WO2023235719A2 - Stereoselective allosteric inhibitors of sarm1 - Google Patents

Stereoselective allosteric inhibitors of sarm1 Download PDF

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WO2023235719A2
WO2023235719A2 PCT/US2023/067646 US2023067646W WO2023235719A2 WO 2023235719 A2 WO2023235719 A2 WO 2023235719A2 US 2023067646 W US2023067646 W US 2023067646W WO 2023235719 A2 WO2023235719 A2 WO 2023235719A2
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sarm1
disease
tryptoline
compound
formula
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PCT/US2023/067646
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French (fr)
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WO2023235719A3 (en
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Benjamin F. Cravatt
Hannah FELDMAN
Minoru Yokoyama
Bruno MELILLO
Stuart Schreiber
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The Scripps Research Institute
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/437—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a five-membered ring having nitrogen as a ring hetero atom, e.g. indolizine, beta-carboline
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
    • A61K31/444—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a six-membered ring with nitrogen as a ring heteroatom, e.g. amrinone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca

Definitions

  • Axonal degeneration is an early hallmark and driver of disease progression in diverse neurodegenerative disorders that affect both the central and peripheral nervous systems, including Alzheimer’s disease, amyotrophic lateral sclerosis (ALS), and chemotherapy-induced peripheral neuropathy (1-3).
  • ALS amyotrophic lateral sclerosis
  • 1-3 chemotherapy-induced peripheral neuropathy
  • SARM1 protein sterile alpha toll/interleukin receptor motif containing-1
  • SARM1 possesses an N-terminal armadillo repeat (ARM) domain followed by tandem sterile alpha motif (SAM) domains and a C-terminal toll/interleukin receptor (TIR) domain.
  • the TIR domain of SARM1 has been found to possess intrinsic nicotinamide adenine dinucleotide (NAD) hydrolase (NADase) activity, converting NAD+ into nicotinamide, adenosine diphosphate ribose (ADPR), and cyclic ADPR, representing a prototype member of a growing class of TIR domains with enzymatic function (5, 6).
  • NAD nicotinamide adenine dinucleotide
  • ADPR adenosine diphosphate ribose
  • cyclic ADPR representing a prototype member of a growing class of TIR domains with enzymatic function
  • SARM1 catalytic function of SARM1 is tightly regulated by a complex autoinhibitory mechanism.
  • SARM1 forms an inactive homo-octameric complex (8, 9).
  • Autoinhibition in this state is achieved through the physical separation of the TIR domains by the ARM domains, preventing TIR-TIR domain dimerization, which is necessary for formation of a composite active site that catalyzes NAD hydrolysis (10, 11).
  • NAD hydrolysis NAD hydrolysis
  • SARM1 also prevents the axonal decline caused by loss of nicotinamide mononucleotide adenyl transferase 2 (NMNAT2) (13), which functions upstream of SARM1 through the enzymatic conversion of NMN to NAD+ (21).
  • NMNAT2 nicotinamide mononucleotide adenyl transferase 2
  • SARM1 hypermorphic risk alleles have recently been discovered in patients with ALS (27, 28), supporting the human biology relevance of the NMNAT2-SARM1 pathway to maintaining neuronal integrity and CNS health.
  • SARM1 genetic disruption in the prevention of axonal degeneration
  • this enzyme is considered an attractive therapeutic target for the treatment of neurodegenerative disorders.
  • Multiple inhibitors of SARM1 have been described (29-32), most of which target the NADase domain (29-31). These include simple isoquinolines that have recently been found to serve as pseudo-substrates, being converted to NAD mimetics by SARM1 to form the active inhibitors (11, 31).
  • cysteines within both the enzymatic (TIR) and allosteric (ARM) domains of SARM1, have been identified as potential targets for electrophilic small molecules (29, 30, 32); however, the mechanisms of action of putative orthosteric (30) and allosteric (32) electrophilic inhibitors of SARM1 remain poorly understood.
  • a nisoldipine derivative, dehydronitrosonisoldipine (dHNN) was recently shown to inhibit SARM1 and found to react with C311 in the ARM domain (32).
  • dHNN dehydronitrosonisoldipine
  • mutagenesis of C311 only modestly impaired ( ⁇ two-fold) the inhibitory activity of dHNN, leading to the conclusion that this compound may engage multiple cysteines in SARM1 (32).
  • the application provides a method of inhibiting the NADase activity of SARM1, comprising contacting SARM1 with a tryptoline acrylamide derivative.
  • the application further provides the above method, wherein the tryptoline acrylamide derivative reacts with C311 in the ARM domain of SARM1. [0011] The application further provides either of the above methods, wherein the tryptoline acrylamide derivative reacts stereospecifically and site-specifically with C311 in the ARM domain of SARM1. [0012] The application further provides any of the above methods, wherein the tryptoline acrylamide derivative covalently binds to C311 in the ARM domain of SARM1. [0013] The application further provides any of the above methods, wherein the reaction of the tryptoline acrylamide derivative with C311 in the ARM domain of SARM1 allosterically inhibits the NADase activity of SARM1.
  • the application further provides any of the above methods, wherein the inhibition of the NADase activity of SARM1 prevents axonal degeneration. [0015] The application further provides the above method, wherein the prevention of axonal degeneration promotes maintenance of neuronal integrity. [0016] The application further provides any of the above methods, wherein the inhibition of the NADase activity of SARM1 prevents or ameliorates a neurodegenerative disorder. [0017] The application further provides the above method, wherein the neurodegenerative disorder is ALS, Alzheimer’s Disease, or chemotherapy-induced peripheral neuropathy.
  • the application provides a tryptoline acrylamide derivative compound of Formula I that inhibits the NADase activity of SARM1, wherein: R is optionally substituted -O(C 2 -C 6 )alkyl, -NH(C 1 -C 6 )alkyl, -NH(C 3 -C 6 )cycloalkyl, -(C 3 - C 6 )heterocycloalkyl, or -NH(C 5 -C 6 )heteroaryl; or a pharmaceutically acceptable salt or prodrug thereof.
  • the application further provides the above electrophilic tryptoline acrylamide derivative compound of Formula I, wherein R is -NHMe, cyclopropylamino, pyridinylamino, or propylamino.
  • R is -NHMe, cyclopropylamino, pyridinylamino, or propylamino.
  • the application further provides the above electrophilic tryptoline acrylamide derivative compound of Formula I, wherein Formula I has either of the following structures: [0021]
  • Compounds of Formula I can inhibit the activity the NADase activity of SARM1.
  • the compounds of the invention can be used to inhibit activity or a function of SARM1 in a cell or in an individual or patient in need of inhibition of the enzyme by administering an inhibiting amount of a compound of Formula I to the cell, individual, or patient.
  • FIG. 1 Discovery of covalent ligands that stereoselectively and site-specifically engage C311 in SARM1.
  • Figure 2. Stereoselective and site-specific engagement of C311 allosterically inhibits SARM1 enzymatic activity.
  • Figure 3. SAR analysis of engagement and inhibition of SARM1 by tryptoline acrylamides.
  • Figure 4. Inhibitory activity and proteome-wide selectivity of chemical probes targeting SARM1_C311.
  • Mouse SARM1 is stereoselectively engaged and inhibited by chemical probes targeting SARM1_C311.
  • Figure 19 Stereoselective inhibition of vacor-induced cADPR production in mouse Neuro-2a cells by chemical probes targeting SARM1_C311.
  • Figure 20 Concentration-dependent inhibition of vacor-induced axonal degeneration by chemical probes targeting SARM1_C311.
  • Figure 21 Acrylamide stereoprobes do not independently affect axonal integrity in mouse DRG neurons.
  • Figure 22 Concentration-dependent inhibition of vincristine-induced axonal degeneration by chemical probes targeting SARM1_C311.
  • Embodiment 1 The application provides a method of inhibiting the NADase activity of SARM1, comprising contacting SARM1 with a tryptoline acrylamide derivative.
  • Embodiment 2 The application provides the method of Embodiment 1, wherein the tryptoline acrylamide derivative reacts with C311 in the ARM domain of SARM1.
  • Embodiment 1 The application provides the method of either Embodiment 1 or Embodiment 2, wherein the tryptoline acrylamide derivative reacts stereoselectively and site- specifically with C311 in the ARM domain of SARM1.
  • Embodiment 4. The application provides the method of any one of Embodiments 1-3, wherein the tryptoline acrylamide derivative covalently binds to C311 in the ARM domain of SARM1.
  • the application provides the method of any one of Embodiments 2-4, wherein the reaction of the tryptoline acrylamide derivative with C311 in the ARM domain of SARM1 allosterically inhibits the NADase activity of SARM1.
  • Embodiment 7 The application provides the method of Embodiment 6, wherein the prevention of axonal degeneration promotes maintenance of neuronal integrity.
  • Embodiment 8 The application provides the method of any one of Embodiments 1-7, wherein the inhibition of the NADase activity of SARM1 prevents or ameliorates neurodegenerative disorders.
  • the neurodegenerative disorder is selected from: spinal muscular atrophy (SMA), Chemotherapy Induced Peripheral Neuropathy, multiple sclerosis (MS), traumatic brain injury (TBI), spinal cord injury, stroke, Parkinson' disease, glaucoma, Huntington's disease, Alzheimer's disease, Charcot-Marie-Tooth disease (CMT), retinitis pigmentosa (RP), age- related macular degeneration (AMD), small fiber neuropathies, peripheral neuropathy (e.g., viral neuropathy), spinocerebellar ataxias, cystic fibrosis, familial amyloidotic polyneuropathy, spongiform encephalopathies, spinal and bulbar muscular atrophy, hereditary dentatorubral-pallidoluysian atrophy, adrenoleukodystrophy, adrenomyeloneuropathy, Alexander's disease, amyotrophic lateral sclerosis (ALS), Bassen- Korn
  • SMA spinal muscular atrophy
  • MS Che
  • Embodiment 10 The application provides the method of Embodiment 9, wherein the neurodegenerative disorder is ALS, Alzheimer’s Disease, or chemotherapy-induced peripheral neuropathy.
  • Embodiment 11 The application provides a tryptoline acrylamide derivative compound of Formula I,
  • the application provides the tryptoline acrylamide derivative compound of Formula I of Embodiment 12, wherein Formula I has the following structure: [0058] Embodiment 14.
  • the application provides the tryptoline acrylamide derivative compound of Formula I of Embodiment 12, wherein Formula I has the following structure: .
  • Embodiment 15 The application provides the tryptoline acrylamide derivative compound of Formula I of Embodiment 12, wherein Formula I has the following structure: wherein R is -O(C 2 -C 6 )alkyl.
  • Embodiment 16 The application provides the tryptoline acrylamide derivative compound of Formula I of Embodiment 12, wherein Formula I has the following structure: . [0061] Embodiment 17.
  • the application provides the tryptoline acrylamide derivative compound of Formula I of Embodiment 12, wherein Formula I has the following structure: .
  • Embodiment 18 The application provides a method of inhibiting the NADase activity of SARM1, comprising contacting the SARM1 with the tryptoline acrylamide derivative compound of Formula I of any one of Embodiments 11-17.
  • Embodiment 19 The application provides the method of Embodiment 18, wherein the tryptoline acrylamide derivative compound covalently binds C311 in the ARM domain of SARM1.
  • Embodiment 20 is
  • Embodiment 19 The application provides the method of Embodiment 19, wherein the tryptoline acrylamide derivative compound site-specifically and covalently binds C311 of SARM1.
  • Embodiment 21 The application provides the method of Embodiment 20, wherein the tryptoline acrylamide derivative compound site-specifically, stereoselectively, and covalently binds C311 of SARM1.
  • Embodiment 22 The application provides the method of Embodiment 21, wherein the tryptoline acrylamide derivative compound allosterically inhibits SARM1.
  • Embodiment 23 Embodiment 19.
  • the application provides a method of inhibiting the NADase activity of SARM1, comprising contacting the SARM1 with the tryptoline acrylamide derivative compound of Formula I of any one of Embodiments 11-17.
  • Embodiment 24 The application provides the method of Embodiment 23, wherein the inhibition of the NADase activity of SARM1 prevents or ameliorates a SARM1-mediated disorder.
  • Embodiment 25 The application provides a method of treating a SARM1- mediated disorder, comprising administering to a patient in need thereof a therapeutically effective amount of the tryptoline acrylamide derivative compound of Formula I of any one of Claims 11-17.
  • Embodiment 26 Embodiment 26.
  • Embodiment 25 provides the method of Embodiment 25, wherein the SARM1-mediated disorder is a neurodegenerative disorder.
  • Embodiment 27 provides the method of Embodiment 26, wherein the neurodegenerative disorder is selected from: spinal muscular atrophy (SMA), Chemotherapy Induced Peripheral Neuropathy, multiple sclerosis (MS), traumatic brain injury (TBI), spinal cord injury, stroke, Parkinson' disease, glaucoma, Huntington's disease, Alzheimer's disease, Charcot-Marie-Tooth disease (CMT), retinitis pigmentosa (RP), age- related macular degeneration (AMD), small fiber neuropathies, peripheral neuropathy (e.g., viral neuropathy), spinocerebellar ataxias, cystic fibrosis, familial amyloidotic polyneuropathy, spongiform encephalopathies, spinal and bulbar muscular atrophy, hereditary dentatorubral-pallidoluysian atrophy, adrenoleukodys
  • Embodiment 28 The application provides the method of Embodiment 27, wherein the neurodegenerative disorder is ALS, Alzheimer’s Disease, or chemotherapy-induced peripheral neuropathy.
  • Embodiment 29 The application provides a composition comprising the compound of Formula I of any one of Embodiments 11-17, admixed with at least one carrier, diluent or excipient.
  • Embodiment 30 The application provides the composition of Embodiment 29, further comprising another pharmaceutically active compound.
  • Embodiment 31 The application provides the composition of either Embodiment 29 or Embodiment 30, further comprising another anti-neurodegenerative compound.
  • Embodiment 32 The application provides the method of Embodiment 27, wherein the neurodegenerative disorder is ALS, Alzheimer’s Disease, or chemotherapy-induced peripheral neuropathy.
  • Embodiment 29 The application provides a composition comprising the compound of Formula I of any one of Embodiments 11-17, admixed with at least one carrier, diluent or excipient.
  • Embodiment 33 The application provides a composition comprising the compound of Formula I of any one of Embodiments 13-15, admixed with at least one carrier, diluent or excipient.
  • Embodiment 34 The application provides the composition of Embodiment 32, further comprising another pharmaceutically active compound.
  • Embodiment 35 The application provides the composition of either Embodiment 33 or Embodiment 34, further comprising another anti-neurodegenerative compound.
  • Embodiment 36 Embodiment 36.
  • Embodiment 37 The application provides any methods of inhibiting the NADase activity of SARM1, electrophilic tryptoline acrylamide derivative compounds, methods of treating SARM1-mediated disorders, or compositions comprising the tryptoline acrylamide derivative compound of Formula I, as described herein. Definitions [0082] As referred to herein, unless otherwise specified, for instance in the Examples or Figures herein disclosed, “SARM1” is human wild-type (WT) SARM1 (Accession No. NP_055892).
  • phrases “a” or “an” entity as used herein refers to one or more of that entity; for example, a compound refers to one or more compounds or at least one compound. As such, the terms “a” (or “an”), “one or more”, and “at least one” can be used interchangeably herein.
  • the phrase "as defined herein above” refers to the broadest definition for each group as provided in the Summary of the Invention, the Detailed Description of the Invention, the Experimentals, or the broadest claim. In all other embodiments provided below, substituents which can be present in each embodiment and which are not explicitly defined retain the broadest definition provided in the Summary of the Invention.
  • the terms “comprise(s)” and “comprising” are to be interpreted as having an open-ended meaning. That is, the terms are to be interpreted synonymously with the phrases “having at least” or “including at least”.
  • the term “comprising” means that the process includes at least the recited steps, but may include additional steps.
  • the term “comprising” means that the compound or composition includes at least the recited features or components, but may also include additional features or components.
  • each R 1 and R 2 is independently selected from carbon and nitrogen
  • both R 1 and R 2 can be carbon, both R 1 and R 2 can be nitrogen, or R 1 or R 2 can be carbon and the other nitrogen or vice versa.
  • any variable occurs more than one time in any moiety or formula depicting and describing compounds employed or claimed in the present invention, its definition on each occurrence is independent of its definition at every other occurrence. Also, combinations of substituents and/or variables are permissible only if such compounds result in stable compounds.
  • Tautomeric compounds can exist as two or more interconvertable species.
  • Prototropic tautomers result from the migration of a covalently bonded hydrogen atom between two atoms.
  • Tautomers generally exist in equilibrium and attempts to isolate individual tautomers usually produce a mixture whose chemical and physical properties are consistent with a mixture of compounds. The position of the equilibrium is dependent on chemical features within the molecule. For example, in many aliphatic aldehydes and ketones, such as acetaldehyde, the keto form predominates while; in phenols, the enol form predominates.
  • Common prototropic tautomers include keto/enol amide/imidic acid and amidine tautomers.
  • phenylalkyl refers to an alkyl group having one to two phenyl substituents, and thus includes benzyl, phenylethyl, and biphenyl.
  • An “alkylaminoalkyl” is an alkyl group having one to two alkylamino substituents.
  • “Hydroxyalkyl” includes 2-hydroxyethyl, 2-hydroxypropyl, 1- (hydroxymethyl)-2-methylpropyl, 2-hydroxybutyl, 2,3-dihydroxybutyl, 2-(hydroxymethyl), 3-hydroxypropyl, and so forth. Accordingly, as used herein, the term “hydroxyalkyl” is used to define a subset of heteroalkyl groups defined below.
  • -(ar)alkyl refers to either an unsubstituted alkyl or an aralkyl group.
  • the term (hetero)aryl or (het)aryl refers to either an aryl or a heteroaryl group.
  • alkyl as used herein denotes an unbranched or branched chain, saturated, monovalent hydrocarbon residue containing 1 to 12 carbon atoms.
  • lower alkyl or “C 1 -C 6 alkyl” as used herein denotes a straight or branched chain hydrocarbon residue containing 1 to 6 carbon atoms.
  • C 1–12 alkyl refers to an alkyl composed of 1 to 12 carbons.
  • alkyl groups include, but are not limited to, lower alkyl groups include methyl, ethyl, propyl, i-propyl, n-butyl, i-butyl, t- butyl or pentyl, isopentyl, neopentyl, hexyl, heptyl, and octyl.
  • alkyl When the term “alkyl” is used as a suffix following another term, as in “phenylalkyl,” or “hydroxyalkyl,” this is intended to refer to an alkyl group, as defined above, being substituted with one to two substituents selected from the other specifically- named group.
  • phenylalkyl denotes the radical R'R"-, wherein R' is a phenyl radical, and R" is an alkylene radical as defined herein with the understanding that the attachment point of the phenylalkyl moiety will be on the alkylene radical.
  • arylalkyl radicals include, but are not limited to, benzyl, phenylethyl, 3-phenylpropyl.
  • arylalkyl or “aralkyl” are interpreted similarly except R' is an aryl radical.
  • the terms "(het)arylalkyl” or “(het)aralkyl” are interpreted similarly except R' is optionally an aryl or a heteroaryl radical.
  • 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 having from 1 to 20 carbon atoms (“C 1–20 alkyl”). In some embodiments, an alkyl group has 1 to 15 carbon atoms (“C 1–15 alkyl”). In some embodiments, an alkyl group has 1 to 14 carbon atoms (“C 1–14 alkyl”). In some embodiments, an alkyl group has 1 to 13 carbon atoms (“C 1–13 alkyl”). In some embodiments, an alkyl group has 1 to 12 carbon atoms (“C 1–12 alkyl”). In some embodiments, an alkyl group has 1 to 11 carbon atoms (“C 1–11 alkyl”).
  • an alkyl group has 1 to 10 carbon atoms (“C 1–10 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 (“C 1–7 alkyl”). In some embodiments, an alkyl group has 1 to 6 carbon atoms (“C 1–6 alkyl”). In some embodiments, 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”).
  • 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 (“C 1–2 alkyl”). In some embodiments, an alkyl group has 1 carbon atom (“C 1 alkyl”). In some embodiments, an alkyl group has 2 to 6 carbon atoms (“C 2–6 alkyl”).
  • C 1–6 alkyl groups include methyl (C 1 ), ethyl (C 2 ), n–propyl (C 3 ), isopropyl (C 3 ), n–butyl (C 4 ), tert–butyl (C 4 ), sec– butyl (C 4 ), iso–butyl (C 4 ), n–pentyl (C 5 ), 3–pentanyl (C 5 ), amyl (C 5 ), neopentyl (C 5 ), 3– methyl–2–butanyl (C 5 ), tertiary amyl (C 5 ), and n–hexyl (C 6 ).
  • alkyl groups include n–heptyl (C 7 ), n–octyl (C 8 ) and the like.
  • Alkenyl or “olefin” refers to a radical of a straight–chain or branched hydrocarbon group having from 2 to 10 carbon atoms and 1, 2, 3, or 4 carbon-carbon double bonds (“C 2–10 alkenyl”).
  • 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”).
  • an alkenyl group has 2 to 7 carbon atoms (“C 2–7 alkenyl”).
  • an alkenyl group has 2 to 6 carbon atoms (“C 2–6 alkenyl”). In some embodiments, an alkenyl group has 2 to 5 carbon atoms (“C 2–5 alkenyl”). In some embodiments, an alkenyl group has 2 to 4 carbon atoms (“C 2–4 alkenyl”). In some embodiments, an alkenyl group has 2 to 3 carbon atoms (“C 2–3 alkenyl”). In some embodiments, an alkenyl group has 2 carbon atoms (“C 2 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 C 2–4 alkenyl groups include ethenyl (C 2 ), 1–propenyl (C 3 ), 2–propenyl (C 3 ), 1–butenyl (C 4 ), 2–butenyl (C 4 ), butadienyl (C 4 ), and the like.
  • Examples of C 2–6 alkenyl groups include the aforementioned C 2–4 alkenyl groups as well as pentenyl (C 5 ), pentadienyl (C 5 ), hexenyl (C 6 ), and the like.
  • alkenyl examples 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 10 carbon atoms and one or more carbon-carbon triple bonds (e.g., 1, 2, 3, or 4 triple bonds) (“C 2–10 alkynyl”). In some embodiments, an alkynyl group has 2 to 9 carbon atoms (“C 2–9 alkynyl”). In some embodiments, an alkynyl group has 2 to 8 carbon atoms (“C 2–8 alkynyl”).
  • an alkynyl group has 2 to 7 carbon atoms (“C 2–7 alkynyl”). In some embodiments, an alkynyl group has 2 to 6 carbon atoms (“C 2–6 alkynyl”). In some embodiments, an alkynyl group has 2 to 5 carbon atoms (“C 2–5 alkynyl”). In some embodiments, an alkynyl group has 2 to 4 carbon atoms (“C 2–4 alkynyl”). In some embodiments, an alkynyl group has 2 to 3 carbon atoms (“C 2–3 alkynyl”). In some embodiments, an alkynyl group has 2 carbon atoms (“C 2 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 C 2–4 alkynyl groups include, without limitation, ethynyl (C 2 ), 1–propynyl (C 3 ), 2–propynyl (C 3 ), 1–butynyl (C 4 ), 2–butynyl (C 4 ), and the like.
  • Examples of C 2–6 alkenyl groups include the aforementioned C 2–4 alkynyl groups as well as pentynyl (C 5 ), hexynyl (C 6 ), and the like.
  • alkynyl examples include heptynyl (C 7 ), octynyl (C 8 ), and the like.
  • haloalkyl or “halo-lower alkyl” or “lower haloalkyl” refers to a straight or branched chain hydrocarbon residue containing 1 to 6 carbon atoms wherein one or more carbon atoms are substituted with one or more halogen atoms.
  • alkylene or "alkylenyl” as used herein denotes a divalent saturated linear hydrocarbon radical of 1 to 10 carbon atoms (e.g., (CH 2 ) n )or a branched saturated divalent hydrocarbon radical of 2 to 10 carbon atoms (e.g., -CHMe- or -CH 2 CH(i-Pr)CH 2 -), unless otherwise indicated. Except in the case of methylene, the open valences of an alkylene group are not attached to the same atom.
  • alkylene radicals include, but are not limited to, methylene, ethylene, propylene, 2-methyl-propylene, 1,1-dimethyl- ethylene, butylene, 2-ethylbutylene.
  • alkoxy as used herein means an -O-alkyl group, wherein alkyl is as defined above such as methoxy, ethoxy, n-propyloxy, i-propyloxy, n-butyloxy, i-butyloxy, t-butyloxy, pentyloxy, hexyloxy, including their isomers.
  • “Lower alkoxy” as used herein denotes an alkoxy group with a “lower alkyl” group as previously defined.
  • “C 1 - 10 alkoxy” as used herein refers to an-O-alkyl wherein alkyl is C 1-10 .
  • hydroxyalkyl denotes an alkyl radical as herein defined wherein one to three hydrogen atoms on different carbon atoms is/are replaced by hydroxyl groups.
  • cycloalkyl refers to a saturated carbocyclic ring containing 3 to 8 carbon atoms, i.e. cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl or cyclooctyl.
  • C 3-7 cycloalkyl refers to an cycloalkyl composed of 3 to 7 carbons in the carbocyclic ring.
  • carboxy-alkyl refers to an alkyl moiety wherein one, hydrogen atom has been replaced with a carboxyl with the understanding that the point of attachment of the heteroalkyl radical is through a carbon atom.
  • carboxy or “carboxyl” refers to a –CO 2 H moiety.
  • heteroaryl or “heteroaromatic” as used herein means a monocyclic or bicyclic radical of 5 to 12 ring atoms having at least one aromatic ring containing four to eight atoms per ring, incorporating one or more N, O, or S heteroatoms, the remaining ring atoms being carbon, with the understanding that the attachment point of the heteroaryl radical will be on an aromatic ring.
  • heteroaryl rings have less aromatic character than their all-carbon counter parts. Thus, for the purposes of the invention, a heteroaryl group need only have some degree of aromatic character.
  • heteroaryl moieties include monocyclic aromatic heterocycles having 5 to 6 ring atoms and 1 to 3 heteroatoms include, but is not limited to, pyridinyl, pyrimidinyl, pyrazinyl, pyrrolyl, pyrazolyl, imidazolyl, oxazol, isoxazole, thiazole, isothiazole, triazoline, thiadiazole and oxadiaxoline which can optionally be substituted with one or more, preferably one or two substituents selected from hydroxy, cyano, alkyl, alkoxy, thio, lower haloalkoxy, alkylthio, halo, lower haloalkyl, alkylsulfinyl, alkylsulfonyl, halogen, amino, alkylamino,dialkylamino, aminoalkyl, alkylaminoalkyl, and dialkylaminoalkyl, nitro, alkoxycarbon
  • bicyclic moieties include, but are not limited to, quinolinyl, isoquinolinyl, benzofuryl, benzothiophenyl, benzoxazole, benzisoxazole, benzothiazole and benzisothiazole.
  • Bicyclic moieties can be optionally substituted on either ring; however the point of attachment is on a ring containing a heteroatom.
  • heterocyclyl denotes a monovalent saturated cyclic radical, consisting of one or more rings, preferably one to two rings, including spirocyclic ring systems, of three to eight atoms per ring, incorporating one or more ring heteroatoms (chosen from N,O or S(O) 0-2 ), and which can optionally be independently substituted with one or more, preferably one or two substituents selected from hydroxy, oxo, cyano, lower alkyl, lower alkoxy, lower haloalkoxy, alkylthio, halo, lower haloalkyl, hydroxyalkyl, nitro, alkoxycarbonyl, amino, alkylamino, alkylsulfonyl, arylsulfonyl, alkylaminosulfonyl, arylaminosulfonyl, alkyls
  • heterocyclic radicals include, but are not limited to, azetidinyl, pyrrolidinyl, hexahydroazepinyl, oxetanyl, tetrahydrofuranyl, tetrahydrothiophenyl, oxazolidinyl, thiazolidinyl, isoxazolidinyl, morpholinyl, piperazinyl, piperidinyl, tetrahydropyranyl, thiomorpholinyl, quinuclidinyl and imidazolinyl.
  • Heterocyclyl refers to a group or radical of a 3– to 14– membered non–aromatic ring system having ring carbon atoms and 1 to 4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“3– 14 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 polycyclic (e.g., a fused, bridged or spiro ring system such as a bicyclic system (“bicyclic heterocyclyl”) or tricyclic system (“tricyclic heterocyclyl”)), and can be saturated or can contain one or more carbon–carbon double or triple bonds.
  • Heterocyclyl polycyclic 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, and sulfur (“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 1 ring heteroatom selected from nitrogen, oxygen, and sulfur.
  • Exemplary 3–membered heterocyclyl groups containing 1 heteroatom include, without limitation, azirdinyl, oxiranyl, and thiiranyl.
  • Exemplary 4–membered heterocyclyl groups containing 1 heteroatom include, without limitation, azetidinyl, oxetanyl and thietanyl.
  • Exemplary 5–membered heterocyclyl groups containing 1 heteroatom include, without limitation, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothiophenyl, dihydrothiophenyl, pyrrolidinyl, dihydropyrrolyl, and pyrrolyl–2,5–dione.
  • Exemplary 5– membered heterocyclyl groups containing 2 heteroatoms include, without limitation, dioxolanyl, oxathiolanyl and dithiolanyl.
  • Exemplary 5–membered heterocyclyl groups containing 3 heteroatoms include, without limitation, triazolinyl, oxadiazolinyl, and thiadiazolinyl.
  • Exemplary 6–membered heterocyclyl groups containing 1 heteroatom include, without limitation, piperidinyl, tetrahydropyranyl, dihydropyridinyl, and thianyl.
  • Exemplary 6–membered heterocyclyl groups containing 2 heteroatoms include, without limitation, piperazinyl, morpholinyl, dithianyl, and dioxanyl.
  • Exemplary 6–membered heterocyclyl groups containing 3 heteroatoms include, without limitation, triazinanyl.
  • Exemplary 7–membered heterocyclyl groups containing 1 heteroatom include, without limitation, azepanyl, oxepanyl and thiepanyl.
  • Exemplary 8–membered heterocyclyl groups containing 1 heteroatom include, without limitation, azocanyl, oxecanyl and thiocanyl.
  • Exemplary bicyclic heterocyclyl groups include, without limitation, indolinyl, isoindolinyl, dihydrobenzofuranyl, dihydrobenzothienyl, tetrahydrobenzothienyl, tetrahydrobenzofuranyl, tetrahydroindolyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, decahydroisoquinolinyl, octahydrochromenyl, octahydroisochromenyl, decahydronaphthyridinyl, decahydro–1,8–naphthyridinyl, octahydropyrrolo[3,2–b]pyrrole, indolinyl, phthalimidyl, naphthalimidyl, chromanyl, chromenyl, 1H–benzo[e][
  • 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 pi 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 6 ring carbon atoms (“C 6 aryl”; e.g., phenyl).
  • an aryl group has 10 ring carbon atoms (“C 10 aryl”; e.g., naphthyl such as 1–naphthyl ( ⁇ -naphthyl) and 2–naphthyl ( ⁇ -naphthyl)).
  • C 10 aryl e.g., naphthyl such as 1–naphthyl ( ⁇ -naphthyl) and 2–naphthyl ( ⁇ -naphthyl)).
  • an aryl group has 14 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.
  • Heteroaryl refers to a radical of a 5–14 membered monocyclic or polycyclic (e.g., bicyclic, tricyclic) 4n+2 aromatic ring system (e.g., having 6, 10, or 14 pi 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 (“5–14 membered heteroaryl”).
  • the point of attachment can be a carbon or nitrogen atom, as valency permits.
  • Heteroaryl polycyclic 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 polycyclic (aryl/heteroaryl) ring system.
  • 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. In some embodiments, the 5–6 membered heteroaryl has 1 ring heteroatom selected from nitrogen, oxygen, and sulfur.
  • Exemplary 5–membered heteroaryl groups containing 1 heteroatom include, without limitation, pyrrolyl, furanyl and thiophenyl.
  • Exemplary 5–membered heteroaryl groups containing 2 heteroatoms include, without limitation, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, and isothiazolyl.
  • Exemplary 5–membered heteroaryl groups containing 3 heteroatoms include, without limitation, triazolyl, oxadiazolyl, and thiadiazolyl.
  • Exemplary 5–membered heteroaryl groups containing 4 heteroatoms include, without limitation, tetrazolyl.
  • Exemplary 6–membered heteroaryl groups containing 1 heteroatom include, without limitation, pyridinyl.
  • Exemplary 6–membered heteroaryl groups containing 2 heteroatoms include, without limitation, pyridazinyl, pyrimidinyl, and pyrazinyl.
  • Exemplary 6–membered heteroaryl groups containing 3 or 4 heteroatoms include, without limitation, triazinyl and tetrazinyl, respectively.
  • Exemplary 7–membered heteroaryl groups containing 1 heteroatom include, without limitation, azepinyl, oxepinyl, and thiepinyl.
  • Exemplary 5,6–bicyclic heteroaryl groups include, without limitation, indolyl, isoindolyl, indazolyl, benzotriazolyl, benzothiophenyl, isobenzothiophenyl, benzofuranyl, benzoisofuranyl, benzimidazolyl, benzoxazolyl, benzisoxazolyl, benzoxadiazolyl, benzthiazolyl, benzisothiazolyl, benzthiadiazolyl, indolizinyl, and purinyl.
  • Exemplary 6,6–bicyclic heteroaryl groups include, without limitation, naphthyridinyl, pteridinyl, quinolinyl, isoquinolinyl, cinnolinyl, quinoxalinyl, phthalazinyl, and quinazolinyl.
  • Exemplary tricyclic heteroaryl groups include, without limitation, phenanthridinyl, dibenzofuranyl, carbazolyl, acridinyl, phenothiazinyl, phenoxazinyl and phenazinyl.
  • “Saturated” refers to a ring moiety that does not contain a double or triple bond, i.e., the ring contains all single bonds.
  • Alkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl groups may be optionally substituted.
  • Optionally substituted refers to a group which may be substituted or unsubstituted.
  • substituted means that at least one hydrogen present on a group is replaced with a non-hydrogen substituent, and 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.
  • Heteroatoms such as nitrogen, oxygen, and sulfur may have hydrogen substituents and/or non-hydrogen substituents which satisfy the valencies of the heteroatoms and results in the formation of a stable compound.
  • Halo or “halogen” refers to fluorine (fluoro, –F), chlorine (chloro, –Cl), bromine (bromo, –Br), or iodine (iodo, –I).
  • composition is intended to encompass a product comprising the specified ingredients, as well as any product which results, directly or indirectly, from combination of the specified ingredients.
  • Salt includes any and all salts.
  • “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.
  • Pharmaceutically acceptable salts are well known in the art. For example, Berge et al., describes pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences (1977) 66:1–19.
  • Pharmaceutically acceptable salts include those derived from 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, dodecylsulfate, 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.
  • prodrug refers to compounds that are transformed in vivo to yield a disclosed compound or a pharmaceutically acceptable salt, hydrate or solvate of the compound. The transformation may occur by various mechanisms (such as by esterase, amidase, phosphatase, oxidative and or reductive metabolism) in various locations (such as in the intestinal lumen or upon transit of the intestine, blood or liver). Prodrugs are well known in the art (for example, see Rautio, Kumpulainen, et al., Nature Reviews Drug Discovery 2008, 7, 255).
  • a prodrug can comprise an ester formed by the replacement of the hydrogen atom of the acid group with a group such as (C 1-8 )alkyl, (C 2- 12 )alkylcarbonyloxymethyl, 1-(alkylcarbonyloxy)ethyl having from 4 to 9 carbon atoms, 1- methyl-1-(alkylcarbonyloxy)-ethyl having from 5 to 10 carbon atoms, alkoxycarbonyloxymethyl having from 3 to 6 carbon atoms, 1-(alkoxycarbonyloxy)ethyl having from 4 to 7 carbon atoms, 1-methyl-1-(alkoxycarbonyloxy)ethyl having from 5 to 8 carbon atoms, N-(alkoxycarbonyl)aminomethyl having from 3 to 9 carbon atoms, 1-(N- (alkoxycarbonyl)amino)e
  • a group such as (C 1-8 )alkyl, (C 2- 12 )alkylcarbonyloxymethyl, 1-(alkyl
  • a prodrug can be formed by the replacement of the hydrogen atom of the alcohol group with a group such as (C 1-6 )alkylcarbonyloxymethyl, 1-((C 1-6 )alkylcarbonyloxy)ethyl, 1-methyl-1-(( C1- 6 )alkylcarbonyloxy)ethyl (C 1-6 )alkoxycarbonyloxymethyl, N-(C 1- 6 )alkoxycarbonylaminomethyl, succinoyl, (C 1-6) alkylcarbonyl, .alpha.-amino(C 1- 4 )alkylcarbonyl, arylalkylcarbonyl and ⁇ -aminoalkylcarbonyl, or .alpha.- aminoalkylcarbonyl- ⁇ -aminoalkylcarbonyl, where each -aminoalkylcarbonyl group is independently selected from the naturally occurring L-amino acids
  • compounds described herein can comprise one or more asymmetric centers, and thus can exist in various stereoisomeric 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).
  • Compounds with such isotopically enriched atoms are useful, for example, as analytical tools or probes in biological assays.
  • Certain isotopically-labelled compounds e.g., those labeled with 3 H and 14 C
  • Tritiated (i.e., 3 H) and carbon-14 (i.e., 14 C) isotopes are particularly preferred for their ease of preparation and detectability.
  • Certain isotopically-labelled compounds of Formula (I) can be useful for medical imaging purposes, for example, those labeled with positron-emitting isotopes like 11 C or 18 F can be useful for application in Positron Emission Tomography (PET) and those labeled with gamma ray emitting isotopes like 123 I can be useful for application in Single Photon Emission Computed Tomography (SPECT). Further, substitution with heavier isotopes such as deuterium (i.e., 2 H) may afford certain therapeutic advantages resulting from greater metabolic stability (e.g., increased in vivo half-life or reduced dosage requirements) and hence may be preferred in some circumstances.
  • PTT Positron Emission Tomography
  • SPECT Single Photon Emission Computed Tomography
  • substitution with heavier isotopes such as deuterium may afford certain therapeutic advantages resulting from greater metabolic stability (e.g., increased in vivo half-life or reduced dosage requirements), and hence, may be preferred in some circumstances.
  • isotopic substitution at a site where epimerization occurs may slow or reduce the epimerization process and thereby retain the more active or efficacious form of the compound for a longer period of time.
  • Isotopically labeled compounds of Formula (I), in particular those containing isotopes with longer half-lives (t 1/2 >1 day), can generally be prepared by following procedures analogous to those disclosed in the Schemes and/or in the Examples herein below, by substituting an appropriate isotopically labeled reagent for a non- isotopically labeled reagent.
  • Methods of Use [0132] As referred to herein, unless otherwise specified, for instance in the Examples or Figures herein disclosed, “SARM1” is human wild-type (WT) SARM1 (Accession No. NP_055892). [0133] Compounds of the invention can inhibit the NADase activity of SARM1.
  • the compounds of the invention can be used to inhibit activity or a function of SARM1 in a cell or in an individual or patient in need of inhibition of the enzyme by administering an inhibiting amount of a compound of Formula I to the cell, individual, or patient.
  • the term "in a cell” includes both inside the cell membrane and on the surface of the cell membrane.
  • the present invention is further directed to a method of increasing the level of NAD+ in a sample or in a patient, comprising contacting the sample or administering to the patient a compound of of the invention, or a pharmaceutically acceptable salt thereof, wherein the increased level of NAD+ is relative to the level of NAD+ prior to the contacting or administering.
  • Compounds of the invention as SARM1 inhibitors, can inhibit axonal degeneration.
  • the present invention is further directed to a method of inhibiting axonal degeneration in a sample or in a patient, comprising contacting the sample or administering to the patient an inhibiting amount of a compound of the invention, or a pharmaceutically acceptable salt thereof.
  • the compounds of the invention are useful in the treatment and prevention of various diseases associated with abnormal expression or activity of SARM1.
  • the compounds of the invention are useful in the treatment and prevention of neurological disorders.
  • the term "neurological disorder” generally refers to a disorder affecting the nervous system, including the central nervous system or the peripheral nervous system.
  • the term “neurological disorder” also includes ocular indications having a nexus to the nervous system.
  • the neurological disorder treatable or preventable by administration of a compound of the invention includes neurodegenerative diseases. Neurodegenerative diseases are characterized by damage to the central nervous system and can be identified by progressive dysfunction, degeneration and death of specific populations of neurons which are often synaptically interconnected.
  • neurodegenerative diseases include Parkinson's disease (PD), Alzheimer's disease (AD), Huntington's disease (HD), prion disease, motor neuron diseases (MND), spinocerebellar ataxia (SCA), spinal muscular atrophy (SMA), amyotrophic lateral sclerosis (ALS), and epilepsy.
  • PD Parkinson's disease
  • AD Alzheimer's disease
  • HD Huntington's disease
  • MND motor neuron diseases
  • SCA spinocerebellar ataxia
  • SMA spinal muscular atrophy
  • ALS amyotrophic lateral sclerosis
  • Examples of neurological disorders treatable or preventable according to the methods of the invention include spinal muscular atrophy (SMA), Chemotherapy Induced Peripheral Neuropathy (representative chemotherapeutic agents include vinca-alkaloids, taxols and platins), multiple sclerosis (MS), traumatic brain injury (TBI), spinal cord injury, stroke, Parkinson' disease, glaucoma, Huntington's disease, Alzheimer's disease, Charcot- Marie-Tooth disease (CMT), retinitis pigmentosa (RP), age-related macular degeneration (AMD), small fiber neuropathies, peripheral neuropathy (e.g., viral neuropathy), spinocerebellar ataxias, cystic fibrosis, familial amyloidotic polyneuropathy, spongiform encephalopathies, spinal and bulbar muscular atrophy, hereditary dentatorubral- pallidoluysian atrophy, adrenoleukodystrophy, adrenomyeloneur
  • the neurological disorder treatable or preventable by administration of a compound of the invention is a neuropathy.
  • the term "neuropathy” refers broadly to diseased conditions of the nervous system, including polyneuropathy; neuropathy, ataxia, and retinosa pigmentosa (NARP); familial amyloid neuropathies; diabetic neuropathy (peripheral neuropathy due to diabetes mellitus); peripheral neuropathy (e.g., chemotherapy-induced peripheral neuropathy (CIPN), including CIPN caused by vinca alkaloids, bortezomib, lxabepilone, thalidomide and its analogs, taxanes, and platinum-based agents); and cranial neuropathy (e.g., auditory neuropathy and optic neuropathy).
  • CIPN chemotherapy-induced peripheral neuropathy
  • cranial neuropathy e.g., auditory neuropathy and optic neuropathy
  • the neurological disorder treatable or preventable by administration of a compound of the invention is an ocular neuropathy (e.g., optic neuropathy).
  • optic neuropathy refers to damage to the optic nerve from a number of causes.
  • optic neuropathy examples include ischemic optic neuropathy (e.g., anterior and posterior ischemic optic neuropathy); optic neuritis (e.g., chronic relapsing inflammatory optic neuropathy (CRION), single isolated optic neuritis (SION), and relapsing isolated optic neuritis); compressive optic neuropathy; infiltrative optic neuropathy; traumatic optic neuropathy; mitochondrial optic neuropathies; and hereditary optic neuropathies (e.g., Leber's hereditary optic neuropathy (LHON), hereditary neuropathy with liability to pressure palsy (HNPP), and dominant optic atrophy).
  • ischemic optic neuropathy e.g., anterior and posterior ischemic optic neuropathy
  • optic neuritis e.g., chronic relapsing inflammatory optic neuropathy (CRION), single isolated optic neuritis (SION), and relapsing isolated optic neuritis
  • compressive optic neuropathy e.g., infiltrative optic neuropathy; traumatic optic neuropathy; mitochondrial optic
  • the neurological disorder treatable or preventable by administration of a compound of the invention is multiple sclerosis (MS), chemotherapy- induced peripheral neuropathy (CIPN), amyotrophic lateral sclerosis (ALS), glaucoma, traumatic brain injury (TBI), or stroke.
  • MS multiple sclerosis
  • CIPN chemotherapy- induced peripheral neuropathy
  • ALS amyotrophic lateral sclerosis
  • TBI traumatic brain injury
  • the term "cell” is meant to refer to a cell that is in vitro, ex vivo or in vivo.
  • an ex vivo cell can be part of a tissue sample excised from an organism such as a mammal.
  • an in vitro cell can be a cell in a cell culture.
  • an in vivo cell is a cell living in an organism such as a mammal.
  • the term “contacting” refers to the bringing together of indicated moieties in an in vitro system or an in vivo system.
  • "contacting" SARM1 or “contacting” a cell with a compound of the invention includes the administration of a compound of the present invention to an individual or patient, such as a human, having SARM1, as well as, for example, introducing a compound of the invention into a sample containing a cellular or purified preparation containing SARM1.
  • the phrase "therapeutically effective amount” refers to the amount of active compound or pharmaceutical agent that elicits the biological or medicinal response in a tissue, system, animal, individual or human that is being sought by a researcher, veterinarian, medical doctor or other clinician.
  • the phrase “inhibiting amount” refers to the amount of active compound or pharmaceutical agent that elicits a measurable SARM1 inhibition or axonal degeneration in a tissue, system, animal, individual or human.
  • treating refers to 1) inhibiting the disease in an individual who is experiencing or displaying the pathology or symptomatology of the disease (i.e., arresting further development of the pathology and/or symptomatology), or 2) ameliorating the disease in an individual who is experiencing or displaying the pathology or symptomatology of the disease (i.e., reversing the pathology and/or symptomatology).
  • preventing refers to preventing the disease in an individual who may be predisposed to the disease but does not yet experience or display the pathology or symptomatology of the disease.
  • the invention is directed to a method of preventing a disease in a patient, by administering to the patient a therapeutically effective amount of a compound of the invention, or a pharmaceutically acceptable salt thereof.
  • Combination Therapy [0149]
  • One or more additional pharmaceutically active agents or treatment methods can be used in combination with the compounds of the present invention.
  • the agents can be combined with the present compounds in a single dosage form, or the agents can be administered simultaneously or sequentially as separate dosage forms.
  • additional agents include acamprosate, agomelatine, almotriptan, amantadine, amisulpride, amitriptyline, apomorphine, aripiprazole, asenapine, atomoxetine, baclofen, botulinum toxin type A, bromocriptine, buccal midazolam, buprenorphine, buspirone, cabergoline, carbamazepine, chlordiazepoxide, chlorpromazine, citalopram, clobazam, clomethiazole, clomipramine, clonazepam, clozapine, denzapine, co-beneldopa, co-careldopa, dantrolene, dexamfetamine, diazepam, divalproex sodium, donepezil, doxepin, duloxetine, eletriptan, entacapone, epinephrine, escita
  • the one or more additional pharmaceutically active agent can include a neuroprotective agent.
  • the neuroprotective agent is a dual leucine-zipper kinase (DLK) inhibitor.
  • the neuroprotective agent is a nicotinamide phosphoribosyltransferase (NAMPT) inhibitor.
  • the one or more additional pharmaceutically active agent can be NAD+ or an NAD+ precursor.
  • NAD+ precursors include, for example, nicotinamide riboside (NR), nicotinic acid (NA), nicotinic acid riboside (NaR), nicotinamide (NAM), nicotinamide mononucleotide (NMN), nicotinic acid mononucleotide (NaMN), tryptophan, vitamin B3, and nicotinic acid adenine dinucleotide (NAAD).
  • NR nicotinamide riboside
  • NA nicotinic acid
  • NaR nicotinic acid riboside
  • NAM nicotinamide
  • NMN nicotinamide mononucleotide
  • NaMN nicotinic acid mononucleotide
  • tryptophan vitamin B3, and nicotinic acid adenine dinucleotide (NAAD).
  • NAAD nicotinic acid
  • a pharmaceutical composition refers to a combination of a compound of the invention, or its pharmaceutically acceptable salt, and at least one pharmaceutically acceptable carrier. These compositions can be prepared in a manner well known in the pharmaceutical art, and can be administered by a variety of routes, depending upon whether local or systemic treatment is desired and upon the area to be treated.
  • Administration may be oral, topical (including ophthalmic and to mucous membranes including intranasal, vaginal and rectal delivery), pulmonary (e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal), ocular (e.g., eye drops or intravitreal, subconjunctival, subtenon, or retrobulbar injection), or parenteral.
  • This invention also includes pharmaceutical compositions which contain, as the active ingredient, one or more of the compounds of the invention above in combination with one or more pharmaceutically acceptable carriers.
  • the active ingredient is typically mixed with an excipient, diluted by an excipient or enclosed within such a carrier in the form of, for example, a capsule, sachet, paper, or other container.
  • an excipient serves as a diluent, it can be a solid, semi-solid, or liquid material, 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, suppositories, sterile injectable solutions, and sterile packaged powders.
  • the compositions can be formulated in a unit dosage form.
  • unit dosage form refers to a physically discrete unit 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.
  • the active compound can be effective over a wide dosage range and is generally administered in a pharmaceutically effective amount. It will be understood, however, that the amount of the compound actually administered will usually be determined by a physician, according to the relevant circumstances, including the condition to be treated, the chosen route of administration, the actual compound administered, the age, weight, and response of the individual patient, the severity of the patient's symptoms, and the like.
  • the principal active ingredient is mixed with a pharmaceutical excipient to form a solid pre-formulation composition containing a homogeneous mixture of a compound of the present invention.
  • the active ingredient is typically dispersed evenly throughout the composition so that the composition can be readily subdivided into equally effective unit dosage forms such as tablets, pills and capsules.
  • This solid pre-formulation is then subdivided into unit dosage forms of the type described above containing from, for example, 0.1 to about 500 mg of the active ingredient of the present invention.
  • the tablets or pills of the present invention can be coated or otherwise compounded to provide a dosage form affording the advantage of prolonged action.
  • 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 which 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 supra. In some embodiments, the compositions are administered by the oral or nasal respiratory route for local or systemic effect.
  • compositions can be nebulized by use of inert gases. Nebulized solutions may be breathed directly from the nebulizing device or the nebulizing device can be attached to a face masks tent, or intermittent positive pressure breathing machine. Solution, suspension, or powder compositions can be administered orally or nasally from devices which deliver the formulation in an appropriate manner. [0160] The amount of compound or composition administered to a patient will vary depending upon what is being administered, the purpose of the administration, such as prophylaxis or therapy, the state of the patient, the manner of administration, and the like. In therapeutic applications, compositions can be administered to a patient already suffering from a disease in an amount sufficient to cure or at least partially arrest the symptoms of the disease and its complications.
  • compositions administered to a patient can be in the form of pharmaceutical compositions described above. These compositions can be sterilized by conventional sterilization techniques, or may be sterile filtered. Aqueous solutions can be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile aqueous carrier prior to administration.
  • the therapeutic dosage of the compounds of the present invention can vary according to, for example, the particular use for which the treatment is made, the manner of administration of the compound, the health and condition of the patient, and the judgment of the prescribing physician.
  • the proportion or concentration of a compound of the invention in a pharmaceutical composition can vary depending upon a number of factors including dosage, chemical characteristics (e.g., hydrophobicity), and the route of administration.
  • the compounds of the invention can be provided in an aqueous physiological buffer solution containing about 0.1 to about 10% w/v of the compound for parenteral administration. Some typical dose ranges are from about 1 .mu.g/kg to about 1 g/kg of body weight per day.
  • the dose range is from about 0.01 mg/kg to about 100 mg/kg of body weight per day.
  • the dosage is likely to depend on such variables as the type and extent of progression of the disease or disorder, the overall health status of the particular patient, the relative biological efficacy of the compound selected, formulation of the excipient, and its route of administration. Effective doses can be extrapolated from dose-response curves derived from in vitro or animal model test systems.
  • DETAILED DESCRIPTION OF THE FIGURES [0163] Figure 1. Discovery of covalent ligands that stereoselectively and site-specifically engage C311 in SARM1. (A) Chemical structures of tryptoline acrylamide stereoprobes EV- 96-EV-99.
  • B, C MS-ABPP quantification of the reactivity of SARM1_C311 and other cysteines in SARM1 in human T cells treated with EV-96-EV-99 (20 ⁇ M, 3 h) or DMSO control.
  • B data represent mean values ⁇ SEM for eight independent experiments.
  • C individual cysteine reactivity data points represent mean values for two-eight independent experiments.
  • D Domain architecture of SARM1 with C311 is highlighted in black and ALS-relevant human hypermorphic mutants (27, 28) are highlighted in green.
  • the ARM-domain is shown in royal blue
  • the SAM domains are shown in navy
  • the TIR is shown in light blue
  • NAD+ is represented as yellow sticks
  • C311 is represented as red spheres
  • ALS-relevant hypermorphic mutants are represented as green spheres.
  • SARM1_C311 is marked in blue. Data represent log 2 mean fold-change values from two independent experiments.
  • A Schematic for neurite degeneration assays.
  • dorsal root ganglia from E13.5-15.5 mice or rats are harvested, isolated, and grown for 7 days in culture before treatment with vacor (50 ⁇ M) or vincristine (40 nM). Neurite morphology is then analyzed via fluorescence microscopy at various time points after vacor or vincristine treatment.
  • B Representative brightfield images from mouse DRG neurons treated with DMSO or 50 ⁇ M vacor +/- MY-9A, MY-9B, WX-02-17, or WX-02-37 (10 ⁇ M each) at 0, 8, 24, and 48 h post-treatment. Images shown are from a single experiment representative of three independent experiments.
  • NAD A
  • ADPR B
  • HEK293T cells recombinantly expressing SARM1- WT or SARM1-C311A and treated with buffer, 100 ⁇ M NAD+, 1 mM NMN, or 100 ⁇ M NAD+ and 1 mM NMN.
  • buffer 100 ⁇ M NAD+, 1 mM NMN, or 100 ⁇ M NAD+ and 1 mM NMN.
  • Data represent mean values ⁇ SD for two-three independent experiments.
  • C, D Relative amounts of NAD (C) and ADPR (D) generated from lysates of HEK293T cells expressing WT-SARM1 that were pretreated with DMSO, EV-98, or EV-99 (in situ, 20 ⁇ M, 3 h) and then, after lysis, supplemented with 100 ⁇ M NAD+ and 1 mM NMN. UT, untransfected HEK293T cells. Significance determined from a one-way ANOVA with Dunnett’s post hoc test. P-values vs. DMSO treatment, *P ⁇ 0.05, ***P ⁇ 0.001.
  • B, C Relative amounts of cADPR (B) and NAD (C) generated from lysates of HEK293T cells expressing SARM1-WT, SARM1-C311A, or SARM1-C311S and treated with buffer, 100 ⁇ M NAD+, 1 mM NMN, or 100 ⁇ M NAD+ and 1 mM NMN.
  • B, C Relative amounts of cADPR (B) and NAD (C) generated from lysates of HEK293T cells expressing SARM1-WT, SARM1-C311A, or SARM1-C311S and treated with buffer, 100 ⁇ M NAD+, 1 mM NMN, or 100 ⁇ M NAD+ and 1 mM NMN.
  • B, C Relative amounts of cADPR (B) and NAD (C) generated from lysates of HEK293T cells expressing SARM1-WT, SARM1-C3
  • FIG. 1 For (A-C), data represent mean values ⁇ SEM for three independent experiments.
  • Figure 15 Proteome-wide reactivity of chemical probes targeting SARM1_C311.
  • (A-D) Waterfall plots of MS-ABPP data showing total numbers of quantified cysteines and their respective reactivity ratios (DMSO/compound) in 22Rv1 (A, C) or Ramos (B, D) cells treated with MY-9B (A, B) or WX-02-37 (C, D) (20 ⁇ M, 3 h). Also shown are heat maps comparing the cysteine reactivity values for cysteines substantially engaged (> 75%) by MY- 9B and/or WX-02-37 in cells treated with each of the corresponding diastereomers.
  • FIG. 16 Proteome-wide selectivity of chemical probes targeting SARM1_C311.
  • A, B Volcano plots comparing global cysteine reactivity profiles for (A) MY-9A versus MY-9B or (B) WX-02-17 versus WX-02-37 (in situ, 20 ⁇ M, 3h) determined by MS-ABPP in Ramos cells, where cysteines that were significantly (log 10 (p-value) ⁇ 1.5 and stereoselectively (log 2 > 1.5) engaged by MY-9B or WX-02-37 are shown in upper-right quadrant of dashed lines.
  • SARM1_C311 is marked in blue.
  • E, F NAD
  • cADPR F
  • E cADPR
  • G Relative amounts of cADPR generated from lysates of HEK293T cells expressing mouse SARM1 that were pretreated with DMSO or the indicated compounds (in situ, 20 ⁇ M, 3 h) and then, after lysis, supplemented with 100 ⁇ M NAD+ and 1 mM NMN.
  • FIG. 1 UT, untransfected HEK293T cells. Significance determined from a one-way ANOVA with Dunnett’s post hoc test. P-values vs. DMSO treatment, **P ⁇ 0.01, ***P ⁇ 0.001.
  • Figure 19 Stereoselective inhibition of vacor-induced cADPR production in mouse Neuro-2a cells by chemical probes targeting SARM1_C311.
  • A Quantification of cADPR (pmol per mg protein) produced from Neuro-2A cells treated with vacor (50 ⁇ M, in situ) for 0, 2, 4, or 8 h. Significance determined from a one-way ANOVA with Dunnett’s post hoc test.
  • Axonal degeneration is an early hallmark and driver of disease progression in diverse neurodegenerative disorders that affect both the central and peripheral nervous system, including Alzheimer’s disease, amyotrophic lateral sclerosis (ALS), and chemotherapy-induced peripheral neuropathy (1-3).
  • SARM1 protein sterile alpha toll/interleukin receptor motif containing-1
  • SARM1 possesses an N-terminal armadillo repeat (ARM) domain followed by tandem sterile alpha motif (SAM) domains and a C-terminal toll/interleukin receptor (TIR) domain.
  • ARM N-terminal armadillo repeat
  • SAM tandem sterile alpha motif
  • TIR C-terminal toll/interleukin receptor
  • the TIR domain of SARM1 has been found to possess intrinsic nicotinamide adenine dinucleotide (NAD) hydrolase (NADase) activity, converting NAD+ into nicotinamide, adenosine diphosphate ribose (ADPR), and cyclic ADPR, representing a prototype member of a growing class of TIR domains with enzymatic function (5, 6).
  • NAD nicotinamide adenine dinucleotide
  • ADPR adenosine diphosphate ribose
  • cyclic ADPR representing a prototype member of a growing class of TIR domains with enzymatic function
  • SARM1 catalytic function of SARM1 is tightly regulated by a complex autoinhibitory mechanism.
  • SARM1 forms an inactive homo-octameric complex (8, 9).
  • Autoinhibition in this state is achieved through the physical separation of the TIR domains by the ARM domains, preventing TIR-TIR domain dimerization, which is necessary for formation of a composite active site that catalyzes NAD hydrolysis (10, 11).
  • NAD hydrolysis NAD hydrolysis
  • SARM1 also prevents the axonal decline caused by loss of nicotinamide mononucleotide adenyl transferase 2 (NMNAT2) (13), which functions upstream of SARM1 through the enzymatic conversion of NMN to NAD+ (21).
  • NMNAT2 nicotinamide mononucleotide adenyl transferase 2
  • SARM1 hypermorphic risk alleles have recently been discovered in patients with ALS (27, 28), supporting the human biology relevance of the NMNAT2-SARM1 pathway to maintaining neuronal integrity and CNS health.
  • SARM1 genetic disruption in the prevention of axonal degeneration
  • this enzyme is considered an attractive therapeutic target for the treatment of neurodegenerative disorders.
  • Multiple inhibitors of SARM1 have been described (29-32), most of which target the NADase domain (29-31). These include simple isoquinolines that have recently been found to serve as pseudo-substrates, being converted to NAD mimetics by SARM1 to form the active inhibitors (11, 31).
  • cysteines within both the enzymatic (TIR) and allosteric (ARM) domains of SARM1, have been identified as potential targets for electrophilic small molecules (29, 30, 32); however, the mechanisms of action of putative orthosteric (30) and allosteric (32) electrophilic inhibitors of SARM1 remain poorly understood.
  • a nisoldipine derivative, dehydronitrosonisoldipine (dHNN) was recently shown to inhibit SARM1 and found to react with C311 in the ARM domain (32).
  • dHNN dehydronitrosonisoldipine
  • mutagenesis of C311 only modestly impaired ( ⁇ two-fold) the inhibitory activity of dHNN, leading to the conclusion that this compound may engage multiple cysteines in SARM1 (32).
  • C311 is located in the ARM domain of SARM1 (Fig.1D), and recent cryo-electron microscopy structures have revealed that this residue sits on a flexible loop located adjacent to the allosteric metabolite binding pocket of the ARM domain (7-9, 12) (Fig.1E).
  • Fig.1E several hypermorphic mutations in human SARM1 that have been linked to ALS (27, 28) are also structurally proximal to C311 (Fig.1E), supporting the functionality of this region for regulating SARM1 activity.
  • Non-electrophilic propanamide analogues of EV-98 and EV-99 did not affect MY-13B reactivity with recombinant SARM1 in HEK293T cells (Fig.7), supporting that the tryptoline acrylamides bind SARM1 through a covalent mechanism.
  • vacor acts as a specific and direct activator of SARM1 through metabolic conversion to the NMN mimetic vacor mononucleotide (VMN) (16). It was found that vacor (50 ⁇ M) induced the robust, time-dependent production of cADPR in the human neuroblastoma cell line SH-SY5Y (Fig.2E), and this effect was absent in SARM1-null SH-SY5Y cells generated by CRISPR-Cas9 genome editing (Fig.10).
  • the cis-tryptoline acrylamides were first analyzed for engagement of WT-SARM1 in HEK293T cells by pre-treatment at 20 ⁇ M for 3 h, followed by exposure of the cells to MY-13B (20 ⁇ M, 1 h) and analysis of the reactions by gel-ABPP.
  • Several of the amide analogues showed equivalent or greater engagement of WT- SARM1 compared to EV-99, including the methylamide MY-9B, the aminopyridine WX-02- 35, and the propylamide WX-02-37 (Fig.3B and 3C). In each case, the stereoselectivity of SARM1 engagement by the R,R cis-tryptoline acrylamides was maintained.
  • SARM1-C311A mutant displayed a modest hypermorphic activity reflected in greater cADPR production compared to WT-SARM1 (Fig.13A-C). This is perhaps not surprising, considering the high density of other gain-of-function mutants regionalized to the ARM domain in spatial proximity to SARM1-C311A.
  • An evaluation of alternative mutations revealed that the SARM1-C311S mutant displayed similar catalytic activity to WT-SARM1 (Fig.13A-C), and it was confirmed that the SARM1_C311S mutant was fully resistant to the inhibitory effects of active tryptoline acrylamide inhibitors of WT-SARM1 (Fig.13D).
  • cysteines in other enzymes that directly consume NAD+ or participate in NAD+ biosynthetic pathways e.g., PARPs, SIRTs, NMNATs, NAMPTs, etc.
  • C119 the catalytic cysteine in CD38
  • MY-9B and WX-02-37 display an attractive combination of cellular potency (low- ⁇ M), well-defined SARs (stereoselective and site-specific reactivity with C311), and proteome-wide selectivity for use as chemical probes of SARM1.
  • Applicant proceeded to treated mouse DRG neurons concurrently with vacor (50 ⁇ M) and DMSO or active and inactive enantiomeric compounds (MY-9B vs MY-9A; WX-02-37 vs WX-02-17; 1-20 ⁇ M each), followed by analysis of neurite morphology over a 48 h period by fluorescent microscopy (Fig.5A).
  • vacor caused ⁇ 60% degeneration by 8 h and complete degeneration by 24 h. This vacor-induced degeneration was near- completely blocked in neurons treated with 10 or 20 ⁇ M of MY-9B or WX-02-37 (Fig.5B-D and Fig.20).
  • Vincristine is a chemotherapeutic that indirectly induces SARM1 activity and axonal degeneration by inhibiting microtubule assembly and axonal transport leading to NMNAT2 depletion (49, 50). Applicant found that both MY-9B and WX-02-37 stereoselectively prevented vincristine-induced neurite degeneration in rat DRG neurons (Fig.
  • Described herein is the chemical proteomic discovery of a series of tryptoline acrylamides that stereoselectively and site-specifically engaged SARM1_C311 and, through doing so, inhibit the NAD+ glucohydrolase activity of this enzyme. This work extends previous studies pointing to SARM1_C311 as a potential site of druggability (32) in important ways.
  • Covalent inhibitors may also offer other advantages, including a durability of enzyme inhibition that lasts until turnover of the SARM1 protein. While our current understanding of the cellular half-life of SARM1 is incomplete, initial studies indicate that this protein is relatively long-lived in mouse neurons (half-life of ⁇ 58-64 h) (53). Consistent with this conclusion, a single treatment of MY-9B and WX-02-37 protected neurons from vacor- induced axonal degeneration for up to 48 h. Aside from their therapeutic potential, chemical probes targeting SARM1_C311 should serve as valuable tools to better understand the allosteric relationship between the protein’s regulatory (ARM) and enzymatic (TIR) domains.
  • ARM regulatory
  • TIR enzymatic
  • NP_055892 1 mvltlllsay klcrffamsg prpgaerlav pgpdggggtg pwwaaggrgp revspgagte 61 vqdaleralp elqqalsalk qaggaravga glaevfqlve eawllpavgr evaqglcdai 121 rldggldlll rllqapelet rvqaarlleq ilvaenrdrv ariglgviln lakerepvel 181 arsvagileh mfkhseetcq rlvaagglda vlywcrrtdp allrhcalal gncalhggqa 241 vqrrmvekra aewlfplafs kedellrlha clavavlatn keverevers gtlalveplv 301
  • Flash chromatography was performed using 20-40 ⁇ m silica gel (60- ⁇ mesh) on a Teledyne ISCO Combiflash Rf or a Biotage Isolera Prime, alternatively in a glass column using SiliaFlash® F6040-63 ⁇ m silica gel (60- ⁇ mesh).
  • Preparative high-pressure liquid chromatography prep-HPLC was performed on a Gilson GX-281 instrument.
  • Analytical thin layer chromatography (TLC) was performed on 0.2 mm or 0.25 mm silica gel 60-F plates and visualized by UV light (254 nm).
  • Preparative thin layer chromatography was performed on GF254 plates (acrylic adhesive, 0.5 ⁇ 200 ⁇ 200 mm, 5–20 ⁇ M particle size, 250 ⁇ M thickness).
  • 1 H NMR spectra were recorded on Bruker Avance III 400, Avance III HD 400, Avance Neo 400 spectrometers ( 1 H, 400 MHz) at 300 K unless otherwise noted.
  • 13 C NMR spectra were recorded on a Bruker Avance III HD 600 spectrometer ( 13 C, 151 MHz) at 298 K.
  • Tandem liquid chromatography/mass spectrometry was performed on an Agilent 1200 series LC/MSD system equipped with an Agilent G6110A mass detector, alternatively a Shimadzu LC-20AD or AB series LC- MS system equipped with Shimadzu SPD-M20A or SPDM40 mass detectors, alternatively a Waters H-Class LC with equipped with diode array and QDa mass detector.
  • LC-MS Tandem liquid chromatography/mass spectrometry
  • Step 2 N-capping [0215] To a precooled (0 oC) solution of amide S7–S10 (1.0 equiv) in dichloromethane (0.1 M) were added triethylamine (1.5 equiv) and acryloyl chloride (1.0 equiv). The mixture was stirred at 0 °C for 10 min. The reaction mixture was then concentrated under reduced pressure and the resulting residue was purified by prep-HPLC to deliver the title compounds.
  • a 10x master mix containing 200 ⁇ M rhodamine-azide, 10 mM tris(2- carboxyethyl)phosphine hydrochloride (TCEP), 1 mM Tris((1-benzyl-4- triazolyl)methyl)amine (TBTA; in 4:1 tBuOH:DMSO), and 10 mM CuSO 4 was made and 6 ⁇ L of CuAAC master mix was then added to 60 ⁇ L of cellular lysate. After 1 hour of click labeling, 4x SDS running buffer was added to samples. Samples were boiled for 2 minutes and then analyzed by SDS-PAGE.
  • Nitrocellulose blots were blocked in milk (5% w/v in TBST (20 mM Tris pH 7.5, 150 mM NaCl, 0.1% Tween-20)) for 30 minutes at room temperature. Primary antibodies were diluted into milk (5% w/v in TBST) and incubated overnight at 4oC with gentle rotation. Blots were washed in TBST for 5 minutes thrice followed by the addition of secondary antibodies (Li-Cor) diluted into milk (5% w/v in TBST) for 1 hour at room temperature (dilution: 1:10,000). Blots were imaged using an Li-Cor Odyssey IR imager and quantitated using ImageStudio Lite software.
  • mice anti-FLAG M2 antibody Sigma, F3165, dilution 1:1,000
  • rabbit anti-GAPDH Cell Signaling, 2118, dilution: 1:1,000
  • rabbit anti- SARM1 Cell Signaling,13022, dilution: 1:1,000
  • hSARM1-C311S plasmid was cloned using hSARM1- FLAG pcDNA5/FRT/TO as a template via Quikchange mutagenesis. All sequences were verified via Sanger sequencing before use. Molecular cloning primers listed below.
  • sgRNA target sequence design and cloning [0220] sgRNAs targeting human SARM1 were designed using CRISPick genetic perturbation platform (https://portals.broadinstitute.org/gppx/crispick/public, Broad Institute). Guide oligos were cloned into lentiCRISPRv2-puro vector (Addgene, 98290) using Golden Gate assembly (New England Bioscience, E1602).
  • SARM1-KO cell lines were maintained following the same protocol as their parental cell lines outlines above. Culturing of mouse primary embryonic DRG neurons [0222] Dorsal root ganglia were dissected from E13.5-E14.5 C57BL/6J mouse embryos (RRID:IMSR_JAX:000664).
  • Explants were cultured in 35 mm tissue culture dishes pre- coated with poly-L-lysine (20 ⁇ g/ml for 1 hr; Merck) and laminin (20 ⁇ g/ml for 1 hr; Merck) in Dulbecco's Modified Eagle's Medium (DMEM, Gibco) with 1% penicillin/streptomycin, 33 ng/ml 2.5S NGF (Invitrogen) and 2% B27 (Gibco).4 ⁇ M aphidicolin (Merck) was used to reduce proliferation and viability of small numbers of non-neuronal cells (54). Culture media was replenished every 3 days. Neurites were allowed to extend for 7 days before treatment.
  • DMEM Dulbecco's Modified Eagle's Medium
  • DRG dorsal root ganglia
  • L-15 Leibovitz's 15
  • FBS TrypLE Express
  • DRGs were then centrifuged at 1000 rpm for 5 min and resuspend in 15 mL complete medium containing neurobasal medium (2% B-27 (Gibco, 17504-044), 2 mM L- glutamine (Gibco, 25030-081), 2 ⁇ M 5-Fluoro-2'-deoxyuridine (Sigma, F0503), 2 ⁇ M uridine (Sigma, U3003), 50 ng/mL 2.5S NGF (Millipore, 01-125-100ug), and 100 U/mL Penicillin- Streptomycin (Gibco, 15140). Cell were counted and diluted in complete medium to a final concentration of 1 x 10 7 cells/mL.
  • neurobasal medium 2% B-27 (Gibco, 17504-044), 2 mM L- glutamine (Gibco, 25030-081), 2 ⁇ M 5-Fluoro-2'-deoxyuridine (Sigma, F0503), 2 ⁇ M
  • HEK293T (ATCC, CRL-3216) cells were plated into a 6-well dish at 3 x 10 5 cells/well in 2 mLs of media and allowed to attach overnight.
  • DMEM/Fugene mixture was then added to a 1.5 mL Eppendorf tube containing 1 ⁇ g of sgRNA-encoding lentiCRISPRv2-puro plasmid, 0.1 ⁇ g CMV-dr8.91 envelope, and 0.9 ⁇ g pCMV-VSV-G (Addgene, 8454) and incubated for 10 minutes before adding dropwise to plated HEK293T cells. Cells were transfected for 48 hours before virus was harvested.
  • the viral supernatant was collected by filtering through a 0.45 ⁇ m PVDF filter and stored at -80oC until ready for use.
  • SH-SY5Y cells were plated into a 6-well plate 5 x 10 5 cells/well in 2 mLs of media.
  • the day of infection 1 mL of media was removed and replaced with 1 mL of virus with 2 ⁇ L polybrene (final concentration: 1 ⁇ g/L, Santa Cruz Biotechnology, SC134220).
  • media was removed and replaced with complete DMEM media supplemented with 0.75 ⁇ g/mL puromycin (Gibco, A1113803).
  • Transient transfection into HEK293T cells was achieved by plating cells (6-well: 3 x 10 5 cells/well in 3 mL media, 10 cm: 2 x 10 6 cells/well in 10 mL media) the day prior to transfection.
  • cells were transfected by adding DNA (2 ⁇ g per 6-well, 10 ⁇ g per 10 cm plate) into a 1.5 mL Eppendorf tube and adding serum-free DMEM (500 ⁇ L for 6-well, 1 mL for 10 cm plate) followed by addition of the transfection reagent polyethylenimine (PEI, 1 mg/mL) using a 1:3 ratio of DNA ( ⁇ g) to PEI ( ⁇ L).
  • DNA 2 ⁇ g per 6-well, 10 ⁇ g per 10 cm plate
  • serum-free DMEM 500 ⁇ L for 6-well, 1 mL for 10 cm plate
  • PEI transfection reagent polyethylenimine
  • Cells were transfected for 48 hours, with a media exchange at 24 hours, before assaying or collection.
  • SARM1 LC-MS/MS NADase assay SARM1 exogenous substrate assay [0226] HEK293T cells expressing recombinant SARM1 (see Tissue culture methods for detailed transfection protocol) were treated in situ with tryptoline acrylamide for 3 hours (0.1% DMSO). Following drug treatment, cells were collected in a 1.5 mL Eppendorf tube, pelleted by centrifugation (800 g, 3 minutes), flash frozen in LN2, and stored at -80oC.
  • Cell lysate concentrations were determined using DC Protein Assay and standardized to 1 mg/mL.
  • Samples were quenched by the addition of 400 ⁇ L ice cold methanol:acetonitrile (1:1) with 100 pmol internal standard 8-Br-cADPR (Enzo Life Sciences, BML-CA417-0500). Samples were vortexed for 30 seconds and frozen in LN2. To precipitate proteins, samples were then thawed at room temperature and sonicated for 15 minutes in an ice-cold ultrasonic bath sonicator. Following sonication, samples were incubated at -20oC for 1 hour and then centrifuged at 16,000 g for 15 minutes at 4oC to pellet precipitated proteins.100 ⁇ L of the supernatant was transferred to an LC-MS/MS vial for metabolomic analysis.
  • SH-SY5Y cells were treated in situ with tryptoline acrylamide for 3 hours (0.1% DMSO) followed by treatment with 50 ⁇ M vacor (Sigma, S668923) for 4 hours. Following drug treatment, cells were collected in a 1.5 mL Eppendorf tube, pelleted by centrifugation (800 g, 3 minutes), flash frozen in LN2, and stored at -80oC. Cells pellets thawed on ice and resuspended in 100 ⁇ L of DPBS.
  • Test compounds were prepared as 3-folds serial dilutions with 100% (v/v) DMSO from a 10 mM stock. Serial dilutions were then subsequently diluted again, 500-fold, using complete medium. Vincristine (MCE, HY-N0488) solution was prepared by diluting a 40 ⁇ M stock solution 100-fold (400 nM) using complete medium.
  • Half the media (50 ⁇ L) from DRG culture plate was removed and replaced with 50 ⁇ L compound solution to each well of cell plate, in which final concentrations of test compounds were 10, 3.33, 1.11, 0.37, 0.12, 0.041, 0.014 and 0.005 ⁇ M.
  • Compounds were incubated with DRG neurons for 3 hours prior to the addition of vincristine to each well (11.1 ⁇ L, 40 nM final concentration).
  • Low control (DMSO treated DRG neurons) and high control (DMSO + vincristine treated neurons) were prepared by dilution of 100% DMSO and 40 ⁇ M Vincristine using complete medium, respectively.
  • Permeabilization solution was aspirated, and cells were briefly washed thrice with 100 ⁇ L DPBS for 5 minutes per wash. Samples were then blocked for 30 minutes in DPBS with 5% FBS (Gibco, 10099141), 2% BSA (Sigma, A1933), and 0.1% Tween-20 (Sigma, P1379) before incubation with anti-beta III Tubulin (Abcam, ab41489) and anti-NeuN (Abcam, ab104225) primary antibodies overnight at 4oC.
  • Protein precipitation, denaturation, reduction, and alkylation [0230] After activity probe treatment, samples were then precipitated with the addition of 600 ⁇ L ice-cold MeOH, 200 ⁇ L CHCl 3 , and 100 ⁇ L water (in order), then vortexed for 10 seconds, and centrifuged (10 min, 16,000 g). Without perturbing the protein disk, both top and bottom layers were aspirated, and the protein disk was washed with 1 mL ice-cold MeOH, followed by centrifugation (10 min, 16,000 g). Protein pellets were allowed to air dry briefly until solvent droplets are no longer visible (5 minutes).
  • Samples were re-suspended in 90 ⁇ L denaturation buffer (9 M urea, 10 mM DTT, 50 mM TEAB pH 8.5). Samples were reduced by heating at 65oC for 20 minutes and water bath sonicated as needed to resuspend the protein pellets, followed by alkylation via addition of 10 ⁇ L (500 mM) iodoacetamide (Sigma, I1149) and incubated at 37oC for 30 min with shaking. Samples were diluted with 300 ⁇ L buffer (50 mM TEAB pH 8.5) to reach final concentration of 2 M urea. To ensure that samples are completely dissolved, samples were briefly centrifuged, and probe sonicated (1x, 10 pulses, 10% power).
  • Tryptic digestion and streptavidin enrichment [0231] Trypsin (4 ⁇ L of 0.25 ⁇ g/ ⁇ L in trypsin resuspension buffer with 25 mM CaCl 2 ) was added to each sample and digested at 37oC with shaking overnight. Streptavidin-agarose beads (Thermo Scientific, 20353) were prepared by washing twice in 10x bead volumes of wash buffer (50 mM TEAB pH 8.5, 150 mM NaCl, 0.2% NP-40) and resuspended to give 25 ⁇ L beads per 300 ⁇ L wash buffer.
  • wash buffer 50 mM TEAB pH 8.5, 150 mM NaCl, 0.2% NP-40
  • Trypsin digested samples were then diluted with 300 ⁇ L wash buffer (50 mM TEAB pH 8.5, 150 mM NaCl, 0.2% NP-40) containing streptavidin- agarose beads (25 ⁇ L beads) and were rotated at room temperature for 2 hours. Enriched samples were transferred to BioSpin columns (BioRad, 732-6204) and washed (3x 1 mL wash buffer, 3x 1 mL DPBS, 3x 1mL water). Enriched peptides were eluted by addition of 400 ⁇ L 50% acetonitrile with 0.1% formic acid and eluate was evaporated to dryness via speedvac.
  • wash buffer 50 mM TEAB pH 8.5, 150 mM NaCl, 0.2% NP-40
  • streptavidin- agarose beads 25 ⁇ L beads
  • Tandem mass tag (TMT) labeling of enriched peptides [0232] IA-DTB labeled and enriched peptides were resuspended in 100 ⁇ L EPPS buffer (140 mM, pH 8.0) with 30% acetonitrile, vortexed, and water bath sonicated. Samples were TMT (Thermo Scientific, 90406) labeled with 3 ⁇ L of corresponding TMT tag (5 mg tag resuspended in 256 ⁇ L acetonitrile), vortexed, and incubated at room temperature for 1 hour. TMT labeling was quenched with the addition of hydroxylamine (5 ⁇ L 5% solution in H 2 O) and incubated for 15 minutes at room temperature.
  • Samples were then acidified with 5 ⁇ L formic acid, combined, and dried via speedvac. Finally, samples were desalted via Sep-Pak C18 cartridge (Waters, WAT051910) and then high pH fractionated before LC-MS/MS/MS analysis. High pH HPLC fractionation [0233] Samples were resuspended in 500 ⁇ L resuspension buffer (95% water, 5% ACN, 0.1% FA) and fractionated into a 96 deep-well plate via HPLC (Agilent Infinity 1260 II LC system). Aqueous stationary phase (Buffer A) used is 10 mM aqueous NH 4 HCO 3, organic mobile phase (Buffer B) used is 100% acetonitrile.
  • Buffer A used is 10 mM aqueous NH 4 HCO 3
  • organic mobile phase (Buffer B) used is 100% acetonitrile.
  • Peptides were loaded onto a capillary column (ZORBAX 300Extend-C18, 3.5 ⁇ m) at a flow rate of 0.5 mL/min and eluted using the following gradient: 0-2 min, 100% Buffer A; 2-3 minutes, 0%–13% buffer B; 3-60 minutes, 13%–42% buffer B; 60-61 minutes, 42%–100% buffer B; 61-65 minutes, 100% buffer B; 65-66 minutes, 100%–0% buffer B; 66-75 minutes, 100% buffer A; 75-78 minutes, 0%–13% buffer B; 78-80 minutes, 13%–80% buffer B; from 80-85 minutes, 80% buffer B; 86-91 minutes, 100% buffer A; 91-94 minutes, 0%–13% buffer B 94-96 minutes, 13%–80% buffer B; 96-101 minutes, 80% buffer; 101-102 minutes, 80%–0% buffer B.
  • Each well in the 96-well plate contained 20 ⁇ L of 20% formic acid to acidify the eluting peptides.
  • the eluents were evaporated to dryness in the plate using via speed vac.
  • the top row (Row A, wells 1-12) were resuspended in 80% acetonitrile, 0.1% formic acid buffer (200 ⁇ L/well). Resuspended peptides solution from row A, was then added to row B, then row C, etc. to combine every 12th fraction. Resuspension process was repeated three times and the twelve fractions were dried via speecvac. The resulting twelve fractions were re-suspended in resuspension buffer and analyzed by LC-MS/MS/MS.
  • the peptides were loaded onto a capillary column (75 ⁇ m inner diameter fused silica, packed with C18 (Waters, Acquity BEH C18, 1.7 ⁇ m, 25 cm)) or an EASY-Spray HPLC column (Thermo ES902, ES903) using an Acclaim PepMap 100 (Thermo 164535) loading column a flow rate of 0.25 ⁇ L/min, and separated using the following gradient: 0-15 minutes, 5% buffer B; 15- 55 minutes, 5%–35% buffer B; 155-160 minutes, 35%–95% buffer B; 160-169 minutes, 95% buffer B; 169-170 minutes, 95%–5% buffer B; 170-200 minutes, 5% buffer B, where buffer A: 95% H2O, 5% acetonitrile, 0.1% FA and buffer B: 5% H2O, 95% CH 3 CN, 0.1% FA.
  • MS3-based TMT method on Orbitrap Fusion or Orbitrap Eclipse Tribrid Mass Spectrometers. Briefly, the scan sequence began with an MS1 master scan (Orbitrap analysis, resolution 120,000, 400 ⁇ 1700 m/z, RF lens 60%, automatic gain control [AGC] target 2E5, maximum injection time 50 ms, centroid mode) with dynamic exclusion enabled (repeat count 1, duration 15 s). The top ten precursors were then selected for MS2/MS3 analysis. MS2 analysis consisted of: quadrupole isolation (isolation window 0.7) of precursor ion followed by collision-induced dissociation (CID) in the ion trap (AGC 1.8E4, normalized collision energy 35%, maximum injection time 120 ms).
  • MS3 precursors were fragmented by HCD and analyzed using the Orbitrap (collision energy 55%, AGC 1.5E5, maximum injection time 120 ms, resolution was 50,000).
  • Orbitrap collision energy 55%, AGC 1.5E5, maximum injection time 120 ms, resolution was 50,000.
  • MS2 and MS3 files were extracted from the raw files using RAW Converter (version 1.1.0.22; available at http://fields.scripps.edu/rawconv/), uploaded to Integrated Proteomics Pipeline (IP2), and searched using the ProLuCID algorithm (this information is readily publicly available at http://fields.scripps.edu/downloads.php) using a reverse concatenated, non-redundant variant of the at human UniProt database (release 07-2016) and mouse UniProt dataset (release 07- 2017). Cysteine residues were searched with a static modification for carboxyamidomethylation (+57.02146 Da).
  • Cysteines were considered significantly liganded if they inhibited IA-DTB labeling ⁇ 75% (equivalent to a cysteine engagement ratio of ⁇ 4). Cysteines were considered stereoselective liganded if they displayed a log 2 (active/inactive) > 1.5 and a -log 10 (p-value) > 1.5.
  • Equation 2 Molecular cloning primers, mutagenesis primers, and sgRNA sequences Human SARM1(WT and C311A) Gibson primers: Forward primer: Reverse primer: Mouse SARM1 Gibson primers: Forward primer: Reverse primer: 3’- ’ SARM1-C311S Quikchange primers: hSARM1-C311S-fwd: hSARM1-C311S-rev: SARM1-targeted sgRNA sequences (all sequences are displayed 5’ to 3’): LC-MS/MS measurement of NAD metabolites [0236] NAD+, ADPR, and cADPR metabolite species were measured using LC-MS/MS.
  • Samples were injected onto HILIC column (Acquity UPLC BEH Amide column; 1.7 ⁇ M; 2.1 x 100 mM; Waters, 186004801) using HPLC (Agilent 1290 Infinity LC) with a flow rate of 0.4 mL/minute.
  • Aqueous buffer (Buffer A) contained 5% acetonitrile, 10 mM ammonium formate, and 0.1% formic acid.
  • Organic buffer (Buffer B) contained 95% acetonitrile, 10 mM ammonium formate, and 0.1% formic acid.
  • Metabolites were eluted using the following gradient: 0-2 minutes, 100% Buffer B; 2-14 minutes 100-65% Buffer B; 14-17 minutes, 65- 40% Buffer B; 17-18 minutes, 40% Buffer B; 18-19 minutes, 40-100% Buffer B; 19-22 minutes, 100% Buffer B.
  • Eluted metabolites were detected using a triple quad mass spectrometer (Agilent 6470 MassHunter; Agilent) via multiple reaction monitoring (MRM) using an electrospray ionization (ESI) source in positive mode.
  • MRM multiple reaction monitoring
  • ESI electrospray ionization
  • MS analysis was performed using ESI with the following parameters: gas temperature: 350oC; gas flow: 11 L/min; nebulizer: 45 psi; sheath gas temperature: 450oC; sheath gas flow: 12 L/min; capillary: 12 V; nozzle voltage/charging: 1500 V.
  • NAD+ precursor ion: 664.1, product ion: 136, dwell: 50, fragmentation (F): 100 V, collision (C): 53 V, collision acceleration (CA) : 4 V
  • ADPR precursor ion: 560.1, product ion: 136, dwell: 50, F: 100 V, C: 41 V, CA: 4 V
  • cADPR precursor ion: 542.1, product ion: 136, dwell: 50, F: 100 V, C: 49 V, CA: 4 V
  • 8-Br-cADPR precursor ion: 620, product ion: 214, dwell: 50, F: 100 V, C: 33 V, CA: 4 V).

Abstract

Described herein is a series of tryptoline acrylamides that site-specifically and stereoselectively covalently bind allosteric cysteine-311 (C311) in the non-catalytic armadillo repeat (ARM) domain of SARM1. These covalently binding inhibitors, show a high degree of proteome-wide selectivity for cysteine-311 of SARM1, and have been shown to stereoselectively block vincristine- and vacor-induced axonal degeneration in dorsal root ganglion neurons. The disclosed stereoselective inhibitor compounds and compositions thereof, that covalently bind allosteric C311 of SARM1, exhibit a potentially attractive therapeutic strategy for treating axon degenerative SARM1 -mediated forms of neurological disease. Methods of inhibiting SARM1 for the treatment of neurodegenerative SARM1- modulated disorders are also described herein.

Description

STEREOSELECTIVE ALLOSTERIC INHIBITORS OF SARM1 CROSS REFERENCE TO RELATED APPLICATION [0001] This application claims priority to U.S. provisional patent application No. 63/347,180, which was filed on May 31, 2022, and which is hereby incorporated by reference in its entirety. GOVERNMENT SUPPORT [0002] This invention was made with government support under grant number DA033760, awarded by the National Institutes of Health. The government has certain rights in the invention. FIELD OF THE INVENTION [0003] Disclosed herein is a series of tryptoline acrylamides that site-specifically and stereoselectively covalently bind the allosteric cysteine-311 (C311) in the non-catalytic armadillo repeat (ARM) domain of SARM1. These compounds stereoselectively inhibit the NADase activity of WT-SARM1, representing an attractive therapeutic strategy for axon degeneration-dependent forms of neurological disease. BACKGROUD OF THE INVENTION [0004] Axonal degeneration is an early hallmark and driver of disease progression in diverse neurodegenerative disorders that affect both the central and peripheral nervous systems, including Alzheimer’s disease, amyotrophic lateral sclerosis (ALS), and chemotherapy-induced peripheral neuropathy (1-3). Efforts to characterize the molecular pathways that contribute to axonal degeneration have led to the identification of the protein sterile alpha toll/interleukin receptor motif containing-1 (SARM1) as a key mediator of this process (4). SARM1 possesses an N-terminal armadillo repeat (ARM) domain followed by tandem sterile alpha motif (SAM) domains and a C-terminal toll/interleukin receptor (TIR) domain. The TIR domain of SARM1 has been found to possess intrinsic nicotinamide adenine dinucleotide (NAD) hydrolase (NADase) activity, converting NAD+ into nicotinamide, adenosine diphosphate ribose (ADPR), and cyclic ADPR, representing a prototype member of a growing class of TIR domains with enzymatic function (5, 6). [0005] The NADase activity of SARM1 is critical to its role in axonal degeneration, as excessive SARM1-dependent consumption of NAD+ results in metabolic crisis, which initiates the cell autonomous axon self-destruction process (5, 7). Accordingly, the catalytic function of SARM1 is tightly regulated by a complex autoinhibitory mechanism. Under homeostatic conditions, SARM1 forms an inactive homo-octameric complex (8, 9). Autoinhibition in this state is achieved through the physical separation of the TIR domains by the ARM domains, preventing TIR-TIR domain dimerization, which is necessary for formation of a composite active site that catalyzes NAD hydrolysis (10, 11). Recently, it was discovered that the ARM domain contains an allosteric pocket that regulates SARM1 activity through differential binding to inhibitory (NAD+) or stimulatory (nicotinamide mononucleotide (NMN)) metabolites (7, 12). In healthy neurons, where NAD+ levels are high and the ratio of NMN/NAD+ is low, the ARM domain is bound to NAD+ and SARM1 remains autoinhibited. Conversely, stress conditions that reduce NAD+ concentrations and increase the ratio of NMN/NAD+ lead to the exchange of NAD+ for NMN in the allosteric ARM pocket, which, in turn, causes a conformational change in the ARM domain that allows for TIR domain dimerization and SARM1 activation (7, 11). [0006] The genetic disruption of SARM1 has been found to be protective in various models of neurological injury, including peripheral neuropathy, traumatic brain injury, axotomy, and exposure to environmental toxins (13-20). Genetic deletion of SARM1 also prevents the axonal decline caused by loss of nicotinamide mononucleotide adenyl transferase 2 (NMNAT2) (13), which functions upstream of SARM1 through the enzymatic conversion of NMN to NAD+ (21). Notably, humans with deleterious mutations in NMNAT2 suffer from pediatric neurological disorders (22-26), and, conversely, SARM1 hypermorphic risk alleles have recently been discovered in patients with ALS (27, 28), supporting the human biology relevance of the NMNAT2-SARM1 pathway to maintaining neuronal integrity and CNS health. [0007] Considering the effectiveness of SARM1 genetic disruption in the prevention of axonal degeneration, this enzyme is considered an attractive therapeutic target for the treatment of neurodegenerative disorders. Multiple inhibitors of SARM1 have been described (29-32), most of which target the NADase domain (29-31). These include simple isoquinolines that have recently been found to serve as pseudo-substrates, being converted to NAD mimetics by SARM1 to form the active inhibitors (11, 31). Additionally, several cysteines, within both the enzymatic (TIR) and allosteric (ARM) domains of SARM1, have been identified as potential targets for electrophilic small molecules (29, 30, 32); however, the mechanisms of action of putative orthosteric (30) and allosteric (32) electrophilic inhibitors of SARM1 remain poorly understood. For instance, a nisoldipine derivative, dehydronitrosonisoldipine (dHNN), was recently shown to inhibit SARM1 and found to react with C311 in the ARM domain (32). However, mutagenesis of C311 only modestly impaired (~two-fold) the inhibitory activity of dHNN, leading to the conclusion that this compound may engage multiple cysteines in SARM1 (32). Thus, there remains a need for site-specific inhibitors of allosteric C311 of SARM1 for the amelioration of neurodegenerative disorders modulated by SARM1. SUMMARY OF THE INVENTION [0008] Herein described is the chemical proteomic discovery of a structurally distinct class of electrophilic compounds that stereoselectively and site-specifically react with a druggable cysteine, C311, in the autoregulatory ARM1 domain of SARM1. It is demonstrated that these tryptoline acrylamide ligands inhibit the NADase activity of WT- SARM1, but not C311A or C311S mutants of this enzyme, block vacor-induced cADPR production in human cells, and display high degrees of selectivity across > 23,000 quantified cysteines in the human proteome. Finally, the covalent SARM1 inhibitors prevented both vacor- and vincristine- induced axonal degeneration in rodent DRG neurons. [0009] The application provides a method of inhibiting the NADase activity of SARM1, comprising contacting SARM1 with a tryptoline acrylamide derivative. [0010] The application further provides the above method, wherein the tryptoline acrylamide derivative reacts with C311 in the ARM domain of SARM1. [0011] The application further provides either of the above methods, wherein the tryptoline acrylamide derivative reacts stereospecifically and site-specifically with C311 in the ARM domain of SARM1. [0012] The application further provides any of the above methods, wherein the tryptoline acrylamide derivative covalently binds to C311 in the ARM domain of SARM1. [0013] The application further provides any of the above methods, wherein the reaction of the tryptoline acrylamide derivative with C311 in the ARM domain of SARM1 allosterically inhibits the NADase activity of SARM1. [0014] The application further provides any of the above methods, wherein the inhibition of the NADase activity of SARM1 prevents axonal degeneration. [0015] The application further provides the above method, wherein the prevention of axonal degeneration promotes maintenance of neuronal integrity. [0016] The application further provides any of the above methods, wherein the inhibition of the NADase activity of SARM1 prevents or ameliorates a neurodegenerative disorder. [0017] The application further provides the above method, wherein the neurodegenerative disorder is ALS, Alzheimer’s Disease, or chemotherapy-induced peripheral neuropathy. [0018] The application provides a tryptoline acrylamide derivative compound of Formula I that inhibits the NADase activity of SARM1,
Figure imgf000006_0001
wherein: R is optionally substituted -O(C2-C6)alkyl, -NH(C1-C6)alkyl, -NH(C3-C6)cycloalkyl, -(C3- C6)heterocycloalkyl, or -NH(C5-C6)heteroaryl; or a pharmaceutically acceptable salt or prodrug thereof. [0019] The application further provides the above electrophilic tryptoline acrylamide derivative compound of Formula I, wherein R is -NHMe, cyclopropylamino, pyridinylamino, or propylamino. [0020] The application further provides the above electrophilic tryptoline acrylamide derivative compound of Formula I, wherein Formula I has either of the following structures:
Figure imgf000006_0002
[0021] Compounds of Formula I can inhibit the activity the NADase activity of SARM1. For example, the compounds of the invention can be used to inhibit activity or a function of SARM1 in a cell or in an individual or patient in need of inhibition of the enzyme by administering an inhibiting amount of a compound of Formula I to the cell, individual, or patient. BRIEF DESCRIPTION OF THE FIGURES [0022] Figure 1. Discovery of covalent ligands that stereoselectively and site-specifically engage C311 in SARM1. [0023] Figure 2. Stereoselective and site-specific engagement of C311 allosterically inhibits SARM1 enzymatic activity. [0024] Figure 3. SAR analysis of engagement and inhibition of SARM1 by tryptoline acrylamides. [0025] Figure 4. Inhibitory activity and proteome-wide selectivity of chemical probes targeting SARM1_C311. [0026] Figure 5. Chemical probes targeting SARM1_C311 prevent vacor- and vincristine-induced axonal degeneration. [0027] Figure 6. In vitro engagement of SARM1 by tryptoline acrylamides. [0028] Figure 7. Non-electrophilic analogs of EV-99 do not engage SARM1_C311. [0029] Figure 8. dHNN does not substantially engage SARM1_C311. [0030] Figure 9. SARM1-dependent changes in NAD and ADPR levels. [0031] Figure 10. Vacor-induced increases in cADPR content of SH-SY5Y cells is dependent on SARM1. [0032] Figure 11. The non-electrophilic compound WX-02-226 does not inhibit vacor- induced increases in cADPR in SH-SY5Y cells. [0033] Figure 12. SAR analysis of inhibition of WT-SARM1 and C311 mutants by tryptoline acrylamides. [0034] Figure 13. Characterization of the activity and inhibitor sensitivity of a SARM1- C311S mutant. [0035] Figure 14. Chemical probes targeting SARM1_C311 stereoselectively inhibit vacor-induced cADPR production in 22Rv1 cells. [0036] Figure 15. Proteome-wide reactivity of chemical probes targeting SARM1_C311. [0037] Figure 16. Proteome-wide selectivity of chemical probes targeting SARM1_C311. [0038] Figure 17. Reactivity values for cysteines in other NAD+ metabolic enzymes from cells treated with chemical probes targeting SARM1_C311. [0039] Figure 18. Mouse SARM1 is stereoselectively engaged and inhibited by chemical probes targeting SARM1_C311. [0040] Figure 19. Stereoselective inhibition of vacor-induced cADPR production in mouse Neuro-2a cells by chemical probes targeting SARM1_C311. [0041] Figure 20. Concentration-dependent inhibition of vacor-induced axonal degeneration by chemical probes targeting SARM1_C311. [0042] Figure 21. Acrylamide stereoprobes do not independently affect axonal integrity in mouse DRG neurons. [0043] Figure 22. Concentration-dependent inhibition of vincristine-induced axonal degeneration by chemical probes targeting SARM1_C311. DETAILED DESCRIPTION OF THE INVENTION [0044] The application provides the following Embodiments of the Invention: Embodiments of the Invention. [0045] Embodiment 1. The application provides a method of inhibiting the NADase activity of SARM1, comprising contacting SARM1 with a tryptoline acrylamide derivative. [0046] Embodiment 2. The application provides the method of Embodiment 1, wherein the tryptoline acrylamide derivative reacts with C311 in the ARM domain of SARM1. [0047] Embodiment 3. The application provides the method of either Embodiment 1 or Embodiment 2, wherein the tryptoline acrylamide derivative reacts stereoselectively and site- specifically with C311 in the ARM domain of SARM1. [0048] Embodiment 4. The application provides the method of any one of Embodiments 1-3, wherein the tryptoline acrylamide derivative covalently binds to C311 in the ARM domain of SARM1. [0049] Embodiment 5. The application provides the method of any one of Embodiments 2-4, wherein the reaction of the tryptoline acrylamide derivative with C311 in the ARM domain of SARM1 allosterically inhibits the NADase activity of SARM1. [0050] Embodiment 6. The application provides the method of any one of Embodiments 1-5, wherein the inhibition of the NADase activity of SARM1 prevents axonal degeneration. [0051] Embodiment 7. The application provides the method of Embodiment 6, wherein the prevention of axonal degeneration promotes maintenance of neuronal integrity. [0052] Embodiment 8. The application provides the method of any one of Embodiments 1-7, wherein the inhibition of the NADase activity of SARM1 prevents or ameliorates neurodegenerative disorders. [0053] Embodiment 9. The application provides the method of Embodiment 8, wherein the neurodegenerative disorder is selected from: spinal muscular atrophy (SMA), Chemotherapy Induced Peripheral Neuropathy, multiple sclerosis (MS), traumatic brain injury (TBI), spinal cord injury, stroke, Parkinson' disease, glaucoma, Huntington's disease, Alzheimer's disease, Charcot-Marie-Tooth disease (CMT), retinitis pigmentosa (RP), age- related macular degeneration (AMD), small fiber neuropathies, peripheral neuropathy (e.g., viral neuropathy), spinocerebellar ataxias, cystic fibrosis, familial amyloidotic polyneuropathy, spongiform encephalopathies, spinal and bulbar muscular atrophy, hereditary dentatorubral-pallidoluysian atrophy, adrenoleukodystrophy, adrenomyeloneuropathy, Alexander's disease, amyotrophic lateral sclerosis (ALS), Bassen- Kornzweig syndrome, Bell's palsy, progressive supra nuclear palsy (PSP), central pontine myelolysis, cluster headache, congenital hypomyelination, corticobasal degeneration, Creutzfeldt-Jakob disease, epilepsy, dementia (e.g., frontotemporal dementia and Lewy body dementia), demyelination disorders (e.g., ischemic demyelination), encephalomyelitis, Friedrich's ataxia, Gaucher's disease, hereditary sensory and autonomic neuropathy (HSAN), Hurler syndrome, Krabbe's disease, metachromatic leukodystrophy, migraine and tension headaches, mild cognitive impairment, motor spinoneuron disease, neuromyelitis optica, Niemann-Pick disease, optic neuritis, Pelizaeus Merzbacher disease, peripheral neuropathy, periventricular leukomalacia, post-herpetic neuralgia, prion disease, progressive supranuclear palsy, progressive multifocal leukoencephalopathy, Tay-Sacks disease, thoracic disc herniation, traverse myelitis, trigeminal neuralgia, Wallerian degeneration, cerebellar degeneration, chiari malformation, dystonia, encephalitis (e.g., pediatric viral encephalitis and La Crosse virus encephalitis), hyperekplexia, multifocal motor neuropathy, muscular dystrophy, myasthenia gravis, myopathy, neurofibromatosis, neuronal ceroid lipofuscinosis, neuropathies (e.g., peripheral neuropathy), pseudobulbar affect, restless legs syndrome, spina bifida, syringomyelia, thoracic outlet syndrome, and transverse myelitis.includes, but is not limited to, amyotrophic lateral sclerosis (ALS), multiple sclerosis, Parkinson's disease, Alzheimer's disease, Huntington's disease, multiple system atrophy, prion diseases, and chemotherapy-induced peripheral neuropathy. [0054] Embodiment 10. The application provides the method of Embodiment 9, wherein the neurodegenerative disorder is ALS, Alzheimer’s Disease, or chemotherapy-induced peripheral neuropathy. [0055] Embodiment 11. The application provides a tryptoline acrylamide derivative compound of Formula I,
Figure imgf000010_0001
wherein: R is optionally substituted -O(C2-C6)alkyl, -NH(C1-C6)alkyl, -NH(C3-C6)cycloalkyl, -(C3- C6)heterocycloalkyl, or -NH(C5-C6)heteroaryl; or a pharmaceutically acceptable salt or prodrug thereof. [0056] Embodiment 12. The application provides the tryptoline acrylamide derivative compound of Formula I of Embodiment 11, wherein R is -NHMe, cyclopropylamino, pyridinylamino, or propylamino. [0057] Embodiment 13. The application provides the tryptoline acrylamide derivative compound of Formula I of Embodiment 12, wherein Formula I has the following structure:
Figure imgf000010_0002
[0058] Embodiment 14. The application provides the tryptoline acrylamide derivative compound of Formula I of Embodiment 12, wherein Formula I has the following structure:
Figure imgf000010_0003
. [0059] Embodiment 15. The application provides the tryptoline acrylamide derivative compound of Formula I of Embodiment 12, wherein Formula I has the following structure:
Figure imgf000011_0003
wherein R is -O(C2-C6)alkyl. [0060] Embodiment 16. The application provides the tryptoline acrylamide derivative compound of Formula I of Embodiment 12, wherein Formula I has the following structure:
Figure imgf000011_0001
. [0061] Embodiment 17. The application provides the tryptoline acrylamide derivative compound of Formula I of Embodiment 12, wherein Formula I has the following structure:
Figure imgf000011_0002
. [0062] Embodiment 18. The application provides a method of inhibiting the NADase activity of SARM1, comprising contacting the SARM1 with the tryptoline acrylamide derivative compound of Formula I of any one of Embodiments 11-17. [0063] Embodiment 19. The application provides the method of Embodiment 18, wherein the tryptoline acrylamide derivative compound covalently binds C311 in the ARM domain of SARM1. [0064] Embodiment 20. The application provides the method of Embodiment 19, wherein the tryptoline acrylamide derivative compound site-specifically and covalently binds C311 of SARM1. [0065] Embodiment 21. The application provides the method of Embodiment 20, wherein the tryptoline acrylamide derivative compound site-specifically, stereoselectively, and covalently binds C311 of SARM1. [0066] Embodiment 22. The application provides the method of Embodiment 21, wherein the tryptoline acrylamide derivative compound allosterically inhibits SARM1. [0067] Embodiment 23. The application provides a method of inhibiting the NADase activity of SARM1, comprising contacting the SARM1 with the tryptoline acrylamide derivative compound of Formula I of any one of Embodiments 11-17. [0068] Embodiment 24. The application provides the method of Embodiment 23, wherein the inhibition of the NADase activity of SARM1 prevents or ameliorates a SARM1-mediated disorder. [0069] Embodiment 25. The application provides a method of treating a SARM1- mediated disorder, comprising administering to a patient in need thereof a therapeutically effective amount of the tryptoline acrylamide derivative compound of Formula I of any one of Claims 11-17. [0070] Embodiment 26. The application provides the method of Embodiment 25, wherein the SARM1-mediated disorder is a neurodegenerative disorder. [0071] Embodiment 27. The application provides the method of Embodiment 26, wherein the neurodegenerative disorder is selected from: spinal muscular atrophy (SMA), Chemotherapy Induced Peripheral Neuropathy, multiple sclerosis (MS), traumatic brain injury (TBI), spinal cord injury, stroke, Parkinson' disease, glaucoma, Huntington's disease, Alzheimer's disease, Charcot-Marie-Tooth disease (CMT), retinitis pigmentosa (RP), age- related macular degeneration (AMD), small fiber neuropathies, peripheral neuropathy (e.g., viral neuropathy), spinocerebellar ataxias, cystic fibrosis, familial amyloidotic polyneuropathy, spongiform encephalopathies, spinal and bulbar muscular atrophy, hereditary dentatorubral-pallidoluysian atrophy, adrenoleukodystrophy, adrenomyeloneuropathy, Alexander's disease, amyotrophic lateral sclerosis (ALS), Bassen- Kornzweig syndrome, Bell's palsy, progressive supra nuclear palsy (PSP), central pontine myelolysis, cluster headache, congenital hypomyelination, corticobasal degeneration, Creutzfeldt-Jakob disease, epilepsy, dementia (e.g., frontotemporal dementia and Lewy body dementia), demyelination disorders (e.g., ischemic demyelination), encephalomyelitis, Friedrich's ataxia, Gaucher's disease, hereditary sensory and autonomic neuropathy (HSAN), Hurler syndrome, Krabbe's disease, metachromatic leukodystrophy, migraine and tension headaches, mild cognitive impairment, motor spinoneuron disease, neuromyelitis optica, Niemann-Pick disease, optic neuritis, Pelizaeus Merzbacher disease, peripheral neuropathy, periventricular leukomalacia, post-herpetic neuralgia, prion disease, progressive supranuclear palsy, progressive multifocal leukoencephalopathy, Tay-Sacks disease, thoracic disc herniation, traverse myelitis, trigeminal neuralgia, Wallerian degeneration, cerebellar degeneration, chiari malformation, dystonia, encephalitis (e.g., pediatric viral encephalitis and La Crosse virus encephalitis), hyperekplexia, multifocal motor neuropathy, muscular dystrophy, myasthenia gravis, myopathy, neurofibromatosis, neuronal ceroid lipofuscinosis, neuropathies (e.g., peripheral neuropathy), pseudobulbar affect, restless legs syndrome, spina bifida, syringomyelia, thoracic outlet syndrome, and transverse myelitis.includes, but is not limited to, amyotrophic lateral sclerosis (ALS), multiple sclerosis, Parkinson's disease, Alzheimer's disease, Huntington's disease, multiple system atrophy, prion diseases, and chemotherapy-induced peripheral neuropathy. [0072] Embodiment 28. The application provides the method of Embodiment 27, wherein the neurodegenerative disorder is ALS, Alzheimer’s Disease, or chemotherapy-induced peripheral neuropathy. [0073] Embodiment 29. The application provides a composition comprising the compound of Formula I of any one of Embodiments 11-17, admixed with at least one carrier, diluent or excipient. [0074] Embodiment 30. The application provides the composition of Embodiment 29, further comprising another pharmaceutically active compound. [0075] Embodiment 31. The application provides the composition of either Embodiment 29 or Embodiment 30, further comprising another anti-neurodegenerative compound. [0076] Embodiment 32. The application provides the composition of any one of Embodiments 29-31, further comprising another SARM1-inhibiting compound. [0077] Embodiment 33. The application provides a composition comprising the compound of Formula I of any one of Embodiments 13-15, admixed with at least one carrier, diluent or excipient. [0078] Embodiment 34. The application provides the composition of Embodiment 32, further comprising another pharmaceutically active compound. [0079] Embodiment 35. The application provides the composition of either Embodiment 33 or Embodiment 34, further comprising another anti-neurodegenerative compound. [0080] Embodiment 36. The application provides the composition of any one of Embodiments 33-35, further comprising another SARM1-inhibiting compound. [0081] Embodiment 37. The application provides any methods of inhibiting the NADase activity of SARM1, electrophilic tryptoline acrylamide derivative compounds, methods of treating SARM1-mediated disorders, or compositions comprising the tryptoline acrylamide derivative compound of Formula I, as described herein. Definitions [0082] As referred to herein, unless otherwise specified, for instance in the Examples or Figures herein disclosed, “SARM1” is human wild-type (WT) SARM1 (Accession No. NP_055892). [0083] The phrase “a” or “an” entity as used herein refers to one or more of that entity; for example, a compound refers to one or more compounds or at least one compound. As such, the terms “a” (or “an”), “one or more”, and “at least one” can be used interchangeably herein. [0084] The phrase "as defined herein above" refers to the broadest definition for each group as provided in the Summary of the Invention, the Detailed Description of the Invention, the Experimentals, or the broadest claim. In all other embodiments provided below, substituents which can be present in each embodiment and which are not explicitly defined retain the broadest definition provided in the Summary of the Invention. [0085] As used in this specification, whether in a transitional phrase or in the body of the claim, the terms "comprise(s)" and "comprising" are to be interpreted as having an open-ended meaning. That is, the terms are to be interpreted synonymously with the phrases "having at least" or "including at least". When used in the context of a process, the term "comprising" means that the process includes at least the recited steps, but may include additional steps. When used in the context of a compound or composition, the term "comprising" means that the compound or composition includes at least the recited features or components, but may also include additional features or components. [0086] As used herein, unless specifically indicated otherwise, the word "or" is used in the "inclusive" sense of "and/or" and not the "exclusive" sense of "either/or". [0087] The term "independently" is used herein to indicate that a variable is applied in any one instance without regard to the presence or absence of a variable having that same or a different definition within the same compound. Thus, in a compound in which “R” appears twice and is defined as "independently selected from” means that each instance of that R group is separately identified as one member of the set which follows in the definition of that R group. For example, “each R1 and R2 is independently selected from carbon and nitrogen" means that both R1 and R2 can be carbon, both R1 and R2 can be nitrogen, or R1 or R2 can be carbon and the other nitrogen or vice versa. [0088] When any variable occurs more than one time in any moiety or formula depicting and describing compounds employed or claimed in the present invention, its definition on each occurrence is independent of its definition at every other occurrence. Also, combinations of substituents and/or variables are permissible only if such compounds result in stable compounds. [0089] The symbols "*" at the end of a bond or a line drawn through a bond or “~~~~” drawn through a bond each refer to the point of attachment of a functional group or other chemical moiety to the rest of the molecule of which it is a part. [0090] A bond drawn into ring system (as opposed to connected at a distinct vertex) indicates that the bond may be attached to any of the suitable ring atoms. The term “optional” or “optionally” as used herein means that a subsequently described event or circumstance may, but need not, occur, and that the description includes instances where the event or circumstance occurs and instances in which it does not. For example, “optionally substituted” means that the “optionally substituted” moiety may incorporate a hydrogen or a substituent. [0091] The phrase “optional bond” means that the bond may or may not be present, and that the description includes single, double, or triple bonds. If a substituent is designated to be a "bond" or "absent", the atoms linked to the substituents are then directly connected. [0092] The term "about" is used herein to mean approximately, in the region of, roughly, or around. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term "about" is used herein to modify a numerical value above and below the stated value by a variance of 20%. [0093] Certain compounds of Formula I may exhibit tautomerism. Tautomeric compounds can exist as two or more interconvertable species. Prototropic tautomers result from the migration of a covalently bonded hydrogen atom between two atoms. Tautomers generally exist in equilibrium and attempts to isolate individual tautomers usually produce a mixture whose chemical and physical properties are consistent with a mixture of compounds. The position of the equilibrium is dependent on chemical features within the molecule. For example, in many aliphatic aldehydes and ketones, such as acetaldehyde, the keto form predominates while; in phenols, the enol form predominates. Common prototropic tautomers include keto/enol amide/imidic acid
Figure imgf000015_0001
Figure imgf000016_0001
and amidine
Figure imgf000016_0002
tautomers. The latter two are particularly common in heteroaryl and heterocyclic rings and the present invention encompasses all tautomeric forms of the compounds. [0094] Technical and scientific terms used herein have the meaning commonly understood by one of skill in the art to which the present invention pertains, unless otherwise defined. Reference is made herein to various methodologies and materials known to those of skill in the art. Standard reference works setting forth the general principles of pharmacology include Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10th Ed., McGraw Hill Companies Inc., New York (2001). Any suitable materials and/or methods known to those of skill can be utilized in carrying out the present invention. However, preferred materials and methods are described. Materials, reagents and the like to which reference are made in the following description and examples are obtainable from commercial sources, unless otherwise noted. [0095] The definitions described herein may be appended to form chemically-relevant combinations, such as “heteroalkylaryl,” “haloalkylheteroaryl,” “arylalkylheterocyclyl,” “alkylcarbonyl,” “alkoxyalkyl,” and the like. When the term “alkyl” is used as a suffix following another term, as in “phenylalkyl,” or “hydroxyalkyl,” this is intended to refer to an alkyl group, as defined above, being substituted with one to two substituents selected from the other specifically-named group. Thus, for example, “phenylalkyl” refers to an alkyl group having one to two phenyl substituents, and thus includes benzyl, phenylethyl, and biphenyl. An “alkylaminoalkyl” is an alkyl group having one to two alkylamino substituents. “Hydroxyalkyl" includes 2-hydroxyethyl, 2-hydroxypropyl, 1- (hydroxymethyl)-2-methylpropyl, 2-hydroxybutyl, 2,3-dihydroxybutyl, 2-(hydroxymethyl), 3-hydroxypropyl, and so forth. Accordingly, as used herein, the term “hydroxyalkyl” is used to define a subset of heteroalkyl groups defined below. The term -(ar)alkyl refers to either an unsubstituted alkyl or an aralkyl group. The term (hetero)aryl or (het)aryl refers to either an aryl or a heteroaryl group. [0096] The term “acyl” as used herein denotes a group of formula -C(=O)R wherein R is hydrogen or lower alkyl as defined herein. The term or "alkylcarbonyl" as used herein denotes a group of formula C(=O)R wherein R is alkyl as defined herein. The term C1-6 acyl refers to a group -C(=O)R contain 6 carbon atoms. The term "arylcarbonyl" as used herein means a group of formula C(=O)R wherein R is an aryl group; the term "benzoyl" as used herein an "arylcarbonyl" group wherein R is phenyl. [0097] The term “alkyl” as used herein denotes an unbranched or branched chain, saturated, monovalent hydrocarbon residue containing 1 to 12 carbon atoms. The term “lower alkyl” or “C1-C6 alkyl” as used herein denotes a straight or branched chain hydrocarbon residue containing 1 to 6 carbon atoms. "C1–12 alkyl" as used herein refers to an alkyl composed of 1 to 12 carbons. Examples of alkyl groups include, but are not limited to, lower alkyl groups include methyl, ethyl, propyl, i-propyl, n-butyl, i-butyl, t- butyl or pentyl, isopentyl, neopentyl, hexyl, heptyl, and octyl. [0098] When the term “alkyl” is used as a suffix following another term, as in “phenylalkyl,” or “hydroxyalkyl,” this is intended to refer to an alkyl group, as defined above, being substituted with one to two substituents selected from the other specifically- named group. Thus, for example, “phenylalkyl” denotes the radical R'R"-, wherein R' is a phenyl radical, and R" is an alkylene radical as defined herein with the understanding that the attachment point of the phenylalkyl moiety will be on the alkylene radical. Examples of arylalkyl radicals include, but are not limited to, benzyl, phenylethyl, 3-phenylpropyl. The terms “arylalkyl” or "aralkyl" are interpreted similarly except R' is an aryl radical. The terms "(het)arylalkyl" or "(het)aralkyl" are interpreted similarly except R' is optionally an aryl or a heteroaryl radical. [0099] 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. [0100] “Alkyl” refers to a radical of a straight–chain or branched saturated hydrocarbon group having from 1 to 20 carbon atoms (“C1–20 alkyl”). In some embodiments, an alkyl group has 1 to 15 carbon atoms (“C1–15 alkyl”). In some embodiments, an alkyl group has 1 to 14 carbon atoms (“C1–14 alkyl”). In some embodiments, an alkyl group has 1 to 13 carbon atoms (“C1–13 alkyl”). In some embodiments, an alkyl group has 1 to 12 carbon atoms (“C1–12 alkyl”). In some embodiments, an alkyl group has 1 to 11 carbon atoms (“C1–11 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”). In some embodiments, an alkyl group has 2 to 6 carbon atoms (“C2–6 alkyl”). Examples of C1–6 alkyl groups include methyl (C1), ethyl (C2), n–propyl (C3), isopropyl (C3), n–butyl (C4), tert–butyl (C4), sec– butyl (C4), iso–butyl (C4), n–pentyl (C5), 3–pentanyl (C5), amyl (C5), neopentyl (C5), 3– methyl–2–butanyl (C5), tertiary amyl (C5), and n–hexyl (C6). Additional examples of alkyl groups include n–heptyl (C7), n–octyl (C8) and the like. [0101] “Alkenyl” or “olefin” refers to a radical of a straight–chain or branched hydrocarbon group having from 2 to 10 carbon atoms and 1, 2, 3, or 4 carbon-carbon double bonds (“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 C2–4 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. [0102] “Alkynyl” refers to a radical of a straight–chain or branched hydrocarbon group having from 2 to 10 carbon atoms and one or more carbon-carbon triple bonds (e.g., 1, 2, 3, or 4 triple bonds) (“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 C2–4 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 C2–4 alkynyl groups as well as pentynyl (C5), hexynyl (C6), and the like. Additional examples of alkynyl include heptynyl (C7), octynyl (C8), and the like. [0103] The terms “haloalkyl” or “halo-lower alkyl” or “lower haloalkyl” refers to a straight or branched chain hydrocarbon residue containing 1 to 6 carbon atoms wherein one or more carbon atoms are substituted with one or more halogen atoms. [0104] The term "alkylene" or "alkylenyl" as used herein denotes a divalent saturated linear hydrocarbon radical of 1 to 10 carbon atoms (e.g., (CH2)n)or a branched saturated divalent hydrocarbon radical of 2 to 10 carbon atoms (e.g., -CHMe- or -CH2CH(i-Pr)CH2-), unless otherwise indicated. Except in the case of methylene, the open valences of an alkylene group are not attached to the same atom. Examples of alkylene radicals include, but are not limited to, methylene, ethylene, propylene, 2-methyl-propylene, 1,1-dimethyl- ethylene, butylene, 2-ethylbutylene. [0105] The term "alkoxy" as used herein means an -O-alkyl group, wherein alkyl is as defined above such as methoxy, ethoxy, n-propyloxy, i-propyloxy, n-butyloxy, i-butyloxy, t-butyloxy, pentyloxy, hexyloxy, including their isomers. "Lower alkoxy" as used herein denotes an alkoxy group with a "lower alkyl" group as previously defined. "C1-10 alkoxy" as used herein refers to an-O-alkyl wherein alkyl is C1-10. [0106] The term "hydroxyalkyl" as used herein denotes an alkyl radical as herein defined wherein one to three hydrogen atoms on different carbon atoms is/are replaced by hydroxyl groups. [0107] The terms "alkylsulfonyl" and "arylsulfonyl" as used herein refers to a group of formula -S(=O)2R wherein R is alkyl or aryl respectively and alkyl and aryl are as defined herein. The term “heteroalkylsulfonyl” as used herein refers herein denotes a group of formula -S(=O)2R wherein R is “heteroalkyl” as defined herein. [0108] The terms "alkylsulfonylamino" and "arylsulfonylamino"as used herein refers to a group of formula -NR'S(=O)2R wherein R is alkyl or aryl respectively, R' is hydrogen or C1-3 alkyl, and alkyl and aryl are as defined herein. [0109] The term “cycloalkyl” as used herein refers to a saturated carbocyclic ring containing 3 to 8 carbon atoms, i.e. cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl or cyclooctyl. "C3-7 cycloalkyl" as used herein refers to an cycloalkyl composed of 3 to 7 carbons in the carbocyclic ring. [0110] The term carboxy-alkyl as used herein refers to an alkyl moiety wherein one, hydrogen atom has been replaced with a carboxyl with the understanding that the point of attachment of the heteroalkyl radical is through a carbon atom. The term “carboxy” or “carboxyl” refers to a –CO2H moiety. [0111] The term "heteroaryl” or "heteroaromatic" as used herein means a monocyclic or bicyclic radical of 5 to 12 ring atoms having at least one aromatic ring containing four to eight atoms per ring, incorporating one or more N, O, or S heteroatoms, the remaining ring atoms being carbon, with the understanding that the attachment point of the heteroaryl radical will be on an aromatic ring. As well known to those skilled in the art, heteroaryl rings have less aromatic character than their all-carbon counter parts. Thus, for the purposes of the invention, a heteroaryl group need only have some degree of aromatic character. Examples of heteroaryl moieties include monocyclic aromatic heterocycles having 5 to 6 ring atoms and 1 to 3 heteroatoms include, but is not limited to, pyridinyl, pyrimidinyl, pyrazinyl, pyrrolyl, pyrazolyl, imidazolyl, oxazol, isoxazole, thiazole, isothiazole, triazoline, thiadiazole and oxadiaxoline which can optionally be substituted with one or more, preferably one or two substituents selected from hydroxy, cyano, alkyl, alkoxy, thio, lower haloalkoxy, alkylthio, halo, lower haloalkyl, alkylsulfinyl, alkylsulfonyl, halogen, amino, alkylamino,dialkylamino, aminoalkyl, alkylaminoalkyl, and dialkylaminoalkyl, nitro, alkoxycarbonyl and carbamoyl, alkylcarbamoyl, dialkylcarbamoyl, arylcarbamoyl, alkylcarbonylamino and arylcarbonylamino. Examples of bicyclic moieties include, but are not limited to, quinolinyl, isoquinolinyl, benzofuryl, benzothiophenyl, benzoxazole, benzisoxazole, benzothiazole and benzisothiazole. Bicyclic moieties can be optionally substituted on either ring; however the point of attachment is on a ring containing a heteroatom. [0112] The term "heterocyclyl", “heterocycloalkyl” or "heterocycle" as used herein denotes a monovalent saturated cyclic radical, consisting of one or more rings, preferably one to two rings, including spirocyclic ring systems, of three to eight atoms per ring, incorporating one or more ring heteroatoms (chosen from N,O or S(O)0-2), and which can optionally be independently substituted with one or more, preferably one or two substituents selected from hydroxy, oxo, cyano, lower alkyl, lower alkoxy, lower haloalkoxy, alkylthio, halo, lower haloalkyl, hydroxyalkyl, nitro, alkoxycarbonyl, amino, alkylamino, alkylsulfonyl, arylsulfonyl, alkylaminosulfonyl, arylaminosulfonyl, alkylsulfonylamino, arylsulfonylamino, alkylaminocarbonyl, arylaminocarbonyl, alkylcarbonylamino, arylcarbonylamino, unless otherwise indicated. Examples of heterocyclic radicals include, but are not limited to, azetidinyl, pyrrolidinyl, hexahydroazepinyl, oxetanyl, tetrahydrofuranyl, tetrahydrothiophenyl, oxazolidinyl, thiazolidinyl, isoxazolidinyl, morpholinyl, piperazinyl, piperidinyl, tetrahydropyranyl, thiomorpholinyl, quinuclidinyl and imidazolinyl. [0113] “Heterocyclyl” or “heterocyclic” refers to a group or radical of a 3– to 14– membered non–aromatic ring system having ring carbon atoms and 1 to 4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“3– 14 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 polycyclic (e.g., a fused, bridged or spiro ring system such as a bicyclic system (“bicyclic heterocyclyl”) or tricyclic system (“tricyclic heterocyclyl”)), and can be saturated or can contain one or more carbon–carbon double or triple bonds. Heterocyclyl polycyclic 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. [0114] 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, and sulfur (“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 1 ring heteroatom selected from nitrogen, oxygen, and sulfur. [0115] Exemplary 3–membered heterocyclyl groups containing 1 heteroatom include, without limitation, azirdinyl, oxiranyl, and thiiranyl. Exemplary 4–membered heterocyclyl groups containing 1 heteroatom include, without limitation, azetidinyl, oxetanyl and thietanyl. Exemplary 5–membered heterocyclyl groups containing 1 heteroatom include, without limitation, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothiophenyl, dihydrothiophenyl, pyrrolidinyl, dihydropyrrolyl, and pyrrolyl–2,5–dione. Exemplary 5– membered heterocyclyl groups containing 2 heteroatoms include, without limitation, dioxolanyl, oxathiolanyl and dithiolanyl. Exemplary 5–membered heterocyclyl groups containing 3 heteroatoms include, without limitation, triazolinyl, oxadiazolinyl, and thiadiazolinyl. Exemplary 6–membered heterocyclyl groups containing 1 heteroatom include, without limitation, piperidinyl, tetrahydropyranyl, dihydropyridinyl, and thianyl. Exemplary 6–membered heterocyclyl groups containing 2 heteroatoms include, without limitation, piperazinyl, morpholinyl, dithianyl, and dioxanyl. Exemplary 6–membered heterocyclyl groups containing 3 heteroatoms include, without limitation, triazinanyl. Exemplary 7–membered heterocyclyl groups containing 1 heteroatom include, without limitation, azepanyl, oxepanyl and thiepanyl. Exemplary 8–membered heterocyclyl groups containing 1 heteroatom include, without limitation, azocanyl, oxecanyl and thiocanyl. Exemplary bicyclic heterocyclyl groups include, without limitation, indolinyl, isoindolinyl, dihydrobenzofuranyl, dihydrobenzothienyl, tetrahydrobenzothienyl, tetrahydrobenzofuranyl, tetrahydroindolyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, decahydroisoquinolinyl, octahydrochromenyl, octahydroisochromenyl, decahydronaphthyridinyl, decahydro–1,8–naphthyridinyl, octahydropyrrolo[3,2–b]pyrrole, indolinyl, phthalimidyl, naphthalimidyl, chromanyl, chromenyl, 1H–benzo[e][1,4]diazepinyl, 1,4,5,7–tetrahydropyrano[3,4–b]pyrrolyl, 5,6– dihydro–4H–furo[3,2–b]pyrrolyl, 6,7–dihydro–5H–furo[3,2–b]pyranyl, 5,7–dihydro–4H– thieno[2,3–c]pyranyl, 2,3–dihydro–1H–pyrrolo[2,3–b]pyridinyl, 2,3–dihydrofuro[2,3– b]pyridinyl, 4,5,6,7–tetrahydro–1H–pyrrolo[2,3–b]pyridinyl, 4,5,6,7–tetrahydrofuro[3,2– c]pyridinyl, 4,5,6,7–tetrahydrothieno[3,2–b]pyridinyl, 1,2,3,4–tetrahydro–1,6– naphthyridinyl, and the like. [0116] “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 pi 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 6 ring carbon atoms (“C6 aryl”; e.g., phenyl). In some embodiments, an aryl group has 10 ring carbon atoms (“C10 aryl”; e.g., naphthyl such as 1–naphthyl (α-naphthyl) and 2–naphthyl (β-naphthyl)). In some embodiments, an aryl group has 14 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. [0117] “Heteroaryl” refers to a radical of a 5–14 membered monocyclic or polycyclic (e.g., bicyclic, tricyclic) 4n+2 aromatic ring system (e.g., having 6, 10, or 14 pi 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 (“5–14 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 polycyclic 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 polycyclic (aryl/heteroaryl) ring system. Polycyclic 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). [0118] 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. [0119] Exemplary 5–membered heteroaryl groups containing 1 heteroatom include, without limitation, pyrrolyl, furanyl and thiophenyl. Exemplary 5–membered heteroaryl groups containing 2 heteroatoms include, without limitation, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, and isothiazolyl. Exemplary 5–membered heteroaryl groups containing 3 heteroatoms include, without limitation, triazolyl, oxadiazolyl, and thiadiazolyl. Exemplary 5–membered heteroaryl groups containing 4 heteroatoms include, without limitation, tetrazolyl. Exemplary 6–membered heteroaryl groups containing 1 heteroatom include, without limitation, pyridinyl. Exemplary 6–membered heteroaryl groups containing 2 heteroatoms include, without limitation, pyridazinyl, pyrimidinyl, and pyrazinyl. Exemplary 6–membered heteroaryl groups containing 3 or 4 heteroatoms include, without limitation, triazinyl and tetrazinyl, respectively. Exemplary 7–membered heteroaryl groups containing 1 heteroatom include, without limitation, azepinyl, oxepinyl, and thiepinyl. Exemplary 5,6–bicyclic heteroaryl groups include, without limitation, indolyl, isoindolyl, indazolyl, benzotriazolyl, benzothiophenyl, isobenzothiophenyl, benzofuranyl, benzoisofuranyl, benzimidazolyl, benzoxazolyl, benzisoxazolyl, benzoxadiazolyl, benzthiazolyl, benzisothiazolyl, benzthiadiazolyl, indolizinyl, and purinyl. Exemplary 6,6–bicyclic heteroaryl groups include, without limitation, naphthyridinyl, pteridinyl, quinolinyl, isoquinolinyl, cinnolinyl, quinoxalinyl, phthalazinyl, and quinazolinyl. Exemplary tricyclic heteroaryl groups include, without limitation, phenanthridinyl, dibenzofuranyl, carbazolyl, acridinyl, phenothiazinyl, phenoxazinyl and phenazinyl. [0120] “Saturated” refers to a ring moiety that does not contain a double or triple bond, i.e., the ring contains all single bonds. [0121] Alkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl groups may be optionally substituted. Optionally substituted refers to a group which may be substituted or unsubstituted. In general, the term “substituted” means that at least one hydrogen present on a group is replaced with a non-hydrogen substituent, and 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. Heteroatoms such as nitrogen, oxygen, and sulfur may have hydrogen substituents and/or non-hydrogen substituents which satisfy the valencies of the heteroatoms and results in the formation of a stable compound. [0122] Exemplary non-hydrogen substituents wherein a moiety is “optionally substituted” as used herein means the moiety may be substituted with any additional moiety selected from, but not limited to, the group consisting of halogen, –CN, –NO2, –N3, –SO2H, –SO3H, –OH, –ORaa, –N(Rbb)2, –N(ORcc)Rbb, –SH, –SRaa, –C(=O)Raa, –CO2H, –CHO, – CO2Raa, –OC(=O)Raa, –OCO2Raa, –C(=O)N(Rbb)2, –OC(=O)N(Rbb)2, –NRbbC(=O)Raa, – NRbbCO2Raa, –NRbbC(=O)N(Rbb)2, –C(=NRbb)Raa, –C(=NRbb)ORaa, –OC(=NRbb)Raa, – OC(=NRbb)ORaa, –C(=NRbb)N(Rbb)2, –OC(=NRbb)N(Rbb)2, –NRbbC(=NRbb)N(Rbb)2, – C(=O)NRbbSO2Raa, –NRbbSO2Raa, –SO2N(Rbb)2, –SO2Raa, –S(=O)Raa, –OS(=O)Raa, - B(ORcc)2, C1–10 alkyl, C2–10 alkenyl, C2–10 alkynyl, C3–14 carbocyclyl, 3– to 14- membered heterocyclyl, C6–14 aryl, and 5– to 14- membered heteroaryl, wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 Rdd groups, or two geminal hydrogens on a carbon atom are replaced with the group =O; each instance of Raa is, independently, selected from the group consisting of C1–10 alkyl, C1–10 perhaloalkyl, C2–10 alkenyl, C2–10 alkynyl, C3–14 carbocyclyl, 3– to 14- membered heterocyclyl, C6–14 aryl, and 5– to 14- membered heteroaryl, or two Raa groups are joined to form a 3– to 14- membered heterocyclyl or 5– to 14- membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 Rdd groups; each instance of Rbb is, independently, selected from the group consisting of hydrogen, –OH, –ORaa, –N(Rcc)2, – CN, –C(=O)Raa, –C(=O)N(Rcc)2, –CO2Raa, –SO2Raa, –SO2N(Rcc)2, –SORaa, C1–10 alkyl, C1– 10 perhaloalkyl, C2–10 alkenyl, C2–10 alkynyl, C3–14 carbocyclyl, 3– to 14- membered heterocyclyl, C6–14 aryl, and 5– to 14- membered heteroaryl, or two Rbb groups are joined to form a 3– to 14- membered heterocyclyl or 5– to 14- membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 Rdd groups; each instance of Rcc is, independently, selected from the group consisting of hydrogen, C1–10 alkyl, C1–10 perhaloalkyl, C2–10 alkenyl, C2–10 alkynyl, C3–14 carbocyclyl, 3– to 14- membered heterocyclyl, C6–14 aryl, and 5– to 14- membered heteroaryl, or two Rcc groups are joined to form a 3– to 14- membered heterocyclyl or 5– to 14- membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 Rdd groups; and each instance of Rdd is, independently, selected from the group consisting of halogen, –CN, –NO2, –N3, –SO2H, –SO3H, –OH, –OC1–6 alkyl, –ON(C1–6 alkyl)2, –N(C1–6 alkyl)2, –N(OC1–6 alkyl)(C1–6 alkyl), –N(OH)(C1–6 alkyl), –NH(OH), –SH, –SC1–6 alkyl, –C(=O)(C1–6 alkyl), –CO2H, –CO2(C1–6 alkyl), –OC(=O)(C1–6 alkyl), – OCO2(C1–6 alkyl), –C(=O)NH2, –C(=O)N(C1–6 alkyl)2, –OC(=O)NH(C1–6 alkyl), – NHC(=O)( C1–6 alkyl), –N(C1–6 alkyl)C(=O)( C1–6 alkyl), –NHCO2(C1–6 alkyl), – NHC(=O)N(C1–6 alkyl)2, –NHC(=O)NH(C1–6 alkyl), –NHC(=O)NH2, –C(=NH)O(C1–6 alkyl),–OC(=NH)(C1–6 alkyl), –OC(=NH)OC1–6 alkyl, –C(=NH)N(C1–6 alkyl)2, – C(=NH)NH(C1–6 alkyl), –C(=NH)NH2, –OC(=NH)N(C1–6 alkyl)2, –OC(NH)NH(C1–6 alkyl), –OC(NH)NH2, –NHC(NH)N(C1–6 alkyl)2, –NHC(=NH)NH2, –NHSO2(C1–6 alkyl), – SO2N(C1–6 alkyl)2, –SO2NH(C1–6 alkyl), –SO2NH2,–SO2C1–6 alkyl, -B(OH)2, -B(OC1–6 alkyl)2,C1–6 alkyl, C1–6 perhaloalkyl, C2–6 alkenyl, C2–6 alkynyl, C3–10 carbocyclyl, C6–10 aryl, 3–to 10- membered heterocyclyl, and 5- to 10- membered heteroaryl; or two geminal Rdd substituents on a carbon atom may be joined to form =O. [0123] “Halo” or “halogen” refers to fluorine (fluoro, –F), chlorine (chloro, –Cl), bromine (bromo, –Br), or iodine (iodo, –I). [0124] As used herein, the term “composition” is intended to encompass a product comprising the specified ingredients, as well as any product which results, directly or indirectly, from combination of the specified ingredients. [0125] “Salt” includes any and all salts. “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. Pharmaceutically acceptable salts are well known in the art. For example, Berge et al., describes pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences (1977) 66:1–19. Pharmaceutically acceptable salts include those derived from 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, dodecylsulfate, 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. [0126] The term "prodrug" refers to compounds that are transformed in vivo to yield a disclosed compound or a pharmaceutically acceptable salt, hydrate or solvate of the compound. The transformation may occur by various mechanisms (such as by esterase, amidase, phosphatase, oxidative and or reductive metabolism) in various locations (such as in the intestinal lumen or upon transit of the intestine, blood or liver). Prodrugs are well known in the art (for example, see Rautio, Kumpulainen, et al., Nature Reviews Drug Discovery 2008, 7, 255). For example, if a compound of the invention or a pharmaceutically acceptable salt, hydrate or solvate of the compound contains a carboxylic acid functional group, a prodrug can comprise an ester formed by the replacement of the hydrogen atom of the acid group with a group such as (C1-8)alkyl, (C2- 12)alkylcarbonyloxymethyl, 1-(alkylcarbonyloxy)ethyl having from 4 to 9 carbon atoms, 1- methyl-1-(alkylcarbonyloxy)-ethyl having from 5 to 10 carbon atoms, alkoxycarbonyloxymethyl having from 3 to 6 carbon atoms, 1-(alkoxycarbonyloxy)ethyl having from 4 to 7 carbon atoms, 1-methyl-1-(alkoxycarbonyloxy)ethyl having from 5 to 8 carbon atoms, N-(alkoxycarbonyl)aminomethyl having from 3 to 9 carbon atoms, 1-(N- (alkoxycarbonyl)amino)ethyl having from 4 to 10 carbon atoms, 3-phthalidyl, 4- crotonolactonyl, gamma-butyrolacton-4-yl, di-N,N-(C1-2)alkylamino(C2-3)alkyl (such as β- dimethylaminoethyl), carbamoyl-(C1-2)alkyl, N,N-di(C1-2)alkylcarbamoyl-(C1-2)alkyl and piperidino-, pyrrolidino- or morpholino(C2-3)alkyl. [0127] Similarly, if a compound contains an alcohol functional group, a prodrug can be formed by the replacement of the hydrogen atom of the alcohol group with a group such as (C1-6)alkylcarbonyloxymethyl, 1-((C1-6)alkylcarbonyloxy)ethyl, 1-methyl-1-((C1- 6)alkylcarbonyloxy)ethyl (C1-6)alkoxycarbonyloxymethyl, N-(C1- 6)alkoxycarbonylaminomethyl, succinoyl, (C1-6)alkylcarbonyl, .alpha.-amino(C1- 4)alkylcarbonyl, arylalkylcarbonyl and α-aminoalkylcarbonyl, or .alpha.- aminoalkylcarbonyl- α -aminoalkylcarbonyl, where each -aminoalkylcarbonyl group is independently selected from the naturally occurring L-amino acids, -P(O)(OH)2, - P(O)(O(C1-6)alkyl)2 or glycosyl (the radical resulting from the removal of a hydroxyl group of the hemiacetal form of a carbohydrate). [0128] Unless otherwise indicated, compounds described herein can comprise one or more asymmetric centers, and thus can exist in various stereoisomeric 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). Compounds described herein can be in the form of individual isomers substantially free of other isomers, and alternatively, as mixtures of various isomers. [0129] Unless otherwise stated, structures depicted herein are also meant to include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, compounds having the present structures except for the replacement of hydrogen by deuterium or tritium, replacement of 19F with 18F, replacement of a carbon by a 13C- or 14C-enriched carbon, and/or replacement of an oxygen atom with 18O, are within the scope of the disclosure. Other examples of isotopes include 15N, 18O, 17O, 31P, 32P, 35S, 18F, 36Cl and 123I. Compounds with such isotopically enriched atoms are useful, for example, as analytical tools or probes in biological assays. [0130] Certain isotopically-labelled compounds (e.g., those labeled with 3H and 14C) are useful in compound and/or substrate tissue distribution assays. Tritiated (i.e., 3H) and carbon-14 (i.e., 14C) isotopes are particularly preferred for their ease of preparation and detectability. [0131] Certain isotopically-labelled compounds of Formula (I) can be useful for medical imaging purposes, for example, those labeled with positron-emitting isotopes like 11C or 18F can be useful for application in Positron Emission Tomography (PET) and those labeled with gamma ray emitting isotopes like 123I can be useful for application in Single Photon Emission Computed Tomography (SPECT). Further, substitution with heavier isotopes such as deuterium (i.e., 2H) may afford certain therapeutic advantages resulting from greater metabolic stability (e.g., increased in vivo half-life or reduced dosage requirements) and hence may be preferred in some circumstances. Further, substitution with heavier isotopes such as deuterium (i.e., 2H) may afford certain therapeutic advantages resulting from greater metabolic stability (e.g., increased in vivo half-life or reduced dosage requirements), and hence, may be preferred in some circumstances. Additionally, isotopic substitution at a site where epimerization occurs may slow or reduce the epimerization process and thereby retain the more active or efficacious form of the compound for a longer period of time. Isotopically labeled compounds of Formula (I), in particular those containing isotopes with longer half-lives (t1/2 >1 day), can generally be prepared by following procedures analogous to those disclosed in the Schemes and/or in the Examples herein below, by substituting an appropriate isotopically labeled reagent for a non- isotopically labeled reagent. Methods of Use [0132] As referred to herein, unless otherwise specified, for instance in the Examples or Figures herein disclosed, “SARM1” is human wild-type (WT) SARM1 (Accession No. NP_055892). [0133] Compounds of the invention can inhibit the NADase activity of SARM1. For example, the compounds of the invention can be used to inhibit activity or a function of SARM1 in a cell or in an individual or patient in need of inhibition of the enzyme by administering an inhibiting amount of a compound of Formula I to the cell, individual, or patient. As used herein, the term "in a cell" includes both inside the cell membrane and on the surface of the cell membrane. [0134] Compounds of the invention, as SARM1 inhibitors, can increase levels of NAD+ in a cell. Accordingly, the present invention is further directed to a method of increasing the level of NAD+ in a sample or in a patient, comprising contacting the sample or administering to the patient a compound of of the invention, or a pharmaceutically acceptable salt thereof, wherein the increased level of NAD+ is relative to the level of NAD+ prior to the contacting or administering. [0135] Compounds of the invention, as SARM1 inhibitors, can inhibit axonal degeneration. Accordingly, the present invention is further directed to a method of inhibiting axonal degeneration in a sample or in a patient, comprising contacting the sample or administering to the patient an inhibiting amount of a compound of the invention, or a pharmaceutically acceptable salt thereof. [0136] The compounds of the invention are useful in the treatment and prevention of various diseases associated with abnormal expression or activity of SARM1. For example, the compounds of the invention are useful in the treatment and prevention of neurological disorders. The term "neurological disorder" generally refers to a disorder affecting the nervous system, including the central nervous system or the peripheral nervous system. The term "neurological disorder" also includes ocular indications having a nexus to the nervous system. [0137] In some embodiments, the neurological disorder treatable or preventable by administration of a compound of the invention includes neurodegenerative diseases. Neurodegenerative diseases are characterized by damage to the central nervous system and can be identified by progressive dysfunction, degeneration and death of specific populations of neurons which are often synaptically interconnected. Examples of neurodegenerative diseases include Parkinson's disease (PD), Alzheimer's disease (AD), Huntington's disease (HD), prion disease, motor neuron diseases (MND), spinocerebellar ataxia (SCA), spinal muscular atrophy (SMA), amyotrophic lateral sclerosis (ALS), and epilepsy. [0138] Examples of neurological disorders treatable or preventable according to the methods of the invention include spinal muscular atrophy (SMA), Chemotherapy Induced Peripheral Neuropathy (representative chemotherapeutic agents include vinca-alkaloids, taxols and platins), multiple sclerosis (MS), traumatic brain injury (TBI), spinal cord injury, stroke, Parkinson' disease, glaucoma, Huntington's disease, Alzheimer's disease, Charcot- Marie-Tooth disease (CMT), retinitis pigmentosa (RP), age-related macular degeneration (AMD), small fiber neuropathies, peripheral neuropathy (e.g., viral neuropathy), spinocerebellar ataxias, cystic fibrosis, familial amyloidotic polyneuropathy, spongiform encephalopathies, spinal and bulbar muscular atrophy, hereditary dentatorubral- pallidoluysian atrophy, adrenoleukodystrophy, adrenomyeloneuropathy, Alexander's disease, amyotrophic lateral sclerosis (ALS), Bassen-Kornzweig syndrome, Bell's palsy, progressive supra nuclear palsy (PSP), central pontine myelolysis, cluster headache, congenital hypomyelination, corticobasal degeneration, Creutzfeldt-Jakob disease, epilepsy, dementia (e.g., frontotemporal dementia and Lewy body dementia), demyelination disorders (e.g., ischemic demyelination), encephalomyelitis, Friedrich's ataxia, Gaucher's disease, hereditary sensory and autonomic neuropathy (HSAN), Hurler syndrome, Krabbe's disease, metachromatic leukodystrophy, migraine and tension headaches, mild cognitive impairment, motor spinoneuron disease, neuromyelitis optica, Niemann-Pick disease, optic neuritis, Pelizaeus Merzbacher disease, peripheral neuropathy, periventricular leukomalacia, post- herpetic neuralgia, prion disease, progressive supranuclear palsy, progressive multifocal leukoencephalopathy, Tay-Sacks disease, thoracic disc herniation, traverse myelitis, trigeminal neuralgia, Wallerian degeneration, cerebellar degeneration, chiari malformation, dystonia, encephalitis (e.g., pediatric viral encephalitis and La Crosse virus encephalitis), hyperekplexia, multifocal motor neuropathy, muscular dystrophy, myasthenia gravis, myopathy, neurofibromatosis, neuronal ceroid lipofuscinosis, neuropathies (e.g., peripheral neuropathy), pseudobulbar affect, restless legs syndrome, spina bifida, syringomyelia, thoracic outlet syndrome, and transverse myelitis. [0139] In other embodiments, the neurological disorder treatable or preventable by administration of a compound of the invention is a neuropathy. As used herein, the term "neuropathy" refers broadly to diseased conditions of the nervous system, including polyneuropathy; neuropathy, ataxia, and retinosa pigmentosa (NARP); familial amyloid neuropathies; diabetic neuropathy (peripheral neuropathy due to diabetes mellitus); peripheral neuropathy (e.g., chemotherapy-induced peripheral neuropathy (CIPN), including CIPN caused by vinca alkaloids, bortezomib, lxabepilone, thalidomide and its analogs, taxanes, and platinum-based agents); and cranial neuropathy (e.g., auditory neuropathy and optic neuropathy). The term also includes other neuropathies associated with genetic disorders (e.g., NMNAT2 genetic mutation disorders). [0140] In still other embodiments, the neurological disorder treatable or preventable by administration of a compound of the invention is an ocular neuropathy (e.g., optic neuropathy). The term "optic neuropathy" refers to damage to the optic nerve from a number of causes. Types of optic neuropathy include ischemic optic neuropathy (e.g., anterior and posterior ischemic optic neuropathy); optic neuritis (e.g., chronic relapsing inflammatory optic neuropathy (CRION), single isolated optic neuritis (SION), and relapsing isolated optic neuritis); compressive optic neuropathy; infiltrative optic neuropathy; traumatic optic neuropathy; mitochondrial optic neuropathies; and hereditary optic neuropathies (e.g., Leber's hereditary optic neuropathy (LHON), hereditary neuropathy with liability to pressure palsy (HNPP), and dominant optic atrophy). [0141] In still other embodiments, the neurological disorder treatable or preventable by administration of a compound of the invention is multiple sclerosis (MS), chemotherapy- induced peripheral neuropathy (CIPN), amyotrophic lateral sclerosis (ALS), glaucoma, traumatic brain injury (TBI), or stroke. [0142] As used herein, the term "cell" is meant to refer to a cell that is in vitro, ex vivo or in vivo. In some embodiments, an ex vivo cell can be part of a tissue sample excised from an organism such as a mammal. In some embodiments, an in vitro cell can be a cell in a cell culture. In some embodiments, an in vivo cell is a cell living in an organism such as a mammal. [0143] As used herein, the term "contacting" refers to the bringing together of indicated moieties in an in vitro system or an in vivo system. For example, "contacting" SARM1 or "contacting" a cell with a compound of the invention includes the administration of a compound of the present invention to an individual or patient, such as a human, having SARM1, as well as, for example, introducing a compound of the invention into a sample containing a cellular or purified preparation containing SARM1. [0144] As used herein, the term "individual" or "patient," used interchangeably, refers to mammals, and particularly humans. The individual or patient can be in need of treatment. [0145] As used herein, the phrase "therapeutically effective amount" refers to the amount of active compound or pharmaceutical agent that elicits the biological or medicinal response in a tissue, system, animal, individual or human that is being sought by a researcher, veterinarian, medical doctor or other clinician. [0146] As used herein, the phrase "inhibiting amount" refers to the amount of active compound or pharmaceutical agent that elicits a measurable SARM1 inhibition or axonal degeneration in a tissue, system, animal, individual or human. [0147] As used herein the term "treating" or "treatment" refers to 1) inhibiting the disease in an individual who is experiencing or displaying the pathology or symptomatology of the disease (i.e., arresting further development of the pathology and/or symptomatology), or 2) ameliorating the disease in an individual who is experiencing or displaying the pathology or symptomatology of the disease (i.e., reversing the pathology and/or symptomatology). [0148] As used herein the term "preventing" or "prevention" refers to preventing the disease in an individual who may be predisposed to the disease but does not yet experience or display the pathology or symptomatology of the disease. In some embodiments, the invention is directed to a method of preventing a disease in a patient, by administering to the patient a therapeutically effective amount of a compound of the invention, or a pharmaceutically acceptable salt thereof. Combination Therapy [0149] One or more additional pharmaceutically active agents or treatment methods can be used in combination with the compounds of the present invention. The agents can be combined with the present compounds in a single dosage form, or the agents can be administered simultaneously or sequentially as separate dosage forms. Examples of additional agents include acamprosate, agomelatine, almotriptan, amantadine, amisulpride, amitriptyline, apomorphine, aripiprazole, asenapine, atomoxetine, baclofen, botulinum toxin type A, bromocriptine, buccal midazolam, buprenorphine, buspirone, cabergoline, carbamazepine, chlordiazepoxide, chlorpromazine, citalopram, clobazam, clomethiazole, clomipramine, clonazepam, clozapine, denzapine, co-beneldopa, co-careldopa, dantrolene, dexamfetamine, diazepam, divalproex sodium, donepezil, doxepin, duloxetine, eletriptan, entacapone, epinephrine, escitalopram, eslicarbazepine, ethosuximide, fingolimod, fluoxetine, flupentixol, flupentixol, fluphenazine long-acting injection (modecate), fluvoxamine (Faverin), frovatriptan, gabapentin, galantamine, haloperidol, imipramine, lacosamide, lamotrigine, levetiracetam, levomepromazine, lisdexamfetamine, lithium, lofepramine, loprazolam, lorazepam, lormetazepam, lurasidone, melatonin, memantine, methylphenidate, mianserin, mirtazapine, moclobemide, modafinil, naratriptan, neostigmine, nitrazepam, nortriptyline, olanzapine, orlistat, orphenadrine, oxazepam, oxcarbazepine, paliperidone, paliperidone, paroxetine, perampanel, pergolide, pericyazine, phenobarbital, phenytoin, piracetam, pizotifen, pramipexole, pregabalin, primidone, prochlorperazine, procyclidine, pyridostigmine, quetiapine, rasagiline, reboxetine, risperidone, rivastigmine, rizatriptan, ropinirole, rotigotine, rufinamide, selegiline, sertraline, sodium oxybate, sodium valproate, sulpiride, sumatriptan, temazepam, tetrabenazine, tiagabine, tizanidine, tolcapone, topiramate, trazodone, trihexyphenidyl, trimipramine, valproate semisodium, venlafaxine, vigabatrin, vortioxetine, zolmitriptan, zolpidem, zonisamide, zopiclone, and zuclopenthixol. [0150] In some embodiments, the one or more additional pharmaceutically active agent can include a neuroprotective agent. In some embodiments, the neuroprotective agent is a dual leucine-zipper kinase (DLK) inhibitor. In some embodiments, the neuroprotective agent is a nicotinamide phosphoribosyltransferase (NAMPT) inhibitor. [0151] In some embodiments, the one or more additional pharmaceutically active agent can be NAD+ or an NAD+ precursor. NAD+ precursors include, for example, nicotinamide riboside (NR), nicotinic acid (NA), nicotinic acid riboside (NaR), nicotinamide (NAM), nicotinamide mononucleotide (NMN), nicotinic acid mononucleotide (NaMN), tryptophan, vitamin B3, and nicotinic acid adenine dinucleotide (NAAD). Pharmaceutical Formulations and Dosage Forms [0152] When employed as pharmaceuticals, the compounds of the invention can be administered in the form of pharmaceutical compositions. A pharmaceutical composition refers to a combination of a compound of the invention, or its pharmaceutically acceptable salt, and at least one pharmaceutically acceptable carrier. These compositions can be prepared in a manner well known in the pharmaceutical art, and can be administered by a variety of routes, depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be oral, topical (including ophthalmic and to mucous membranes including intranasal, vaginal and rectal delivery), pulmonary (e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal), ocular (e.g., eye drops or intravitreal, subconjunctival, subtenon, or retrobulbar injection), or parenteral. [0153] This invention also includes pharmaceutical compositions which contain, as the active ingredient, one or more of the compounds of the invention above in combination with one or more pharmaceutically acceptable carriers. In making the compositions of the invention, the active ingredient is typically mixed with an excipient, diluted by an excipient or enclosed within such a carrier in the form of, for example, a capsule, sachet, paper, or other container. When the excipient serves as a diluent, it can be a solid, semi-solid, or liquid material, 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, suppositories, sterile injectable solutions, and sterile packaged powders. [0154] The compositions can be formulated in a unit dosage form. The term "unit dosage form" refers to a physically discrete unit 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. [0155] The active compound can be effective over a wide dosage range and is generally administered in a pharmaceutically effective amount. It will be understood, however, that the amount of the compound actually administered will usually be determined by a physician, according to the relevant circumstances, including the condition to be treated, the chosen route of administration, the actual compound administered, the age, weight, and response of the individual patient, the severity of the patient's symptoms, and the like. [0156] For preparing solid compositions such as tablets, the principal active ingredient is mixed with a pharmaceutical excipient to form a solid pre-formulation composition containing a homogeneous mixture of a compound of the present invention. When referring to these pre-formulation compositions as homogeneous, the active ingredient is typically dispersed evenly throughout the composition so that the composition can be readily subdivided into equally effective unit dosage forms such as tablets, pills and capsules. This solid pre-formulation is then subdivided into unit dosage forms of the type described above containing from, for example, 0.1 to about 500 mg of the active ingredient of the present invention. [0157] The tablets or pills of the present invention can be coated or otherwise compounded to provide a dosage form affording the advantage of prolonged action. 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 which 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. [0158] The liquid forms in which the compounds and compositions of the present invention can be incorporated for administration orally or by injection include aqueous solutions, suitably flavored syrups, aqueous or oil suspensions, and flavored emulsions with edible oils such as cottonseed oil, sesame oil, coconut oil, or peanut oil, as well as elixirs and similar pharmaceutical vehicles. [0159] 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 supra. In some embodiments, the compositions are administered by the oral or nasal respiratory route for local or systemic effect. Compositions can be nebulized by use of inert gases. Nebulized solutions may be breathed directly from the nebulizing device or the nebulizing device can be attached to a face masks tent, or intermittent positive pressure breathing machine. Solution, suspension, or powder compositions can be administered orally or nasally from devices which deliver the formulation in an appropriate manner. [0160] The amount of compound or composition administered to a patient will vary depending upon what is being administered, the purpose of the administration, such as prophylaxis or therapy, the state of the patient, the manner of administration, and the like. In therapeutic applications, compositions can be administered to a patient already suffering from a disease in an amount sufficient to cure or at least partially arrest the symptoms of the disease and its complications. Effective doses will depend on the disease condition being treated as well as by the judgment of the attending clinician depending upon factors such as the severity of the disease, the age, weight and general condition of the patient, and the like. [0161] The compositions administered to a patient can be in the form of pharmaceutical compositions described above. These compositions can be sterilized by conventional sterilization techniques, or may be sterile filtered. Aqueous solutions can be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile aqueous carrier prior to administration. [0162] The therapeutic dosage of the compounds of the present invention can vary according to, for example, the particular use for which the treatment is made, the manner of administration of the compound, the health and condition of the patient, and the judgment of the prescribing physician. The proportion or concentration of a compound of the invention in a pharmaceutical composition can vary depending upon a number of factors including dosage, chemical characteristics (e.g., hydrophobicity), and the route of administration. For example, the compounds of the invention can be provided in an aqueous physiological buffer solution containing about 0.1 to about 10% w/v of the compound for parenteral administration. Some typical dose ranges are from about 1 .mu.g/kg to about 1 g/kg of body weight per day. In some embodiments, the dose range is from about 0.01 mg/kg to about 100 mg/kg of body weight per day. The dosage is likely to depend on such variables as the type and extent of progression of the disease or disorder, the overall health status of the particular patient, the relative biological efficacy of the compound selected, formulation of the excipient, and its route of administration. Effective doses can be extrapolated from dose-response curves derived from in vitro or animal model test systems. DETAILED DESCRIPTION OF THE FIGURES [0163] Figure 1. Discovery of covalent ligands that stereoselectively and site-specifically engage C311 in SARM1. (A) Chemical structures of tryptoline acrylamide stereoprobes EV- 96-EV-99. (B, C) MS-ABPP quantification of the reactivity of SARM1_C311 and other cysteines in SARM1 in human T cells treated with EV-96-EV-99 (20 µM, 3 h) or DMSO control. For B, data represent mean values ± SEM for eight independent experiments. For C, individual cysteine reactivity data points represent mean values for two-eight independent experiments. (D) Domain architecture of SARM1 with C311 is highlighted in black and ALS-relevant human hypermorphic mutants (27, 28) are highlighted in green. (E) Crystal structure of SARM1 (PDB: 7ANW) bound to NAD+, illustrating proximity of C311 to the NAD+/NMN-binding pocket in the ARM domain that allosterically regulates SARM1 activity. The ARM-domain is shown in royal blue, the SAM domains are shown in navy, the TIR is shown in light blue, NAD+ is represented as yellow sticks, C311 is represented as red spheres, and ALS-relevant hypermorphic mutants are represented as green spheres. (F) Chemical structures of alkynylated tryptoline acrylamide stereoprobes MY-13A and MY- 13B. The alkyne handle appended to the 3-position of the tryptoline ring and used for CuAAC to reporter tags is highlighted in red. (G) Gel-ABPP showing reactivity of SARM1- WT and a SARM1-C311A mutant recombinantly expressed in HEK293T cells with MY-13A or MY-13B (20 µM, 1 h, in situ). Reactions were visualized by CuAAC to a rhodamine-azide (Rh-N3) reporter tag, followed by SDS-PAGE and in-gel fluorescence scanning UT, untransfected HEK293T cells. Recombinant SARM1 proteins were expressed with N- terminal FLAG epitope tags and expression confirmed by immunoblotting (IB: FLAG). GAPDH served as an IB loading control. Results are from a single experiment representative of two independent experiments. (H) Quantitation of (G). Data represent mean values ± SD for two independent experiments. (I) Gel-ABPP showing effects of pre-treatment with DMSO, EV-98 or EV-99 (5, 20, or 50 µM, 3 h, in situ) on the reactivity of MY-13B (20 µM, 3 h, in situ) with WT-SARM1 recombinantly expressed in HEK293T cells. Results are from a single experiment representative of two independent experiments. (J) Quantitation of (I). Data represent mean values ± SEM for three independent experiments. Significance determined from a one-way ANOVA with Dunnett’s post hoc test where *P < 0.05. [0164] Figure 2. Stereoselective and site-specific engagement of C311 allosterically inhibits SARM1 enzymatic activity. (A) NAD+ hydrolysis reaction catalyzed by SARM1 to form nicotinamide, ADPR, and cADPR. (B) cADPR produced from lysates of HEK293T cells recombinantly expressing SARM1-WT or SARM1-C311A and treated with buffer, 100 µM NAD+, 1 mM NMN, or 100 µM NAD+ and 1 mM NMN. UT, untransfected HEK cells. (C) Relative amounts of cADPR generated from lysates of HEK293T cells expressing WT- SARM1 that were pretreated with DMSO, EV-98, or EV-99 (in situ, 20 µM, 3 h) and then, after lysis, supplemented with 100 µM NAD+ and 1 mM NMN. UT, untransfected HEK293T cells. Significance determined from a one-way ANOVA with Dunnett’s post hoc test. P- values vs. DMSO treatment, ***P < 0.001. (D) Relative amounts of cADPR generated from lysates of HEK293T cells expressing SARM1-C311A that were pretreated with DMSO or EV-99 (in situ, 20 µM, 3 h) and then, after lysis, supplemented with 100 µM NAD+ and 1 mM NMN. UT, untransfected HEK293T cells. (E) Quantification of cADPR (pmol per mg protein) produced from SH-SY5Y cells treated with vacor (50 µM, in situ) for 0, 2, 4, or 8 h. Significance determined from a one-way ANOVA with Dunnett’s post hoc test. P-values vs. 0 h treatment where ***P < 0.001, **P < 0.01, *P < 0.05. (F) Concentration-dependent effects of EV-98 or EV-99 (3 h pre-treatment) on vacor-induced cADPR production in SH- SY5Y cells (vacor: 50 µM, 4 h). For (B-F), data represent mean values ± SEM for three independent experiments. [0165] Figure 3. SAR analysis of engagement and inhibition of SARM1 by tryptoline acrylamides. (A) General structure of tryptoline acrylamide stereoprobes profiled where the structural element varied in this study is denoted by the R-group at the 3-position of the tryptoline ring. (B) Gel-ABPP showing effects of pre-treatment with DMSO or the indicated tryptoline acrylamides (20 µM, 3 h, in situ) on the reactivity of MY-13B (20 µM, 3 h, in situ) with WT-SARM1 recombinantly expressed in HEK293T cells. MY-13B reactivity with the SARM1-C311A mutant is also shown for comparison. UT, untransfected HEK293T cells. Results are from a single experiment representative of three independent experiments. (C) Quantitation of (B). Data represent mean values ± SEM for three independent experiments. Significance determined from a one-way ANOVA with Dunnett’s post hoc test where ***P < 0.001. (D) Relative amounts of cADPR generated from lysates of HEK293T cells expressing WT-SARM1 that were pretreated with DMSO or the indicated tryptoline acrylamides (20 µM, 3 h, in situ) and then, after lysis, supplemented with 100 µM NAD+ and 1 mM NMN. UT, untransfected HEK293T cells. Data represent mean values ± SEM for three independent experiments. Significance in determined from a one-way ANOVA with Dunnett’s post hoc test. P-values vs. DMSO treatment where ***P < 0.001, **P < 0.01, *P <0.05. (E) Correlation between SARM1 engagement (shown in (C) and SARM1 inhibition (shown in (D)).95% confidence interval is displayed by blue lines. WX-02-36, which falls well outside of the 95% confidence interval is marked. [0166] Figure 4. Inhibitory activity and proteome-wide selectivity of chemical probes targeting SARM1_C311. (A) Chemical structures of tryptoline acrylamides that engage SARM1_C311 (MY-9B and WX-02-37) along with their inactive enantiomers (MY-9A and WX-02-17, respectively). (B) Concentration-dependent effects of MY-9A and MY-9B (top) or WX-02-17 and WX-02-37 (bottom) (3 h, in situ) on vacor-induced cADPR production in SH-SY5Y cells (vacor: 50 µM, 4 h). data represent mean values ± SEM for three independent experiments. (C, D) Volcano plots comparing global cysteine reactivity profiles for (C) MY- 9A versus MY-9B or (D) WX-02-17 versus WX-02-37 (in situ, 20 µM, 3h) determined by MS-ABPP in human 22Rv1 cells, where cysteines that were significantly (log10(p-value) < 1.5 and stereoselectively (log2 > 1.5) engaged by MY-9B or WX-02-37 are shown in upper- right quadrant of dashed lines. SARM1_C311 is marked in blue. Data represent log2 mean fold-change values from two independent experiments. (E, F) MS-ABPP quantification of the reactivity of SARM1_C311 and other cysteines in SARM1 in 22Rv1 cells treated with MY- 9B (E) or WX-02-37 (F) (20 µM, 3 h) along with the corresponding full set of diastereomers (MY-9A, MY-10A, and MY-10B; or WX-02-17, WX-02-27, WX-02-47) relative to DMSO control. SARM1_C311 is marked in blue. Individual cysteine reactivity data points represent mean values for two independent experiments. [0167] Figure 5. Chemical probes targeting SARM1_C311 prevent vacor- and vincristine-induced neurite degeneration. (A) Schematic for neurite degeneration assays. In brief, dorsal root ganglia from E13.5-15.5 mice or rats are harvested, isolated, and grown for 7 days in culture before treatment with vacor (50 µM) or vincristine (40 nM). Neurite morphology is then analyzed via fluorescence microscopy at various time points after vacor or vincristine treatment. (B) Representative brightfield images from mouse DRG neurons treated with DMSO or 50 µM vacor +/- MY-9A, MY-9B, WX-02-17, or WX-02-37 (10 µM each) at 0, 8, 24, and 48 h post-treatment. Images shown are from a single experiment representative of three independent experiments. (C) Quantitation of neurite degeneration as shown in (B). Neurite degeneration was quantified by calculating the total fragmented neurite area versus total neurite area and reported as a degeneration index (see Methods for more details). Significance determined from a two-way ANOVA with Dunnett’s post hoc test. P- values for vacor versus vacor + MY-9B: ***P < 0.001. P-values for vacor versus vacor + WX-02-37: ###P <0.001. (D) Quantitation of neurite degeneration as described in (C) for mouse DRG neurons treated with DMSO or 50 µM vacor +/- MY-9A, MY-9B, WX-02-17, or WX-02-37 (10 µM) at 48 h post-treatment. Significance determined from a one-way ANOVA with Dunnett’s post hoc test. ***P <0.001. For (C, D), data represent mean values ± SEM for three independent experiments. (E) Representative images of beta III tubulin immunostained rat DRG neurites treated with DMSO or 40 nM vincristine +/- MY-9A, MY- 9B, WX-02-17, or WX-02-37 (1.1 µM) at 48 h post-treatment. Images shown are from a single experiment representative of at least two independent experiments. (F) Quantitation of axonal degeneration in (E). Neurite degeneration was monitored using high content imaging and quantified by calculating the degeneration of the nerve fibers versus total area of nerve fibers (see Methods section for more details). Data represent mean values ± SD for two-eight independent experiments. [0168] Figure 6. In vitro engagement of SARM1 by tryptoline acrylamides. (A) Gel- ABPP showing in vitro reactivity of SARM1-WT and a SARM1-C311A mutant recombinantly expressed in HEK293T cells with MY-13A or MY-13B (20 µM, 1 h). Reactions were visualized by CuAAC to a rhodamine-azide (Rh-N3) reporter tag, followed by SDS-PAGE and in-gel fluorescence scanning UT, untransfected HEK293T cells. Results are from a single experiment representative of two independent experiments. (B) Quantitation of (A). Data represent mean values ± SD for two independent experiments. (C) Gel-ABPP showing effects of pre-treatment with DMSO, EV-98 or EV-99 (20 µM, 3 h, in vitro) on the reactivity of MY-13B (20 µM, 1 h, in vitro) with WT-SARM1 recombinantly expressed in HEK293T cells. Reactivity of MY-13B with the SARM1-C311A mutant shown for comparison. Results are from a single experiment representative of two independent experiments. (D) Quantitation of (C). Data represent mean values ± SEM for three independent experiments. Data represent mean values ± SD for two independent experiments. [0169] Figure 7. Non-electrophilic analogs of EV-99 do not engage SARM1_C311. (A) Chemical structures of non-electrophilic tryptoline propanamides WX-02-225 and WX-02- 226. (B) Gel-ABPP showing effects of pre-treatment with DMSO, WX-02-225, or WX-02- 226 (20 µM, 3 h, in situ) on the reactivity of MY-13B (20 µM, 1 h, in situ) with WT-SARM1 recombinantly expressed in HEK293T cells. Results are from a single experiment representative of two independent experiments. (C) Quantitation of (B). Data represent mean values ± SD for two independent experiments. [0170] Figure 8. dHNN does not substantially engage SARM1_C311. (A) Chemical structure of dHNN. (B) Gel-ABPP showing effects of pre-treatment with DMSO or indicated concentrations of dHNN (5-50 µM, 3 h, in situ) on the reactivity of MY-13B (20 µM, 1 h, in situ) with WT-SARM1 recombinantly expressed in HEK293T cells. Results are from a single experiment representative of two independent experiments. (C) Quantitation of (B). Data represent mean values ± SD for two independent experiments. [0171] Figure 9. SARM1-dependent changes in NAD and ADPR levels. (A, B) NAD (A) and ADPR (B) produced from lysates of HEK293T cells recombinantly expressing SARM1- WT or SARM1-C311A and treated with buffer, 100 µM NAD+, 1 mM NMN, or 100 µM NAD+ and 1 mM NMN. UT, untransfected HEK cells. Data represent mean values ± SD for two-three independent experiments. (C, D) Relative amounts of NAD (C) and ADPR (D) generated from lysates of HEK293T cells expressing WT-SARM1 that were pretreated with DMSO, EV-98, or EV-99 (in situ, 20 µM, 3 h) and then, after lysis, supplemented with 100 µM NAD+ and 1 mM NMN. UT, untransfected HEK293T cells. Significance determined from a one-way ANOVA with Dunnett’s post hoc test. P-values vs. DMSO treatment, *P < 0.05, ***P < 0.001. (E, F) Relative amounts of NAD (C) and ADPR (D) generated from lysates of HEK293T cells expressing SARM1-C311A that were pretreated with DMSO or EV-99 (in situ, 20 µM, 3 h) and then, after lysis, supplemented with 100 µM NAD+ and 1 mM NMN. UT, untransfected HEK293T cells. For (C-F), data represent mean values ± SEM for three independent experiments. [0172] Figure 10. Vacor-induced increases in cADPR content of SH-SY5Y cells is dependent on SARM1. (A) Western blot showing SARM1 expression in WT SH-SY5Y cells (lane 1) or populations of SH-SY5Y cells with SARM1 gene disruption (SARM1-KO) achieved via CRISPR/Cas9-mediated genome editing with the indicated SARM1 targeting guide RNAs (lanes 2-6). (B) cADPR content (pmol per mg protein) of SH-SY5Y WT cells or SH-SY5Y SARM1-KO cells treated with vacor (in situ, 50 µM, 4 h). Data represent mean values ± SEM for three independent experiments. [0173] Figure 11. The non-electrophilic compound WX-02-226 does not inhibit vacor- induced increases in cADPR in SH-SY5Y cells. Effect of WX-02-226 (20 µM, 3 h pre- treatment) on vacor-induced cADPR production in SH-SY5Y cells (vacor: 50 µM, 4 h). For (B-F), data represent mean values ± SEM for three independent experiments. Data represent mean values ± SEM for three independent experiments. [0174] Figure 12. SAR analysis of inhibition of WT-SARM1 and C311 mutants by tryptoline acrylamides. (A) Relative amounts of NAD generated from lysates of HEK293T cells expressing WT-SARM1 that were pretreated with DMSO or the indicated tryptoline acrylamides (20 µM, 3 h, in situ) and then, after lysis, supplemented with 100 µM NAD+ and 1 mM NMN. UT, untransfected HEK293T cells. Significance in determined from a one-way ANOVA with Dunnett’s post hoc test. P-values vs. DMSO treatment where ***P < 0.001, **P < 0.01, *P <0.05. (B, C) Relative amounts of cADPR (C) and NAD (D) generated from lysates of HEK293T cells expressing the SARM1-C311A that were pretreated with DMSO or the indicated compounds (in situ, 20 µM, 3 h) and then, after lysis, supplemented with 100 µM NAD+ and 1 mM NMN. UT, untransfected HEK293T cells. For (A-C), data represent mean values ± SEM for three independent experiments. [0175] Figure 13. Characterization of the activity and inhibitor sensitivity of a SARM1- C311S mutant. (A) Western blot showing SARM1 expression in transiently transfected HEK293T cells. UT, untransfected cells. (B, C) Relative amounts of cADPR (B) and NAD (C) generated from lysates of HEK293T cells expressing SARM1-WT, SARM1-C311A, or SARM1-C311S and treated with buffer, 100 µM NAD+, 1 mM NMN, or 100 µM NAD+ and 1 mM NMN. UT, untransfected HEK cells. (D) Relative amounts of cADPR (C) and NAD (D) generated from lysates of HEK293T cells expressing SARM1-WT or the SARM1-C311S mutant that were pretreated with DMSO or the indicated compounds (in situ, 20 µM, 3 h) and then, after lysis, supplemented with 100 µM NAD+ and 1 mM NMN. UT, untransfected HEK293T cells. For (B-D), data represent mean values ± SEM for three independent experiments. [0176] Figure 14. Chemical probes targeting SARM1_C311 stereoselectively inhibit vacor-induced cADPR production in 22Rv1 cells. (A) Quantification of cADPR (pmol per mg protein) produced from 22Rv1 cells treated with vacor (50 µM, in situ) for 0, 2, 4, or 8 h. Significance determined from a one-way ANOVA with Dunnett’s post hoc test. P-values vs. 0 h treatment where ***P < 0.001, **P < 0.01, . (B, C) Concentration-dependent effects of MY-9A and MY-9B (B) or WX-02-17 and WX-02-37 (C) (3 h pre-treatment) on vacor- induced cADPR production in 22Rv1 cells (vacor: 50 µM, 4 h). For (A-C), data represent mean values ± SEM for three independent experiments. [0177] Figure 15. Proteome-wide reactivity of chemical probes targeting SARM1_C311. (A-D) Waterfall plots of MS-ABPP data showing total numbers of quantified cysteines and their respective reactivity ratios (DMSO/compound) in 22Rv1 (A, C) or Ramos (B, D) cells treated with MY-9B (A, B) or WX-02-37 (C, D) (20 µM, 3 h). Also shown are heat maps comparing the cysteine reactivity values for cysteines substantially engaged (> 75%) by MY- 9B and/or WX-02-37 in cells treated with each of the corresponding diastereomers. Data represent mean values from two independent experiments. [0178] Figure 16. Proteome-wide selectivity of chemical probes targeting SARM1_C311. (A, B) Volcano plots comparing global cysteine reactivity profiles for (A) MY-9A versus MY-9B or (B) WX-02-17 versus WX-02-37 (in situ, 20 µM, 3h) determined by MS-ABPP in Ramos cells, where cysteines that were significantly (log10(p-value) < 1.5 and stereoselectively (log2 > 1.5) engaged by MY-9B or WX-02-37 are shown in upper-right quadrant of dashed lines. SARM1_C311 is marked in blue. Data represent log2 mean fold- change values from two independent experiments. (C, D) MS-ABPP quantification of the reactivity of SARM1_C311 and other cysteines in SARM1 in Ramos cells treated with MY- 9B (C) or WX-02-37 (D) (20 µM, 3 h) along with the corresponding full set of diastereomers (MY-9A, MY-10A, and MY-10B; or WX-02-17, WX-02-27, WX-02-47) relative to DMSO control. SARM1_C311 is marked in blue. Individual cysteine reactivity data points represent mean values for two independent experiments. [0179] Figure 17. Reactivity values for cysteines in other NAD+ metabolic enzymes from cells treated with chemical probes targeting SARM1_C311. Reactivity values for cysteines from diverse NAD+ metabolic enzymes as determined by MS-ABPP of 22Rv1 and Ramos cells treated with the indicated tryptoline acrylamide stereoprobes (20 µM, 3 h). Data represent mean values from two-four independent experiments, where data for cysteines quantified in both 22Rv1 and Ramos cells were combined and averaged. [0180] Figure 18. Mouse SARM1 is stereoselectively engaged and inhibited by chemical probes targeting SARM1_C311. (A) Gel-ABPP showing reactivity of human and mouse recombinantly expressed in HEK293T cells with MY-13A or MY-13B (20 µM, 1 h, in situ). Reactions were visualized by CuAAC to a rhodamine-azide (Rh-N3) reporter tag, followed by SDS-PAGE and in-gel fluorescence scanning UT, untransfected HEK293T cells. (B) Quantitation of (A). Data represent mean values ± SD for two independent experiments. (C) Gel-ABPP showing effects of pre-treatment with DMSO or indicated compounds (20 µM, 3 h, in situ) on the reactivity of MY-13B (20 µM, 1 h, in situ) with mouse SARM1 recombinantly expressed in HEK293T cells. Results are from a single experiment representative of two independent experiments. (D) Quantitation of (C). Data represent mean values ± SD for two independent experiments. (E, F) NAD (E) cADPR (F) produced from lysates of HEK293T cells recombinantly expressing human or mouse SARM1 and treated with buffer, 100 µM NAD+, 1 mM NMN, or 100 µM NAD+ and 1 mM NMN. UT, untransfected HEK cells. (G) Relative amounts of cADPR generated from lysates of HEK293T cells expressing mouse SARM1 that were pretreated with DMSO or the indicated compounds (in situ, 20 µM, 3 h) and then, after lysis, supplemented with 100 µM NAD+ and 1 mM NMN. UT, untransfected HEK293T cells. Significance determined from a one-way ANOVA with Dunnett’s post hoc test. P-values vs. DMSO treatment, **P < 0.01, ***P < 0.001. [0181] Figure 19. Stereoselective inhibition of vacor-induced cADPR production in mouse Neuro-2a cells by chemical probes targeting SARM1_C311. (A) Quantification of cADPR (pmol per mg protein) produced from Neuro-2A cells treated with vacor (50 µM, in situ) for 0, 2, 4, or 8 h. Significance determined from a one-way ANOVA with Dunnett’s post hoc test. P-values vs.0 h treatment where ***P < 0.001, **P < 0.01, *P < 0.05. (B, C) Concentration-dependent effects of MY-9A and MY-9B (B) or WX-02-17 and WX-02-37 (C) (3 h pre-treatment) on vacor-induced cADPR production in Neuro-2A cells (vacor: 50 µM, 4 h). For (A-C), data represent mean values ± SEM for three independent experiments. [0182] Figure 20. Concentration-dependent inhibition of vacor-induced neurite degeneration by chemical probes targeting SARM1_C311. Representative brightfield images of mouse DRG neurons treated with DMSO or 50 µM vacor +/- MY-9B and WX-02-37 (1, 10, or 20 µM) at 0, 8, 24, and 48 h post-treatment. Images shown are from a single experiment. [0183] Figure 21. Acrylamide stereoprobes do not independently affect neurite integrity in mouse DRG neurons. (A) Representative brightfield images from mouse DRG neurons treated with DMSO, 50 µM vacor, MY-9A, MY-9B, WX-02-17, or WX-02-37 (10 µM each) at 0, 8, 24, and 48 h post-treatment. Images shown are from a single experiment representative of three independent experiments. (B) Quantitation of neurite degeneration as shown in (A). Neurite degeneration was quantified by calculating the total fragmented neurite area versus total neurite area and reported as a degeneration index (see Methods for more details) Data represents mean values ± SEM for three independent experiments. [0184] Figure 22. Concentration-dependent inhibition of vincristine-induced neurite degeneration by chemical probes targeting SARM1_C311. (A) Representative images of beta III tubulin-immunostained rat DRG neurites treated with DMSO or 40 nM vincristine +/- MY-9A, MY-9B, WX-02-17, or WX-02-37 (10-0.041 µM) at 48 h post-treatment. Images shown are from a single experiment representative of at least two biologically independent experiments. (B, C) Quantitation of neurite degeneration in (A). Neurite degeneration was monitored using high content imaging and was quantified by calculating the degeneration of the nerve fibers versus total area of nerve fibers (see Methods section for more details). Data represent mean values ± SD for two-eight independent experiments. Effects of Inhibition of NADase Activity of SARM1 [0185] Axonal degeneration is an early hallmark and driver of disease progression in diverse neurodegenerative disorders that affect both the central and peripheral nervous system, including Alzheimer’s disease, amyotrophic lateral sclerosis (ALS), and chemotherapy-induced peripheral neuropathy (1-3). Efforts to characterize the molecular pathways that contribute to axonal degeneration have led to the identification of the protein sterile alpha toll/interleukin receptor motif containing-1 (SARM1) as a key mediator of this process (4). SARM1 possesses an N-terminal armadillo repeat (ARM) domain followed by tandem sterile alpha motif (SAM) domains and a C-terminal toll/interleukin receptor (TIR) domain. The TIR domain of SARM1 has been found to possess intrinsic nicotinamide adenine dinucleotide (NAD) hydrolase (NADase) activity, converting NAD+ into nicotinamide, adenosine diphosphate ribose (ADPR), and cyclic ADPR, representing a prototype member of a growing class of TIR domains with enzymatic function (5, 6). [0186] The NADase activity of SARM1 is critical to its role in axonal degeneration, as excessive SARM1-dependent consumption of NAD+ results in metabolic crisis, which initiates the cell autonomous axon self-destruction process (5, 7). Accordingly, the catalytic function of SARM1 is tightly regulated by a complex autoinhibitory mechanism. Under homeostatic conditions, SARM1 forms an inactive homo-octameric complex (8, 9). Autoinhibition in this state is achieved through the physical separation of the TIR domains by the ARM domains, preventing TIR-TIR domain dimerization, which is necessary for formation of a composite active site that catalyzes NAD hydrolysis (10, 11). Recently, it was discovered that the ARM domain contains an allosteric pocket that regulates SARM1 activity through differential binding to inhibitory (NAD+) or stimulatory (nicotinamide mononucleotide (NMN)) metabolites (7, 12). In healthy neurons, where NAD+ levels are high and the ratio of NMN/NAD+ is low, the ARM domain is bound to NAD+ and SARM1 remains autoinhibited. Conversely, stress conditions that reduce NAD+ concentrations and increase the ratio of NMN/NAD+ lead to the exchange of NAD+ for NMN in the allosteric ARM pocket, which, in turn, causes a conformational change in the ARM domain that allows for TIR domain dimerization and SARM1 activation (7, 11). [0187] The genetic disruption of SARM1 has been found to be protective in various models of neurological injury, including peripheral neuropathy, traumatic brain injury, axotomy, and exposure to environmental toxins (13-20). Genetic deletion of SARM1 also prevents the axonal decline caused by loss of nicotinamide mononucleotide adenyl transferase 2 (NMNAT2) (13), which functions upstream of SARM1 through the enzymatic conversion of NMN to NAD+ (21). Notably, humans with deleterious mutations in NMNAT2 suffer from pediatric neurological disorders (22-26), and, conversely, SARM1 hypermorphic risk alleles have recently been discovered in patients with ALS (27, 28), supporting the human biology relevance of the NMNAT2-SARM1 pathway to maintaining neuronal integrity and CNS health. [0188] Considering the effectiveness of SARM1 genetic disruption in the prevention of axonal degeneration, this enzyme is considered an attractive therapeutic target for the treatment of neurodegenerative disorders. Multiple inhibitors of SARM1 have been described (29-32), most of which target the NADase domain (29-31). These include simple isoquinolines that have recently been found to serve as pseudo-substrates, being converted to NAD mimetics by SARM1 to form the active inhibitors (11, 31). Additionally, several cysteines, within both the enzymatic (TIR) and allosteric (ARM) domains of SARM1, have been identified as potential targets for electrophilic small molecules (29, 30, 32); however, the mechanisms of action of putative orthosteric (30) and allosteric (32) electrophilic inhibitors of SARM1 remain poorly understood. For instance, a nisoldipine derivative, dehydronitrosonisoldipine (dHNN), was recently shown to inhibit SARM1 and found to react with C311 in the ARM domain (32). However, mutagenesis of C311 only modestly impaired (~two-fold) the inhibitory activity of dHNN, leading to the conclusion that this compound may engage multiple cysteines in SARM1 (32). Herein described is the chemical proteomic discovery of a structurally distinct class of electrophilic compounds that stereoselectively and site-specifically react with C311 of SARM1. Herein it is demonstrated that these tryptoline acrylamide ligands inhibit the NADase activity of WT-SARM1, but not C311A or C311S mutants of this enzyme, block vacor-induced cADPR production in human cells, and display high degrees of selectivity across > 23,000 quantified cysteines in the human proteome. Finally, the covalent SARM1 inhibitors prevented both vacor- and vincristine- induced neurite degeneration in rodent DRG neurons. [0189] Chemical proteomic methods such as activity-based protein profiling (ABPP) have emerged as a powerful strategy to globally map small molecule-protein interactions in native biological systems (33-36) and have specifically revealed the broad potential for electrophilic small molecules to engage (or ‘ligand’) cysteine residues on structurally and functionally diverse classes of proteins (37-43). Understanding how such electrophile- cysteine interactions affect protein function in cells, however, remains technically challenging, especially if the electrophilic ligands are promiscuous and lacking stringent structure-activity relationships (SARs). Recently introduced was a way to address this problem by generating focused libraries of stereochemically defined electrophilic compounds (or ‘stereoprobes’), which can reveal cysteine interactions in cells that depend on the absolute stereochemistry of compounds (39), thereby helping to identify liganding events that occur in well-defined binding pockets with tractable SARs. Additionally, cysteines displaying stereoselective reactivity can be paired with physicochemically matched active and inactive enantiomeric stereoprobes to facilitate well-controlled pharmacological studies in cells (39). [0190] In the course of mapping the cysteine reactivity of a set of tryptoline acrylamide stereprobes (Fig.1A) by mass spectrometry (MS)-ABPP in primary human T cells (39), it was discovered that compound EV-99 that stereoselectively engaged C311 on SARM1 (Fig. 1B and 1C). Other quantified cysteines in SARM1 were unaffected by EV-99 (Fig.1B and 1C), providing additional support for a specific interaction of this compound with C311. C311 is located in the ARM domain of SARM1 (Fig.1D), and recent cryo-electron microscopy structures have revealed that this residue sits on a flexible loop located adjacent to the allosteric metabolite binding pocket of the ARM domain (7-9, 12) (Fig.1E). Notably, several hypermorphic mutations in human SARM1 that have been linked to ALS (27, 28) are also structurally proximal to C311 (Fig.1E), supporting the functionality of this region for regulating SARM1 activity. Finally, recent efforts to discover inhibitors of SARM1 identified an electrophilic derivative of the calcium channel blocking agent nisoldipine – dehydronitrosonisoldipine (dHNN) – that blocked SARM1 activity possibly, in part, by covalent modification of C311 (32). These various past findings motivated us to further characterize tryptoline acrylamides as ligands and potential inhibitors of SARM1. [0191] The site-specific and stereoselective labeling of SARM1_C31, utilizing alkynylated analogues of EV-98 and EV-99 (MY-13A and MY-13B, respectively (Fig.1F)) that can be modified with a rhodamine-azide (Rh-N3) reporter tag via copper-catalyzed azide- alkyne cycloaddition (CuAAC) (44) and the corresponding SARM1-alkyne probe adduct conveniently quantified by gel-ABPP methods, was confirmed (45). Analysis of HEK293T cells recombinantly expressing FLAG epitope-tagged SARM1 variants revealed that WT- SARM1 reacted strongly with MY-13B, but not MY-13A, while the SARM1-C311A showed very low reactivity with either alkyne probe (Fig.1G and 1H). The reactivity of WT-SARM1 with MY-13B was blocked in a concentration-dependent manner by pre-treatment with EV- 99, but not the enantiomer EV-98 (Fig.1I and 1J), mirroring the stereoselective interactions with the tryptoline acrylamides observed for endogenous SARM1 (Fig.1B). While stereoselective reactivity with recombinant SARM1 was observed for MY-13B following in situ or in vitro treatment, it was observed that there was a much stronger competitive blockade of this interaction by EV-99 in situ (Fig.1G-I and Fig.6), suggesting that the tryptoline acrylamides showed greater reactivity with SARM1 in cells versus cell lysates. [0192] Non-electrophilic propanamide analogues of EV-98 and EV-99 (WX-02-225 and WX-02-226, respectively) did not affect MY-13B reactivity with recombinant SARM1 in HEK293T cells (Fig.7), supporting that the tryptoline acrylamides bind SARM1 through a covalent mechanism. Finally, substantial alterations in MY-13B reactivity with SARM1 in cells pre-treated with increasing concentrations of dHNN (5-50 µM, 3 h pre-treatment was not observed; Fig.8), suggesting that this compound may engage C311 at low stoichiometry and produce its inhibitory effects through modifying multiple SARM1 cysteines, as previously posited (32). [0193] Taken together, these initial findings demonstrate that tryptoline acrylamide EV- 99 can stereoselectively and site-specifically react with C311 of SARM1. Next, the effects of EV-99 on SARM1 activity were determined. [0194] Recognizing that EV-99 showed superior engagement of SARM1 in cells, a protocol where HEK293T cells expressing SARM1 variants were first treated with compounds in situ, followed by lysis and monitoring SARM1 enzyme activity in vitro using an LC-MS/MS assay that quantified the consumption of NAD+ and production of ADPR and cADPR (Fig.2A) was established. Consistent with previous findings (7), the co-addition of NAD+ and NMN dramatically increased SARM1 activity (compared to exposure of cell lysates to NAD+ or NMN alone), and it was found that both WT-SARM1 and the SARM1- C311A mutant showed similar activities that were > 10-fold higher than the activity observed in untransfected HEK293T cell lysates (Fig.2B and Fig.9A and 9B). Next, the increases in cADPR, as this proved to be the most sensitive measure of SARM1 activity were monitored. Treatment of WT-SARM1-expressing cells with EV-99 (in situ, 20 µM, 3 h) resulted in substantial inhibition of SARM1 activity (Fig.2C, Fig.9C, and 9D). In contrast, the inactive enantiomer EV-98 did not inhibit SARM1 activity (Fig.2C, Fig.9C, and 9D). EV-99 did not alter the activity of the SARM1-C311A mutant (Fig.2D, Fig.9E, and 9F), supporting that this compound acts as a SARM1 inhibitor principally through covalent modification of C311. [0195] Next evaluated were the effects of EV-99 on endogenous SARM1 activity leveraging the recent discovery that the rodenticide vacor acts as a specific and direct activator of SARM1 through metabolic conversion to the NMN mimetic vacor mononucleotide (VMN) (16). It was found that vacor (50 µM) induced the robust, time- dependent production of cADPR in the human neuroblastoma cell line SH-SY5Y (Fig.2E), and this effect was absent in SARM1-null SH-SY5Y cells generated by CRISPR-Cas9 genome editing (Fig.10). Pre-treatment of SH-SY5Y cells with EV-99 (0.25-20 µM, 3 h) produced a concentration-dependent and complete blockade of vacor-induced cADPR production with an apparent IC50 value of 4.7 ± 0.6 µM (Fig.2F). In contrast, the inactive analogues – enantiomer EV-98 and non-electrophilic propanamide WX-02-226 – did not affect vacor-induced cADPR production in SH-SY5Y cells (Fig.2F and Fig.11). [0196] Having established that EV-99 acts as a stereoselective and site-specific inhibitor of SARM1 activity, next evaluated was the SAR for this interaction by screening a focused library of cis-tryptoline acrylamides where the methyl ester group was replaced with various carbonyl substituents (Fig.3A). It was reasoned that these analogues would not only assess the impact of larger groups in the 3-position, but might also allow for replacement of the hydrolytically labile methyl ester in EV-99. The cis-tryptoline acrylamides were first analyzed for engagement of WT-SARM1 in HEK293T cells by pre-treatment at 20 µM for 3 h, followed by exposure of the cells to MY-13B (20 µM, 1 h) and analysis of the reactions by gel-ABPP. Several of the amide analogues showed equivalent or greater engagement of WT- SARM1 compared to EV-99, including the methylamide MY-9B, the aminopyridine WX-02- 35, and the propylamide WX-02-37 (Fig.3B and 3C). In each case, the stereoselectivity of SARM1 engagement by the R,R cis-tryptoline acrylamides was maintained. Among the amide analogues, only the morpholino compound WX-02-33 showed substantially reduced reactivity with SARM1, while the free carboxylic acid analogue WX-02-247 was completely inactive. In the case of WX-02-247, it was not possible to exclude the possibility that inactivity may reflect a lack of cell penetrance for the compound. [0197] The SAR for SARM1 engagement determined by gel-ABPP was next compared to the profile of compound activities in the SARM1 exogenous substrate assay (Fig.3D and Fig.12A), where it was observed that a generally strong correlation (R2 = 0.78; Fig.3E). One notable exception, however, was the propylamide analogue WX-02-36, which, despite robustly reacting with SARM1 (~80% engagement), showed only modest inhibition (~20%) of SARM1 enzymatic activity. These data point to the potential for a divergence in the binding/reactivity and functional effects of covalent ligands engaging SARM1_C311, a pharmacological complexity that is not uncommon for small molecules that act at allosteric sites (46-48). The active compounds did not inhibit the SARM1-C311A mutant (Fig.17 and 12C). In the course of performing these studies, it was noticed that the SARM1-C311A mutant displayed a modest hypermorphic activity reflected in greater cADPR production compared to WT-SARM1 (Fig.13A-C). This is perhaps not surprising, considering the high density of other gain-of-function mutants regionalized to the ARM domain in spatial proximity to SARM1-C311A. An evaluation of alternative mutations revealed that the SARM1-C311S mutant displayed similar catalytic activity to WT-SARM1 (Fig.13A-C), and it was confirmed that the SARM1_C311S mutant was fully resistant to the inhibitory effects of active tryptoline acrylamide inhibitors of WT-SARM1 (Fig.13D). [0198] Based on a combination of the SARM1 engagement and inhibition data, two sets of stereoprobes for further studies – MY-9A/MY-9B and WX-02-17/WX-02-37 (Fig.4A) were prioritized. It was first verified that both active enantiomers, MY-9B and WX-02-37, stereoselectively inhibited vacor-induced, SARM1-dependent cADPR production in two human cell lines – SH-SY5Y and 22Rv1 cells – with low-µM IC50 values that were ~three- four-fold more potent than EV-99 (Fig.4B and Fig.14). It was next assessed that the broader proteome-wide reactivity of MY-9B and WX-02-37 (20 µM, 3 h, in situ), along with their full set of stereoisomers (cis and trans) in two representative human cell lines (the adherent cell line 22Rv1 and suspension cell line Ramos) by cysteine-directed MS-ABPP using the iodoacetamide-desthiobiotin (IA-DTB) probe (39). Of more than 23,000 quantified cysteines across the two cell lines, only 25 sites were substantially engaged (> 75% reductions in IA- DTB labeling) by MY-9B or WX-02-37 (Fig.15). Cysteines that were stereoselectively engaged by MY-9B and WX-02-37 (i.e., a log2 fold change >1.5 in engagement by both compounds over their inactive stereoisomers) were of particular interest. As expected, SARM1_C311 met these criteria, and it was observed that only a handful of additional cysteines across the proteome that were stereoselectively engaged by MY-9B and/or WX-02- 37 (Fig.4C, 4D, Fig.16A, and 16B). Several additional cysteines in SARM1 were also quantified in these MS-ABPP experiments, and none showed changes in IA-DTB reactivity (Fig.4E, 4F, Fig.16C, and 16D). Numerous cysteines in other enzymes that directly consume NAD+ or participate in NAD+ biosynthetic pathways (e.g., PARPs, SIRTs, NMNATs, NAMPTs, etc.), including the catalytic cysteine in CD38 (C119), and none of these cysteines was substantially engaged by MY-9B or WX-02-37, were quantified (Fig.17). [0199] Taken together, these data support that MY-9B and WX-02-37 display an attractive combination of cellular potency (low-µM), well-defined SARs (stereoselective and site-specific reactivity with C311), and proteome-wide selectivity for use as chemical probes of SARM1. [0200] Previous studies with orthosteric SARM1 inhibitors or genetically disrupted SARM1 systems have revealed that the loss of this enzyme protects against axonal degeneration (13, 14, 18, 19, 30-32). Via evaluating the allosteric inhibitors MY-9B and WX- 02-37 in chemical toxicity-induced rodent dorsal root ganglion (DRG) neuronal models of axonal degeneration (Fig.5A), it was first confirmed that recombinant mouse SARM1 was stereoselectively engaged by MY-9B and WX-02-37, and that these compounds also inhibited mouse SARM1 activity, albeit to a less dramatic degree than observed for recombinant human SARM1 (Fig.18). However, it was interpreted that this apparent difference in the inhibitor sensitivity of mouse and human SARM1 to reflect a feature specific to the recombinant proteins, as both MY-9B and WX-02-37 produced complete, stereoselective blockade of vacor-induced cADPR production in the mouse Neuro-2A cell line with IC50 values similar to those measured in human cell lines (Fig.19). Applicant proceeded to treated mouse DRG neurons concurrently with vacor (50 µM) and DMSO or active and inactive enantiomeric compounds (MY-9B vs MY-9A; WX-02-37 vs WX-02-17; 1-20 µM each), followed by analysis of neurite morphology over a 48 h period by fluorescent microscopy (Fig.5A). In DMSO-treated control neurons, vacor caused ~60% degeneration by 8 h and complete degeneration by 24 h. This vacor-induced degeneration was near- completely blocked in neurons treated with 10 or 20 µM of MY-9B or WX-02-37 (Fig.5B-D and Fig.20). In contrast, the inactive enantiomers MY-9A and WX-02-17 were ineffective at blocking vacor-induced neurite degeneration (Fig.5B-D and Fig.20). In separate control experiments, Applicant also confirmed that none of the tested stereoprobes were independently cytotoxic or caused changes in neurite morphology (Fig.21). [0201] Vincristine is a chemotherapeutic that indirectly induces SARM1 activity and axonal degeneration by inhibiting microtubule assembly and axonal transport leading to NMNAT2 depletion (49, 50). Applicant found that both MY-9B and WX-02-37 stereoselectively prevented vincristine-induced neurite degeneration in rat DRG neurons (Fig. 5E, 5F, and Fig.22A). MY-9B and WX-02-37 displayed striking potency in the vincristine model, with EC50 values of ~ 300 nM that were ~10-fold more potent than the corresponding enantiomers (Fig.22B and 22C). It was unclear why the SARM1 inhibitors displayed such high potency in vincristine-induced neurite degeneration model, although this outcome might relate to the slower and indirect mechanism of SARM1 activation, which could provide greater time for compound engagement of SARM1_C311 before axonal degeneration takes place (indeed, previous reports have shown that granular axon degeneration does not emerge until ~24-36 h after vincristine treatment (5, 32, 50)). This hypothesis might also explain the weak activity observed for control enantiomers MY-9A and WX-02-17, as it could not be excluded that, at higher concentrations and longer time points, these compounds may begin to show some SARM1_C311 engagement. [0202] Taken together, these data support that covalent, allosteric inhibitors targeting SARM1_C311 display striking protective effects in multiple models of axonal degeneration. [0203] The discovery that SARM1 is required for axonal degeneration has motivated research into the molecular mechanisms of SARM1 function and regulation, as well as the pursuit of small-molecule inhibitors of this enzyme. Much of this latter effort has focused on the identification of competitive, orthosteric inhibitors, as reflected by the screening of compounds against a constitutively active SARM1 construct that contains only the enzymatic TIR domain (29-31). Considering, however, that SARM1 is also subject to intricate autoregulatory mechanisms, the discovery of druggable pockets on other domains of the protein, such as the ARM domain, may provide a path to allosteric inhibitors. Such allosteric agents may have advantages over orthosteric inhibitors in terms of achieving greater selectivity over the many other proteins in the human proteome that also bind NAD+. [0204] Described herein is the chemical proteomic discovery of a series of tryptoline acrylamides that stereoselectively and site-specifically engaged SARM1_C311 and, through doing so, inhibit the NAD+ glucohydrolase activity of this enzyme. This work extends previous studies pointing to SARM1_C311 as a potential site of druggability (32) in important ways. First, unlike the original covalent ligand described for SARM1 – dHNN – which engaged C311 along with apparently additional cysteines in the protein, it is convincingly demonstrated that the tryptoline acrylamides exhibit site-specificity for SARM1_C311 and a well-defined SAR that led to the identification two compounds – MY- 9B and WX-02-37 – with low-µM cellular activity and excellent proteome-wide selectivity. MY-9B and WX-02-37 are viewed as suitable cellular probes for biological investigations of SARM1 and highlight that such studies can also benefit from the use of physicochemically matched inactive enantiomeric control compounds (MY-9A and WX-02-17). Future goals include to improve the potency of the SARM1_C311 ligands to enable in vivo studies, as well as to better understand the relationship between modification of C311 and functional effects on SARM1. This latter objective is underscored by the discovery of at least one compound (WX-02-36) for which C311 engagement and SARM1 inhibition were disconnected. Considering the close structural similarity between WX-02-36 and active inhibitors MY-9B and WX-02-37, it is concluded that subtle differences in the structure of covalent ligands targeting SARM1_C311 may lead to substantial changes in inhibitory activity. Slight structural modifications causing dramatic changes in compound activity are precedented for other types of allosteric agents, including those targeting G-protein coupled receptors (51), the guanine nucleotide exchange factor SOS1 (46), the protein kinase/endoribonuclease IRE1α (47, 48), and TEM β-lactamase (52). In the case of SARM1, it is speculated that the greater flexibility of the propyl group of WX-02-36 may lead to a mode of engagement that is neutral as opposed to the methyl or cyclopropyl groups, where the rigidity of these motifs may enforce a mode of engagement that is antagonistic. [0205] These studies also point to some peculiar features of the human and mouse SARM1 proteins, at least in recombinant form, that may impinge upon the apparent activity of covalent ligands engaging C311. First, the tryptoline acrylamides displayed much greater engagement of SARM1_C311 in cells versus in cell lysates. Similarly, it was found that recombinant human and mouse SARM1, despite showing similar levels of catalytic activity, demonstrated differential sensitivity to inhibition by MY-9B and WX-02-37. Interestingly, the weaker inhibition of recombinant mouse SARM1 was also reflected in a lower extent of labeling by the corresponding alkyne probe MY-13B, despite this labeling retaining stereoselectivity and being fully competed by MY-9B/WX-02-37. It was interpreted these data, taken together, to indicate that recombinant forms of full-length SARM1 may exist in multiple conformations, each of which may display catalytic activity, but only a subset of which are sensitive to electrophilic ligands targeting C311. While it is not yet known what the postulated electrophilic ligand-insensitive conformations of SARM1 might represent, it is encouraging that both MY-9B and WX-02-37 completely suppressed vacor-induced cADPR production in both human (SH-SY5Y, 22Rv1) and mouse (Neuro-2A) cells, indicating that endogenous SARM1 can be fully inhibited by these compounds. These findings thus underscore, in the investigation of allosteric inhibitors of SARM1, the importance of assays that measure the activity of endogenous forms of this enzyme. [0206] While it is not yet understood how the modification of C311 by electrophilic small molecules leads to SARM1 inhibition, a recent cryo-EM structure of activated SARM1 revealed that NMN causes a conformational change in a flexible loop containing C311 (residues 310-325), and movement of this loop leads to reorientation of the ARM domain, dislodging the TIR domain and allowing for dimerization-induced activation (11). It is accordingly speculated that modification of C311 by tryptoline acrylamides may prevent NMN-mediated allosteric activation of SARM1. This may occur if, for instance, covalent ligands targeting C311 act as gatekeepers to block binding of NMN to the ARM domain. An alternative possible mechanism of allosteric inhibition could allow for simultaneous binding of NMN and C311-directed covalent ligands, with the latter then preventing NMN-driven reorientation of the ARM domain required for SARM1 activation. Determining the mechanism of allosteric inhibition would benefit, in the future, from structural studies of tryptoline acrylamide-SARM1 complexes. [0207] In considering the potential translational implications of this disclosure, it is believed that the full protection furnished by MY-9B and WX-02-37 in multiple cellular assays of axonal degeneration supports that allosteric inhibitors targeting SARM1_C311 have the capacity to match the pharmacological activity of orthosteric inhibitors of this enzyme. Covalent inhibitors may also offer other advantages, including a durability of enzyme inhibition that lasts until turnover of the SARM1 protein. While our current understanding of the cellular half-life of SARM1 is incomplete, initial studies indicate that this protein is relatively long-lived in mouse neurons (half-life of ~58-64 h) (53). Consistent with this conclusion, a single treatment of MY-9B and WX-02-37 protected neurons from vacor- induced axonal degeneration for up to 48 h. Aside from their therapeutic potential, chemical probes targeting SARM1_C311 should serve as valuable tools to better understand the allosteric relationship between the protein’s regulatory (ARM) and enzymatic (TIR) domains. Lastly, the discovery of allosteric SARM1 inhibitors showcases the versatility of chemical proteomic methods for identifying cysteines in the human proteome that are proximal to druggable and functional pockets, especially those that may go unrecognized because they are allosteric in mechanism and distal from protein active sites. Toward this end, it is believed that the further expansion and screening of stereochemically defined libraries of electrophilic compounds has the potential to uncover many additional cryptic druggable and functional pockets in the human proteome. EXAMPLES [0208] The invention will be described in greater detail by way of specific examples. The following examples are offered for illustrative purposes and are not intended to limit the invention in any manner. Those of skill in the art will readily recognize a variety of non-critical parameters which can be changed or modified to yield essentially the same results. The compounds of the Examples were found to be inhibitors of SARM1 according to one or more of the assays provided herein. Sequence of human WT SARM-1 (Accession No. NP_055892): 1 mvltlllsay klcrffamsg prpgaerlav pgpdggggtg pwwaaggrgp revspgagte 61 vqdaleralp elqqalsalk qaggaravga glaevfqlve eawllpavgr evaqglcdai 121 rldggldlll rllqapelet rvqaarlleq ilvaenrdrv ariglgviln lakerepvel 181 arsvagileh mfkhseetcq rlvaagglda vlywcrrtdp allrhcalal gncalhggqa 241 vqrrmvekra aewlfplafs kedellrlha clavavlatn keverevers gtlalveplv 301 asldpgrfar clvdasdtsq grgpddlqrl vplldsnrle aqcigafylc aeaaikslqg 361 ktkvfsdiga iqslkrlvsy stngtksala kralrllgee vprpilpsvp swkeaevqtw 421 lqqigfskyc esfreqqvdg dlllrlteee lqtdlgmksg itrkrffrel telktfanys 481 tcdrsnladw lgsldprfrq ytyglvscgl drsllhrvse qqlledcgih lgvhrarilt 541 aaremlhspl pctggkpsgd tpdvfisyrr nsgsqlasll kvhlqlhgfs vfidveklea 601 gkfedkliqs vmgarnfvlv lspgaldkcm qdhdckdwvh keivtalscg knivpiidgf 661 ewpepqvlpe dmqavltfng ikwsheyqea tiekiirflq grssrdssag sdtslegaap 721 mgpt Abbreviations [0209] Commonly used abbreviations include: acetyl (Ac), azo-bis-isobutyrylnitrile (AIBN), atmospheres (Atm), 9-borabicyclo[3.3.1]nonane (9-BBN or BBN), tert- butoxycarbonyl (Boc), di-tert-butyl pyrocarbonate or boc anhydride (BOC2O), benzyl (Bn), butyl (Bu), Chemical Abstracts Registration Number (CASRN), benzyloxycarbonyl (CBZ or Z), carbonyl diimidazole (CDI), 1,4-diazabicyclo[2.2.2]octane (DABCO), diethylaminosulfur trifluoride (DAST), dibenzylideneacetone (dba), 1,5- diazabicyclo[4.3.0]non-5-ene (DBN), 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), N,N'- dicyclohexylcarbodiimide (DCC), 1,2-dichloroethane (DCE), dichloromethane (DCM), diethyl azodicarboxylate (DEAD), di-iso-propylazodicarboxylate (DIAD), di-iso- butylaluminumhydride (DIBAL or DIBAL-H), di-iso-propylethylamine (DIPEA), N,N- dimethyl acetamide (DMA), 4-N,N-dimethylaminopyridine (DMAP), N,N- dimethylformamide (DMF), dimethyl sulfoxide (DMSO), 1,1'-bis- (diphenylphosphino)ethane (dppe), 1,1'-bis-(diphenylphosphino)ferrocene (dppf), 1-(3- dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), ethyl (Et), ethyl acetate (EtOAc), ethanol (EtOH), 2-ethoxy-2H-quinoline-1-carboxylic acid ethyl ester (EEDQ), diethyl ether (Et2O), O-(7-azabenzotriazole-1-yl)-N, N,N’N’-tetramethyluronium hexafluorophosphate acetic acid (HATU), acetic acid (HOAc), 1-N-hydroxybenzotriazole (HOBt), high pressure liquid chromatography (HPLC), iso-propanol (IPA), lithium hexamethyl disilazane (LiHMDS), methanol (MeOH), melting point (mp), MeSO2- (mesyl or Ms), , methyl (Me), acetonitrile (MeCN), m-chloroperbenzoic acid (MCPBA), mass spectrum (ms), methyl t-butyl ether (MTBE), N-bromosuccinimide (NBS), N- carboxyanhydride (NCA), N-chlorosuccinimide (NCS), N-methylmorpholine (NMM), N- methylpyrrolidone (NMP), pyridinium chlorochromate (PCC), pyridinium dichromate (PDC), phenyl (Ph), propyl (Pr), iso-propyl (i-Pr), pounds per square inch (psi), pyridine (pyr), room temperature (rt or RT), tert-butyldimethylsilyl or t-BuMe2Si (TBDMS), triethylamine (TEA or Et3N), 2,2,6,6-tetramethylpiperidine 1-oxyl (TEMPO), triflate or CF3SO2- (Tf), trifluoroacetic acid (TFA), 1,1'-bis-2,2,6,6-tetramethylheptane-2,6-dione (TMHD), O-benzotriazol-1-yl-N,N,N',N'-tetramethyluronium tetrafluoroborate (TBTU), thin layer chromatography (TLC), tetrahydrofuran (THF), trimethylsilyl or Me3Si (TMS), p-toluenesulfonic acid monohydrate (TsOH or pTsOH), 4-Me-C6H4SO2- or tosyl (Ts), N- urethane-N-carboxyanhydride (UNCA),. Conventional nomenclature including the prefixes normal (n), iso (i-), secondary (sec-), tertiary (tert-) and neo have their customary meaning when used with an alkyl moiety. (J. Rigaudy and D. P. Klesney, Nomenclature in Organic Chemistry, IUPAC 1979 Pergamon Press, Oxford.). Chemistry Methods [0210] General considerations [0211] All reagents and solvents were purchased and used as received from commercial vendors or synthesized according to cited procedures. Yields refer to chromatographically and spectroscopically pure compounds, unless otherwise stated. Flash chromatography was performed using 20-40 µm silica gel (60-Å mesh) on a Teledyne ISCO Combiflash Rf or a Biotage Isolera Prime, alternatively in a glass column using SiliaFlash® F6040-63 µm silica gel (60-Å mesh). Preparative high-pressure liquid chromatography (prep-HPLC) was performed on a Gilson GX-281 instrument. Analytical thin layer chromatography (TLC) was performed on 0.2 mm or 0.25 mm silica gel 60-F plates and visualized by UV light (254 nm). Preparative thin layer chromatography (prep-TLC) was performed on GF254 plates (acrylic adhesive, 0.5×200×200 mm, 5–20 µM particle size, 250 µM thickness).1H NMR spectra were recorded on Bruker Avance III 400, Avance III HD 400, Avance Neo 400 spectrometers (1H, 400 MHz) at 300 K unless otherwise noted.13C NMR spectra were recorded on a Bruker Avance III HD 600 spectrometer (13C, 151 MHz) at 298 K.1H NMR data are reported as follows: chemical shift (δ), multiplicity (s = singlet, d = doublet, t = triplet, m = multiplet; br = broad), coupling constants, and integration. Chemical shifts are reported in parts per million (ppm) using the appropriate solvent as reference. Analytical supercritical fluid chromatography (SFC) was performed on a Shimadzu LC system (flow rate: 3 mL/min, back pressure: 100 Bar, column temperature: 35 ºC, phase A: supercritical CO2, phase B as indicated) equipped with a polydiode array detector. Tandem liquid chromatography/mass spectrometry (LC-MS) was performed on an Agilent 1200 series LC/MSD system equipped with an Agilent G6110A mass detector, alternatively a Shimadzu LC-20AD or AB series LC- MS system equipped with Shimadzu SPD-M20A or SPDM40 mass detectors, alternatively a Waters H-Class LC with equipped with diode array and QDa mass detector. Procedures for the synthesis of stereoisomeric tryptolines [0212] EV-96, EV-97, EV-98, EV-99 were prepared as reported previously (Vinogradova, E. V. et al. An Activity-Guided Map of Electrophile-Cysteine Interactions in Primary Human T Cells. Cell 182, 1009-1026 e1029, doi:10.1016/j.cell.2020.07.001 (2020)). Synthesis of MY-9B and stereoisomers
Figure imgf000057_0001
(1R,3R)-1-(benzo[d][1,3]dioxol-5-yl)-N-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4- b]indole-3-carboxamide (S2) A solution of S1 (1.50 g, 4.28 mmol) 1 in ethanol (10 mL) was degassed by purging with nitrogen (×3), and methylamine (20.3 g, 40% w/w in water) was added. The mixture was stirred at 0 °C for 1 hour, then 80 °C for 2 hours under nitrogen atmosphere. The reaction was monitored by LC-MS. Upon
Figure imgf000057_0002
completion, the reaction mixture was concentrated under reduced pressure to give a residue, which was purified by column chromatography (SiO2, petroleum ether/EtOAc = 1:0 to 1:1) to give S2 (1.45 g, 4.03 mmol, 94% yield) as a yellow solid. 1H NMR (400 MHz, CD3OD): δ 7.44 (d, J = 7.7 Hz, 1H), 7.22 (d, J = 7.9 Hz, 1H), 7.10 – 6.94 (m, 2H), 6.93 – 6.74 (m, 3H), 5.91 (s, 2H), 5.16 – 5.01 (m, 1H), 3.69 (dd, J = 11.1, 4.3 Hz, 1H), 3.11 (ddd, J = 15.1, 4.4, 1.9 Hz, 1H), 2.86 (ddd, J = 15.2, 11.2, 2.6 Hz, 1H), 2.79 (s, 3H), 3 exchangeable protons not observed. LC-MS m/z calculated for C20H20N3O3 [M+H]+ 350.1. Found 350.1. (1R,3R)-2-acryloyl-1-(benzo[d][1,3]dioxol-5-yl)-N-methyl-2,3,4,9-tetrahydro-1H- pyrido[3,4-b]indole-3-carboxamide (MY-9B) To a solution of S2 (88.0 mg, 0.252 mmol) in dichloromethane (2 mL) were added triethylamine (38 mg, 0.38 mmol, 53 μL) and acryloyl chloride (23 mg, 0.25 mmol, 21 μL). The mixture was stirred at 0 °C for 10 min. Upon reaction completion, the reaction mixture was concentrated under reduced
Figure imgf000058_0001
pressure. The resulting residue was purified by prep-HPLC (column: Waters Xbridge 150×25mm×5µm; mobile phase: [water (10mM ammonium bicarbonate)-CH3CN]; B%: 27%-57%,10 min) to obtain MY-9B (47 mg, 46% yield) as a white solid. 1H NMR (400 MHz, CD3OD): δ 7.53 (d, J = 7.8 Hz, 1H), 7.28 (d, J = 8.0 Hz, 1H), 7.13 – 7.07 (m, 1H), 7.06 – 7.00 (m, 1H), 7.03 – 6.65 (m, 5H), 6.30 (d, J = 16.7 Hz, 1H), 5.89 (s, 2H), 5.83 (d, J = 10.7 Hz, 1H), 5.24 (br. s, 1H), 3.75 – 3.45 (m, 1H), 2.96 (dd, J = 15.9, 6.6 Hz, 1H), 2.27 (br. s, 3H), 2 exchangeable protons not observed. 13C NMR (151 MHz, CD3OD) δ 169.08, 147.68, 147.28, 136.91, 134.03, 128.93, 126.19, 121.44, 118.58, 117.71, 110.65, 107.25, 106.85, 101.06, 25.15, 20.86. HRMS ESI-TOF m/z calculated for C23H22N3O4 [M+H]+ 404.1610. Found 404.1608. (1S,3S)-2-acryloyl-1-(benzo[d][1,3]dioxol-5-yl)-N-methyl-2,3,4,9-tetrahydro-1H- pyrido[3,4-b]indole-3-carboxamide (MY-9A) Prepared in analogous fashion from S2. 1H NMR (400 MHz, CD3OD): δ 7.52 (dd, J = 7.7, 1.2 Hz, 1H), 7.27 (d, J = 7.9 Hz, 1H), 7.10 (ddd, J = 8.1, 7.0, 1.3 Hz, 1H), 7.06 – 7.00 (m, 1H), 7.02 – 6.66 (m, 5H), 6.29 (d, J = 16.8 Hz, 1H), 5.89 (s, 2H), 5.83 (d, J = 10.7 Hz,
Figure imgf000058_0002
1H), 5.23 (s, 1H), 3.79 – 3.45 (m, 1H), 2.95 (dd, J = 15.8, 6.7 Hz, 1H), 2.27 (s, 3H), 2 exchangeable protons not observed. HRMS ESI-TOF m/z calculated for C23H22N3O4 [M+H]+ 404.1610. Found 404.1612. (1S,3R)-2-acryloyl-1-(benzo[d][1,3]dioxol-5-yl)-N-methyl-2,3,4,9-tetrahydro-1H- pyrido[3,4-b]indole-3-carboxamide (MY-10B) Prepared in analogous fashion from S2. 1H NMR (400 MHz, CD3OD): δ 7.41 (d, J = 7.8 Hz, 1H), 7.26 (d, J = 8.1 Hz, 1H), 7.04 (t, J = 7.5 Hz, 1H), 7.01 – 6.95 (m, 1H), 6.96 – 6.85 (m, 2H), 6.82 – 6.64 (m, 2H), 6.32 (s, 1H), 6.17 (d, J = 16.7 Hz, 1H), 5.88 (br. s, 2H),
Figure imgf000058_0003
5.75 – 5.54 (m, 1H), 5.25 (dd, J = 5.6, 3.6 Hz, 1H), 4.76 – 4.49 (m, 2H), 3.61 – 3.37 (m, 1H), 2.59 (br. s, 3H), 1 exchangeable proton not observed. LC-MS m/z calculated for C23H22N3O4 [M+H]+ 404.2. Found 404.2. HRMS ESI-TOF m/z calculated for C23H22N3O4 [M+H]+ 404.1610. Found 404.1606. (1R,3S)-2-acryloyl-1-(benzo[d][1,3]dioxol-5-yl)-N-methyl-2,3,4,9-tetrahydro-1H- pyrido[3,4-b]indole-3-carboxamide (MY-10A) Prepared in analogous fashion from S2. 1H NMR (400 MHz, CD3OD): δ 7.41 (d, J = 7.8 Hz, 1H), 7.26 (d, J = 8.1 Hz, 1H), 7.04 (t, J = 7.5 Hz, 1H), 7.01 – 6.95 (m, 1H), 6.95 – 6.86 (m, 2H), 6.81 – 6.61 (m, 2H), 6.32 (s, 1H), 6.17 (d, J = 16.7 Hz, 1H), 5.88 (br. s, 2H),
Figure imgf000059_0001
5.76 – 5.53 (m, 1H), 5.25 (t, J = 4.5 Hz, 1H), 4.59 (s, 1H), 3.47 (d, J = 13.8 Hz, 1H), 2.59 (br. s, 3H). LC-MS m/z calculated for C23H22N3O4 [M+H]+ 404.2. Found 404.2. HRMS ESI-TOF m/z calculated for C23H22N3O4 [M+H]+ 404.1610. Found 404.1604. Synthesis of propanamide controls
Figure imgf000059_0002
(1R,3R)-1-(benzo[d][1,3]dioxol-5-yl)-N-methyl-2-propionyl-2,3,4,9-tetrahydro-1H- pyrido[3,4-b]indole-3-carboxamide (WX-02-226) To a solution of S2 (50.0 mg, 0.143 mmol) in dichloromethane (2 mL) were added triethylamine (22 mg, 0.22 mmol, 30 μL) and propionyl chloride (13 mg, 0.14 mmol, 13 μL). The mixture was stirred at 0 °C for 10 min. Upon completion, the reaction mixture was concentrated under reduced pressure.
Figure imgf000059_0003
The resulting residue was purified by prep-HPLC (column: Waters Xbridge 150 mm×25 mm×5 µm; mobile phase: [water (10 mM NH4HCO3)-CH3CN]; B%: 29%-59%, 10 min) to obtain WX-02-226 (37 mg, 64% yield) as a white solid. 1H NMR (400 MHz, CD3OD): δ 7.53 (d, J = 7.8 Hz, 1H), 7.27 (d, J = 8.0 Hz, 1H), 7.12-6.95 (m, 1H), 7.09 (ddd, J = 8.2, 7.0, 1.3 Hz, 1H), 7.03 (td, J = 7.5, 1.1 Hz, 1H), 6.82 (br. s, 1H), 6.69 (br. s, 2H), 5.89 (s, 2H), 5.08 (br. s, 1H), 4.60 (br. s, 2H), 3.75-3.55 (m, 1H), 2.97 (ddd, J = 15.7, 6.4, 1.0 Hz, 1H), 2.76 – 2.54 (m, 2H), 2.20 (br. s, 3H), 1.21 (t, J = 7.3 Hz, 3H). HRMS ESI-TOF m/z calculated for C23H24N3O4 [M+H]+ 406.1761. Found 406.1756. (1S,3S)-1-(benzo[d][1,3]dioxol-5-yl)-N-methyl-2-propionyl-2,3,4,9-tetrahydro-1H- pyrido[3,4-b]indole-3-carboxamide (WX-02-225) Prepared in analogous fashion from S2. 1H NMR (400 MHz, CD3OD): δ 7.52 (d, J = 7.8 Hz, 1H), 7.26 (d, J = 8.1 Hz, 1H), 7.09 (td, J = 8.1, 7.6, 1.3 Hz, 1H), 7.06 – 6.99 (m, 2H), 6.83 (br. s, 1H), 6.76 – 6.62 (m, 2H), 5.89 (s, 2H), 5.06 (br. s, 1H), 3.79 – 3.50 (m, 1H),
Figure imgf000060_0001
2.96 (dd, J = 15.9, 6.8 Hz, 1H), 2.79 – 2.50 (m, 2H), 2.20 (br. s, 3H), 1.20 (t, J = 7.3 Hz, 3H), 2 exchangeable protons not observed. HRMS ESI-TOF m/z calculated for C23H24N3O4 [M+H]+ 406.1761. Found 406.1762. Synthesis of analogs of MY-9B Synthesis of carboxylic acid intermediates
Figure imgf000060_0002
(1R,3R)-1-(benzo[d][1,3]dioxol-5-yl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole-3- carboxylic acid (S3) To a solution of S1 (3.50 g, 9.99 mmol in MeOH (20 mL) and water (1.80 mL) was added LiOH•H2O (503 mg, 12.0 mmol).1 The mixture was stirred at 20 °C for 12 hours. Upon completion, the reaction mixture was concentrated in vacuo to give S3 (3.6 g, crude) as a white solid used in
Figure imgf000060_0003
subsequent steps without further purification. 1H NMR (400 MHz, DMSO-d6): δ 7.38 (d, J = 7.2 Hz, 1H), 7.18 (d, J = 7.2 Hz, 1H), 7.00 – 6.85 (m, 4H), 6.83 (d, J = 1.4 Hz, 1H), 5.98 (dd, J = 2.5, 0.9 Hz, 2H), 5.04 (dt, J = 4.6, 2.2 Hz, 1H), 3.23 (dt, J = 11.1, 3.5 Hz, 1H), 2.99 – 2.89 (m, 1H), 2.71 (dd, J = 4.9, 3.0 Hz, 1H), 2.59 (ddd, J = 15.1, 11.1, 2.5 Hz, 1H), 2 exchangeable protons not observed. LC-MS m/z calculated for C19H17N2O4 [M+H]+ 337.1. Found 337.1. (1S,3S)-1-(benzo[d][1,3]dioxol-5-yl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole-3- carboxylic acid (S4) Prepared in analogous fashion from S1.1 Purified by prep-HPLC (column: Phenomenex luna C18 150×40 mm×15 µm; mobile phase: [water(HCl)- acetonitrile]; B%: 9%-39%, 11 min).
Figure imgf000061_0001
1H NMR (400 MHz, DMSO-d6): δ 10.68 (s, 1H), 7.51 (d, J = 7.6 Hz, 1H), 7.26 (d, J = 8.0 Hz, 1H), 7.11 – 7.06 (m, 1H), 7.05 – 6.98 (m, 3H), 6.94 – 6.92 (m, 1H), 6.08 (d, J = 2.1 Hz, 2H), 5.69 (s, 1H), 4.32 (dd, J = 12.0, 4.7 Hz, 1H), 3.29 (dd, J = 15.4, 4.8 Hz, 1H), 3.10 (ddd, J = 15.4, 12.1, 2.4 Hz, 1H), 2 exchangeable protons not observed. LC-MS m/z calculated for C19H17N2O4 [M+H]+ 337.1. Found 337.1. (1S,3R)-1-(benzo[d][1,3]dioxol-5-yl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole-3- carboxylic acid (S5) To a solution of S1 (0.20 g, 0.57 mmol)1 in MeOH (4 mL) were added LiOH•H2O (29 mg, 0.69 mmol) and water (0.4 mL). The mixture was stirred at 20 °C for 12 hours. Upon completion, the reaction mixture was
Figure imgf000061_0002
concentrated in vacuo to give S5 (180 mg, crude) as a white solid used in subsequent steps without further purification. 1H NMR (400 MHz, DMSO-d6): δ 10.71 (s, 1H), 7.47 (d, J = 7.6 Hz, 1H), 7.25 (d, J = 8.0 Hz, 1H), 7.09 – 7.03 (m, 1H), 7.02 – 6.96 (m, 1H), 6.92 – 6.82 (m, 2H), 6.70 (dd, J = 8.0, 1.8 Hz, 1H), 6.00 (s, 2H), 5.47 (s, 1H), 3.74 (dd, J = 7.9, 5.3 Hz, 1H), 3.18 – 3.10 (m, 1H), 2.94 (dd, J = 15.4, 7.9 Hz, 1H), 2 exchangeable protons not observed. LC-MS m/z calculated for C19H17N2O4 [M+H]+ 337.1. Found 337.1. (1R,3S)-1-(benzo[d][1,3]dioxol-5-yl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole-3- carboxylic acid (S6) Prepared in analogous fashion from S1.1 . 1H NMR (400 MHz, DMSO-d6): δ 10.57 (s, 1H), 7.40 (d, J = 7.7 Hz, 1H), 7.28 – 7.12 (m, 2H), 7.03 – 6.91 (m, 2H), 6.80 (d, J = 8.0 Hz, 1H), 6.77 – 6.73 (m, 1H), 6.57 (dd, J = 8.0, 1.7 Hz, 1H), 5.96 (d, J = 2.3 Hz, 2H), 5.15 (s, 1H),
Figure imgf000061_0003
3.22 – 3.08 (m, 2H), 2.91 (dd, J = 15.3, 4.5 Hz, 1H), 2.60 (dd, J = 15.3, 9.8 Hz, 1H). LC-MS m/z calculated for C19H17N2O4 [M+H]+ 337.1. Found 337.1 Diversification of carboxylic acid intermediates The diversification of carboxylic acid intermediates S3-S6 was achieved by adapting as indicated the standard two-step procedures described below.
Figure imgf000062_0001
Step 1: amide coupling [0213] Method A. To a solution of carboxylic acid intermediate (S3–S6, 1.0 equiv) in acetonitrile/ethyl acetate (2:1, 0.3 M) were added the corresponding amine building block (1.1 equiv), pyridine (4.0 equiv) and T3P (50% w/w in ethyl acetate, 2.0 equiv). The mixture was stirred at 0 °C for 3 hours. Upon completion, the reaction mixture was diluted with water and extracted with ethyl acetate. The organic layers were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The resulting residue was purified by prep-HPLC to deliver amide S7–S10 for use in Step 2. [0214] Method B. To a solution of carboxylic acid intermediate (S3–S6, 1.0 equiv) in DMF (0.15 M) were added DIPEA (4.0 equiv), the corresponding amine building block (1.5 equiv), and HATU (2.0 equiv). The mixture was stirred at 25 °C for 2 hours. Upon completion, the reaction mixture was filtered and concentrated under reduced pressure. The resulting residue was purified by prep-HPLC to deliver amide S7–S10 for use in Step 2. Step 2: N-capping [0215] To a precooled (0 ºC) solution of amide S7–S10 (1.0 equiv) in dichloromethane (0.1 M) were added triethylamine (1.5 equiv) and acryloyl chloride (1.0 equiv). The mixture was stirred at 0 °C for 10 min. The reaction mixture was then concentrated under reduced pressure and the resulting residue was purified by prep-HPLC to deliver the title compounds. (1R,3R)-2-acryloyl-1-(benzo[d][1,3]dioxol-5-yl)-N-(prop-2-yn-1-yl)-2,3,4,9-tetrahydro- 1H-pyrido[3,4-b]indole-3-carboxamide (MY-13B) Prepared from S3 using Method B: propargyl amine (1.5 equiv), HATU (2 equiv), DIPEA (4 equiv). Title compound purified by prep-HPLC: (column: Waters Xbridge 150×25mm×5µm; mobile phase: [water (ammonium hydroxide v/v)-CH3CN]; B%: 28%-56%, 9 min).
Figure imgf000063_0001
1H NMR (400 MHz, CD3OD): δ 7.52 (d, J = 7.8 Hz, 1H), 7.28 (d, J = 8.0 Hz, 1H), 7.10 (t, J = 7.5 Hz, 1H), 7.03 (t, J = 7.3 Hz, 1H), 7.01 – 6.83 (m, 3H), 6.83 – 6.62 (m, 2H), 6.30 (d, J = 16.7 Hz, 1H), 5.92 – 5.88 (m, 2H), 5.84 (dt, J = 10.6, 2.1 Hz, 1H), 5.25 (br. s, 1H), 3.85 – 3.39 (m, 2H), 3.38 – 3.12 (m, 1H), 3.07 – 2.88 (m, 1H), 2.48 (s, 1H), 2 exchangeable protons not observed. 13C NMR (151 MHz, CD3OD) δ 169.14, 147.80, 147.43, 136.87, 133.94, 128.84, 126.13, 121.45, 118.62, 117.68, 110.68, 107.32, 106.66, 101.10, 78.71, 70.86, 28.49, 20.95. HRMS ESI-TOF m/z calculated for C25H22N3O4 [M+H]+ 428.1610. Found 428.1617. (1S,3S)-2-acryloyl-1-(benzo[d][1,3]dioxol-5-yl)-N-(prop-2-yn-1-yl)-2,3,4,9-tetrahydro- 1H-pyrido[3,4-b]indole-3-carboxamide (MY-13A) Prepared from S4 using Method A: propargyl amine (5 equiv), T3P (4 equiv), pyridine (20 equiv). Title compound purified by prep-HPLC (column: Phenomenex Gemini-NX C18 75×30mm×3µm; mobile phase: [water (0.05% ammonium hydroxide v/v)-CH3CN]; B%: 28%-58%, 7 min). 1H NMR (400 MHz, CD3OD): δ 7.52 (dt, J = 7.8, 1.0 Hz, 1H), 7.28 (dt, J = 8.2, 1.0 Hz, 1H), 7.10 (ddd, J = 8.1, 7.0, 1.3 Hz, 1H), 7.03 (ddd, J = 8.0, 7.1, 1.1 Hz, 1H), 7.02 – 6.83 (m, 3H), 6.82 – 6.62 (m, 2H), 6.31 (d, J = 16.8 Hz, 1H), 5.96 – 5.89 (m, 2H), 5.84 (dd, J = 10.7, 1.8 Hz, 1H), 5.26 (br. s, 1H), 3.90 – 3.39 (m, 2H), 3.33 – 3.10 (m, 1H), 3.11 – 2.90 (m, 1H), 2.49 (s, 1H), 2 exchangeable protons not observed. HRMS ESI-TOF m/z calculated for C25H22N3O4 [M+H]+ 428.1610. Found 428.1606. (1R,3R)-2-acryloyl-1-(benzo[d][1,3]dioxol-5-yl)-N-cyclopropyl-2,3,4,9-tetrahydro-1H- pyrido[3,4-b]indole-3-carboxamide (WX-02-37) Prepared from S3 using Method A: cyclopropyl amine (1.1 equiv), T3P (2 equiv), pyridine (4 equiv). Title compound purified by prep-HPLC (column: Phenomenex Gemini-NX C18 75×30mm×3µm; mobile phase: [water (0.05% ammonium hydroxide v/v)-CH3CN]; B%: 25%-55%, 7 min).
Figure imgf000064_0001
1H NMR (400 MHz, CD3OD): δ 7.51 (d, J = 7.9 Hz, 1H), 7.29 (dt, J = 8.1, 1.0 Hz, 1H), 7.10 (ddd, J = 8.2, 7.1, 1.3 Hz, 1H), 7.03 (ddd, J = 8.0, 7.1, 1.1 Hz, 1H), 7.00 – 6.77 (m, 3H), 6.73 (d, J = 8.0 Hz, 1H), 6.29 (d, J = 16.8 Hz, 1H), 5.91 (s, 2H), 5.82 (dd, J = 10.7, 1.8 Hz, 1H), 5.24 – 5.11 (m, 1H), 4.59 (s, 1H), 3.77 – 3.36 (m, 1H), 3.11 – 2.90 (m, 1H), 2.36 – 2.12 (m, 1H), 0.67 – 0.36 (m, 2H), 0.32 – 0.08 (m, 2H), 2 exchangeable protons not observed. 13C NMR (151 MHz, CD3OD) δ 169.28, 147.82, 147.44, 147.44, 136.84, 134.27, 128.94, 126.10, 121.43, 118.64, 117.64, 110.70, 107.47, 101.10, 22.11, 21.10, 5.06, 4.48. HRMS ESI-TOF m/z calculated for C25H24N3O4 [M+H]+ 430.1767. Found 430.1766. (1S,3S)-2-acryloyl-1-(benzo[d][1,3]dioxol-5-yl)-N-cyclopropyl-2,3,4,9-tetrahydro-1H- pyrido[3,4-b]indole-3-carboxamide (WX-02-17) Prepared from S4 using Method A: cyclopropyl amine (5 equiv), T3P (4 equiv), pyridine (20 equiv). Title compound purified by prep-HPLC (column: Phenomenex luna C18 150×25 mm×10 µm; mobile phase: [water(0.1%TFA)-CH3CN]; B%: 34%-64%,10 min and column: Waters
Figure imgf000064_0002
Xbridge 150×25 mm×5 µm; mobile phase: [water (10 mM ammonium bicarbonate)-ACN]; B%: 35%-65%,10 min) and further purified by prep-TLC (petroleum ether/ethyl acetate = 1:1). HRMS ESI-TOF m/z calculated for C25H24N3O4 [M+H] + 430.1767. Found 430.1766. 1H NMR (400 MHz, CD3OD): δ 7.51 (d, J = 7.8 Hz, 1H), 7.29 (d, J = 8.0 Hz, 1H), 7.10 (ddd, J = 8.2, 7.0, 1.3 Hz, 1H), 7.03 (td, J = 7.5, 7.1, 1.1 Hz, 1H), 7.00 – 6.79 (m, 3H), 6.73 (d, J = 8.0 Hz, 1H), 6.29 (d, J = 16.8 Hz, 1H), 5.91 (s, 2H), 5.82 (dd, J = 10.7, 1.8 Hz, 1H), 5.22 – 5.09 (m, 1H), 4.59 (s, 1H), 3.69 – 3.36 (m, 1H), 3.14 – 2.89 (m, 1H), 2.44 – 2.12 (m, 1H), 0.67 – 0.37 (m, 2H), 0.34 – 0.09 (m, 2H), 2 exchangeable protons not observed. (1S,3R)-2-acryloyl-1-(benzo[d][1,3]dioxol-5-yl)-N-cyclopropyl-2,3,4,9-tetrahydro-1H- pyrido[3,4-b]indole-3-carboxamide (WX-02-47) Prepared from S5 using Method B: cyclopropyl amine (5 equiv), HATU (1.2 equiv), DIPEA (1.5 equiv). Title compound purified by prep-HPLC (column: Phenomenex Gemini-NX C18 75×30 mm×3 µm; mobile phase: [water (0.05% ammonium hydroxide v/v)-CH3CN]; B%: 21%-51%, 11.5 min and
Figure imgf000065_0001
column: Waters Xbridge 150×25 mm×5 µm; mobile phase: [water (10 mM ammonium bicarbonate)-CH3CN]; B%: 28%-58%, 10 min). 1H NMR (400 MHz, CD3OD): δ 7.40 (d, J = 7.8 Hz, 1H), 7.26 (d, J = 8.1 Hz, 1H), 7.04 (t, J = 7.5 Hz, 1H), 6.98 (t, J = 7.5 Hz, 1H), 6.92 (br. d, J = 7.0 Hz, 2H), 6.82 – 6.73 (m, 1H), 6.69 (dd, J = 16.7, 10.6 Hz, 1H), 6.29 (s, 1H), 6.16 (d, J = 16.7 Hz, 1H), 5.88 (br. s, 2H), 5.70 – 5.56 (m, 1H), 5.38 – 5.14 (m, 1H), 3.50 – 3.33 (m, 2H), 2.53 – 2.29 (m, 1H), 0.66 – 0.47 (m, 2H), 0.45 – 0.01 (m, 2H), 2 exchangeable protons not observed. HRMS ESI-TOF m/z calculated for C25H24N3O4 [M+H]+ 430.1767. Found 430.1773. (1R,3S)-2-acryloyl-1-(benzo[d][1,3]dioxol-5-yl)-N-cyclopropyl-2,3,4,9-tetrahydro-1H- pyrido[3,4-b]indole-3-carboxamide (WX-02-27) Prepared from S6 using Method A: cyclopropyl amine (5 equiv), T3P (4 equiv), pyridine (5 equiv). Title compound purified by prep-HPLC (column: Phenomenex Synergi C18 150×25 mm×10 µm; mobile phase: [water (0.225%formic acid)-CH3CN]; B%: 35%-65%,10 min).
Figure imgf000065_0002
1H NMR (600 MHz, CD3OD): δ 7.40 (d, J = 7.9 Hz, 1H), 7.26 (d, J = 8.1 Hz, 1H), 7.04 (t, J = 7.6 Hz, 1H), 6.98 (ddd, J = 8.0, 7.0, 1.0 Hz, 1H), 6.92 (d, J = 8.0 Hz, 2H), 6.81 – 6.72 (m, 1H), 6.69 (dd, J = 16.7, 10.6 Hz, 1H), 6.29 (s, 1H), 6.22 – 6.10 (m, 1H), 5.94 – 5.83 (m, 2H), 5.69 – 5.58 (m, 1H), 5.36 – 5.11 (m, 1H), 3.60 – 3.33 (m, 2H), 2.55 – 2.36 (m, 1H), 0.64 – 0.49 (m, 2H), 0.44 – 0.07 (m, 2H), 2 exchangeable protons not observed. HRMS ESI-TOF m/z calculated for C25H24N3O4 [M+H]+ 430.1767. Found 430.1773. 1-((1R,3R)-1-(benzo[d][1,3]dioxol-5-yl)-3-(morpholine-4-carbonyl)-1,3,4,9-tetrahydro- 2H-pyrido[3,4-b]indol-2-yl)prop-2-en-1-one (WX-02-33) Prepared from S3 using Method B: morpholine (10 equiv), HATU (2 equiv), DIPEA (2 equiv). Title compound purified by prep-HPLC (column: Waters Xbridge 150×25 mm×5 µm; mobile phase: [water (10 mM ammonium bicarbonate)-CH3CN]; B%: 30%-60%, 9 min).
Figure imgf000065_0003
1H NMR (400 MHz, CD3OD): δ 7.61 – 7.43 (m, 1H), 7.29 (d, J = 8.0 Hz, 1H), 7.21 – 7.10 (m, 1H), 7.14 – 7.08 (m, 1H), 7.03 (ddd, J = 8.0, 7.1, 1.1 Hz, 1H), 6.89 – 6.73 (m, 3H), 6.56 – 6.42 (m, 1H), 6.39 (d, J = 16.6 Hz, 1H), 6.00 – 5.88 (m, 3H), 5.90 – 5.73 (m, 1H), 3.61 – 3.32 (m, 6H), 3.16 – 3.04 (m, 1H), 2.99 (ddd, J = 15.4, 6.2, 1.5 Hz, 2H), 2.59 – 2.34 (m, 1H), 1 exchangeable proton not observed. HRMS ESI-TOF m/z calculated for C26H26N3O5 [M+H]+ 460.1872. Found 460.1869. 1-((1S,3S)-1-(benzo[d][1,3]dioxol-5-yl)-3-(morpholine-4-carbonyl)-1,3,4,9-tetrahydro- 2H-pyrido[3,4-b]indol-2-yl)prop-2-en-1-one (WX-02-13) Prepared from S4 using Method B: morpholine (5 equiv), HATU (2 equiv), DIPEA (3 equiv). Title compound purified by prep-HPLC (column: Phenomenex Gemini-NX C18 75×30mm×3µm; mobile phase: [water (10 mM ammonium bicarbonate)-CH3CN]; B%: 24%-54%, 8 min).
Figure imgf000066_0001
1H NMR (400 MHz, CD3OD): δ 7.53 (d, J = 7.8 Hz, 1H), 7.29 (d, J = 8.0 Hz, 1H), 7.24 – 7.07 (m, 1H), 7.14 – 7.07 (m, 1H), 7.03 (td, J = 7.5, 1.1 Hz, 1H), 6.92 – 6.73 (m, 3H), 6.64 – 6.43 (m, 1H), 6.39 (d, J = 16.6 Hz, 1H), 5.97 – 5.89 (m, 3H), 5.90 – 5.72 (m, 1H), 3.62 – 3.34 (m, 6H), 3.18 – 3.04 (m, 1H), 3.04 – 2.94 (m, 2H), 2.60 – 2.32 (m, 1H), 1 exchangeable proton not observed. HRMS ESI-TOF m/z calculated for C26H26N3O5 [M+H]+ 460.1872. Found 460.1862. 1-((1R,3R)-1-(benzo[d][1,3]dioxol-5-yl)-3-(4-methylpiperazine-1-carbonyl)-1,3,4,9- tetrahydro-2H-pyrido[3,4-b]indol-2-yl)prop-2-en-1-one (WX-02-34) Prepared from S3 using Method A: N-methyl piperazine (1.1 equiv), T3P (2 equiv), pyridine (4 equiv). Title compound purified by prep-HPLC (column: Phenomenex Gemini-NX C18 75×30mm×3µm; mobile phase: [water (0.05%ammonium hydroxide v/v)-CH3CN]; B%: 25%-55%, 7 min). 1H NMR (400 MHz, CD3OD): δ 7.52 (d, J = 7.8 Hz, 1H), 7.32 (d, J = 8.0
Figure imgf000066_0002
Hz, 1H), 7.16 – 7.00 (m, 3H), 6.98 – 6.84 (m, 2H), 6.78 (d, J = 7.9 Hz, 1H), 6.55 (br. s, 1H), 6.37 (dd, J = 16.6, 1.8 Hz, 1H), 5.98 – 5.84 (m, 3H), 5.80 – 5.60 (m, 1H), 4.01 – 3.53 (m, 2H), 3.48 – 3.23 (m, 3H + CH3OH), 3.26 – 2.90 (m, 4H), 3.06 (dd, J = 15.5, 6.2 Hz, 1H), 2.82 (s, 3H), 1 exchangeable proton not observed. HRMS ESI-TOF m/z calculated for C27H29N4O4 [M+H]+ 473.2189. Found 473.2180. 1-((1S,3S)-1-(benzo[d][1,3]dioxol-5-yl)-3-(4-methylpiperazine-1-carbonyl)-1,3,4,9- tetrahydro-2H-pyrido[3,4-b]indol-2-yl)prop-2-en-1-one (WX-02-14) Prepared from S4 using Method A: N-methyl piperazine (5 equiv), T3P (4 equiv), pyridine (20 equiv). Title compound purified by prep-HPLC (column: Phenomenex Gemini-NX C18 75×30mm×3µm; mobile phase: [water (10mM ammonium bicarbonate)-CH3CN]; B%: 22%-52%, 8 min). 1H NMR (400 MHz, CD3OD): δ 7.52 (d, J = 7.8 Hz, 1H), 7.29 (d, J = 8.0
Figure imgf000067_0001
Hz, 1H), 7.18 – 7.07 (m, 2H), 7.07 – 7.00 (m, 1H), 6.87 – 6.79 (m, 2H), 6.73 (d, J = 8.0 Hz, 1H), 6.64 (s, 1H), 6.36 (dd, J = 16.6, 1.8 Hz, 1H), 5.97 – 5.85 (m, 3H), 5.84 – 5.60 (m, 1H), 3.54 – 3.29 (m, 3H), 3.20 – 3.04 (m, 1H), 2.98 (ddd, J = 15.5, 6.2, 1.6 Hz, 1H), 2.71 – 2.55 (m, 1H), 2.34 – 2.20 (m, 1H), 2.18 (s, 3H), 2.16 – 2.01 (m, 3H), 1 exchangeable proton not observed. HRMS ESI-TOF m/z calculated for C27H29N4O4 [M+H]+ 473.2189. Found 473.2180. (1R,3R)-2-acryloyl-1-(benzo[d][1,3]dioxol-5-yl)-N-(pyridin-2-yl)-2,3,4,9-tetrahydro-1H- pyrido[3,4-b]indole-3-carboxamide (WX-02-35) Prepared from S3 using Method B: 2-aminopyridine (1 equiv), HATU (1.2 equiv), DIPEA (1.5 equiv). Title compound purified by prep-HPLC (column: Phenomenex Luna C18 75×30mm×3µm; mobile phase: [water (0.1%TFA)-CH3CN]; B%: 32%-62%, 7 min).
Figure imgf000067_0002
1H NMR (400 MHz, CD3OD): δ 8.15 (d, J = 5.1 Hz, 1H), 7.92 (ddd, J = 9.0, 7.4, 1.9 Hz, 1H), 7.60 (d, J = 8.6 Hz, 1H), 7.56 (dt, J = 7.7, 1.1 Hz, 1H), 7.33 – 7.29 (m, 1H), 7.23 (ddd, J = 7.1, 5.5, 1.1 Hz, 1H), 7.18 – 7.02 (m, 3H), 6.76 – 6.72 (m, 1H), 6.70 (d, J = 8.0 Hz, 1H), 6.49 – 6.38 (m, 2H), 5.94 (dd, J = 10.7, 1.8 Hz, 1H), 5.71 – 5.55 (m, 3H), 4.73 – 4.44 (m, presumed 4H + H2O), 3.75 – 3.53 (m, 1H), 3.08 (dd, J = 15.7, 6.7 Hz, 1H). HRMS ESI-TOF m/z calculated for C27H23N4O4 [M+H]+ 467.1719. Found 467.1718. (1S,3S)-2-acryloyl-1-(benzo[d][1,3]dioxol-5-yl)-N-(pyridin-2-yl)-2,3,4,9-tetrahydro-1H- pyrido[3,4-b]indole-3-carboxamide (WX-02-15) Prepared from S4 using Method B: 2-aminopyridine (20 equiv), HATU (1.2 equiv), DIPEA (1.5 equiv). Title compound purified by prep-HPLC (column: Phenomenex Synergi C18150×25mm×10µm; mobile phase: [water (0.225% formic acid)-CH3CN]; B%: 36%-66%, 10 min). 1
Figure imgf000068_0001
H NMR (400 MHz, CD3OD): δ 8.09 (s, 1H), 7.82 (d, J = 8.4 Hz, 1H), 7.67 – 7.50 (m, 2H), 7.30 (d, J = 8.0 Hz, 1H), 7.38 – 6.92 (m, 5H), 6.67 (s, 2H), 6.74 – 6.22 (m, 3H), 5.95 (s, 1H), 5.84 – 5.32 (m, 3H), 3.94 – 3.46 (m, 1H), 3.20 – 2.90 (m, 1H), 2 exchangeable protons not observed. HRMS ESI-TOF m/z calculated for C27H23N4O4 [M+H]+ 467.1719. Found 467.1718. (1R,3R)-2-acryloyl-1-(benzo[d][1,3]dioxol-5-yl)-N-propyl-2,3,4,9-tetrahydro-1H- pyrido[3,4-b]indole-3-carboxamide (WX-02-36) Prepared from S3 using Method A: n-propyl amine (1.1 equiv), T3P (2 equiv), pyridine (4 equiv). Title compound purified by prep-HPLC (column: Phenomenex Gemini-NX C18 75×30mm×3µm; mobile phase: [water (0.05% ammonium hydroxide v/v)-CH3CN]; B%: 31%-61%, 7 min).
Figure imgf000068_0002
1H NMR (400 MHz, CD3OD): δ 7.52 (dt, J = 7.7, 1.1 Hz, 1H), 7.28 (dt, J = 8.1, 1.0 Hz, 1H), 7.10 (ddd, J = 8.2, 7.0, 1.3 Hz, 1H), 7.03 (ddd, J = 8.0, 7.1, 1.1 Hz, 1H), 7.04 – 6.76 (m, 4H), 6.72 (d, J = 8.0 Hz, 1H), 6.31 (d, J = 17.0 Hz, 1H), 5.90 (s, 2H), 5.83 (dd, J = 10.7, 1.8 Hz, 1H), 5.34 – 5.15 (m, 1H), 3.68 – 3.37 (m, 1H), 3.02 (dd, J = 15.5, 6.7 Hz, 1H), 2.95 – 2.82 (m, 1H), 2.74 – 2.35 (m, 1H), 1.35 – 1.14 (m, 2H), 0.76 (t, J = 7.4 Hz, 3H), 2 exchangeable protons not observed. HRMS ESI-TOF m/z calculated for C25H26N3O4 [M+H]+ 432.1923. Found 432.1918. (1S,3S)-2-acryloyl-1-(benzo[d][1,3]dioxol-5-yl)-N-propyl-2,3,4,9-tetrahydro-1H- pyrido[3,4-b]indole-3-carboxamide (WX-02-16) Prepared from S4 using Method A: n-propyl amine (5 equiv), T3P (4 equiv), pyridine (20 equiv). Title compound purified by prep-HPLC (column: Phenomenex luna C18 150×25mm×10µm; mobile phase: [water (0.1%TFA)-CH3CN]; B%: 38%-68%, 10 min and column: Waters Xbridge
Figure imgf000068_0003
150×25mm×5µm; mobile phase: [water (10mM ammonium bicarbonate)- CH3CN]; B%: 39%-69%, 10 min). 1H NMR (400 MHz, CD3OD): δ 7.52 (d, J = 7.6 Hz, 1H), 7.28 (dt, J = 8.0, 0.8 Hz, 1H), 7.10 (ddd, J = 8.2, 7.1, 1.3 Hz, 1H), 7.03 (ddd, J = 8.1, 7.0, 1.1 Hz, 1H), 7.07 – 6.78 (m, 4H), 6.72 (d, J = 8.0 Hz, 1H), 6.31 (d, J = 16.6 Hz, 1H), 5.90 (s, 2H), 5.84 (dd, J = 10.7, 1.8 Hz, 1H), 5.33 – 5.13 (m, 1H), 3.65 – 3.43 (m, 1H), 3.02 (dd, J = 15.7, 6.8 Hz, 1H), 2.96 – 2.83 (m, 1H), 2.77 – 2.35 (m, 1H), 1.39 – 1.15 (m, 2H), 0.76 (t, J = 7.5 Hz, 3H), 2 exchangeable protons not observed. HRMS ESI-TOF m/z calculated for C25H26N3O4 [M+H]+ 432.1923. Found 432.1920. (1R,3R)-2-acryloyl-1-(benzo[d][1,3]dioxol-5-yl)-2,3,4,9-tetrahydro-1H-pyrido[3,4- b]indole-3-carboxylic acid (WX-02-247) Prepared from S3 (Step 2 only). Title compound purified by prep-HPLC (column: Phenomenex Gemini-NX C18 75×30mm×3µm; mobile phase: [water (10mM ammonium bicarbonate)-CH3CN]; B%: 6%-36%, 8 min). 1
Figure imgf000069_0001
H NMR (400 MHz, CD3OD): δ 7.51 (d, J = 7.8 Hz, 1H), 7.26 (d, J = 8.0 Hz, 1H), 7.07 (ddd, J = 8.1, 7.1, 1.3 Hz, 1H), 7.01 (td, J = 7.5, 1.1 Hz, 1H), 7.05 – 6.94 (m, 1H), 6.88 (dd, J = 16.9, 10.8 Hz, 1H), 6.89 – 6.74 (m, 2H), 6.67 (d, J = 8.1 Hz, 1H), 6.20 (d, J = 16.7 Hz, 1H), 5.87 (s, 2H), 5.74 (dd, J = 10.8, 1.9 Hz, 1H), 5.13 – 5.02 (m, 1H), 3.74 – 3.45 (m, 1H), 3.20 – 2.92 (m, 1H), 2 exchangeable protons not observed. HRMS ESI-TOF m/z calculated for C22H19N2O5 [M–H]– 389.1137. Found 389.1132. (1S,3S)-2-acryloyl-1-(benzo[d][1,3]dioxol-5-yl)-2,3,4,9-tetrahydro-1H-pyrido[3,4- b]indole-3-carboxylic acid (WX-02-245) Prepared from S4 (Step 2 only). Title compound purified by prep-HPLC (column: Phenomenex Gemini-NX C18 75×30mm×3µm; mobile phase: [water (10mM ammonium bicarbonate)-CH3CN]; B%: 5%-35%, 8 min). 1
Figure imgf000069_0002
H NMR (400 MHz, CD3OD): δ 7.51 (d, J = 7.7 Hz, 1H), 7.27 (d, J = 8.0 Hz, 1H), 7.09 (ddd, J = 8.1, 7.0, 1.3 Hz, 1H), 7.06 – 7.00 (m, 1H), 6.90 (dd, J = 16.9, 10.7 Hz, 1H), 6.98 – 6.56 (m, 4H), 6.23 (d, J = 16.8 Hz, 1H), 5.88 (s, 2H), 5.77 (dd, J = 10.8, 1.9 Hz, 1H), 5.25 – 5.08 (m, 1H), 3.68 – 3.52 (m, 1H), 3.14 – 2.93 (m, 1H), 2 exchangeable protons not observed. HRMS ESI-TOF m/z calculated for C22H19N2O5 [M–H]– 389.1137. Found 389.1134. Cysteine ligandability chemical proteomic platform Sample preparation and activity probe treatment [0216] Cells (22Rv1 cells were plated into 15 cm dishes at a density of 15 million cells/dish and drug treated the next day, Ramos cells were adjusted to a density of 3x106 cells/mL into a T-25 culture flask and allowed to recover for 30 minutes in the incubator before drug treatment) were treated with 20 μM tryptoline acrylamides in situ for 3 hours, washed with ice-cold DPBS (Gibco, 14190144), flash frozen in liquid nitrogen (LN2), and stored at -80ºC until use. Cell pellets thawed on ice and lysed by probe sonication (8 pulses, 10% power, three times) in 500 μL ice-cold PBS supplemented with cOmplete protease inhibitors (Roche, 4693159001). Protein concentration of cell lysates was measured using a Pierce BCA protein assay kit (Thermo Scientific, 23225) and 500 μL (2 mg/mL protein content) were treated with 5 μL of 10 mM IA-DTB (in DMSO) for 1 hour at room temperature in the dark with intermittent vortexing. Sample preparation and activity probe treatment performed in human T-cells proteomes was previously reported (39). Further processing of proteomic samples for LC-MS/MS/MS analysis are described in detail below. Gel-based ABPP for SARM1 cysteine engagement [0217] N-terminally FLAG epitope-tagged SARM1 WT or C311A were transiently expressed in HEK293T cells, 24 hours later media was exchanged, after another 24 hours (48 hours post transfection) cells were treated in situ with compounds. Compound stocks were prepared in DMSO and sufficiently concentration so that total DMSO did not exceed 0.2%. For competition experiments, cells were first treated in situ with tryptoline acrylamides or dHNN (MedChemExpress, HY-Z0816) for 3 hours followed by treatment with 20 μM click probe for 1 hour. For non-competition experiments, cells were treated with just 20 μM click probe for 1 hour. Cells were then collected, pelleted, flash frozen in LN2, and stored at -80ºC. On the day of the experiment, cells were thawed, lysed by sonication in ice-cold DPBS supplemented with protease inhibitors, and their protein concentration normalized to 1.0 mg/mL using DC Protein Assay (BioRad, 5000112) kit. Reagents for the CuAAC click reaction were pre-mixed prior to addition to the samples, as previously described (37). Briefly, a 10x master mix containing 200 µM rhodamine-azide, 10 mM tris(2- carboxyethyl)phosphine hydrochloride (TCEP), 1 mM Tris((1-benzyl-4- triazolyl)methyl)amine (TBTA; in 4:1 tBuOH:DMSO), and 10 mM CuSO4 was made and 6 μL of CuAAC master mix was then added to 60 μL of cellular lysate. After 1 hour of click labeling, 4x SDS running buffer was added to samples. Samples were boiled for 2 minutes and then analyzed by SDS-PAGE. In gel fluorescence was performed using a BioRad ChemiDoc MP, ImageStudio Lite (Version 5.2.5) was used to quantify rhodamine band intensities, and GraphPad Prism Version 9.0.0 was used to generate IC50 curves (four- parameter variable slope least squares regression). Following in-gel fluorescence, gels were transferred to nitrocellulose at 25 V and 1.3 Å for 7 minutes. Nitrocellulose membranes were then subjected to standard western blotting procedures. Western blotting and antibodies [0218] For protein expression analysis, cells pellets were lysed by probe sonication into ice cold DPBS (Gibco, 14190144) supplemented with protease inhibitor (Roche, 4693159001) and 1 mM phenylmethylsulfonyl fluoride (PMSF). Cell lysate concentrations were determined using DC Protein Assay (BioRad, 5000112) and standardized to 1 mg/mL before the addition of 4x SDS loading dye. Western blots were performed using either pre- cast Novex™ WedgeWell™ 10%, Tris-Glycine, 1.0 mm, Mini Protein Gel (Invitrogen, XP00100PK2) or hand-poured 10% acrylamide gels and ran using tris-glycine running buffer (25 mM Tris pH 8.6, 192 mM glycine, 0.1% SDS (w/v)) at 175 V for 1 hour (pre-cast) or 275 V for 2 hours (hand-poured). Gels were transferred to nitrocellulose using a Power Blotter Cassette (Thermo Fisher) at 25 V and 1.3 A for 7 minutes. Nitrocellulose blots were blocked in milk (5% w/v in TBST (20 mM Tris pH 7.5, 150 mM NaCl, 0.1% Tween-20)) for 30 minutes at room temperature. Primary antibodies were diluted into milk (5% w/v in TBST) and incubated overnight at 4ºC with gentle rotation. Blots were washed in TBST for 5 minutes thrice followed by the addition of secondary antibodies (Li-Cor) diluted into milk (5% w/v in TBST) for 1 hour at room temperature (dilution: 1:10,000). Blots were imaged using an Li-Cor Odyssey IR imager and quantitated using ImageStudio Lite software. Primary antibodies used in this study: mouse anti-FLAG M2 antibody (Sigma, F3165, dilution 1:1,000), rabbit anti-GAPDH (Cell Signaling, 2118, dilution: 1:1,000), rabbit anti- SARM1 (Cell Signaling,13022, dilution: 1:1,000). Molecular cloning and mutagenesis Gibson assembly and Quikchange mutagenesis [0219] Human SARM1-FLAG (hSARM1), hSARM1-C311A-FLAG, and mouse SARM1-FLAG (murSARM1) plasmids used for protein overexpression were cloned into Not1 digested pcDNA5/FRT/TO mammalian expression vector (Invitrogen, V652020) via Gibson assembly (New England Biolabs, E2611S) using codon optimized inserts amplified from gBlocks obtained from IDT. hSARM1-C311S plasmid was cloned using hSARM1- FLAG pcDNA5/FRT/TO as a template via Quikchange mutagenesis. All sequences were verified via Sanger sequencing before use. Molecular cloning primers listed below. sgRNA target sequence design and cloning [0220] sgRNAs targeting human SARM1 were designed using CRISPick genetic perturbation platform (https://portals.broadinstitute.org/gppx/crispick/public, Broad Institute). Guide oligos were cloned into lentiCRISPRv2-puro vector (Addgene, 98290) using Golden Gate assembly (New England Bioscience, E1602). All guide sequences were verified via Sanger sequencing before use. Sequences for SARM1-targeted sgRNA are listedbelow. Tissue culture Cell lines and culturing methods [0221] HEK293T (ATCC, CRL-3216), SH-SY5Y (ATCC, CRL-2266), and Neuro-2a (ATCC, CCL-131) cells were maintained in DMEM supplemented with 10% v/v fetal bovine serum (FBS), 2 mM L-alanyl-L-glutamine (GlutaMax, Gibco, 35050061), penicillin (100 U mL-1), and streptomycin (100 μg mL-1).22rv1 cells (ATCC, CRL-2505) were maintained in RPMI media supplemented with 10% v/v FBS, 2 mM GlutaMax, penicillin (100 U mL-1), and streptomycin (100 μg mL-1). SARM1-KO cell lines were maintained following the same protocol as their parental cell lines outlines above. Culturing of mouse primary embryonic DRG neurons [0222] Dorsal root ganglia were dissected from E13.5-E14.5 C57BL/6J mouse embryos (RRID:IMSR_JAX:000664). Explants were cultured in 35 mm tissue culture dishes pre- coated with poly-L-lysine (20 µg/ml for 1 hr; Merck) and laminin (20 µg/ml for 1 hr; Merck) in Dulbecco's Modified Eagle's Medium (DMEM, Gibco) with 1% penicillin/streptomycin, 33 ng/ml 2.5S NGF (Invitrogen) and 2% B27 (Gibco).4 µM aphidicolin (Merck) was used to reduce proliferation and viability of small numbers of non-neuronal cells (54). Culture media was replenished every 3 days. Neurites were allowed to extend for 7 days before treatment. Culturing of rat primary embryonic DRG neurons [0223] Pregnant female Sprague Dawley rat at 15.5 days post-coitus was asphyxiated by CO2 prior to cervical dislocation. The dorsal root ganglia (DRG) were isolated from embryos and kept on ice in Leibovitz's 15 (L-15) medium (Gibco, 11415-064). DRGs were dissociated by incubation in TrypLE Express (Gibco, 12604) at 37℃ for about 30 min. L-15 medium containing 10% FBS was added to the solution and DRG solution was filtered through a 100 μm cell strainer. DRGs were then centrifuged at 1000 rpm for 5 min and resuspend in 15 mL complete medium containing neurobasal medium (2% B-27 (Gibco, 17504-044), 2 mM L- glutamine (Gibco, 25030-081), 2 μM 5-Fluoro-2'-deoxyuridine (Sigma, F0503), 2 μM uridine (Sigma, U3003), 50 ng/mL 2.5S NGF (Millipore, 01-125-100ug), and 100 U/mL Penicillin- Streptomycin (Gibco, 15140). Cell were counted and diluted in complete medium to a final concentration of 1 x 107 cells/mL. Next, 0.5 μL cell suspension was dripped into each well of poly-D-lysine/laminin pre-coated 96-well plate. Plates were incubated at 37ºC for 10-15 minutes before adding 100 μL of complete medium to each well. DRG cultures were incubated at 37ºC with 5% CO2 for 7 days before use in neurite degeneration assays. Generation of SH-SY5Y SARM1 knockout cells [0224] HEK293T (ATCC, CRL-3216) cells were plated into a 6-well dish at 3 x 105 cells/well in 2 mLs of media and allowed to attach overnight. To 200 μL of serum-free DMEM, 6 μL of Fugene (Promega, E2693) was added and incubated for 5 minutes. DMEM/Fugene mixture was then added to a 1.5 mL Eppendorf tube containing 1 μg of sgRNA-encoding lentiCRISPRv2-puro plasmid, 0.1 μg CMV-dr8.91 envelope, and 0.9 μg pCMV-VSV-G (Addgene, 8454) and incubated for 10 minutes before adding dropwise to plated HEK293T cells. Cells were transfected for 48 hours before virus was harvested. To harvest virus, the viral supernatant was collected by filtering through a 0.45 μm PVDF filter and stored at -80ºC until ready for use. The day prior to infection, SH-SY5Y cells were plated into a 6-well plate 5 x 105 cells/well in 2 mLs of media. The day of infection, 1 mL of media was removed and replaced with 1 mL of virus with 2 μL polybrene (final concentration: 1 μg/L, Santa Cruz Biotechnology, SC134220). The following day, media was removed and replaced with complete DMEM media supplemented with 0.75 μg/mL puromycin (Gibco, A1113803). Cells were grown in puromycin selection media until cells from un-transduced SH-SY5Y wells were completely dead (~4-6 days) before assaying for SARM1 knockout via Western Blot. Transfection of epitope tagged and mutant proteins of interest [0225] Transient transfection into HEK293T cells was achieved by plating cells (6-well: 3 x 105 cells/well in 3 mL media, 10 cm: 2 x 106 cells/well in 10 mL media) the day prior to transfection. The following day, cells were transfected by adding DNA (2 μg per 6-well, 10 μg per 10 cm plate) into a 1.5 mL Eppendorf tube and adding serum-free DMEM (500 μL for 6-well, 1 mL for 10 cm plate) followed by addition of the transfection reagent polyethylenimine (PEI, 1 mg/mL) using a 1:3 ratio of DNA (μg) to PEI (μL). Cells were transfected for 48 hours, with a media exchange at 24 hours, before assaying or collection. SARM1 LC-MS/MS NADase assay SARM1 exogenous substrate assay [0226] HEK293T cells expressing recombinant SARM1 (see Tissue culture methods for detailed transfection protocol) were treated in situ with tryptoline acrylamide for 3 hours (0.1% DMSO). Following drug treatment, cells were collected in a 1.5 mL Eppendorf tube, pelleted by centrifugation (800 g, 3 minutes), flash frozen in LN2, and stored at -80ºC. Cells pellets thawed on ice and lysed by probe sonication into ice cold DPBS supplemented with protease inhibitor and 1 mM phenylmethanesulfonyl fluoride (PMSF; Sigma, 10837091001). Cell lysate concentrations were determined using DC Protein Assay and standardized to 1 mg/mL. Cell lysates (100 μL) were added to a 1.5 mL Eppendorf tube and incubated with 1 mM NMN (Sigma, N3501) for 10 minutes followed by the addition of 100 μM NAD+ (Sigma, N1511) for 45 minutes. Samples were quenched by the addition of 400 μL ice cold methanol:acetonitrile (1:1) with 100 pmol internal standard 8-Br-cADPR (Enzo Life Sciences, BML-CA417-0500). Samples were vortexed for 30 seconds and frozen in LN2. To precipitate proteins, samples were then thawed at room temperature and sonicated for 15 minutes in an ice-cold ultrasonic bath sonicator. Following sonication, samples were incubated at -20ºC for 1 hour and then centrifuged at 16,000 g for 15 minutes at 4ºC to pellet precipitated proteins.100 μL of the supernatant was transferred to an LC-MS/MS vial for metabolomic analysis. SARM1 endogenous substrate assay [0227] SH-SY5Y cells were treated in situ with tryptoline acrylamide for 3 hours (0.1% DMSO) followed by treatment with 50 μM vacor (Sigma, S668923) for 4 hours. Following drug treatment, cells were collected in a 1.5 mL Eppendorf tube, pelleted by centrifugation (800 g, 3 minutes), flash frozen in LN2, and stored at -80ºC. Cells pellets thawed on ice and resuspended in 100 μL of DPBS. Immediately after resuspension, 400 μL ice cold methanol:acetonitrile (1:1) with the internal standard 8-Br-cADPR (100 pmol) was added to the samples. Samples were vortexed for 30 seconds and frozen in an LN2 bath. Samples were then thawed at room temperature and sonicated for 15 minutes in an ice-cold ultrasonic bath sonicator. Freezing in LN2 and ice-cold sonication was repeated twice more before incubating at -20ºC for 1 hour and finally centrifuging at 16,000 g for 15 minutes at 4ºC to pellet precipitated proteins. The supernatant was transferred to a new tube and dried in vacuo using a SpeedVac vacuum concentrator. Dried samples were then resuspended in 75 μL methanol:acetonitrile:water (2:2:1) and bath sonicated for 15 minutes before transferring into an LC-MS/MS vial. To account for total protein input per sample, protein pellets were resuspended in DPBS and lysed by probe sonication. Total protein concentrations were then determined using DC Protein Assay. Metabolite measurements were from LC-MS/MS analysis were normalized by the total amount of protein per sample. Details on LC-MS/MS measurement of NAD metabolites can be found below. Axonal Degeneration Studies Vacor induced axonal degeneration studies (16) [0228] Vacor was dissolved in DMSO; quantitation of the dissolved stock was performed spectrophotometrically (ɛ340nm 17.8 mM-1cm-1). DRG explants were treated at day in vitro (DIV) 7 with vacor (Greyhound chromatography) or vehicle (DMSO) just prior to imaging (time 0h). Phase contrast images were acquired on a DMi8 upright fluorescence microscope (Leica microsystems) using a HCXPL 20X/0.40 objective coupled to a monochrome digital camera (Hamamatsu C4742-95). The degeneration index was determined using a Fiji plugin (55). For each experiment, the average was calculated from three fields per condition; the total number of experiments and the drug concentrations are indicated in the figures and figure legends Vincristine induced axonal degeneration studies [0229] Test compounds were prepared as 3-folds serial dilutions with 100% (v/v) DMSO from a 10 mM stock. Serial dilutions were then subsequently diluted again, 500-fold, using complete medium. Vincristine (MCE, HY-N0488) solution was prepared by diluting a 40 μM stock solution 100-fold (400 nM) using complete medium. Half the media (50 μL) from DRG culture plate was removed and replaced with 50 μL compound solution to each well of cell plate, in which final concentrations of test compounds were 10, 3.33, 1.11, 0.37, 0.12, 0.041, 0.014 and 0.005 μM. Compounds were incubated with DRG neurons for 3 hours prior to the addition of vincristine to each well (11.1 μL, 40 nM final concentration). Low control (DMSO treated DRG neurons) and high control (DMSO + vincristine treated neurons) were prepared by dilution of 100% DMSO and 40 μM Vincristine using complete medium, respectively. Then 11.1 μL was added to each well of a cell plate (10% of final culture volume), in which final concentration of DMSO was 0.1%. Drug treated DRG cultures were incubated for 48 hours at 37ºC. After 48 hours, media was aspirated from the plate and each well was washed twice with 100 μL DPBS. Cells were then fixed with 40 μL 4% PFA (Sorblab, P1110) for 10 min at room temperature. Following fixation, PFA solution was aspirated, and cells were washed thrice with 100 μL ice-cold DPBS. Cells were permeabilized with 40 μL 0.5% Triton-X (Sigma, T9284) for 10 minutes. Permeabilization solution was aspirated, and cells were briefly washed thrice with 100 μL DPBS for 5 minutes per wash. Samples were then blocked for 30 minutes in DPBS with 5% FBS (Gibco, 10099141), 2% BSA (Sigma, A1933), and 0.1% Tween-20 (Sigma, P1379) before incubation with anti-beta III Tubulin (Abcam, ab41489) and anti-NeuN (Abcam, ab104225) primary antibodies overnight at 4ºC. After primary antibody incubation, cells were washed thrice with 100 μL DPBS for 5 minutes per wash and then incubated with Goat Anti-Chicken IgY H&L (Abcam, ab150169) and Goat Anti-Rabbit IgG H&L (Abcam, ab150080) secondary antibodies for 2 hours at room temperature. Cells were then washed four times with 100 μL DPBS for 5 minutes per wash and each well was sealed with 50 μL of 90% glycerol. Images from the DRG neurons were then acquired using high content a High Content Analysis System (Perkin Elmer, Operetta CLSTM). Details on the calculation of axonal degeneration can be found below. Protein precipitation, denaturation, reduction, and alkylation [0230] After activity probe treatment, samples were then precipitated with the addition of 600 μL ice-cold MeOH, 200 μL CHCl3, and 100 μL water (in order), then vortexed for 10 seconds, and centrifuged (10 min, 16,000 g). Without perturbing the protein disk, both top and bottom layers were aspirated, and the protein disk was washed with 1 mL ice-cold MeOH, followed by centrifugation (10 min, 16,000 g). Protein pellets were allowed to air dry briefly until solvent droplets are no longer visible (5 minutes). Samples were re-suspended in 90 μL denaturation buffer (9 M urea, 10 mM DTT, 50 mM TEAB pH 8.5). Samples were reduced by heating at 65ºC for 20 minutes and water bath sonicated as needed to resuspend the protein pellets, followed by alkylation via addition of 10 μL (500 mM) iodoacetamide (Sigma, I1149) and incubated at 37ºC for 30 min with shaking. Samples were diluted with 300 μL buffer (50 mM TEAB pH 8.5) to reach final concentration of 2 M urea. To ensure that samples are completely dissolved, samples were briefly centrifuged, and probe sonicated (1x, 10 pulses, 10% power). Tryptic digestion and streptavidin enrichment [0231] Trypsin (4 μL of 0.25 μg/μL in trypsin resuspension buffer with 25 mM CaCl2) was added to each sample and digested at 37ºC with shaking overnight. Streptavidin-agarose beads (Thermo Scientific, 20353) were prepared by washing twice in 10x bead volumes of wash buffer (50 mM TEAB pH 8.5, 150 mM NaCl, 0.2% NP-40) and resuspended to give 25 μL beads per 300 μL wash buffer. Trypsin digested samples were then diluted with 300 μL wash buffer (50 mM TEAB pH 8.5, 150 mM NaCl, 0.2% NP-40) containing streptavidin- agarose beads (25 μL beads) and were rotated at room temperature for 2 hours. Enriched samples were transferred to BioSpin columns (BioRad, 732-6204) and washed (3x 1 mL wash buffer, 3x 1 mL DPBS, 3x 1mL water). Enriched peptides were eluted by addition of 400 μL 50% acetonitrile with 0.1% formic acid and eluate was evaporated to dryness via speedvac. Tandem mass tag (TMT) labeling of enriched peptides [0232] IA-DTB labeled and enriched peptides were resuspended in 100 μL EPPS buffer (140 mM, pH 8.0) with 30% acetonitrile, vortexed, and water bath sonicated. Samples were TMT (Thermo Scientific, 90406) labeled with 3 μL of corresponding TMT tag (5 mg tag resuspended in 256 μL acetonitrile), vortexed, and incubated at room temperature for 1 hour. TMT labeling was quenched with the addition of hydroxylamine (5 μL 5% solution in H2O) and incubated for 15 minutes at room temperature. Samples were then acidified with 5 μL formic acid, combined, and dried via speedvac. Finally, samples were desalted via Sep-Pak C18 cartridge (Waters, WAT051910) and then high pH fractionated before LC-MS/MS/MS analysis. High pH HPLC fractionation [0233] Samples were resuspended in 500 μL resuspension buffer (95% water, 5% ACN, 0.1% FA) and fractionated into a 96 deep-well plate via HPLC (Agilent Infinity 1260 II LC system). Aqueous stationary phase (Buffer A) used is 10 mM aqueous NH4HCO3, organic mobile phase (Buffer B) used is 100% acetonitrile. Peptides were loaded onto a capillary column (ZORBAX 300Extend-C18, 3.5 μm) at a flow rate of 0.5 mL/min and eluted using the following gradient: 0-2 min, 100% Buffer A; 2-3 minutes, 0%–13% buffer B; 3-60 minutes, 13%–42% buffer B; 60-61 minutes, 42%–100% buffer B; 61-65 minutes, 100% buffer B; 65-66 minutes, 100%–0% buffer B; 66-75 minutes, 100% buffer A; 75-78 minutes, 0%–13% buffer B; 78-80 minutes, 13%–80% buffer B; from 80-85 minutes, 80% buffer B; 86-91 minutes, 100% buffer A; 91-94 minutes, 0%–13% buffer B 94-96 minutes, 13%–80% buffer B; 96-101 minutes, 80% buffer; 101-102 minutes, 80%–0% buffer B. Each well in the 96-well plate contained 20 μL of 20% formic acid to acidify the eluting peptides. The eluents were evaporated to dryness in the plate using via speed vac. In the plate, the top row (Row A, wells 1-12) were resuspended in 80% acetonitrile, 0.1% formic acid buffer (200 μL/well). Resuspended peptides solution from row A, was then added to row B, then row C, etc. to combine every 12th fraction. Resuspension process was repeated three times and the twelve fractions were dried via speecvac. The resulting twelve fractions were re-suspended in resuspension buffer and analyzed by LC-MS/MS/MS. TMT liquid chromatography-mass-spectrometry (LC-MS) analysis [0234] Samples were analyzed by liquid chromatography tandem mass-spectrometry using an Orbitrap Fusion mass spectrometer (Thermo Scientific) coupled to an UltiMate 3000 Series Rapid Separation LC system and autosampler (Thermo Scientific Dionex). The peptides were loaded onto a capillary column (75 μm inner diameter fused silica, packed with C18 (Waters, Acquity BEH C18, 1.7 μm, 25 cm)) or an EASY-Spray HPLC column (Thermo ES902, ES903) using an Acclaim PepMap 100 (Thermo 164535) loading column a flow rate of 0.25 μL/min, and separated using the following gradient: 0-15 minutes, 5% buffer B; 15- 55 minutes, 5%–35% buffer B; 155-160 minutes, 35%–95% buffer B; 160-169 minutes, 95% buffer B; 169-170 minutes, 95%–5% buffer B; 170-200 minutes, 5% buffer B, where buffer A: 95% H2O, 5% acetonitrile, 0.1% FA and buffer B: 5% H2O, 95% CH3CN, 0.1% FA. Data was acquired using an MS3-based TMT method on Orbitrap Fusion or Orbitrap Eclipse Tribrid Mass Spectrometers. Briefly, the scan sequence began with an MS1 master scan (Orbitrap analysis, resolution 120,000, 400−1700 m/z, RF lens 60%, automatic gain control [AGC] target 2E5, maximum injection time 50 ms, centroid mode) with dynamic exclusion enabled (repeat count 1, duration 15 s). The top ten precursors were then selected for MS2/MS3 analysis. MS2 analysis consisted of: quadrupole isolation (isolation window 0.7) of precursor ion followed by collision-induced dissociation (CID) in the ion trap (AGC 1.8E4, normalized collision energy 35%, maximum injection time 120 ms). Following the acquisition of each MS2 spectrum, synchronous precursor selection (SPS) enabled the selection of up to 10 MS2 fragment ions for MS3 analysis. MS3 precursors were fragmented by HCD and analyzed using the Orbitrap (collision energy 55%, AGC 1.5E5, maximum injection time 120 ms, resolution was 50,000). For MS3 analysis, charge state–dependent isolation windows were used. For charge state z = 2, the MS isolation window was set at 1.2; for z = 3-6, the MS isolation window was set at 0.7. The MS2 and MS3 files were extracted from the raw files using RAW Converter (version 1.1.0.22; available at http://fields.scripps.edu/rawconv/), uploaded to Integrated Proteomics Pipeline (IP2), and searched using the ProLuCID algorithm (this information is readily publicly available at http://fields.scripps.edu/downloads.php) using a reverse concatenated, non-redundant variant of the at human UniProt database (release 07-2016) and mouse UniProt dataset (release 07- 2017). Cysteine residues were searched with a static modification for carboxyamidomethylation (+57.02146 Da). For cysteine profiling experiments, a dynamic modification for IA-DTB labeling (+398.25292 Da) was included with a maximum number of 2 differential modifications. N-termini and lysine residues were also searched with a static modification corresponding to the TMT tag (+229.1629 Da). Peptides were required to be at least 6 amino acids long, to have at least one tryptic terminus, and to contain the DTB modification. ProLuCID data was filtered through DTASelect (version 2.0) to achieve a peptide false-positive rate below 1%. The MS3-based peptide quantification was performed with reporter ion mass tolerance set to 20 ppm with Integrated Proteomics Pipeline (IP2). Data processing [0235] Inhibition of IA-DTB labeling (DMSO vs. compound treatment) was calculated for each peptide-spectra match by dividing each TMT reporter ion intensity by the average intensity for the channels corresponding to DMSO treatment (see Equation 1). Peptide- spectra matches were then grouped based on protein ID and residue number (e.g. SARM1_C311). The following filters were applied to remove low-quality peptides: peptides with summed reporter ion intensities for the DMSO channels < 10,000, coefficient of variation for DMSO channels > 0.5, and non-tryptic peptide sequences were excluded. Cysteine engagement ratios for waterfall plots were determined via Equation 2. Cysteines were considered significantly liganded if they inhibited IA-DTB labeling ≥ 75% (equivalent to a cysteine engagement ratio of ≥ 4). Cysteines were considered stereoselective liganded if they displayed a log2(active/inactive) > 1.5 and a -log10(p-value) > 1.5.
Figure imgf000079_0001
Equation 2:
Figure imgf000079_0002
Molecular cloning primers, mutagenesis primers, and sgRNA sequences Human SARM1(WT and C311A) Gibson primers: Forward primer:
Figure imgf000080_0001
Reverse primer:
Figure imgf000080_0002
Mouse SARM1 Gibson primers: Forward primer:
Figure imgf000080_0003
Reverse primer: 3’- ’
Figure imgf000080_0004
SARM1-C311S Quikchange primers: hSARM1-C311S-fwd:
Figure imgf000080_0005
hSARM1-C311S-rev:
Figure imgf000080_0006
SARM1-targeted sgRNA sequences (all sequences are displayed 5’ to 3’):
Figure imgf000080_0007
LC-MS/MS measurement of NAD metabolites [0236] NAD+, ADPR, and cADPR metabolite species were measured using LC-MS/MS. Samples were injected onto HILIC column (Acquity UPLC BEH Amide column; 1.7 μM; 2.1 x 100 mM; Waters, 186004801) using HPLC (Agilent 1290 Infinity LC) with a flow rate of 0.4 mL/minute. Aqueous buffer (Buffer A) contained 5% acetonitrile, 10 mM ammonium formate, and 0.1% formic acid. Organic buffer (Buffer B) contained 95% acetonitrile, 10 mM ammonium formate, and 0.1% formic acid. Metabolites were eluted using the following gradient: 0-2 minutes, 100% Buffer B; 2-14 minutes 100-65% Buffer B; 14-17 minutes, 65- 40% Buffer B; 17-18 minutes, 40% Buffer B; 18-19 minutes, 40-100% Buffer B; 19-22 minutes, 100% Buffer B. Eluted metabolites were detected using a triple quad mass spectrometer (Agilent 6470 MassHunter; Agilent) via multiple reaction monitoring (MRM) using an electrospray ionization (ESI) source in positive mode. MS analysis was performed using ESI with the following parameters: gas temperature: 350ºC; gas flow: 11 L/min; nebulizer: 45 psi; sheath gas temperature: 450ºC; sheath gas flow: 12 L/min; capillary: 12 V; nozzle voltage/charging: 1500 V. MRM transitions were specific for each measure metabolite, NAD+ (precursor ion: 664.1, product ion: 136, dwell: 50, fragmentation (F): 100 V, collision (C): 53 V, collision acceleration (CA) : 4 V), ADPR (precursor ion: 560.1, product ion: 136, dwell: 50, F: 100 V, C: 41 V, CA: 4 V), cADPR (precursor ion: 542.1, product ion: 136, dwell: 50, F: 100 V, C: 49 V, CA: 4 V), 8-Br-cADPR (precursor ion: 620, product ion: 214, dwell: 50, F: 100 V, C: 33 V, CA: 4 V). Metabolites were quantified by Masshunter quantitative analysis (version 10.0, Agilent) by integrating their peak area and normalizing relative to the peak area of the internal standard, 8-Br-cADPR as follows:
Figure imgf000081_0001
Quantification of vincristine-induced axonal degeneration The % axonal degeneration of each well was calculated following the formula below:
Figure imgf000081_0002
P= % degeneration of each well Sdeg = degeneration area (spots) of nerve fibers Stotal = total area (whole fiber + spots) of nerve fibers Percent degeneration was then corrected following the formula below:
Figure imgf000082_0001
Protection EC50 was determined by fitting % degeneration against compound concentration and fit using the Hill equation in GraphPad Prism (v.8.0) and fit to a sigmoid-dose response curve with a variable slope. References: 1. J. T. Wang, Z. A. Medress, B. A. Barres, Axon degeneration: molecular mechanisms of a self-destruction pathway. J Cell Biol 196, 7-18 (2012). 2. L. Conforti, J. Gilley, M. P. Coleman, Wallerian degeneration: an emerging axon death pathway linking injury and disease. Nat Rev Neurosci 15, 394-409 (2014). 3. M. P. Coleman, A. Hoke, Programmed axon degeneration: from mouse to mechanism to medicine. Nat Rev Neurosci 21, 183-196 (2020). 4. Y. J. Osterloh JM, Rooney TM, Fox AN, Adalbert R, Powell EH, Sheehan AE, Avery MA, Hackett R, Logan MA, MacDonald JM, Ziegenfuss JS, Milde S, Hou YJ, Nathan C, Ding A, Brown RH Jr, Conforti L, Coleman M, Tessier-Lavigne M, Züchner S, Freeman MR. , dSarm/Sarm1 Is Required for Activation of an Injury-Induced Axon Death Pathway. 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The scope of the disclosure should, therefore, be determined not with reference to the above description, but should instead be determined with reference to the following appended claims, along with the full scope of equivalents to which such claims are entitled. [0238] This application refers to various issued patents, published patent applications, journal articles, and other publications, each of which are incorporated herein by reference.

Claims

WHAT IS CLAIMED IS: 1. A method of inhibiting the NADase activity of SARM1, comprising contacting SARM1 with a tryptoline acrylamide derivative.
2. The method of Claim 1, wherein the tryptoline acrylamide derivative reacts with C311 in the ARM domain of SARM1.
3. The method of either Claim 1 or Claim 2, wherein the tryptoline acrylamide derivative reacts stereospecifically and site-specifically with C311 in the ARM domain of SARM1.
4. The method of any one of Claims 1-3, wherein the tryptoline acrylamide derivative covalently binds to C311 in the ARM domain of SARM1.
5. The method of any one of Claims 2-4 wherein the reaction of the tryptoline acrylamide derivative with C311 in the ARM domain of SARM1 allosterically inhibits the NADase activity of SARM1.
6. The method of any one of Claims 1-5, wherein the inhibition of the NADase activity of SARM1 prevents axonal degeneration.
7. The method of Claim 6, wherein the prevention of axonal degeneration promotes maintenance of neuronal integrity.
8. The method of any one of Claims 1-7, wherein the inhibition of the NADase activity of SARM1 prevents or ameliorates a neurodegenerative disorder.
9. The method of Claim 8, wherein the neurodegenerative disorder is selected from: spinal muscular atrophy (SMA), Chemotherapy Induced Peripheral Neuropathy, multiple sclerosis (MS), traumatic brain injury (TBI), spinal cord injury, stroke, Parkinson' disease, glaucoma, Huntington's disease, Alzheimer's disease, Charcot-Marie-Tooth disease (CMT), retinitis pigmentosa (RP), age-related macular degeneration (AMD), small fiber neuropathies, peripheral neuropathy (e.g., viral neuropathy), spinocerebellar ataxias, cystic fibrosis, familial amyloidotic polyneuropathy, spongiform encephalopathies, spinal and bulbar muscular atrophy, hereditary dentatorubral-pallidoluysian atrophy, adrenoleukodystrophy, adrenomyeloneuropathy, Alexander's disease, amyotrophic lateral sclerosis (ALS), Bassen- Kornzweig syndrome, Bell's palsy, progressive supra nuclear palsy (PSP), central pontine myelolysis, cluster headache, congenital hypomyelination, corticobasal degeneration, Creutzfeldt-Jakob disease, epilepsy, dementia (e.g., frontotemporal dementia and Lewy body dementia), demyelination disorders (e.g., ischemic demyelination), encephalomyelitis, Friedrich's ataxia, Gaucher's disease, hereditary sensory and autonomic neuropathy (HSAN), Hurler syndrome, Krabbe's disease, metachromatic leukodystrophy, migraine and tension headaches, mild cognitive impairment, motor spinoneuron disease, neuromyelitis optica, Niemann-Pick disease, optic neuritis, Pelizaeus Merzbacher disease, peripheral neuropathy, periventricular leukomalacia, post-herpetic neuralgia, prion disease, progressive supranuclear palsy, progressive multifocal leukoencephalopathy, Tay-Sacks disease, thoracic disc herniation, traverse myelitis, trigeminal neuralgia, Wallerian degeneration, cerebellar degeneration, chiari malformation, dystonia, encephalitis (e.g., pediatric viral encephalitis and La Crosse virus encephalitis), hyperekplexia, multifocal motor neuropathy, muscular dystrophy, myasthenia gravis, myopathy, neurofibromatosis, neuronal ceroid lipofuscinosis, neuropathies (e.g., peripheral neuropathy), pseudobulbar affect, restless legs syndrome, spina bifida, syringomyelia, thoracic outlet syndrome, and transverse myelitis.includes, but is not limited to, amyotrophic lateral sclerosis (ALS), multiple sclerosis, Parkinson's disease, Alzheimer's disease, Huntington's disease, multiple system atrophy, prion diseases, and chemotherapy-induced peripheral neuropathy.
10. The method of claim 9, wherein the neurodegenerative disorder is ALS, Alzheimer’s Disease, or chemotherapy-induced peripheral neuropathy.
11. A tryptoline acrylamide derivative compound of Formula I,
Figure imgf000088_0001
wherein: R is optionally substituted -O(C2-C6)alkyl, -NH(C1-C6)alkyl, -NH(C3-C6)cycloalkyl, -(C3- C6)heterocycloalkyl, or -NH(C5-C6)heteroaryl; or a pharmaceutically acceptable salt or prodrug thereof.
12. The tryptoline acrylamide derivative compound of Formula I of Claim 11, wherein R is -NHMe, cyclopropylamino, pyridinylamino, or propylamino.
13. The tryptoline acrylamide derivative compound of Formula I of Claim 12, wherein Formula I has the following structure:
Figure imgf000089_0003
14. The tryptoline acrylamide derivative compound of Formula I of Claim 12, wherein Formula I has the following structure:
Figure imgf000089_0002
.
15. The tryptoline acrylamide derivative compound of Formula I of Claim 12, wherein Formula I has the following structure:
Figure imgf000089_0001
wherein R is -O(C2-C6)alkyl.
16. The tryptoline acrylamide derivative compound of Formula I of Claim 12, wherein Formula I has the following structure:
Figure imgf000090_0002
17. The tryptoline acrylamide derivative compound of Formula I of Claim 12, wherein Formula I has the following structure:
Figure imgf000090_0001
18. A method of inhibiting the NADase activity of SARM1, comprising contacting the SARM1 with the tryptoline acrylamide derivative compound of Formula I of any one of Claims 11-17.
19. The method of Claim 18, wherein the tryptoline acrylamide derivative compound covalently binds C311 in the ARM domain of SARM1.
20. The method of Claim 19, wherein the tryptoline acrylamide derivative compound site- specifically and covalently binds C311 of SARM1.
21. The method of Claim 20, wherein the tryptoline acrylamide derivative compound site- specifically, stereoselectively, and covalently binds C311 of SARM1.
22. The method of Claim 21, wherein the tryptoline acrylamide derivative compound allosterically inhibits SARM1.
23. A method of inhibiting the NADase activity of SARM1, comprising contacting the SARM1 with the tryptoline acrylamide derivative compound of Formula I of any one of Claims 11-17.
24. The method of Claim 23, wherein the inhibition of the NADase activity of SARM1 prevents or ameliorates a SARM1-mediated disorder.
25. A method of treating a SARM1-mediated disorder, comprising administering to a patient in need thereof a therapeutically effective amount of the tryptoline acrylamide derivative compound of Formula I of any one of Claims 11-17.
26. The method of claim 25, wherein the SARM1-mediated disorder is a neurodegenerative disorder.
27. The method of Claim 26, wherein the neurodegenerative disorder is selected from: spinal muscular atrophy (SMA), Chemotherapy Induced Peripheral Neuropathy, multiple sclerosis (MS), traumatic brain injury (TBI), spinal cord injury, stroke, Parkinson' disease, glaucoma, Huntington's disease, Alzheimer's disease, Charcot-Marie-Tooth disease (CMT), retinitis pigmentosa (RP), age-related macular degeneration (AMD), small fiber neuropathies, peripheral neuropathy (e.g., viral neuropathy), spinocerebellar ataxias, cystic fibrosis, familial amyloidotic polyneuropathy, spongiform encephalopathies, spinal and bulbar muscular atrophy, hereditary dentatorubral-pallidoluysian atrophy, adrenoleukodystrophy, adrenomyeloneuropathy, Alexander's disease, amyotrophic lateral sclerosis (ALS), Bassen- Kornzweig syndrome, Bell's palsy, progressive supra nuclear palsy (PSP), central pontine myelolysis, cluster headache, congenital hypomyelination, corticobasal degeneration, Creutzfeldt-Jakob disease, epilepsy, dementia (e.g., frontotemporal dementia and Lewy body dementia), demyelination disorders (e.g., ischemic demyelination), encephalomyelitis, Friedrich's ataxia, Gaucher's disease, hereditary sensory and autonomic neuropathy (HSAN), Hurler syndrome, Krabbe's disease, metachromatic leukodystrophy, migraine and tension headaches, mild cognitive impairment, motor spinoneuron disease, neuromyelitis optica, Niemann-Pick disease, optic neuritis, Pelizaeus Merzbacher disease, peripheral neuropathy, periventricular leukomalacia, post-herpetic neuralgia, prion disease, progressive supranuclear palsy, progressive multifocal leukoencephalopathy, Tay-Sacks disease, thoracic disc herniation, traverse myelitis, trigeminal neuralgia, Wallerian degeneration, cerebellar degeneration, chiari malformation, dystonia, encephalitis (e.g., pediatric viral encephalitis and La Crosse virus encephalitis), hyperekplexia, multifocal motor neuropathy, muscular dystrophy, myasthenia gravis, myopathy, neurofibromatosis, neuronal ceroid lipofuscinosis, neuropathies (e.g., peripheral neuropathy), pseudobulbar affect, restless legs syndrome, spina bifida, syringomyelia, thoracic outlet syndrome, and transverse myelitis.includes, but is not limited to, amyotrophic lateral sclerosis (ALS), multiple sclerosis, Parkinson's disease, Alzheimer's disease, Huntington's disease, multiple system atrophy, prion diseases, and chemotherapy-induced peripheral neuropathy.
28. The method of claim 27, wherein the neurodegenerative disorder is ALS, Alzheimer’s Disease, or chemotherapy-induced peripheral neuropathy.
29. A composition comprising the compound of Formula I of any one of Claims 11-17, admixed with at least one carrier, diluent or excipient.
30. The composition of Claim 29, further comprising another pharmaceutically active compound.
31. The composition of either Claim 29 or Claim 30, further comprising another anti- neurodegenerative compound.
32. The composition of any one of Claims 29-31, further comprising another SARM1- inhibiting compound.
33. A composition comprising the compound of Formula I of any one of Claims 13-15, admixed with at least one carrier, diluent or excipient.
34. The composition of Claim 34, further comprising another pharmaceutically active compound.
35. The composition of either Claim 33 or Claim 34, further comprising another anti- neurodegenerative compound.
36. The composition of any one of Claims 33-35, further comprising another SARM1- inhibiting compound.
37. Any methods of inhibiting the NADase activity of SARM1, electrophilic tryptoline acrylamide derivative compounds, methods of treating SARM1-mediated disorders, or compositions comprising the tryptoline acrylamide derivative compound of Formula I, as described herein.
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