Hz, 1H), 8.67 (d, J= 5.0 Hz, 1H), 7.95 (s, 1H), 7.73 (dd, J= 1.6, 5.1 Hz, 1H), 7.44 (s, 1H), 5.36 (quin, J= 7.2 Hz, 1H), 4.14 (s, 3H), 2.42 - 2.23 (m, 1H), 1.62 (d, J= 7.1 Hz, 3H), 1.06 - 0.96 (m, 4H).
Example 7 - Synthesis of l-methyl-3-(trifluoromethyl)-N-((3-(2-(trifluoromethyl)pyridin-4- yl)isoxazol-5-yl)methyl)-lH-pyrazole-5-carboxamide (Formula (I-IBi))
Figure imgf000070_0001
Step 1: Synthesis of (2-(trifluoromethyl)pyridin-4-yl)methanol (2)
Figure imgf000070_0002
[0181] To a stirred solution of compound 1 (10.0 g, 52.33 mmol) in THF (40 mL) was added BH3 DMS (2M in THF, 52.3 mL, 104.65 mmol) at 0 °C and stirred at room temperature for 3 hours. The reaction mixture was then heated to 50 °C and continued stirring for 12 hours. After completion of the reaction (monitored by TLC), the reaction mixture was cooled to room temperature, slowly quenched using MeOH (100 mL) at 0 °C, and stirred at room temperature for 30 minutes. The solvent was then evaporated at 40°C under reduced pressure, and residue thus obtained was cooled to 0 °C. Solution was rendered alkaline with 1A sodium hydroxide (50 mL) and diluted with EtOAc (150 mL). The organic layer was separated, washed with water (25 mL) followed by brine (25 mL), dried over anhydrous NarSO4, filtered and concentrated under reduced pressure to obtain a crude residue. The obtained crude compound was purified by 100-200 silica gel column chromatography using 0-40% EtOAc/hexane as an eluent to afford the titled compound 2 (6.3 g, 35.569 mmol, 68% yield). Step 2: Synthesis of 2-(trifluoromethyl)isonicotinaldehyde (3)
Figure imgf000071_0002
[0182] To a stirred solution of compound 2 (6. 2 g, 35 mmol) in DCM (60 mL) was added Dess Martin periodinane (29.68 g, 70.01 mmol) at 0 °C. The reaction mixture was stirred at room temperature for 3 hours. After completion of the reaction (monitored by TLC), the reaction mixture was diluted with DCM (100 mL) followed by addition of saturated sodium thiosulphate (100 mL) and saturated sodium bicarbonate (100 mL). The organic layer was separated, washed with water (2x50 mL) followed by saturated brine solution (1x50 mL), dried over MgSO4, and concentrated under reduced pressure to afford compound 3 (4800 mg, 27.411 mmol, 78% yield) as a brown oil, thus used directly for further organic reaction transformations.
Step 3: Synthesis of (Z)-2-(trifluoromethyl)isonicotinaldehyde oxime (4)
Figure imgf000071_0003
[0183] To a stirred solution of compound 3 (4.8 g, 27.41 mmol) in ethanol (10 mL) and water (30 mL) were added hydroxylamine hydrochloride (3809.63 mg, 54.82 mmol) followed by sodium carbonate (5810.11 mg, 54.82 mmol). The reaction mixture was stirred at room temperature for 12 hours. After completion of the reaction (monitored by TLC), the reaction mixture was concentrated under reduced pressure, diluted with EtOAc (50 mL), washed with water (2x10 mL), saturated brine solution (10 mL), separated, dried over MgSO4, and concentrated under reduced pressure. The crude product was then purified by flash column chromatography using 30 % EtOAc in hexane as an eluent to afford as an off-white solid compound 4 (3000 mg, 15.78 mmol, 57% yield).
[0184] Step 4: Synthesis of (E)-N-hydroxy-2-(trifluoromethyl)isonicotinimidoyl chloride (5)
Figure imgf000071_0001
[0185] To a stirred solution of compound 4 (3000 mg, 15.78 mmol) in DMF (10 mL) was added N-chlorosuccinimide (4214.08 mg, 31.56 mmol) at 65 °C, and then stirring was continued further for 30 minutes at 80 °C. After completion of the reaction (monitored by TLC), the reaction mixture was allowed to cool to room temperature, quenched by adding a minimum quantity of crushed ice, and then diluted with Et2O (40 mL). The organic layer was separated and dried over anhydrous Na2SO4 and concentrated under reduced pressure, giving crude compound 5 (3 g). The obtained crude was then used directly for further reaction transformations.
Step 5: Synthesis of 2-((3-(2-(trifluoromethyl)pyridin-4-yl)isoxazol-5-yl)methyl)isoindoline- 1, 3-dione (7)
Figure imgf000072_0001
[0186] To a stirred solution of compound 5 (1.455 g, 6.48 mmol) and compound 6 (1 g, 5.4 mmol) in toluene (1 mL) was added K2CO3 (2.238 g, 16.2 mmol) at room temperature, and then stirring was continued for 16 hours at 100 °C. After completion of the reaction (monitored by TLC), the reaction mixture was allowed to cool to room temperature, quenched by adding water (1 mL), and then diluted with Et2O (5 mL). The organic layer was separated and dried over anhydrous Na2SO4 and concentrated under reduced pressure, giving crude compound 7 (550 mg, crude). The obtained crude was then used directly for further reaction transformations.
Step 6: Synthesis of (3-(2-(trifluoromethyl)pyridin-4-yl)isoxazol-5-yl)methanamine (8)
Figure imgf000072_0002
[0187] To a stirred solution of compound 7 (500 mg, 1.34 mmol) in DCM (10 mL) and EtOH (2.0 mL) was added N2H4 H2O (0.39 mL, 8.04 mmol) dropwise at room temperature, and then stirring was continued for 16 hours at the same temperature. The reaction mixture was fdtered, and the filtered cake was washed with DCM (3x10 mL). The filtrate was concentrated to afford compound 8 (300 mg, 90.256% yield) as a pale-yellow solid which was used directly for the next step without purification. Step 7: Synthesis of l-methyl-3-(trifluoromethyl)-N-((3-(2-(trifluoromethyl)pyridin-4- yl)isoxazol-5-yl)methyl)-lH-pyrazole-5-carboxamide (Formula (I-IBi)):
[0188] To a stirred solution of compound 8 (0.15 g, 0.7300 mmol) and compound 9 (0.14 g, 0.7400 mmol) in DCM (5.00 mL) was added HATU (0.35 g, 0.9300 mmol) followed by DIPEA (0.21 mL, 1.23 mmol) at 0 °C, and then stirring was continued further for 1 hour at 0 °C. Progress of the reaction was monitored by TLC. After completion of the reaction, the reaction mixture was concentrated under reduced pressure, and then diluted by adding water (10 mL). Then the reaction mixture was extracted with EtOAc (2x25 mL), the combined extracts were dried over anhydrous NarSO4, filtered, and concentrated under reduced pressure to obtain the crude residue (250 mg) as a colorless, viscous liquid. The obtained crude was purified by Combi-Flash column chromatography (100-200 silica gel) by eluting 0-40% EtOAc in hexanes to obtain Formula (lOIBi) (76 mg, 0.1810 mmol, 29% yield) as an off-white solid.
[0189] Data for Formula (I-IBi): HPLC: Rt 11.40 min, 99.88%. Method Name : D:\METHODS\XSELECT_10-90_12MIN_TFA-REG-MET.met; Column: X SELECT CSH C18 (150 X 4.6mm, 3.5μ); Mobile Phase A; 0.05% TFA;ACN(95;05); Mobile Phase B : 0.05% TFA;ACN(05:95); Program:T/B% :.0.01/20„12/90, 16/90; Flow : 1 mL/min; Diluent: WATER:ACN.
[0190] LCMS : 420.50 (M+H), Rt 2.021 min, 99.49%. Method:- LCMS X-BRIDGE (Ammonium Bicarbonate); Column : X-Bridge BEH C18 (50*3) mm 2.5u; Mobile Phase: A: 2.5 mM Ammonium Bicarbonate in water; B: Acetonitrile; Inj volume: 2μL, Flow Rate: 1.2 mL/minute; Column oven temp. 50 °C; Gradient program: 0% B to 98 % B in 2.0 minute, hold until 3.0 min, at 3.2 min B cone is 0 % up to 4.0 min.
[0191] 1HNMR (400 MHz, DMSO-d6) 6 9.49 (br t, J= 5.7 Hz, 1H), 8.93 (d, J= 5.0 Hz, 1H), 8.32 (s, 1H), 8.21 (d, J= 4.9 Hz, 1H), 7.43 - 7.31 (m, 2H), 4.70 (d, J= 5.8 Hz, 2H), 4.16 (s, 3H).
Step A: Synthesis of l-methyl-3-(trifluoromethyl)-N-((3-(2-(trifluoromethyl)pyridin-4- yl)isoxazol-5-yl)methyl)-lH-pyrazole-5-carboxamide (6):
Figure imgf000074_0003
[0192] To a mixture of compound 6B (0.35 mL, 5.89 mmol), compound 6A (1.04 g, 7.06 mmol), PPh3 (2.32 g, 8.83 mmol) in THF (100 mL) was added DEAD (1.54 g, 8.83 mol) at 20 °C, and the reaction mixture was stirred at 20 °C for 16 hours. After completion of the reaction, the mixture was poured into water (100 mL) and extracted with EtOAc (2x100 mL). The combined organic layer was washed with brine (2x50 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The crude was purified by flash column (0-20% of EtOAc in Heptane) to afford compound 6 (0.3 g, 1.62 mmol, 27.521% yield).
Example 8 - Synthesis of (S)-3-(difluoromethyl)-N-(l-(3-(2-ethylpyridin-4-yl)isoxazol-5- yl)ethyl)-l-methyl-lH-pyrazole-5-carboxamide hydrochloride [Formula I-IBj)] and (R)-3- (difluoromethyl)-N-(l-(3-(2-ethylpyridin-4-yl)isoxazol-5-yl)ethyl)-l-methyl-lH-pyrazole-5- carboxamide hydrochloride [Formula I-IBk)]:
Figure imgf000074_0001
Figure imgf000074_0002
Step-1: Synthesis of (Z)-2-bromoisonicotinaldehyde oxime (2)
Figure imgf000075_0002
[0193] To a stirred solution of compound 1 (20 g, 107.52 mmol) in ethanol (60 mL) and water (300 mL) was added hydroxylamine hydrochloride (7.47 g, 107.52 mmol). The reaction mixture was stirred at room temperature for 14 hours. The progress of the reaction was monitored by TLC. After completion of reaction, the reaction mixture was concentrated under reduced pressure, diluted with EtOAc (200 mL) and washed with water (2x100 mL) followed by saturated brine solution (100 mL). The combined organic fractions were then separated, dried over MgSO4 and concentrated under reduced pressure to afford compound 2 (20 g, 99.49 mmol, 92% yield).
Step-2: Synthesis of (E)-2-bromo-N-hydroxyisonicotinimidoyl chloride (3)
Figure imgf000075_0003
[0194] To a stirred solution of compound 2 (20 g, 99.49 mmol) in DMF (200 mL) was added NCS (26.57 g, 198.99 mmol), and the reaction mixture was stirred at room temperature for 3 days. Progress of the reaction was monitored by TLC. After completion, the reaction mixture was concentrated under vacuo, the obtained residue was diluted with EtOAc (200 mL) and washed with water (2x100 mL) followed by saturated brine solution (1x100 mL). The combined organic fractions were then separated, dried over MgSO4 and concentrated under reduced pressure to afford compound 3 (23 g, 97.67 mmol, 98% yield).
Step-3: Synthesis of 2-(l-(3-(2-bromopyridin-4-yl)isoxazol-5-yl)ethyl)isoindoline-1, 3-dione (5)
Figure imgf000075_0001
[0195] To a stirred solution of compound 3 (11.5 g, 48.84 mmol) and compound 4 (10.7 g, 53.72 mmol) in toluene (50 mL) was added K2CO3 (22.27 g, 161.17 mmol), and the mixture was stirred at 120 °C for 3 hours. Progress of the reaction was monitored by TLC. After completion of reaction, the reaction mixture was concentrated under reduced pressure, diluted with EtOAc (100 mL), washed with water (2x 100 mL) followed by brine solution (1x100 mL), dried over MgSO4 and concentrated under reduced pressure. The obtained crude was then purified by flash column chromatography eluting with 20% EtOAc in hexane. The desired fractions were concentrated to dryness to afford compound 5 (9 g, 22.6 mmol, 46% yield).
Step-4: Synthesis of 2-(l-(3-(2-vinylpyridin-4-yl)isoxazol-5-yl)ethyl)isoindoline-l, 3-dione (7)
Figure imgf000076_0001
[0196] To a stirred solution of compound 5 (3 g, 7.55 mmol) and compound 6 (3.49 g, 22.66 mmol) in 1,4-dioxane (24 mL) and water (6 mL) was added CsF (3.44 g, 22.66 mmol). The resultant mixture was degassed under nitrogen atmosphere for 15 minutes followed by the addition of Pd(PPh3)2C12 (529.43 mg, 0.76 mmol) and stirred at 100 °C for 14 hours. Progress of the reaction was monitored by TLC. After completion of the reaction, the reaction mixture was concentrated under reduced pressure. The obtained residue was diluted with EtOAc (50 mL), washed with water (30 mL), brine solution (30 mL), dried over MgSCL and concentrated under reduced pressure. The obtained crude was then purified by flash column chromatography eluting 30% EtOAc in hexane. The desired fractions were concentrated to dryness to afford compound 7 (1.9 g, 5.32 mmol, 70% yield).
Step-5: Synthesis of l-(3-(2-ethylpyridin-4-yl)isoxazol-5-yl)ethan-l-amine (8)
Figure imgf000076_0002
[0197] To a stirred solution of compound 8 (1.9 g, 5.5 mmol) in methanol (20 mL) was added hydrazine hydrate (1.62 mL, 33.01 mmol). The reaction mass was stirred at 75 °C for 14 hours. The progress of the reaction was monitored by TLC. After completion of the reaction, the reaction mass was concentrated under reduced pressure, diluted with DCM (20 mL), and triturated to obtain a solid material, which was filtered through Buchner funnel. The filtrate was concentrated under vacuo to get afford compound 8 (1 g, 2.99 mmol, 52% yield).
Step-7: (S)-3-(difluoromethyl)-N-(l-(3-(2-ethylpyridin-4-yl)isoxazol-5-yl)ethyl)-l-methyl- lH-pyrazole-5-carboxamide hydrochloride [Formula I-IBj)] and (R)-3-(difluoromethyl)-N- (l-(3-(2-ethylpyridin-4-yl)isoxazol-5-yl)ethyl)-l-methyl-lH-pyrazole-5-carboxamide hydrochloride [Formula I-IBk)] :
[0198] To a stirred solution of compound 9 (243.18 mg, 1.38 mmol) and compound 8 (300 mg, 1.38 mmol) in DCM (10 mL) were added HATU (787.52 mg, 2.07 mmol) followed by DIPEA (0.48 mL, 2.76 mmol) at 0 °C, and then stirring was continued at room temperature for 2 hours. After completion of the reaction (monitored by TLC and LCMS), the reaction mixture was quenched with water (10 mL) and extracted with DCM (2x25 mL). The combined organic layers were washed with water (20 mL), brine (20 mL), dried over anhydrous Na2SO4 and concentrated under reduced pressure. The obtained crude product was purified by flash chromatography on silica gel using 30-50% EtOAc in Isohexane as an eluent followed by chiral HPLC to afford Formula (LIBj) (40 mg, 0.1056 mmol, 7.6% yield) and Formula I-IBk (35 mg, 0.0930 mmol, 6.7% yield).
[0199] Data for Formula (I-IBj): HPLC: Rt 5.701 min, 99.146%. Method File Name : Formic acid X Select 18 min-REG.lcm; Column : X Select CSH C18(150 x4.6)mm,3.5μ; Mobile phase A:0.1% FA in Water: ACN(95:05); Mobile phase B : Acetonitrile; Gradient Programme : T/B% :0.01/5, 1/5, 8/100, 12/100, 14/5, 18/5; Flow rate : 1.2 ml/min.
[0200] LCMS : 376.15 (M+H), Rt 1.397 min, 98.717%. Column : X-Select CSH C18 (3.0*50) mm 2.5um; Mobile Phase: A: 0.05% Formic acid in water: ACN (95:5); B: ACN; Inj Volume: 2.0μL; Flow Rate : 1.2. mL/minute; Column oven temperature: 50 °C; Gradient program: 0% B to 98 % B in 2.0 min, hold till 3.0 min, at 3.2 min B cone is 0 % up to 4.0 min.
[0201] 1HNMR (400 MHz, DMSO-d6) δ 9.18 (d, J= 7.9 Hz, 1H), 8.61 (d,J= 5.1 Hz, 1H), 7.73 (s, 1H), 7.66 (dd, J= 1.6, 5.1 Hz, 1H), 7.26 (s, 1H), 7.15 (d, J= 0.9 Hz, 1H), 7.04 (s, 1H), 5.38 (quin, J= 7.3 Hz, 1H), 4.11 (s, 3H), 2.82 (q, J= 7.6 Hz, 2H), 1.59 (d, J= 7.1 Hz, 3H), 1.26 (t, J = 7.6 Hz, 3H).
[0202] Chiral method: Rt 6.692 min, 100%. Method File Name : CHIRAL-MET-B.lcm; Description : Column: CHIRALPAK IC(250*4.6mm 3um); Mobile Phase A:0.1%DEA in n- HEXANE; Mobile Phase B::IPA; A:B::70:30; Flow 0.7ml/min. [0203] Data for Formula (I-IBk): HPLC: Rt 5.689 min, 99.77%. Method File Name : Formic acid X Select 18 min-REG.lcm; Column : X Select CSH C188150 x4.6)mm,3.5μ; Mobile phase A:0.1% FA in Water: ACN(95:05); Mobile phase B : Acetonitrile; Gradient Programme : T/B% :0.01/5, 1/5, 8/100, 12/100, 14/5, 18/5; Flow rate :1.2 ml/min.
[0204] LCMS : 376.0 (M+H), Rt 1.395 min, 99.69%. Column : X-Select CSH C18 (3.0*50) mm 2.5um; Mobile Phase: A: 0.05% Formic acid in water : ACN (95:5); B: ACN; Inj Volume: 2.0μL; Flow Rate : 1.2. mL/minute; Column oven temperature: 50 °C; Gradient program: 0% B to 98 % B in 2.0 min, hold till 3.0 min, at 3.2 min B cone is 0 % up to 4.0 min.
[0205] 1HNMR (400 MHz, DMSO-d6) δ 9.19 (d, J= 7.9 Hz, 1H), 8.61 (d,J= 5.0 Hz, 1H), 7.73 (s, 1H), 7.66 (dd, J= 1.6, 5.1 Hz, 1H), 7.26 (s, 1H), 7.15 (d, J= 0.6 Hz, 1H), 7.04 (s, 1H), 5.38 (quin, J= 7.2 Hz, 1H), 4.11 (s, 3H), 2.82 (q, ./ = 7.5 Hz, 2H), 1.59 (d, J= 7.1 Hz, 3H), 1.26 (t, J= 7.6 Hz, 3H).
[0206] Chiral method: Rt 7.736 min, 99.51%. Method File Name : CHIRAL MET-B30- 0.70mL.lcm; Description : Column: CHIRALPAK IC (250*4.6mm 3um); Moblile Phase A:0.1%DEA in n-HEXANE; Moblile Phase B::IPA; A:B::70:30 Flow 0.7ml/min.
Step A: Synthesis of 2-(but-3-yn-2-yl)isoindoline- 1,3-dione (4)
Figure imgf000078_0001
[0207] To a stirred solution of compound 4A (31.49 g, 214.01 mmol) and compound 4B (15 g, 214.01 mmol) in THF (150 mL) was added and DEAD (55.9 g, 321.02 mmol) followed by triphenylphosphine (84.2 g, 321.02 mmol) at 0 °C, and the mixture was stirred at room temperature for 16 hours. Progress of the reaction was monitored by TLC. After completion of reaction, the reaction mixture was concentrated to dryness, and the residue was diluted with EtOAc (100 mL) and washed with water (2x100 mL) followed by saturated brine solution (1x100 mL). The combined organic fractions were then separated, dried over MgSO4 and concentrated under reduced pressure. The crude was purified by flash column chromatography eluting with 30% EtOAc in Isohexane. The desired fractions were concentrated to afford compound 4 (25 g, 125.5 mmol, 58% yield). [0208] Example 9 - Synthesis of (S)-N-(l-(3-(2-ethylpyridin-4-yl)isoxazol-5- yl)ethyl)benzamide hydrochloride [Formula (I-IBn) HC1] and (R)-N-(l-(3-(2-ethylpyridin-4- yl)isoxazol-5-yl)ethyl)benzamide hydrochloride [Formula (I-IBo) HC1]:
Figure imgf000079_0001
Figure imgf000079_0002
Step-1: Synthesis of (Z)-2-bromoisonicotinaldehyde oxime (2)
Figure imgf000079_0003
[0209] To a stirred solution of compound 1 (20 g, 107.52 mmol) in ethanol (60 mL) and water (300 mL) was added hydroxylamine hydrochloride (7.47 g, 107.52 mmol). The reaction mixture was stirred at room temperature for 14 hours. The progress of the reaction was monitored by TLC. After completion of reaction, the reaction mixture was concentrated under reduced pressure, diluted with EtOAc (200 mL) and washed with water (2x100 mL) followed by saturated brine solution (100 mL). The combined organic fractions were then separated, dried over MgSO4 and concentrated under reduced pressure to afford compound 2 (20 g, 99.49 mmol, 92% yield).
Step-2: Synthesis of (E)-2-bromo-N-hydroxyisonicotinimidoyl chloride (3)
Figure imgf000080_0003
[0210] To a stirred solution of compound 2 (20 g, 99.49 mmol) in DMF (200 mL) was added NCS (26.57 g, 198.99 mmol), and the reaction mixture was stirred at room temperature for 3 days. Progress of the reaction was monitored by TLC. After completion, the reaction mixture was concentrated under vacuo, and the obtained residue was diluted with EtOAc (200 mL) and washed with water (2x100 mL) followed by saturated brine solution (1x100 mL). The combined organic fractions were then separated, dried over MgSCL and concentrated under reduced pressure to afford compound 3 (23 g, 97.67 mmol, 98% yield).
Step-3: Synthesis of 2-(l-(3-(2-bromopyridin-4-yl)isoxazol-5-yl)ethyl)isoindoline-l, 3-dione (5)
Figure imgf000080_0001
[0211] To a stirred solution of compound 3 (11.5 g, 48.84 mmol) and compound 4 (10.7 g, 53.72 mmol) in toluene (50 mL) was added K2CO3 (22.27 g, 161.17 mmol), and the mixture was stirred at 120 °C for 3 hours. Progress of the reaction was monitored by TLC. After completion of reaction, the reaction mixture was concentrated under reduced pressure, diluted with EtOAc (100 mL), washed with water (2x 100 mL) followed by brine solution (1x100 mL), dried over MgSO4 and concentrated under reduced pressure. The obtained crude was then purified by flash column chromatography eluting with 20% EtOAc in hexane. The desired fractions were concentrated to dryness to afford compound 5 (9 g, 22.6 mmol, 46% yield).
Step-4: Synthesis of 2-(l-(3-(2-vinylpyridin-4-yl)isoxazol-5-yl)ethyl)isoindoline-l, 3-dione (7)
Figure imgf000080_0002
[0212] To a stirred solution of compound 5 (3 g, 7.55 mmol) and compound 6 (3.49 g, 22.66 mmol) in 1,4-dioxane (24 mL) and water (6 mL) was added CsF (3.44 g, 22.66 mmol). The resultant mixture was degassed under nitrogen atmosphere for 15 minutes followed by the addition of Pd(PPh3)2Cl2 (529.43 mg, 0.76 mmol) and stirred at 100 °C for 14 hours. Progress of the reaction was monitored by TLC. After completion of the reaction, the reaction mixture was concentrated under reduced pressure. The obtained residue was diluted with EtOAc (50 mL), washed with water (30 mL), brine solution (30 mL), dried over MgSO4 and concentrated under reduced pressure. The obtained crude was then purified by flash column chromatography eluting 30% EtOAc in hexane. The desired fractions were concentrated to dryness to afford compound 7 (1.9 g, 5.32 mmol, 70% yield).
Step-5: Synthesis of l-(3-(2-ethylpyridin-4-yl)isoxazol-5-yl)ethan-l-amine (8)
Figure imgf000081_0001
[0213] To a stirred solution of compound 8 (1.9 g, 5.5 mmol) in methanol (20 mL) was added hydrazine hydrate (1.62 mL, 33.01 mmol). The reaction mass was stirred at 75 °C for 14 hours. The progress of the reaction was monitored by TLC. After completion of the reaction, the reaction mass was concentrated under reduced pressure, diluted with DCM (20 mL) and triturated to obtain a solid material, which was filtered through Buchner funnel. The filtrate was concentrated under vacuo to afford compound 8 (1 g, 2.99 mmol, 52% yield).
Step-7 : (S)-N-(l-(3-(2-ethylpyridin-4-yl)isoxazol-5-yl)ethyl)benzamide hydrochloride [Formula (I-IBn) HC1] and (R)-N-(l-(3-(2-ethylpyridin-4-yl)isoxazol-5-yl)ethyl)benzamide hydro-chloride [Formula (I-IBo) HCI]:
[0214] To a stirred solution of compound 8 (330 mg, 0.9700 mmol) and compound 9 (94.97 mg, 0.7800 mmol) in DCM (3 mL) were added HATU (554.41 mg, 1.46 mmol) followed by DIPEA (0.25 mL, 1.46 mmol) at 0 °C, and then stirring was continued at room temperature for 1 hour. After completion of the reaction (monitored by TLC and LCMS), the reaction mixture was quenched with water (10 mL) and extracted with EtOAc (2x25 mL). The combined organic layers were washed with water (20 mL) and brine (20 mL), dried over anhydrous Na2SO4 and concentrated under reduced pressure. The obtained crude product was purified by flash chromatography on silica gel using 35% EtOAc in hexane as an eluent followed by chiral HPLC to afford Formula (I-IBn) HC1 (75 mg, 0.2317 mmol, 23.84% yield) and Formula (Llbo) HC1 (40 mg, 0.1243 mmol, 12.79% yield).
[0215] Data for Formula (I-IBn) HC1: HPLC: Rt 5.347 min, 99.300 %. Method File Name : Formic acid_X Select I 8 min-REG.lcm; Column : X Select CSH C 18(150 x4.6)mm,3.5μ; Mobile phase A:0.1% FA in Water:ACN(95:05); Mobile phase B Acetonitrile; Gradient Programme : T/B% :0.01/5, 1/5, 8/100, 12/100, 14/5, 18/5; Flow rate : 1.2 ml/min.
[0216] LCMS : 322.2 (M+H), Rt 1.511 min, 98.118 %. Column : X-Select CSH (3.0*50) mm 2.5u; Mobile Phase: A: 0.025% Formic acid in water; B: ACN; Inj Volume: 2.0μL, Flow Rate : 1.2. mL/minute; Column oven temperature: 50 °C; Gradient program: 0% B to 98 % B in 2.0 min, hold till 3.0 min, at 3.2 min B cone is 0 % up to 4.0 min.
[0217] 1H NMR (400 MHz, DMSO-d6) δ 9.06 (br d, J= 7.9 Hz, 1H), 8.71 (br d, J= 5.3 Hz, 1H), 7.98 - 7.90 (m, 3H), 7.88 (br s, 1H), 7.57 (br t, J= 7.2 Hz, 1H), 7.50 (br t, J = 7.2 Hz, 1H), 7.18 (s, 2H), 5.47-5.43 (m, 1H), 2.90 (q, J= 7.5 Hz, 2H), 1.62 (d, J= 7.0 Hz, 3H), 1.29 (t, J = 7.7 Hz, 3H).
[0218] Chiral method: Rt 7.699 min, 100%. Method File Name : CHIRAL-A.lcm; COLUMN: :CHIRAL PAK IG(250X 4.6mm, 5μm); Mobile Phase A : 0.1%DEA in n-Hexane; Mobile Phase B :DCM:MEOH(1:1); A:B:70:30; Flow: 1.0mL/min.
[0219] Data for Formula (I-IBo) HC1: HPLC: Rt 5.331 min, 99.90%. Method File Name
: Formic acid X Select_18 min-REG.lcm; Column : X Select CSH C18(150 x4.6)mm,3.5μ; Mobile phase A:0.1% FA in Water:ACN(95:05); Mobile phase B Acetonitrile; Gradient Programme : T/B% :0.01/5, 1/5, 8/100, 12/100, 14/5, 18/5; Flow rate : 1.2 ml/min.
[0220] LCMS : 322.10 (M+H), Rt 1.516 min, 98.934%. Column : X-Select CSH (3.0*50) mm 2.5u; Mobile Phase: A: 0.025% Formic acid in water; B: ACN; Inj Volume: 2.0μL; Flow Rate : 1.2. mL/minute; Column oven temperature: 50 °C; Gradient program: 0% B to 98 % B in 2.0 min, hold till 3.0 min, at 3.2 min B cone is 0 % up to 4.0 min.
[0221] 1H NMR (400 MHz, DMSO-d6): δ 9.07 (br d, J= 7.7 Hz, 1H), 8.74 (br d, J= 5.5 Hz, 1H), 8.03 (br s, 1H), 7.93 (br d, ./ = 7.2 Hz, 3H), 7.57 (br t, ./ = 7.3 Hz, 1H), 7.53 - 7.47 (m, 2H), 7.21 (s, 1H), 5.49 - 5.41 (m, 1H), 2.93 (q, J= 7.5 Hz, 2H), 1.62 (d, J= 7.0 Hz, 3H), 1.30 (t, J= 7.7 Hz, 3H).
[0222] Chiral method: Rt 9.652 min, 99.415%. Method File Name : CHIRAL-A.lcm; COLUMN: :CHIRAL PAK IG(250X 4.6mm, 5μm); Mobile Phase A : 0.1%DEA in n-Hexane; Mobile Phase B :DCM:MEOH(1 :1); A:B:70:30; Flow: LOmL/min.
Step A: Synthesis of (S)-2-(but-3-yn-2-yl)isoindoline-l, 3-dione (4)
Figure imgf000083_0001
[0223] To a stirred solution of compound 4A (31.49 g, 214.01 mmol) and compound 4B (15 g, 214.01 mmol) in THF (150 mL) was added and DEAD (55.9 g, 321.02 mmol) followed by triphenylphosphine (84.2 g, 321.02 mmol) at 0 °C, and the mixture was stirred at room temperature for 16 hours. Progress of the reaction was monitored by TLC. After completion of reaction, the reaction mixture was concentrated to dryness, and the residue was diluted with EtOAc (100 mL) and washed with water (2x100 mL) followed by saturated brine solution (1x100 mL). The combined organic fractions were then separated, dried over MgSO4 and concentrated under reduced pressure. The crude was purified by flash column chromatography eluting with 30% EtOAc in hexane. The desired fractions were concentrated to afford compound 4 (25 g, 125,5 mmol, 58% yield).
Example 10 - Synthesis of (R)-3-chloro-N-(l-(3-(2-ethylpyridin-4-yl)isoxazol-5- yl)ethyl)benzamide hydrochloride [Formula (I-IBm) HC1] and ((S)-3-chloro-N-(l-(3-(2- ethylpyridin-4-yl)isoxazol-5-yl)ethyl)benzamide hydrochloride [Formula (I-IBp) HC1]:
Figure imgf000084_0001
Figure imgf000084_0002
Step-1: Synthesis of (Z)-2-bromoisonicotinaldehyde oxime (2)
Figure imgf000084_0003
[0224] To a stirred solution of compound 1 (20 g, 107.52 mmol) in ethanol (60 mL) and water (300 mL) was added hydroxylamine hydrochloride (7.47 g, 107.52 mmol). The reaction mixture was stirred at room temperature for 14 hours. The progress of the reaction was monitored by TLC. After completion of reaction, the reaction mixture was concentrated under reduced pressure, diluted with EtOAc (200 mL), and washed with water (2x100 mL) followed by saturated brine solution (100 mL). The combined organic fractions were then separated, dried over MgSO4 and concentrated under reduced pressure to afford compound 2 (20 g, 99.49 mmol, 92% yield).
Step-2: Synthesis of (E)-2-bromo-N-hydroxyisonicotinimidoyl chloride (3)
Figure imgf000084_0004
[0225] To a stirred solution of compound 2 (20 g, 99.49 mmol) in DMF (200 mL) was added NCS (26.57 g, 198.99 mmol), and the reaction mixture was stirred at room temperature for 3 days. Progress of the reaction was monitored by TLC. After completion, the reaction mixture was concentrated under vacuo, the obtained residue was diluted with EtOAc (200 mL) and washed with water (2x100 mL) followed by saturated brine solution (1x100 mL). The combined organic fractions were then separated, dried over MgSO4 and concentrated under reduced pressure to afford compound 3 (23 g, 97.67 mmol, 98% yield).
Step-3: Synthesis of 2-(l-(3-(2-bromopyridin-4-yl)isoxazol-5-yl)ethyl)isoindoline-l, 3-dione (5)
Figure imgf000085_0001
[0226] To a stirred solution of compound 3 (11.5 g, 48.84 mmol) and compound 4 (10.7 g, 53.72 mmol) in toluene (50 mL) was added K2CO3 (22.27 g, 161.17 mmol), and the mixture was stirred at 120 °C for 3 hours. Progress of the reaction was monitored by TLC. After completion of reaction, the reaction mixture was concentrated under reduced pressure, diluted with EtOAc (100 mL), washed with water (2x 100 mL) followed by brine solution (1x100 mL), dried over MgSO4 and concentrated under reduced pressure. The obtained crude was then purified by flash column chromatography eluting with 20% EtOAc in hexane. The desired fractions were concentrated to dryness to afford compound 5 (9 g, 22.6 mmol, 46% yield).
Step-4: Synthesis of 2-(l-(3-(2-vinylpyridin-4-yl)isoxazol-5-yl)ethyl)isoindoline-1, 3-dione (7)
Figure imgf000085_0002
[0227] To a stirred solution of compound 5 (3 g, 7.55 mmol) and compound 6 (3.49 g, 22.66 mmol) in 1,4-dioxane (24 mL) and water (6 mL) was added CsF (3.44 g, 22.66 mmol). The resultant mixture was degassed under nitrogen atmosphere for 15 minutes followed by the addition of Pd(PPh3)2Cl2 (529.43 mg, 0.76 mmol) and stirred at 100 °C for 14 hours. Progress of the reaction was monitored by TLC. After completion of the reaction, the reaction mixture was concentrated under reduced pressure. The obtained residue was diluted with EtOAc (50 mL), washed with water (30 mL), brine solution (30 mL), dried over MgSO4 and concentrated under reduced pressure. The obtained crude was then purified by flash column chromatography eluting 30% EtOAc in hexane. The desired fractions were concentrated to dryness to afford compound 7 (1.9 g, 5.32 mmol, 70% yield).
Step-5: Synthesis of l-(3-(2-ethylpyridin-4-yl)isoxazol-5-yl)ethan-l-amine (8)
Figure imgf000086_0001
[0228] To a stirred solution of compound 8 (1.9 g, 5.5 mmol) in methanol (20 mL) was added hydrazine hydrate (1.62 mL, 33.01 mmol). The reaction mass was stirred at 75 °C for 14 hours. The progress of the reaction was monitored by TLC. After completion of the reaction, the reaction mass was concentrated under reduced pressure, diluted with DCM (20 mL) and triturated to obtain a solid material, which was filtered through Buchner funnel. The filtrate was concentrated under vacuo to afford compound 8 (1 g, 2.99 mmol, 52% yield).
Step-7: Synthesis of (R)-3-chloro-N-(l-(3-(2-ethylpyridin-4-yl)isoxazol-5-yl)ethyl)benzamide hydrochloride [Formula (I-IBm) HC1] and ((S)-3-chloro-N-(l-(3-(2-ethylpyridin-4- yl)isoxazol-5-yl)ethyl)benz-amide hydrochloride [Formula (I-IBp) HCI]:
[0229] To a stirred solution of compound 9 (136.2 mg, 0.87 mmol) and compound 8 (136.2 mg, 0.87 mmol) in DCM (3 mL) were added HATU (496.13 mg, 1.3 mmol) followed by DIPEA (0.45 mL, 2.61 mmol) at 0 °C and then stirring was continued at same temperature for 1 h. After completion of the reaction (monitored by TLC and LCMS), the reaction mixture was quenched with water (10 mL) and extracted with EtOAc (2x25 mL). The combined organic layers were washed with water (20 mL), brine (20 mL), dried over anhydrous Na2SO4 and concentrated under reduced pressure. The obtained crude product was purified by flash chromatography on silica gel using 40% EtOAc in heptane as an eluent followed by chiral HPLC to afford Formula (LIBm) HC1 (21 mg, 0.0589 mmol, 6.7758% yield) and Formula (I-IBp) HC1 (40 mg, 0.1121 mmol, 12.89% yield) as HC1 salt.
[0230] Data for Formula (I-IBm) HC1: HPLC: Rt 5.49 min, 99.87%. Method Name : D:\METHODS\XSELECT_20-90_12MIN_TFA-REG.met; Column: X SELECT CSH C18 (150 X 4.6mm, 3.5μ); Mobile Phase A; 0.05% TFA;ACN(95;05); Mobile Phase B : 0.05% TFA;ACN(05;95); Programme :T/B% :.0.01/20, 12/90, 16/90; Flow : 1 mL/min; Diluent: WATER:ACN(80:20). [0231] LCMS : 355.95 (M+H), Rt 1.797 min, 97.84%. Column : X-Select CSH (3.0*50) mm 2.5u; Mobile Phase: A: 0.05% Formic acid in water; B: ACN Inj Volume: 2.0μL; Flow Rate : 1.2. mL/minute; Gradient program: 0% B to 98 % B in 2.0 minute, Hold till 3.0 min, At 3.2 min B cone is 0 % up to 4.0 min.
[0232] 1HNMR (400 MHz, DMSO-d6) δ 9.18 (d, J= 7.9 Hz, 1H), 8.62 (d, J= 5.0 Hz, 1H), 7.98 (t, ./ = 1.8 Hz, 1H), 7.88 (td, J= 1.3, 7.8 Hz, 1H), 7.76 (s, 1H), 7.69 (br d, J= 5.6 Hz, 1H), 7.66 - 7.63 (m, 1H), 7.56 - 7.51 (m, 1H), 7.14 (d, J= 0.6 Hz, 1H), 5.42 (quin, J = 7.2 Hz, 1H), 2.83 (q, J= 7.7 Hz, 2H), 1.61 (d, J= 7.1 Hz, 3H), 1.26 (t, J= 7.6 Hz, 3H).
[0233] Chiral method: Rt 12.501 min, 100%. Method File Name : CHIRAL-A.lcm; COLUMN: :CHIRAL PAK IG(250X 4.6mm, 5μm); Mobile Phase A : 0.1%DEA in n-Hexane; Mobile Phase B :DCM:MEOH(1:1); A:B:70:30; Flow: 1.0mL/min.
[0234] Data for Formula (I-IBp) HC1: HPLC: Rt 6.180 min, 99.740%. Method File Name : Formic acid_X Select_18 min-REG.lcm; Column : X Select CSH C 18(150 x4.6)mm,3.5μ; Mobile phase A:0.1% FA in Water:ACN(95:05); Mobile phase B Acetonitrile; Gradient Programme : T/B% :0.01/5, 1/5, 8/100, 12/100, 14/5, 18/5; Flow rate : 1.2 ml/min.
[0235] LCMS : 355.95 (M+H), Rt 1.810 min, 99.506%, Column : X-Select CSH (3.0*50) mm 2.5u; Mobile Phase: A: 0.05% Formic acid in water; B: ACN; Inj Volume: 2.0μL; Flow Rate : 1.2. mL/minute; Gradient program: 0% B to 98 % B in 2.0 minute, Hold till 3.0 min, At 3.2 min B cone is 0 % up to 4.0 min.
[0236] 1H NMR (400 MHz, DMSO-d6) δ 9.19 (br d, J= 7.5 Hz, 1H), 8.68 (br d, J = 5.3 Hz, 1H), 7.98 (br s, 1H), 7.88 (br d, J= 6.8 Hz, 2H), 7,81 (br d, J = 2.6 Hz, 1H), 7.65 (br d, J= 8.8 Hz, 1H), 7.54 (br t, J= 8.0 Hz, 1H), 7.18 (s, 1H), 5.45 - 5.1 (m, 1H), 2.91 - 2.84 (q, J = 7.5 Hz, 2H), 1.61 (br d, J= 7.2 Hz, 3H), 1.32 - 1.25 (m, 3H).
[0237] Chiral method: Rt 7.293 min, 100%. Method File Name : CHIRAL-A.lcm; COLUMN: :CHIRAL PAK IG(250X 4.6mm, 5μm); Mobile Phase A : 0.1%DEA in n-Hexane; Mobile Phase B :DCM:MEOH(1 :1); A:B:70:30; Flow: LOmL/min.
Step A: Synthesis of (S)-2-(but-3-yn-2-yl)isoindoline-l, 3-dione (4)
Figure imgf000087_0001
[0238] To a stirred solution of compound 4A (31.49 g, 214.01 mmol) and compound 4B (15 g, 214.01 mmol) in THF (150 mL) was added and DEAD (55.9 g, 321.02 mmol) followed by triphenylphosphine (84.2 g, 321.02 mmol) at 0 °C, and the mixture was stirred at room temperature for 16 hours. Progress of the reaction was monitored by TLC. After completion of reaction, the reaction mixture was concentrated to dryness, and the residue was diluted with EtOAc (100 mL) and washed with water (2x100 mL) followed by saturated brine solution (1x100 mL). The combined organic fractions were then separated, dried over MgSO4 and concentrated under reduced pressure. The crude was purified by flash column chromatography eluting with 30% EtOAc in hexane. The desired fractions were concentrated to afford compound 4 (25 g, 125.5 mmol, 58% yield).
Example 11 - Synthesis of (S)-N-(l-(3-(2-(ethylamino)pyridin-4-yl)isoxazol-5- yl)ethyl)benzamide [Formula (I-IBq)] and (R)-N-(l-(3-(2-(ethylamino)pyridin-4-yl)isoxazol- 5-yl)ethyl)benzamide [Formula (I-IBr)]:
Figure imgf000088_0001
Figure imgf000088_0002
Step-1: Synthesis of (Z)-2-bromoisonicotinaldehyde oxime (2)
Figure imgf000089_0002
[0239] To a stirred solution of compound 1 (20 g, 107.52 mmol) in ethanol (60 mL) and water (300 mL) was added hydroxylamine hydrochloride (7.47 g, 107.52 mmol). The reaction mixture was stirred at room temperature for 14 hours. The progress of the reaction was monitored by TLC. After completion of reaction, the reaction mixture was concentrated under reduced pressure, diluted with EtOAc (200 mL), and washed with water (2x100 mL) followed by saturated brine solution (100 mL). The combined organic fractions were then separated, dried over MgSO4 and concentrated under reduced pressure to afford compound 2 (20 g, 99.49 mmol, 92% yield).
Step-2: Synthesis of (E)-2-bromo-N-hydroxyisonicotinimidoyl chloride (3)
Figure imgf000089_0003
[0240] To a stirred solution of compound 2 (20 g, 99.49 mmol) in DMF (200 mL) was added NCS (26.57 g, 198.99 mmol), and the reaction mixture was stirred at room temperature for 3 days. Progress of the reaction was monitored by TLC. After completion, the reaction mixture was concentrated under vacuo, and the obtained residue was diluted with EtOAc (200 mL) and washed with water (2x100 mL) followed by saturated brine solution (1x100 mL). The combined organic fractions were then separated, dried over MgSO4 and concentrated under reduced pressure to afford compound 3 (23 g, 97.67 mmol, 98% yield).
Step-3: Synthesis of 2-(l-(3-(2-bromopyridin-4-yl)isoxazol-5-yl)ethyl)isoindoline-l, 3-dione (5)
Figure imgf000089_0001
[0241] To a stirred solution of compound 3 (11.5 g, 48.84 mmol) and compound 4 (10.7 g, 53.72 mmol) in toluene (50 mL) was added K2CO3 (22.27 g, 161.17 mmol), and the mixture was stirred at 120 °C for 3 hours. Progress of the reaction was monitored by TLC. After completion of reaction, the reaction mixture was concentrated under reduced pressure, diluted with EtOAc (100 mL), washed with water (2x 100 mL) followed by brine solution (1x100 mL), dried over MgSO4 and concentrated under reduced pressure. The obtained crude was then purified by flash column chromatography eluting with 20% EtOAc in hexane. The desired fractions were concentrated to dryness to afford compound 5 (9 g, 22.6 mmol, 46% yield).
Step-4: Synthesis of l-(3-(2-bromopyridin-4-yl)isoxazol-5-yl)ethan-l-amine (6)
Figure imgf000090_0002
[0242] To a stirred solution of compound 5 (2 g, 5.02 mmol) in ethanol (10 mL) and DCM (10 mL) was added N2H4.H2O (1.51 g, 30.13 mmol) at room temperature, and then the mixture was stirred for 12 hours. After completion of the reaction (monitored by TLC), the reaction mixture was concentrated under reduced pressure, diluted with EtOH, and filtered, and the filtered cake was washed with ethanol (50 mL). The filtrate was concentrated under reduced pressure to afford compound 6 (1.3 g, 3.05 mmol, 60% yield), as a colorless liquid.
Step-5: Synthesis of N-(l-(3-(2-bromopyridin-4-yl)isoxazol-5-yl)ethyl)benzamide (8)
Figure imgf000090_0001
[0243] To a stirred solution of compound 6 (600 mg, 2.24 mmol) and compound 7 (273.29 mg, 2.24 mmol) in DCM (5 mL) were added HATU (850.91 mg, 2.24 mmol) and DIPEA (0.52 mL, 2.98 mmol) at room temperature, and stirring was continued for 2 hours. Progress of the reaction was monitored by TLC. After completion, reaction mixture was quenched with water (10 mL) and extracted with DCM (2x20 mL). Combined organic layers were dried over anhydrous Na2SO4 and concentrated under reduced pressure. The obtained crude product was purified by column chromatography (100-200 silica) using 30-50% EtOAc/hexane as an eluent to afford compound 3 (500 mg, 1.23 mmol, 55% yield), as a white solid.
Step-6: Synthesis of (S)-N-(l-(3-(2-(ethylamino)pyridin-4-yl)isoxazol-5-yl)ethyl)benzamide [Formula (I-IBq)] and (R)-N-(l-(3-(2-(ethylamino)pyridin-4-yl)isoxazol-5- yl)ethyl)benzamide [Formula (I-IBr)]:
[0244] To a stirred solution of compound 8 (500 mg, 1.34 mmol) in DMSO (2 mL) and water (6 mL) were added Cui (7.67 mg, 0.0400 mmol) and ethylamine (6.72 mL, 13.43 mmol) at room temperature, and stirring was continued at 180 °C for 2 hours under microwave irradiation. After completion of the reaction (monitored by LCMS), the reaction mixture was quenched with water (10 mL) and extracted with EtOAc (2x25 mL). The combined organic layers were washed with water (20 mL) and brine (20 mL), dried over anhydrous Na2SO4 and concentrated under reduced pressure. The obtained crude was purified by column chromatography (100-200 silica) using 50% EtOAc: heptane as eluent followed by chiral preparative HPLC to afford Formula (I- IBq) (50 mg, 0.1481 mmol, 11% yield) and Formula (I-IBr) (50 mg, 0.1474 mmol, 11% yield).
[0245] Data for Formula (I-IBq): HPLC: Rt 4.739 min, 99.66%. Method File Name : Formic acid X Select 18 min-REG.lcm; Column : X Select CSH C18(150 x4.6)mm,3.5μ; Mobile phase A:0.1% FA in Water: ACN(95:05); Mobile phase B : Acetonitrile; Gradient Programme : T/B% :0.01/5, 1/5, 8/100, 12/100, 14/5, 18/5; Flow rate : 1.2 ml/min.
[0246] LCMS : 337.0 (M+H), Rt 1.310 min, 99.492%. Column : X-Select CSH C18 (3.0*50) mm 2.5um; Mobile Phase: A: 0.05% Formic acid in water:ACN(95:05); B: ACN; Inj Volume: 2.0μL; Flow Rate : 1.2. mL/minute; Gradient program: 0% B to 98 % B in 2.0 minute, Hold till 3.0 min, At 3.2 min B cone is 0 % up to 4.0 min.
[0247] 1HNMR (400 MHz, DMSO-d6): δ 9.02 (d, J= 8 Hz, 1H), 8.09 - 8.05 (m, 1H), 7.93 - 7.89 (m, 2H), 7.59 - 7.53 (m, 1H), 7.52 - 7.46 (m, 2H), 6.88 (d, J= 5.9 Hz, 3H), 6.66 (t, J= 5.2 Hz, 1H), 5.42 (quin, J= 7.5 Hz, 1H), 3.29 - 3.24 (m, 2H), 1.59 (d, J= 7.1 Hz, 3H), 1.13 (t, J= 7.1 Hz, 3H).
[0248] Chiral method: Rt 18.748 min, 100%. Method File Name : met-A.lcm; COULMN : CHIRAL PAK IG (250*4.6mm*5um); Mobile phase A : 0.1% DEA in n-Hexane; Mobile phase B : ETOH; A:B : 80:20; Flow: 1.0 ml/min.
[0249] Data for Formula (I-IBr): HPLC: Rt 4.739 min, 99.203%. Method File Name : Formic acid X Select 18 min-REG.lcm; Column : X Select CSH C18(150 x4.6)mm,3.5μ; Mobile phase A:0.1% FA in Water: ACN(95:05); Mobile phase B : Acetonitrile; Gradient Programme : T/B% :0.01/5, 1/5, 8/100, 12/100, 14/5, 18/5; Flow rate : 1.2 ml/min.
[0250] LCMS : 337.0 (M+H), Rt 1.308 min, 99.357%. Column : X-Select CSH C18 (3.0*50) mm 2.5um; Mobile Phase: A: 0.05% Formic acid in water:ACN(95:05); B: ACN; Inj Volume: 2.0μL; Flow Rate : 1.2. mL/minute; Gradient program: 0% B to 98 % B in 2.0 minute, Hold till 3.0 min, At 3.2 min B cone is 0 % up to 4.0 min. [0251] 1H NMR (400 MHz, DMSO-d6): δ 9.02 (d, J = 7.9 Hz, 1H), 8.07 (d, J = 6.0 Hz, 1H), 7.94 - 7.89 (m, 2H), 7.59 - 7.53 (m, 1H), 7.52 - 7.45 (m, 2H), 6.90 - 6.85 (m, 3H), 6.66 (t, J = 5.4 Hz, 1H), 5.42 (quin, J= 7.3 Hz, 1H), 3.29 - 3.24 (m, 2H), 1.59 (d, J= 7.1 Hz, 3H), 1.13 (t, .7= 7.1 Hz, 3H).
[0252] Chiral method: Rt 23.251 min, 99.836%. Method File Name : met-A.lcm; COULMN : CHIRAL PAK IG (250*4.6mm*5um); Mobile phase A : 0.1% DEA in n-Hexane; Mobile phase B : ETOH; A:B : 80:20; Flow: 1.0 ml/min.
Step A: Synthesis of 2-(but-3-yn-2-yl)isoindoline- 1,3-dione (4)
Figure imgf000092_0001
[0253] To a stirred solution of compound 4A (31.49 g, 214.01 mmol) and compound 4B (15 g, 214.01 mmol) in THF (150 mL) was added and DEAD (55.9 g, 321.02 mmol) followed by triphenylphosphine (84.2 g, 321.02 mmol) at 0 °C, and the mixture was stirred at room temperature for 16 hours. Progress of the reaction was monitored by TLC. After completion of reaction, the reaction mixture was concentrated to dryness, and the residue was diluted with EtOAc (100 mL) and washed with water (2x100 mL) followed by saturated brine solution (1x100 mL). The combined organic fractions were then separated, dried over MgSO4 and concentrated under reduced pressure. The crude was purified by flash column chromatography eluting with 30% EtOAc in hexane. The desired fractions were concentrated to afford compound 4 (25 g, 125.5 mmol, 58% yield).
[0254] Example 12 - Synthesis of (S)-N-(l-(3-(2-(ethylamino)pyridin-4-yl)isoxazol-5- yl)ethyl)-3-isopropyl-l-methyl-lH-pyrazole-5-carboxamide [Formula (I-IBs)] and (R)-N-(l- (3-(2-(ethylamino)pyridin-4-yl)isoxazol-5-yl)ethyl)-3-isopropyl-l-methyl-lH-pyrazole-5- carboxamide [Formula (I-IBt)] :
Figure imgf000093_0001
Figure imgf000093_0002
[0255] Step-1: Synthesis of (E)-2-bromoisonicotinaldehyde oxime (2)
Figure imgf000093_0003
[0256] To a stirred solution of compound 1 (20 g, 107.52 mmol) in ethanol (60 mL) and water (20 mL) was added hydroxylamine hydrochloride (7.47 g, 107.52 mmol). The reaction mixture was stirred at room temperature for 14 hours. The progress of the reaction was monitored by TLC. After completion of reaction, the reaction mixture was concentrated under reduced pressure, diluted with EtOAc (200 mL), and washed with water (2x100 mL) followed by saturated brine solution (100 mL). The combined organic fractions were then separated, dried over MgSO4 and concentrated under reduced pressure to afford compound 2 (20 g, 99.49 mmol, 92% yield).
Step-2: Synthesis of (E)-2-bromo-N-hydroxyisonicotinimidoyl chloride (3)
Figure imgf000093_0004
[0257] To a stirred solution of compound 2 (20 g, 99.49 mmol) in DMF (200 mL) was added NCS (26.57 g, 198.99 mmol), and the reaction mixture was stirred at room temperature for 3 days. Progress of the reaction was monitored by TLC. After completion, the reaction mixture was concentrated under vacuo, and the obtained residue was diluted with EtOAc (200 mL) and washed with water (2x100 mL) followed by saturated brine solution (1x100 mL). The combined organic fractions were then separated, dried over MgSO4 and concentrated under reduced pressure to afford compound 3 (23 g, 97.67 mmol, 98% yield).
Step-3: Synthesis of 2-(l-(3-(2-bromopyridin-4-yl)isoxazol-5-yl)ethyl)isoindoline-l, 3-dione (5)
Figure imgf000094_0001
[0258] To a stirred solution of compound 3 (11.5 g, 48.84 mmol) and compound 4 (10.7 g, 53.72 mmol) in toluene (50 mL) was added K2CO3 (22.27 g, 161.17 mmol), and the mixture was stirred at 120 °C for 3 hours. Progress of the reaction was monitored by TLC. After completion of reaction, the reaction mixture was concentrated under reduced pressure, diluted with EtOAc (100 mL), washed with water (2x 100 mL) followed by brine solution (1x100 mL), dried over MgSO4 and concentrated under reduced pressure. The obtained crude was then purified by flash column chromatography eluting with 20% EtOAc in hexane. The desired fractions were concentrated to dryness to afford compound 5 (9 g, 22.6 mmol, 46% yield).
Step-4: Synthesis of l-(3-(2-bromopyridin-4-yl)isoxazol-5-yl)ethan-l-amine (6)
Figure imgf000094_0002
[0259] To a stirred solution of compound 5 (2 g, 5.02 mmol) in ethanol (10 mL) and DCM (10 mL) was added N2H4.H2O (1.51 g, 30.13 mmol) at room temperature, and the mixture was stirred for 12 hours. After completion of the reaction (monitored by TLC), the reaction mixture was concentrated under reduced pressure, diluted with EtOH, and filtered, and the filtered cake was washed with ethanol (50 mL). The filtrate was concentrated under reduced pressure to afford compound 6 (1.3 g, 3.05 mmol, 60% yield), as a colorless liquid. Step-5: Synthesis of N-(l-(3-(2-bromopyridin-4-yl)isoxazol-5-yl)ethyl)-3-isopropyl-l- methyl-lH-pyrazole-5-carboxamide (8)
Figure imgf000095_0001
[0260] To a stirred solution of compound 6 (600 mg, 2.24 mmol) and compound 7 (376.39 mg, 2.24 mmol) in DCM (10 mL) were added HATU (1276.37 mg, 3.36 mmol) and DIPEA (0.78 mL, 4.48 mmol) at room temperature, and then stirring was continued for 2 hours. Progress of the reaction was monitored by TLC. After completion, reaction mixture was quenched with water (10 mL) and extracted with DCM (2x20 mL). Combined organic layers were dried over anhydrous Na2SO4 and concentrated under reduced pressure. The obtained crude product was purified by column chromatography (100-200 silica) using 30-50% EtOAc/hexane as an eluent to afford compound 8 (650 mg, 1.4607 mmol, 65% yield).
Step-6: Synthesis of (S)-N-(l-(3-(2-(ethylamino)pyridin-4-yl)isoxazol-5-yl)ethyl)-3- isopropyl-l-methyl-lH-pyrazole-5-carboxamide [Formula (I-IBs)] and (R)-N-(l-(3-(2- (ethylamino)pyridin-4-yl)isoxazol-5-yl)ethyl)-3-isopropyl-l-methyl-lH-pyrazole-5- carboxamide [Formula (I-IBt)] :
[0261] To a stirred solution of compound 8 (600. mg, 1.43mmol) in DMSO (3 mL) and water (9 mL) were added Cui (8.2mg, 0.0400mmol) and ethylamine (7.17mL, 14.34mmo) at room temperature, and stirring was continued at 180 °C for 2 hours under microwave irradiation. After completion of the reaction (monitored by LCMS), the reaction mixture was quenched with water (10 mL) and extracted with EtOAc (2x25 mL). The combined organic layers were washed with water (20 mL) and brine (20 mL), dried over anhydrous Na2SO4 and concentrated under reduced pressure. The obtained crude was purified by column chromatography (100-200 silica) using 40- 50% EtOAc: heptane as eluent followed by chiral preparative HPLC to afford Formula (LIBs) (80 mg, 0.21 mmol, 14% yield) and Formula (LIBt) (50 mg, 0.13 mmol, 9% yield).
[0262] Data for Formula (I-IBs): HPLC: Rt 4.293 min, 99.91%. Method File : HPLC- FORMIC ACID-XSELECT- 10-90- 100.1cm; Column : X-Select CSH C18 (4.6*150) mm 3.5u; Mobile Phase: A - 0.1% Formic acid in water : Acetonitrile(95:05); B - Acetonitrile; Flow Rate: 1.0. mL/minute; Gradient program: Time(min)/ B Cone. : 0.01/10, 6.0/90, 10.0/100, 12.0/100, 14/10, 18.0/10. [0263] LCMS : 383.10 (M+H), Rt 1.475 min, 99.65%. Column: X-Select CSH C18 (3.0*50) mm 2.5um; Mobile Phase: A: 0.05% Formic acid in water:ACN(95:05); B: ACN; Inj Volume: 2.0μL; Flow Rate : 1.2. mL/minute; Column oven Temp: 50°C; Gradient program: 0% B to 98 % B in 2.0 minute, Hold till 3.0 min, At 3.2 min B cone is 0 % up to 4.0 min.
[0264] 1H NMR (400 MHz, DMSO-d6): 5 8.92 (d, J= 7.9 Hz, 1H), 8.08 (d, J = 5.9 Hz, 1H), 6.92 - 6.85 (m, 3H), 6.80 (s, 1H), 6.67 (br t, J= 5.3 Hz, 1H), 5.35 (quin, J = 7.2 Hz, 1H), 3.99 (s, 3H), 3.29 - 3.21 (m, 2H), 2.87 (td, J= 6.9, 13.8 Hz, 1H), 1.56 (d, J= 7.1 Hz, 3H), 1.20 (d, J= 7.0 Hz, 6H), 1.13 (t, J= 7.2 Hz, 3H).
[0265] Chiral method: Rt 6.328 min, 100%. Method File Name : CHIRAL-MET-B.lcm; Column: CHIRAL PAK IC (150*4.6mm, 3um); Moblile Phase A:0.1%DEAin n-Hexane; Moblile Phase B: DCM:MEOH (1 :1); A:B::75:25; Flow:0.7ml/min.
[0266] Data for Formula (I-IBt): HPLC: Rt 4.294 min, 98.88%. Method File : HPLC- FORMIC ACID-XSELECT- 10-90- 100.1cm; Column : X-Select CSH C18 (4.6*150) mm 3.5u; Mobile Phase: A - 0.1% Formic acid in water : Acetonitrile(95:05); B - Acetonitrile; Flow Rate: 1.0. mL/minute; Gradient program: Time(min)/ B Cone. : 0.01/10, 6.0/90, 10.0/100, 12.0/100, 14/10, 18.0/10.
[0267] LCMS : 383.10 (M+H), Rt 1.475 min, 99.73%. Column : X-Select CSH C18 (3.0*50) mm 2.5um; Mobile Phase: A: 0.05% Formic acid in water:ACN(95:05); B: ACN; Inj Volume: 2.0μL; Flow Rate : 1.2. mL/minute; Column oven Temp: 50°C; Gradient program: 0% B to 98 % B in 2.0 minute, Hold till 3.0 min, At 3.2 min B cone is 0 % up to 4.0 min.
[0268] 1H NMR (400 MHz, DMSO-d6): δ 8.92 (d, J = 8.1 Hz, 1H), 8.08 (d, J = 6.0 Hz, 1H), 6.91 - 6.86 (m, 3H), 6.80 (s, 1H), 6.67 (t, J= 5.4 Hz, 1H), 5.35 (quin, J= 7.2 Hz, 1H), 3.99 (s, 3H), 3.30 - 3.24 (m, 2H), 2.87 (td, J= 7.0, 13.8 Hz, 1H), 1.56 (d, J= 7.1 Hz, 3H), 1.20 (d, J= 7.0 Hz, 6H), 1.13 (t, J= 7.2 Hz, 3H).
[0269] Chiral method: Rt 7.564 min, 100%. Method File Name : CHIRAL-MET-B.lcm; Column: CHIRAL PAK IC (150*4.6mm, 3um); Moblile Phase A:0.1%DEAin n-Hexane; Moblile Phase B: DCM:MEOH (1 :1); A:B::75:25; Flow:0.7ml/min.
Step A: Synthesis of 2-(but-3-yn-2-yl)isoindoline- 1,3-dione (4)
Figure imgf000097_0001
[0270] To a stirred solution of compound 4A (31.49 g, 214.01 mmol) and compound 4B (15 g, 214.01 mmol) in THF (150 mL) was added and DEAD (55.9 g, 321.02 mmol) followed by triphenylphosphine (84.2 g, 321.02 mmol) at 0 °C, and the mixture was stirred at room temperature for 16 hours. Progress of the reaction was monitored by TLC. After completion of reaction, the reaction mixture was concentrated to dryness, and the residue was diluted with EtOAc (100 mL) and washed with water (2x100 mL) followed by saturated brine solution (1x100 mL). The combined organic fractions were then separated, dried over MgSO4 and concentrated under reduced pressure. The crude was purified by flash column chromatography eluting with 30% EtOAc in hexane. The desired fractions were concentrated to afford compound 4 (25 g, 125.5 mmol, 58% yield).

Claims

What is claimed is:
1. A compound having an isoxazole core chosen from the following compounds:
Figure imgf000098_0001
Figure imgf000099_0001
-Bi),
Figure imgf000100_0001
Figure imgf000101_0001
Figure imgf000102_0001
or a pharmaceutically acceptable salt thereof.
2. A compound having an oxadiazole core chosen from Formula (I-a), Formula (Ib), Formula
(I-c), Formula (Id), Formula (II), Formula (III), Formula (IV), Formula (V), or Formula (VI):
Figure imgf000102_0002
Figure imgf000103_0001
Figure imgf000104_0001
Figure imgf000105_0001
or a pharmaceutically acceptable salt thereof.
3. A compound of Formula (VII) having an isoxazole core:
Figure imgf000105_0002
or a pharmaceutically acceptable salt thereof, wherein:
R1 is chosen from -H or a C1-6alkyl, such a methyl;
R2 is chosen from -H or a C1-6alkyl, such a methyl;
R3 is independently chosen from a halogen, a C1-6alkyl, a carbocyclyl, or an alkoxy, wherein the C1-6alkyl, carbocyclyl or alkoxy optionally comprises at least one halogen substituent; and n is 0, 1, 2, 3, or 4.
4. The compound of claim 4, wherein the compound is chosen from:
Figure imgf000106_0001
Figure imgf000107_0001
or a pharmaceutically acceptable salt thereof.
5. The compound of claim 1, wherein the compound is Formula (I-IBa) or a pharmaceutically acceptable salt thereof.
6. The compound of any of claims 1-5, wherein the compound is a hydrochloride salt.
7. A pharmaceutical composition, comprising: a compound of any of claims 1-5, or a pharmaceutically acceptable salt thereof; and at least one pharmaceutically acceptable excipient.
8. A method of treating a neurological disorder, a disorder associated with excessive neuronal excitability, or a disorder associated with a gain-of-function mutation of a gene, wherein the method comprises administering to a subject in need thereof an effective amount of a compound having an isoxazole core chosen from:
Figure imgf000108_0001
Figure imgf000109_0001
Figure imgf000110_0001
Figure imgf000111_0001
Figure imgf000112_0001
or a pharmaceutically acceptable salt thereof.
9. A method of treating a neurological disorder, a disorder associated with excessive neuronal excitability, or a disorder associated with a gain-of-function mutation of a gene, wherein the method comprises administering to a subject in need thereof an effective amount of a compound having an oxadiazole core chosen from Formula (I-a), Formula (Ib), Formula (Ic), Formula (I- d), Formula (II), Formula (III), Formula (IV), Formula (V), or Formula (VI):
Figure imgf000112_0002
Figure imgf000113_0001
Figure imgf000114_0001
Figure imgf000115_0001
or a pharmaceutically acceptable salt thereof.
10. A method of treating a neurological disorder, a disorder associated with excessive neuronal excitability, or a disorder associated with a gain-of-function mutation of a gene, wherein the method comprises administering to a subject in need thereof an effective amount of a compound of Formula (VII):
Figure imgf000116_0001
or a pharmaceutically acceptable salt thereof, wherein:
R1 is chosen from -H or a C i-6alkyl, such a methyl;
R2 is chosen from -H or a C1-6alkyl, such a methyl;
R3 is independently chosen from a halogen, a C1-6alkyl, a carbocyclyl, or an alkoxy, wherein the C1-6alkyl, carbocyclyl or alkoxy optionally comprises at least one halogen substituent; and n is 0, 1, 2, 3, or 4.
11. The method of claim 10, wherein the compound of Formula (VII) is chosen from:
Figure imgf000116_0002
(VII-b),
Figure imgf000117_0001
or a pharmaceutically acceptable salt thereof.
12. The method of claim 8, wherein the compound is or a pharmaceutically
Figure imgf000118_0001
acceptable salt thereof.
13. The method of any one of claims 8-12, wherein the disorder is a disorder associated with a gain-of-function mutation of KCNT1.
14. The method of any one of claims 8-13, wherein the neurological disorder, the disorder associated with excessive neuronal excitability, or the disorder associated with a gain-of-function mutation of a gene (e.g., KCNT1) is epilepsy, an epilepsy syndrome, or an encephalopathy.
15. The method of any one of claims 8-13, wherein the neurological disorder, the disorder associated with excessive neuronal excitability, or the disorder associated with a gain-of-function mutation of a gene (e.g., KCNT1) is a genetic or pediatric epilepsy or a genetic or pediatric epilepsy syndrome.
16. The method of any one of claims 8-13, wherein the neurological disorder, the disorder associated with excessive neuronal excitability, or the disorder associated with a gain-of-function mutation of a gene (e.g., KCNT1) is a cardiac dysfunction.
17. The method of any one of claims 8-13, wherein the neurological disorder, the disorder associated with excessive neuronal excitability, or the disorder associated with a gain-of-function mutation of a gene (e.g., KCNT1) is chosen from epilepsy and other encephalopathies (e.g., malignant migrating focal seizures of infancy (MMFSI) or epilepsy of infancy with migrating focal seizures (EIMFS), autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE), West syndrome, infantile spasms, epileptic encephalopathy, focal epilepsy, Ohtahara syndrome, developmental and epileptic encephalopathy, and Lennox-Gastaut syndrome), seizures (e.g., Generalized tonic clonic seizures, Asymmetric Tonic Seizures), leukodystrophy, leukoencephalopathy, intellectual disability, Multifocal Epilepsy, drug-resistant epilepsy, Temporal lobe epilepsy, or cerebellar ataxia.
18. The method of any one of claims 8-13, wherein the neurological disorder, the disorder associated with excessive neuronal excitability, or the disorder associated with a gain-of-function mutation of a gene (e.g., KCNT1) is chosen from cardiac arrhythmia, Brugada syndrome, and myocardial infarction.
19. The method of any one of claims 8-13, wherein the neurological disorder, the disorder associated with excessive neuronal excitability, or the disorder associated with a gain-of-function mutation of a gene (e.g., KCNT1) is selected from pain and related conditions (e.g., neuropathic pain, acute/chronic pain, migraine).
20. The method of any one of claims 8-13, wherein the neurological disorder, the disorder associated with excessive neuronal excitability, or the disorder associated with a gain-of-function mutation of a gene (e.g., KCNT1) is a muscle disorder (e.g., myotonia, neuromyotonia, cramp muscle spasms, spasticity).
21. The method of any one of claims 8-13, wherein the neurological disorder, the disorder associated with excessive neuronal excitability, or the disorder associated with a gain-of-function mutation of a gene (e.g., KCNT1) is chosen from itch and pruritis, ataxia, or cerebellar ataxias.
22. The method of any one of claims 8-13, wherein the neurological disorder, the disorder associated with excessive neuronal excitability, or the disorder associated with a gain-of-function mutation of a gene (e.g., KCNT1) is a psychiatric disorder (e.g., major depression, anxiety, bipolar disorder, schizophrenia).
23. The method of any one of claims 8-13, wherein the neurological disorder, the disorder associated with excessive neuronal excitability, or the disorder associated with a gain-of-function mutation of a gene (e.g., KCNT1) is chosen from a learning disorder, Fragile X, neuronal plasticity, or an autism spectrum disorder.
24. The method of any one of claims 8-13, wherein the neurological disorder, the disorder associated with excessive neuronal excitability, or the disorder associated with a gain-of-function mutation of a gene (e.g., KCNT1) is chosen from epileptic encephalopathy with SCN1A, SCN2A, and/or SCN8A mutations, early infantile epileptic encephalopathy, Dravet syndrome, Dravet syndrome with SCN1A mutation, generalized epilepsy with febrile seizures, intractable childhood epilepsy with generalized tonic-clonic seizures, infantile spasms, benign familial neonatal- infantile seizures, SCN2A epileptic encephalopathy, focal epilepsy with SCN3A mutation, cryptogenic pediatric partial epilepsy with SCN3A mutation, SCN8A epileptic encephalopathy, Rasmussen encephalitis, malignant migrating partial seizures of infancy, autosomal dominant nocturnal frontal lobe epilepsy, KCNQ2 epileptic encephalopathy, or KCNT1 epileptic encephalopathy.
PCT/US2023/019648 2022-04-25 2023-04-24 Kcnt1 inhibitors comprising an isoxazole or oxadiazole core and methods of use WO2023211850A1 (en)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20180072708A1 (en) * 2015-03-25 2018-03-15 National Center For Geriatrics And Gerontology Novel oxadiazole derivative and pharmaceutical containing same
WO2020227101A1 (en) * 2019-05-03 2020-11-12 Praxis Precision Medicines, Inc. Kcnt1 inhibitors and methods of use
WO2021195066A2 (en) * 2020-03-23 2021-09-30 Praxis Precision Medicines, Inc. Kcnt1 inhibitors and methods of use

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20180072708A1 (en) * 2015-03-25 2018-03-15 National Center For Geriatrics And Gerontology Novel oxadiazole derivative and pharmaceutical containing same
US10538516B2 (en) * 2015-03-25 2020-01-21 National Center For Geriatrics And Gerontology Oxadiazole derivative and pharmaceutical containing same
WO2020227101A1 (en) * 2019-05-03 2020-11-12 Praxis Precision Medicines, Inc. Kcnt1 inhibitors and methods of use
WO2021195066A2 (en) * 2020-03-23 2021-09-30 Praxis Precision Medicines, Inc. Kcnt1 inhibitors and methods of use

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