Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D215/00—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems
  • C07D215/02—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen atoms or carbon atoms directly attached to the ring nitrogen atom
  • C07D215/16—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen atoms or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
  • C07D215/38—Nitrogen atoms
  • C07D215/42—Nitrogen atoms attached in position 4
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D215/00—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems
  • C07D215/02—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen atoms or carbon atoms directly attached to the ring nitrogen atom
  • C07D215/04—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen atoms or carbon atoms directly attached to the ring nitrogen atom with only hydrogen atoms or radicals containing only hydrogen and carbon atoms, directly attached to the ring carbon atoms
  • C07D215/10—Quaternary compounds
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D215/00—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems
  • C07D215/02—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen atoms or carbon atoms directly attached to the ring nitrogen atom
  • C07D215/16—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen atoms or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
  • C07D215/20—Oxygen atoms
  • C07D215/24—Oxygen atoms attached in position 8
  • C07D215/26—Alcohols; Ethers thereof
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D215/00—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems
  • C07D215/02—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen atoms or carbon atoms directly attached to the ring nitrogen atom
  • C07D215/16—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen atoms or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
  • C07D215/38—Nitrogen atoms
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D221/00—Heterocyclic compounds containing six-membered rings having one nitrogen atom as the only ring hetero atom, not provided for by groups C07D211/00 - C07D219/00
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
  • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
  • C07D401/06—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings linked by a carbon chain containing only aliphatic carbon atoms
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
  • C07D405/02—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings
  • C07D405/06—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings linked by a carbon chain containing only aliphatic carbon atoms

Definitions

• the field of the invention relates generally to quinoline derived small molecule inhibitors of nicotinamide N-methyltransferase (NNMT), the preparation thereof, and the uses thereof.
• NMT nicotinamide N-methyltransferase
• Nicotinamide N-methyltransferase is a key enzyme located in the cytosolic milieu that catalyzes the transfer of methyl group from the co-factor S- (5'-Adenosyl)-L-methionine (SAM) to substrates such as nicotinamide (NCA), pyridine, and related analogs, such as quinoline, isoquinoline, and the aliphatic amine 1,2,3,4 tetrahydroisoquinoline.
• SAM co-factor S- (5'-Adenosyl)-L-methionine
• NCA nicotinamide
• pyridine pyridine
• related analogs such as quinoline, isoquinoline, and the aliphatic amine 1,2,3,4 tetrahydroisoquinoline.
• NNMT directly regulates the detoxification of endogenous and exogenous drugs/xenobiotics by the formation of methylated metabolic products, such as 1- methyl nicotinamide (1-MNA), methylated pyridiniums, and methylated related analogs.
• 1-MNA 1- methyl nicotinamide
• NNMT is predominantly expressed in the liver, but modest levels of the enzyme are also present in other tissues, including the adipose tissue, kidney, brain, lung, heart, and muscle.
• NNMT activity is also upregulated in the brain tissue of patients with Parkinson' s disease (See e.g., K. Aoyama, K. Matsubara, M. Kondo, Y.
• N- methylpyridinium ions leading to excess production of N- methylpyridinium ions in the brain that act as neurotoxins linked to the pathogenesis of neurodegeneration (See e.g., Herraiz T. N- methyltetrahydropyridines and pyridinium cations as toxins and comparison with naturally-occurring alkaloids. Food Chem Toxicol. 97, 23-39, 2016).
• NNMT is known to modulate intracellular metabolite turnover in the methionine-homocysteine cycle and the nicotinamide adenine dinucleotide (NAD+) synthesis pathways critical for cellular energy expenditure. Therefore, targeted small molecule inhibitors of the NNMT could be significantly beneficial as molecular probes for mechanistic investigations and for the development of therapeutics for the treatment of metabolic and chronic disease conditions that are characterized by abnormal NNMT activity.
• NAD+ nicotinamide adenine dinucleotide
• NNMT is required for low SAM levels and H3K27me3 repressive state. See e.g., Sperber, H., et al, Nat Cell Biol. 17: 1523-1535 (2015). This link between NNMT and stem cells makes development of therapeutics to treat regenerative- related diseases a relevant target.
• NNMT inhibitors may be used to inhibit NNMT and to treat related diseases or conditions. Further, the inventors have discovered that NNMT inhibitors may be used for muscular therapy.
• One aspect of the invention pertains to small molecule quinoline derived cations of Formula I, wherein:
• R 1 is Ci-4 alkyl
• R 2 , R 3 , R 4 , and R 5 are independently selected from the group consisting of: H, C1-4 alkyl, halogen-substituted CM alkyl, R 9 R 10 , and CN;
• R 6 is H or halogen
• R 7 is H, methyl, or R U R 12 ;
• R 8 is H, Ci-4 alkyl, halogen-substituted C 1-4 alkyl
• R 9 , R 10 , R 11 , and R 12 are independently selected from H and C1-4 alkyl
• the compound has at least two non-hydrogen substituents at positions R 2 -R 8 and wherein at least one of the non-hydrogen substituents at positions R 2 -R 8 is H 2 .
• Another aspect of the invention pertains to small molecule quinoline derived cations of Formula IA, wherein:
• Formula IA the cation of Formula IA includes two or more non-hydrogen substituents, and wherein:
• R 5 is H or H 2 ,
• R 6 is H or F
• R 7 is H or H 2 .
• R 8 is H or methyl.
• a further aspect of the invention pertains to use of the cations of the invention to inhibit N MT and to treat related diseases or conditions.
• the invention encompasses use of one or more cations of the invention to inhibit N MT in vitro or in vivo by contacting a cell expressing N MT.
• the invention encompasses use of one or more cations of the invention to treat obesity or related chronic metabolic condition, including metabolic syndrome, pre-diabetes, type-2 diabetes, obesity-linked diseases (e.g., non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, CVDs, and the like).
• obesity-linked diseases e.g., non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, CVDs, and the like.
• the invention encompasses use of one or more cations of the invention to treat an N MT-expressing cancer. In further embodiments, the invention encompasses use of one or more cations of the invention to treat tumorigenesis and metastasis of NNMT -positive cancers.
• the invention encompasses use of one or more cations of the invention to treat Parkinson and related neurological diseases.
• the invention encompasses use of one or more cations of the invention to modulate stem cell differentiation.
• One aspect of the invention pertains to the use of small molecule quinoline derived cations of Formula I for muscular therapy, wherein:
• R 1 is Ci-4 alkyl
• R 2 , R 3 , R 4 , and R 5 are independently selected from the group consisting of: H, Ci-4 alkyl, halogen-substituted CM alkyl, NR 9 R 10 , and CN;
• R 6 is H or halogen
• R 7 is H, methyl, or NR U R 12 ;
• R 8 is H, Ci-4 alkyl, halogen-substituted C 1-4 alkyl
• R 9 , R 10 , R 11 , and R 12 are independently selected from H and Ci-4 alkyl;
• Another aspect of the invention pertains to the use of small molecule quinoline derived cations of Formula IA for muscular therapy, wherein:
• Formula IA the cation of Formula IA includes two or more non-hydrogen substituents, and wherein:
• R 5 is H or NH 2 ,
• R 6 is H or F
• R 7 is H or H 2 .
• R 8 is H or methyl.
• FIG IA Schematic illustration of pathways regulated by NNMT, including the NAD+ biosynthesis salvage pathway starting from NA as a precursor that feeds into energy metabolism, methionine cycle that regulates intracellular SAM concentrations and thus cellular epigenetic modifications and polyamine flux, and clearance of NA by conversion to 1-MNA and excretory products.
• Pathway enzyme abbreviations include NMNAT (nicotinamide mononucleotide adenylyltransferase), NAMPT (nicotinamide phosphoribosyltransferase), MTase (SAM-dependent methyltransferases), PARPs (poly-ADP-ribose polymerases), and CD38 (cluster of differentiation
• FIG IB Effects of the NNMT inhibitor 5-amino-lMQ on intracellular levels of (B) NAD+, NA, NAD+:NA ratio in differentiated adipocytes (3T3 cells) treated with the inhibitor (30 ⁇ ) for 24 h.
• Data represent mean metabolite levels measured by LC/MS/MS in 5-amino-lMQ-treated adipocytes (open bar) normalized to control untreated adipocyte (closed bar) levels in biological duplicates ( ⁇ SD).
• FIG 1C Effects of the NNMT inhibitor 5-amino-lMQ on intracellular levels of SAM, SAH, SAM:SAH ratio in differentiated adipocytes (3T3 cells) treated with the inhibitor (30 ⁇ ) for 24 h.
• Data represent mean metabolite levels measured by LC/MS/MS in 5-amino-lMQ-treated adipocytes (open bar) normalized to control untreated adipocyte (closed bar) levels in biological duplicates ( ⁇ SD).
• FIG. Correlation between the Vina docking scores determined using AutoDock Vina Program and experimentally established IC50 values for all analogs with methyl substitution at the ⁇ -position in each of the core scaffolds (-40 compounds). Pearson's correlation analysis indicated a modest positive linear correlation between calculated docking score (indicative of energetic interactions between the analog and the NNMT active site) and inhibitor potency
• FIG 3 Schematic of the NNMT active substrate-binding site with substrates
• A 5-amino-l-methylquinolinium (lj).
• Ligand interacting hydrophobic NNMT residues labels are red.
• Ligand interacting hydrogen bonding NNMT residue bonds are brown; residue/bonding distance labels are green.
• Schematics were produced with LIGPLOT.
• FIG 4. The chemical structure of 5-amino-l-methylquinolin-l- ium iodide (lj)
• FIG 7. Data represent mean metabolite levels measured by LC/MS/MS in 5-amino-lMQ-treated adipocytes (open bar) normalized to control untreated adipocyte (closed bar) levels in biological duplicates ( ⁇ SD). *, P ⁇
• 0.05 vs. control untreated adipocytes determined by one-way ANOVA analyses followed by Dunnett's postests comparisons.
• adipocyte size ⁇ 2 determined in mean number of 20.7 ⁇ 1.8
• FIG 9A-C Effects of 5-amino-lMQ on lipogenesis in differentiating
• NNMT protein is highly expressed in aged (28 mo) tibialis anterior (TA) skeletal muscle tissue vs. young (4 mo) TA muscle tissue.
• muSC muscle stem cell
• FIG. 12 Treatment with NNMT inhibitor increased mitochondrial respiration capacity and oxidative phosphorylation in the quadriceps skeletal muscle of aged mice (> 24 mo old).
• alkyl refers to both straight and branched chain radicals.
• the alkyl group has 1-12 carbons.
• the alkyl group has 1-7 carbons.
• the alkyl group has 1-6 carbons.
• the alkyl group has 1-4 carbons (also referred to as “C 1-4 alkyl” or "CI -4 alkyl”).
• alkyl may include methyl, ethyl, propyl, isopropyl, butyl, t-butyl, isobutyl, pentyl, hexyl, isohexyl, heptyl, 4,4-dimethylpentyl, octyl, 2,2,4- trimethylpentyl, nonyl, decyl, undecyl, and dodecyl.
• alkylene as used herein refers to straight and branched chain alkyl linking groups, i.e., an alkyl group that links one group to another group in a molecule.
• the term “alkylene” may include - (CH 2 )n— where n is 2-8.
• aryl refers to an aromatic group having at least one carbocyclic aromatic group or heterocyclic aromatic group, which may be unsubstituted or substituted by one or more groups selected from halogen, haloalkyl, hydroxy, alkoxy, carbonyl, alkylamido, nitro, amino, dialkylamino, carboxy, thio or thioalkyl.
• Non-limiting examples of aryl rings are phenyl, naphthyl, pyranyl, pyrrolyl, pyrazinyl, pyrimidinyl, pyrazolyl, pyridinyl, furanyl, thiophenyl, thiazolyl, imidazolyl, isoxazolyl, and the like.
• amino refers to an -NH2 group.
• An "amido” group refers to an -CONH 2 group.
• An alkylamido group refers to an -CONHR group wherein R is as defined above.
• a dialkylamido group refers to an -CONRR group wherein R and R are as defined above.
• halogen or "halo” as used herein by itself or as part of another group refers to chlorine, bromine, fluorine or iodine.
• hydroxy or "hydroxyl” as used herein by itself or as part of another group refers to an— OH group.
• alkoxy group refers to an -O-alkyl group wherein “alkyl” is as defined above.
• a "thio" group refers to an -SH group.
• alkylthio refers to an -SR group wherein R is alkyl as defined above.
• heteroaryl refers to groups having 5 to 14 ring atoms; 6, 10 or 14 7 -electrons shared in a cyclic array; and containing carbon atoms and 1, 2 or 3 oxygen, nitrogen or sulfur heteroatoms.
• the heteroaryl moiety may be unsubstituted or substituted by one or more groups selected from halogen, haloalkyl, hydroxy, alkoxy, carbonyl, alkylamido, nitro, amino, dialkylamino, carboxy, thio or thioalkyl.
• heteroaryl groups include thienyl, imadizolyl, oxadiazolyl, isoxazolyl, triazolyl, pyridyl, pyrimidinyl, pyridazinyl, furyl, pyranyl, thianthrenyl, pyrazolyl, pyrazinyl, indolizinyl, isoindolyl, isobenzofuranyl, benzoxazolyl, xanthenyl, 2H-pyrrolyl, pyrrolyl, 3H-indolyl, indolyl, indazolyl, purinyl, 4H-quinolizinyl, isoquinolyl, quinolyl, phthalazinyl, naphthyridinyl, quinazolinyl, phenanthridinyl, acridinyl, perimidinyl, phenanthrolinyl, phenazinyl, isothiazolyl
• heteroaryl groups include 1,2,3-triazole, 1,2,4-triazole, 5-amino 1,2,4-triazole, imidazole, oxazole, isoxazole, 1,2,3-oxadiazole, 1,2,4-oxadiazole, 3 -amino- 1,2,4- oxadiazole, 1,2,5-oxadiazole, 1,3,4-oxadiazole, pyridine, and 2-aminopyridine.
• heterocycle or "heterocyclic ring”, as used herein except where noted, represents a stable 5- to 7-membered monocyclic-, or stable 7- to 11-membered bicyclic heterocyclic ring system, any ring of which may be saturated or unsaturated, and which consists of carbon atoms and from one to three heteroatoms selected from the group consisting of N, O and S, and wherein the nitrogen and sulfur heteroatoms may optionally be oxidized, and the nitrogen heteroatom may optionally be quaternized, and including any bicyclic group in which any of the above-defined heterocyclic rings is fused to a benzene ring.
• Rings may contain one oxygen or sulfur, one to three nitrogen atoms, or one oxygen or sulfur combined with one or two nitrogen atoms.
• the heterocyclic ring may be attached at any heteroatom or carbon atom that results in the creation of a stable structure.
• heterocycle or “heterocyclic ring” moiety may be unsubstituted or substituted by one or more groups selected from halogen, haloalkyl, hydroxy, alkoxy, carbonyl, alkylamido, nitro, amino, dialkylamino, carboxy, thio or thioalkyl.
• heterocyclic groups include piperidinyl, piperazinyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolodinyl, 2-oxoazepinyl, azepinyl, pyrrolyl, 4-piperidonyl, pyrrolidinyl, pyrazolyl, pyrazolidinyl, imidazolyl, imidazolinyl, imidazolidinyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, oxazolyl, oxazolidinyl, isoxazolyl, isoxazolidinyl, morpholinyl, thiazolyl, thiazolidinyl, isothiazolyl, quinuclidinyl, isothiazolidinyl, indolyl, quinolinyl, isoquinolinyl, benzimidazo
• alkylamino refers to an amino group which is substituted with one alkyl group having from 1 to 6 carbon atoms.
• dialkylamino refers to an amino group which is substituted with two alkyl groups, each having from 1 to 6 carbon atoms.
• alkylthio as used herein by itself or as part of another group refers to an thio group which is substituted with one alkyl group having from 1 to 6 carbon atoms.
• cells refer to one or cells, from any animal, which expresses NNMT, such as, without limitation, rat, mice, monkey, horse, dog, cat, and human.
• cells can be progenitor cells, such as stem cells, or differentiated cells, such as endothelial cells, smooth muscle cells.
• cells for medical procedures can be obtained from the patient for autologous procedures or from other donors for allogeneic procedures.
• a “therapeutically effective amount” is an amount sufficient to decrease, prevent or ameliorate the symptoms associated with a medical condition.
• non-hydrogen substituent refers to a substituent that is not made up solely of hydrogen.
• examples of non-hydrogen substituents includes halogen, CI -4 alkyl, halogen-substituted CI -4 alkyl, NR 9 R 10 , NR U R 12 , and CN.
• non-hydrogen substituent includes methyl.
• non-hydrogen substituent includes fluoride (F).
• non-hydrogen substituent includes NH2.
• quinoline derived small molecule cation have been used interchangeably throughout the application to refer to embodiments of the invention and doing so is not meant in any way to limit the scope of the invention.
• the term "muscular therapy” as used herein refers to contacting one or more cells of a subject with one or more NNMT inhibitors to treat and/or prevent muscular disorders; improve neuromuscular function; reduce the time required to restore neuromuscular function; prevent neuromuscular injury; and/or improve muscle regeneration. This term also encompasses administration of NNMT inhibitors to treat and/or prevent muscular disorders; improve neuromuscular function; reduce the time required to restore neuromuscular function; prevent neuromuscular injury; and/or improve muscle regeneration.
• NNMT inhibitors refers small molecule chemical entities that inhibit the enzymatic activity of NNMT, and includes the compounds of Formula I and Formula IA as well as compounds in Tables 1-3.
• administering refers to contacting one or more cells of a subject, (including human, horse, cat, dog, monkey, rat, and mice) with one or more NNMT inhibitors.
• administration may occur in vitro. In further embodiments, administration may occur in vivo.
• the invention encompasses cations of Formula I, wherein:
• R 1 is Ci-4 alkyl
• R 2 , R 3 , R 4 , and R 5 are independently selected from the group consisting of: H, CI -4 alkyl, halogen-substituted CM alkyl, NR 9 R 10 , and CN;
• R 6 is H or halogen
• R 7 is H, methyl, or NR U R 12 ;
• R 8 is H, CM alkyl, halogen-substituted CM alkyl
• R 9 , R 10 , R 11 , and R 12 are independently selected from H and CM alkyl;
• R 2 -R 8 is NH 2 .
• R 1 may be methyl or ethyl.
• R 1 is methyl.
• R 2 and R 3 is H2.
• R 5 is H 2 .
• R 2 , R 3 , and R 4 are hydrogen.
• R 6 is halogen
• R 6 is F.
• R 7 is H 2 .
• R 8 is methyl or CF3.
• R 8 is methyl
• the invention encompasses a cation of
• Formula IA the cation of Formula IA includes two or more non-hydrogen substituents at positions R 2 -R 8 , and wherein:
• R 5 is H or H 2 ,
• R 6 is H or F
• R 7 is H or H 2 .
• R 8 is H or methyl.
• R 1 is methyl or ethyl.
• R 6 is F.
• the cation of Formula IA is one of:
• R 5 is H or NH 2 ;
• R 6 is H or F
• R 8 is H or methyl.
• the small molecule cations of the invention may be accompanied by a counter anion (X " ).
• the counter ion may be chosen from sulfonate (e.g., trifluoromethanesulfonate, mesylate, tosylate, besylate, and the like); halide (e.g., fluoride, bromide, chloride or iodide); acetate; sulfate; bisulfate; nitrate; oxalate; valerate; oleate; palmitate; stearate; laurate; borate; benzoate; lactate; phosphate; citrate; maleate; fumarate; succinate; tartrate; glucoheptonate; and lactobionate.
• sulfonate e.g., trifluoromethanesulfonate, mesylate, tosylate, besylate, and the like
• halide e.g., fluoride
• Another aspect of the invention pertains generally to the use of the cations of the invention to inhibit N MT and diseases or conditions involving NNMT.
• NNMT has been linked to a number of chronic diseases/conditions.
• several studies support a causal relationship between augmented NNMT activity in cancer cells and tumor proliferation/progression in a variety of cancerous states with potential implications for NNMT as a biomarker for cancer prognosis and a relevant target for anti-cancer therapeutic development.
• NNMT was preferentially expressed by mesenchymal glioblastoma stem cells (GSCs). See e.g., Figures 5 and 9 of Jung, J., et al, Nicotinamide metabolism regulates glioblastoma stem cell maintenance JCI Insight, 2: 1-23 (2017).
• NNMT activity also plays a role in Parkinson's disease and in modulating stem cell differentiation. Furthermore, emerging reports in both animals and humans indicate that NNMT plays a role in obesity and related chronic metabolic conditions (e.g., type-2 diabetes).
• the invention encompasses a method of inhibiting NNMT in vitro or in vivo by contacting a cell expressing NNMT with one or more cations of the invention. In further embodiments, the invention encompasses a method of inhibiting NNMT in vitro or in vivo by contacting a cell expressing NNMT with one or more cations chosen from lc, If, 11, lm, 2j, 2k, 21, 2m, 2aa, lc', If, 11', lm', 2j ⁇ 2k', 21', 2m', and 2aa' [00098] In further embodiments, the invention encompasses a method of inhibiting N MT in vitro or in vivo by contacting a cell expressing N MT with one or more cations chosen from Tables 1, 2, 3a, and 3b.
• the invention encompasses a method of inhibiting NNMT in vitro or in vivo by contacting a cell expressing NNMT with one or more cations of the invention and with one or more cations chosen from Tables 3a and 3b.
• one or more cations of the invention is contacted with a cell expressing NNMT concurrently with one or more cations chosen from Tables 3a and 3b.
• one or more cations of the invention is contacted with a cell expressing NNMT followed by contacting said cell expressing NNMT with one or more cations chosen from Tables 3a and 3b.
• one or more cations chosen from Tables 3a and 3b is contacted with a cell expressing NNMT followed by contacting said cell expressing NNMT with one or more cations of the invention.
• the invention encompasses a method of treating obesity or related chronic metabolic condition by administering a therapeutically effective amount of one or more cations of the invention. In further embodiments, the invention encompasses a method of treating obesity or related chronic metabolic condition by administering a therapeutically effective amount of one or more cations chosen from lc, If, 11, lm, 2j, 2k, 21, 2m, 2aa, lc ⁇ IP, IF, lm', 2j ⁇ 2k',
• the invention encompasses a method of treating obesity or related chronic metabolic condition by administering a therapeutically effective amount of one or more cations chosen from Tables 1, 2, 3a, and 3b. In further embodiments, the invention encompasses a method of treating obesity or related chronic metabolic condition by administering a therapeutically effective amount of one or more cations of the invention and one or more cations chosen from Tables 3a and 3b.
• One aspect of the invention pertains to treating obesity or related chronic metabolic condition by administering a therapeutically effective amount of one or more cations of the invention with concurrent administration of one or more cations chosen from Tables 3a and 3b.
• Another aspect of the invention pertains to treating obesity or related chronic metabolic condition by administering a therapeutically effective amount of one or more cations of the invention followed by administration of one or more cations chosen from Tables 3a and 3b.
• a further aspect of the invention pertains to treating obesity or related chronic metabolic condition by administering a therapeutically effective amount of one or more cations chosen from Tables 3a and 3b followed by the administration of one or more cations of the invention.
• the invention encompasses a method of treating an N MT-expressing cancer, such as glioblastoma, by administering a therapeutically effective amount of one or more cations of the invention.
• the invention encompasses a method of treating an NNMT- expressing cancer such as glioblastoma, by administering a therapeutically effective amount of one or more cations chosen from lc, If, 11, lm, 2j, 2k, 21, 2m, 2aa, lc',
• the invention encompasses a method of treating an NNMT-expressing cancer, such as glioblastoma, by administering a therapeutically effective amount of one or more cations chosen from Tables 1, 2, 3a, and 3b.
• the invention encompasses a method of treating an NNMT-expressing cancer by administering a therapeutically effective amount of one or more cations of the invention and one or more cations chosen from Tables 3a and 3b.
• One aspect of the invention pertains to treating an NNMT- expressing cancer, such as glioblastoma, by administering a therapeutically effective amount of one or more cations of the invention with concurrent administration of one or more cations chosen from Tables 3a and 3b.
• Another aspect of the invention pertains to treating an NNMT-expressing cancer, such as glioblastoma, by administering a therapeutically effective amount of one or more cations of the invention followed by administration of one or more cations chosen from Tables 3a and 3b.
• a further aspect of the invention pertains to treating an NNMT-expressing cancer, such as glioblastoma
• NNMT-expressing cancer such as glioblastoma
• administering a therapeutically effective amount of one or more cations chosen from Tables 3a and 3b followed by the administration of one or more cations of the invention.
• the invention encompasses a method of treating Parkinson and related neurological diseases by administering a therapeutically effective amount of one or more cations of the invention.
• the invention encompasses a method of treating Parkinson and related neurological diseases by administering a therapeutically effective amount of one or more cations chosen from lc, If, 11, lm, 2j, 2k, 21, 2m, 2aa, lc', If, IP, lm ⁇ 2j', 2k', 21', 2m', and 2aa' .
• the invention encompasses a method of treating Parkinson and related neurological diseases by administering a therapeutically effective amount of one or more cations chosen from Tables 1, 2, 3a, and 3b. In further embodiments, the invention encompasses a method of treating Parkinson and related neurological diseases by administering a therapeutically effective amount of one or more cations of the invention and one or more cations chosen from Tables 3a and 3b.
• One aspect of the invention pertains to treating Parkinson and related neurological diseases by administering a therapeutically effective amount of one or more cations of the invention with concurrent administration of one or more cations chosen from Tables 3a and 3b.
• Another aspect of the invention pertains to treating Parkinson and related neurological diseases by administering a therapeutically effective amount of one or more cations of the invention followed by administration of one or more cations chosen from Tables 3a and 3b.
• a further aspect of the invention pertains to treating Parkinson and related neurological diseases by administering a therapeutically effective amount of one or more cations chosen from Tables 3a and 3b followed by the administration of one or more cations of the invention.
• the invention encompasses a method of modulating stem cell differentiation by contacting a stem cell expressing N MT with one or more cations of the invention. In further embodiments, the invention encompasses a method of modulating stem cell differentiation by contacting a stem cell expressing NNMT with one or more cations chosen from lc, If, 11, lm, 2j, 2k, 21, 2m, 2aa, lc', If, 11', lm', 2j ⁇ 2k', 21', 2m', and 2aa'
• the invention encompasses a method of modulating stem cell differentiation by contacting a stem cell expressing NNMT with one or more cations chosen from Tables 1, 2, 3a, and 3b. In further embodiments, the invention encompasses a method of modulating stem cell differentiation by contacting a stem cell expressing NNMT with one or more cations of the invention and with one or more cations chosen from Tables 3a and 3b. In one aspect of the invention, one or more cations of the invention is contacted with a stem cell expressing NNMT concurrently with one or more cations chosen from Tables 3a and 3b.
• one or more cations of the invention is contacted with a stem cell expressing NNMT followed by contacting said stem cell expressing NNMT with one or more cations chosen from Tables 3a and 3b.
• one or more cations chosen from Tables 3a and 3b is contacted with a stem cell expressing NNMT followed by contacting said stem cell expressing NNMT with one or more cations of the invention.
• Certain cations of Formula I and IA can be prepared via N-alkylation of a substituted quinoline derivative.
• preparation of certain cations of Formulas I and IA may occur by alkylating the N-positions of the quinoline scaffold using, for example, iodomethane or methyl trifluoromethanesulfonate (see Scheme 1).
• °Reagents and conditions (a) iodomethane, isopropanol, 90 °C, 12 h;
• preparation of certain cations of the invention may occur via reductive amination followed by alkylation.
• Scheme 2 Synthesis of certain C3-amino-alkylated quinolinium derivatives of the invention* [000114] a Reagents and conditions: (a) triethyl orthoformate, TFA, 125 °C, 12 h then NaBH 4 , EtOH, room temperature, 12 h; (b) Mel, IP A, 90 °C, 12 h.
• Certain cations of the invention may be prepared via the two-step process outlined in Scheme 2, as exemplified with the preparation of cation If. Specifically, alkylation of a C3-amino-quinoline derived precursor, such as compound 8, may be achieved via reductive amination with, for example, triethyl orthoformate in TFA followed by treatment with NaBFLt to give the corresponding secondary C3 -amine derivative (such as N-methyl-C3-amino- quinoline 9). The secondary C3-amine derivative intermediate (e.g., compound 9) may then be methylated to obtain the desired cation (e.g., compound If).
• preparation of certain cations of the invention may occur via a one-pot procedure reported by Venkatesan et al. involving a SnCl 2 mediated Friedlander synthesis followed by Curtius rearrangement and deprotection with subsequent alkylation, as exemplified with the preparation of cation 2j (Scheme 3).
• cation 2j may be prepared from 5-fluoro-2-nitro- benzaldehyde 11 via a one-pot procedure reported by Venkatesan et al. involving a SnCl 2 mediated Friedlander synthesis to construct the desired C2-ethyl- carboxylate quinoline 12 (Scheme 3).
• the resulting ester group may then be hydrolyzed and converted into acyl azide with DPP A, followed by Curtius rearrangement with an alcohol, such as tert-butanol, to provide the corresponding N-Boc protected substrate (not depicted).
• N-Boc deprotection with TFA provides the corresponding fluorinated C3-amino-quinoline intermediate 13.
• Methylation of precursor 13 may then occur using, for example, the general method outlined in Scheme 1 to obtain cation 2j.
• preparation of certain cations of the invention may occur via oxidation of quinoline (using e.g., mCPBA) followed by nitration and chlorination, respectively and further followed by amination and alkylation, respectively, as exemplified with the preparation of cation 2k (Scheme 4).
• cation 2k may be prepared from quinoline-N-oxide 14 or the like, which may be derived from quinoline via a mCPBA oxidation (Scheme 4).
• a regioselective nitration of 14 may be used to selectively install a nitro group at the C3 position of compound 14 or the like, followed by chlorination of the quinoline-N-oxide moiety in the presence of POCI3 to give intermediate 15.
• the desired C2/3-diamino-group may be introduced via a two- step sequence involving an amination of C2-chloro- group with, for example, ammonia and reduction of C3 -nitro group (via, e.g., hydrogenation) to give the precursor 16.
• Compound 16 may be methylated using, for example, the general method outlined in Scheme 1 to give cation 2k.
• a Reagents and conditions (a) mCPBA, CH2CI2, 0 °C to room temperature, 12 h; (b) tert-butyl nitrite, MeCN, 100 °C, 24 h; (c) POCI3, 95 °C, 12 h; (d) NH 3 (7N in MeOH), 90 °C, 12 h; (e) Pd/C, H 2 , MeOH/THF, room temperature, 12 h; (f) Mel, IP A, 90 °C, 12 h.
• NNMT has been linked to a number of diseases/conditions. For example, it has been shown that NNMT activity plays a role in certain neurological diseases/conditions. The inventors surprisingly discovered that NNMT inhibitors may be used for muscular therapy, including treatment of certain muscular dystrophy diseases.
• the invention encompasses use of one or more NNMT inhibitors for muscular therapy comprising contacting one or more cells with one or more NNMT inhibitors. In other embodiments, the invention encompasses use of NNMT inhibitors for treating a muscular dystrophy disease comprising contacting one or more cells with one or more NNMT inhibitors.
• the invention encompasses a method of providing muscular therapy by administering a cation of Formula I, wherein:
• R 1 is Ci-4 alkyl
• R 2 , R 3 , R 4 , and R 5 are independently selected from the group consisting of: H, CI -4 alkyl, halogen-substituted CM alkyl, NR 9 R 10 , and CN;
• R 6 is H or halogen
• R 7 is H, methyl, or NR U R 12 ;
• R 8 is H, Ci-4 alkyl, halogen-substituted C 1-4 alkyl
• R 9 , R 10 , R 11 , and R 12 are independently selected from H and Ci-4 alkyl;
• R 1 may be methyl or ethyl.
• R 1 is methyl
• At least one of R 2 and R 3 is NH 2 .
• R 5 is NH 2 .
• R 2 , R 3 , and R 4 are hydrogen.
• R 6 is halogen. [000143] In some embodiments, R 6 is F.
• R 7 is H2.
• R 8 is methyl or CF3.
• R 8 is methyl
• the invention encompasses a method of
• Formula IA the cation of Formula IA includes two or more non-hydrogen substituents at positions R 2 -R 8 , and wherein:
• R 6 is H or F
• R 8 is H or methyl.
• R 1 is methyl or ethyl.
• R 6 is F.
• the invention encompasses a method of
• R 5 is H or NH 2 ;
• R 6 is H or F
• R 8 is H or methyl.
• the small molecule cations of described herein may be accompanied by a counter anion (X " ).
• the counter ion may be chosen from sulfonate (e.g., trifluoromethanesulfonate, mesylate, tosylate, besylate, and the like); halide (e.g., fluoride, bromide, chloride or iodide); acetate; sulfate; bisulfate; nitrate; oxalate; valerate; oleate; palmitate; stearate; laurate; borate; benzoate; lactate; phosphate; citrate; maleate; fumarate; succinate; tartrate; glucoheptonate; and lactobionate.
• sulfonate e.g., trifluoromethanesulfonate, mesylate, tosylate, besylate, and the like
• halide e.g., fluor
• the invention encompasses a method of providing muscular therapy by administering a cation of chosen from Tables 1, 2, 3a, and 3b, described herein.
• the invention encompasses a method of providing muscular therapy by contacting a cell expressing N MT with one or more cations of the invention.
• the invention encompasses a method of providing muscular therapy by contacting a cell expressing NNMT with one or more cations chosen from lc, If, 11, lm, 2j, 2k, 21, 2m, 2aa, lc', 1 ⁇ , IP, lm', 2j', 2k', 21', 2m', and 2aa ⁇
• the invention encompasses a method of providing muscular therapy by contacting a cell expressing NNMT with one or more cations chosen from Tables 1, 2, 3a, and 3b. In further embodiments, the invention encompasses a method of providing muscular therapy by contacting a cell expressing N MT with one or more cations of the invention and with one or more cations chosen from Tables 3a and 3b. In one aspect of the invention, one or more cations of the invention is contacted with a cell expressing NNMT concurrently with one or more cations chosen from Tables 3a and 3b.
• one or more cations of the invention is contacted with a cell expressing NNMT followed by contacting said cell expressing NNMT with one or more cations chosen from Tables 3a and 3b.
• one or more cations chosen from Tables 3a and 3b is contacted with a cell expressing NNMT followed by contacting said cell expressing NNMT with one or more cations of the invention.
• the invention encompasses a method of providing muscular therapy by administering a therapeutically effective amount of one or more NNMT inhibitors. In some embodiments, the invention encompasses a method of providing muscular therapy by administering a therapeutically effective amount of one or more cations of the invention. Another aspect of the invention pertains to a method of providing muscular therapy by administering a therapeutically effective amount of one or more cations.
• the invention encompasses a method of providing muscular therapy by administering a therapeutically effective amount of one or more cations chosen from lc, If, 11, lm, 2j, 2k, 21, 2m, 2aa, lc', If, 11', lm', 2j ⁇ 2k', 21', 2m', and 2aa'
• the invention encompasses a providing muscular therapy by administering a therapeutically effective amount of one or more cations chosen from Tables 1, 2, 3a, and 3b.
• the invention encompasses a method of providing muscular therapy by administering a therapeutically effective amount of one or more cations of the invention and one or more cations chosen from Tables 3a and 3b.
• One aspect of the invention pertains to treating providing muscular therapy by administering a therapeutically effective amount of one or more cations of the invention with concurrent administration of one or more cations chosen from Tables 3a and 3b.
• Another aspect of the invention pertains to providing muscular therapy by administering a therapeutically effective amount of one or more cations of the invention followed by administration of one or more cations chosen from Tables 3a and 3b.
• a further aspect of the invention pertains to providing muscular therapy by administering a therapeutically effective amount of one or more cations chosen from Tables 3a and 3b followed by the administration of one or more cations of the invention.
• One aspect of the invention pertains to the administration of one or more N MT inhibitors by, for example, contacting one or more cells of an animal to:
• muscular disorders including, but not limited to, sarcopenia, muscle atrophy, Duchenne muscular dystrophy, Becker muscular dystrophy, Limb-girdle muscular dystrophies, Pompe disease, cardiac myopathies, pulmonary disorders;
• (c) reduce the time required to restore neuromuscular function, including, but not limited to, following acute muscle injury, following overuse muscle injury, and/or following chronic muscle injury;
• administration of the NNMT inhibitor is in vitro. In further embodiments, administration of the NNMT inhibitor is in vivo.
• NNMT inhibitors may be used with one or more chemical entities (e.g., nicotinamide riboside, nicotinamide mononucleotide) that increase intracellular NAD+ levels, to produce synergistic or additive effects to provide muscular therapy.
• chemical entities e.g., nicotinamide riboside, nicotinamide mononucleotide
• the invention encompasses a method of providing muscular therapy by co-administering a therapeutically effective amount of one or more NNMT inhibitors with one or more chemical entities (e.g., nicotinamide riboside, nicotinamide mononucleotide) that increase intracellular
• one or more chemical entities e.g., nicotinamide riboside, nicotinamide mononucleotide
• the invention encompasses a method of providing muscular therapy by contacting a cell expressing NNMT with a NNMT inhibitor (such as a cation of Formula I or IA, or otherwise disclosed herein) and one or more chemical entities (e.g., nicotinamide riboside, nicotinamide mononucleotide) that modulates intracellular NAD+ levels to provide muscular therapy.
• a NNMT inhibitor such as a cation of Formula I or IA, or otherwise disclosed herein
• chemical entities e.g., nicotinamide riboside, nicotinamide mononucleotide
• the invention encompasses a method of providing muscular therapy by contacting a cell expressing N MT with a N MT inhibitor
• nicotinamide riboside nicotinamide mononucleotide
• one or more NNMT inhibitors (such as a cation of Formula I or IA, or otherwise disclosed herein) is contacted with a cell expressing NNMT concurrently with one or more chemical entities (e.g., nicotinamide riboside, nicotinamide mononucleotide) that increase intracellular NAD+ levels to provide muscular therapy.
• one or more chemical entities e.g., nicotinamide riboside, nicotinamide mononucleotide
• the invention encompasses contacting one or more NNMT inhibitors (such as a cation of Formula I or IA, or otherwise disclosed herein) is contacted with a cell expressing NNMT concurrently with one or more chemical entities (e.g., nicotinamide riboside, nicotinamide mononucleotide) that modulate intracellular NAD+ levels to provide muscular therapy.
• one or more NNMT inhibitors such as a cation of Formula I or IA, or otherwise disclosed herein
• one or more chemical entities e.g., nicotinamide riboside, nicotinamide mononucleotide
• one or more NNMT inhibitors (such as a cation of Formula I or IA, or otherwise disclosed herein) is contacted with a cell expressing NNMT followed by contacting said cell expressing NNMT with one or more chemical entities (e.g., nicotinamide riboside, nicotinamide mononucleotide) that modulate intracellular NAD+ levels to provide muscular therapy.
• one or more chemical entities e.g., nicotinamide riboside, nicotinamide mononucleotide
• one or more NNMT inhibitors (such as a cation of Formula I or IA, or otherwise disclosed herein) is contacted with a cell expressing NNMT followed by contacting said cell expressing NNMT with one or more chemical entities (e.g., nicotinamide riboside, nicotinamide mononucleotide) that increase intracellular NAD+ levels to provide muscular therapy.
• one or more chemical entities e.g., nicotinamide riboside, nicotinamide mononucleotide
• one or more chemical entities e.g., nicotinamide riboside, nicotinamide mononucleotide
• a cell expressing NNMT followed by contacting said cell expressing NNMT one or more NNMT inhibitors (such as a cation of Formula I or IA, or otherwise disclosed herein).
• one or more chemical entities e.g., nicotinamide riboside, nicotinamide mononucleotide
• a cell expressing one or more chemical entities e.g., nicotinamide riboside, nicotinamide mononucleotide
• NNMT followed by contacting said cell expressing NNMT one or more NNMT inhibitors (such as a cation of Formula I or IA, or otherwise disclosed herein).
• NNMT inhibitors such as a cation of Formula I or IA, or otherwise disclosed herein.
• SAM was obtained from Sigma Aldrich and nicotinamide from Fluka Analytical (Kwazulu Natal, South Africa; distributed by Sigma Aldrich in the
• MNA chloride and S-adenosylhomocysteine (SAH) were obtained from Cayman Chemical Company (Ann Arbor, MI). All compounds were made in double distilled water.
• HPLC-MS HPLC-MS. All samples were analyzed on Agilent 1290 series HPLC system comprised of binary pumps, degasser and UV detector, equipped with an auto-sampler that is coupled with Agilent 6150 mass spectrometer. Purity was determined via UV detection with a bandwidth of 170nm in the range from 230-
• mt-hNNMT A modified mutant human NNMT [lacking 3 amino acid residues from the C-terminus of the NNMT protein that was not observed in crystal structure] (3ROD, PDB accession code) cloned into an IPTG-inducible plasmid pJ401 expression vector was purchased from DNA 2.0 (Menlo Park, CA). The expression and purification of mt-hNNMT was modified from a previously reported protocol. Briefly, the expression vector was used to transform chemically competent E. coli BL21/DE3 cells.
• the BL21 transformants were plated on LB agar plate with kanamycin (KAN) (30 ⁇ g/mL) and incubated overnight at 37°C that was used to inoculate 1L media along with 0.5 mM each of magnesium and calcium chloride for protein over-expression.
• KAN kanamycin
• the culture was placed in a shaker at 37°C to an ODeoo of 0.7-0.8 ( ⁇ 2- 3 h) before induction with 0.5 mM IPTG and incubated for an additional 3 h.
• Cells were harvested by centrifugation at 10°C and 4000 rpm for 20 min and removal of the supernatant.
• harvested cells were first re- suspended in chilled lysis buffer (20 mM Tris [pH 7.9], 0.5 M NaCl, 5 mM imidazole, 10% glycerol, 1 mM DTT, 1 mM PMSF) and the lysis mixture was sonicated on ice.
• Cell lysates were centrifuged at 4°C and 15000 rpm for 30 min. The soluble fraction was loaded onto a nickel affinity column formed from nickel sepharose beads (GE Biosciences) pre-equilibrated with lysis buffer.
• NNMT Activity Assay HPLC Instrumentation and Chromatographic Conditions.
• An HPLC-UV method for the detection of NNMT catalyzed product, 1 -methyl nicotinamide (MNA) was developed by modifying a previously reported protocol (Patel et al. 2013).
• Shimatzu 10AVP HPLC System Shimatzu, Kyoto, Japan
• manual sample injector was used to run the HPLC-UV method on an isocratic gradient with mobile phase comprising of lOmM 1 -heptane sulfonate, 20 mM potassium phosphate monobasic [pH 3.1], 4% methanol, and 3% acetonitrile.
• MNA Calibration Curve and NNMT Activity Assay To establish a linear curve for the detection of MNA peak, a 10 - 0.3125 uM/100 ⁇ L ⁇ half-fold serially diluted samples of MNA were prepared in reaction buffer containing 1 mM Tris [pH 8.6], 1 mM DTT, 10% trichloroacetic acid, 4% methanol, and water.
• substrate nicotinamide at 100 ⁇ , methyl donor S-adenosyl-L- methionine (SAM) at 5 ⁇ , and S-adenosyl methionine (SAH) at 5 ⁇ concentration) samples were also run individually in the reaction buffer [1 mM Tris [pH 8.6], 1 mM DTT, 10% trichloroacetic acid, 4% methanol, and water] to identify elution time and define substrate, co-factor, and product peaks.
• MNA, nicotinamide, SAM, and SAH peaks were detected using a wavelength of 265 nm.
• 5 ⁇ _ of 10 mM nicotinamide made in water
• reaction buffer 2.5 ⁇ _, of 1 mM SAM made in water were added/500 ⁇ _, of the reaction buffer.
• the reaction was initiated by adding 4 ⁇ _, of 25 ⁇ stock purified NNMT protein (final concentration of NNMT in the reaction was 200 nM) and incubated on a heat block at 37°C for 6 min, following which the reaction was terminated by the addition of a mixture of 10% trichloroacetic acid and 4% methanol, vortexing for
• Peak area and peak height for MNA were determined by running 100 ⁇ _, of the supernatant using the chromatographic conditions described above. Reactions were run in the absence of NNMT as control samples in each experiment.
• NNMT ICso Curves for Inhibitors were analyzed by HPLC as described above, and used to construct inhibition curves for 1-MQ and 1-MQ analogs. Compounds were initially tested for NNMT inhibition activity at 100 ⁇ or 1 mM concentration (compounds with no activity at 100 uM were tested at lmM concentration). Compounds with >50% inhibitory activity at 1 mM were advanced to comprehensive concentration-response analysis (concentration range of 100 nM-lmM/100 ⁇ _, reaction). Otherwise, IC50 values are reported as either >1000 uM or no observable inhibition (NI). Data were normalized and reported as % NNMT activity against concentrations tested (uM). IC50 values were determined by three parameters non-linear regression [inhibitor cone. vs. normalized % NNMT activity] fitted by least squares method
• Example 2 Biological evaluation of certain embodiments of the invention (and analogs thereof).
• IC 5 o values are represented at mean ⁇ SD of duplicate or triplicate measurements.
• the inventors also surprisingly found inhibitory activity with certain embodiments of the invention where R 1 is methyl with dual positional substitutions (see e.g., compounds 2j, 2m, 2k, and 21, Table 2).
• IC 5 o values are represented at mean ⁇ SD of duplicate or triplicate measurements.
• IC 5 o values are represented at mean ⁇ SD of duplicate or triplicate measurements.
• IC 5 o values are represented at mean ⁇ SD of duplicate or triplicate measurements.
• Vina docking calculations are useful to predict the binding modes, orientations, and conformations of small molecule inhibitors within the catalytic domain of the target protein.
• the predicted inhibitor-binding mode of lj with an orientation and conformation that favors most negative docking score when superimposed with the endogenous substrate NCA of the NNMT enzyme indicated that the analog binds consistent with the binding mode of NCA, i.e., the Nl-atom of both ligands aligned almost identical, conferring similar molecular interactions with key residues within the active site of the enzyme.
• the binding mode for lj permits the formation of strong hydrophobic interactions within the apolar pocket surrounding the quinolinium Nl-atom, consisting of Tyr20, Tyr204, Tyr242, Leul64, Alal98, and Ala247 residues
• the predicted binding mode of lj indicates the C5 '-amino substituent forms hydrogen bonding interaction with the carboxylic backbone of the Ser201 residue and a hydrophobic bonding with the Ser213 residue unlike the NCA amide group that is in hydrogen bonding distance from with the NNMT Ser213 residue.
• These interactions for lj might promote tighter binding affinity compared to the endogenous substrate NCA, further indicated by a much lower calculated Vina docking score for lj (-8.1 vs for NCA) that suggests improved energetic interactions for lj in the NNMT active site.
• Example 4 It has been observed that NNMT protein expression in muscle tissue was significantly greater in aged (27-mo old C57BL/6 mice) compared to young (3-mo old C57BL/6 mice) individuals ( Figure 5). Thus, NNMT inhibitors should reduce NNMT activity in aged muscles such that the NAD+ salvage cycle in aged muscle cells is returned to the functioning observed in young muscle cells.
• Example 5 Small molecule N MT inhibitors as highly membrane- permeable, selective inhibitors, which reduce intracellular 1-MNA levels and prevent lipogenesis in vitro were investigated. Furthermore, a proof-of-concept in vivo study in diet-induced obese mice to test the hypothesis that the most potent inhibitor when administered systemically, would reverse obesity by causing substantial loss of body weight and adiposity without causing any observable adverse effects was conducted.
• PAMPA Parallel artificial membrane permeability assay
• Passive membrane transport properties were measured using a 96-well pre-coated PAMPA plate system with membrane pore size 0.4 ⁇ (GentestTM, Corning; Bedford, MA, USA). Briefly, 1 mM stock solution of each compound was prepared in deionized water, diluted to a final concentration of 400 ⁇ in PBS (Sigma Aldrich; St. Louis, MO), and placed in the plate bottom well (donor well). After 4 h incubation at room temperature, the sample concentration in the donor and acceptor wells were measured using a UV-Vis spectrophotometer (Beckman, DU640) set at the wavelength corresponding to the maximum absorption of each compound. Compound concentration in the donor and acceptor wells were calculated from calibration curves spanning 400-3.125 ⁇ . Samples were tested in triplicates in three separate experiments.
• a ⁇ B and basolateral to apical (B ⁇ A) permeability compounds were added at 10 ⁇ concentration to the apical (A) side and basolateral (B) side, respectively, and the corresponding amount of permeation was determined by measuring compound concentration on the B or A side.
• the A-side buffer contained 100 ⁇ Lucifer yellow dye, in transport buffer (1.98 g/L glucose in 10 mM HEPES, lx Hank's balanced salt solution, pH 7.4), and the B-side buffer was transport buffer at pH 7.4. Caco-2 cells were incubated with these buffers for 2 h, and the receiver side buffer was removed for analysis by LC/MS/MS (using bucetin as an analytical internal standard). Data were expressed as permeability
• 3T3-L1 pre-adipocytes cells (catalog CL-173, American Type Culture Collection; Manassas, VA, USA) were seeded at a density of 2 x 103 cells per well in 96-well plates, cultured with standard culture media [DMEM, 4.5 g/L glucose, L-glutamine, sodium pyruvate (Mediatech Inc.; Tewksbury, MA, USA), 10% FBS (Sigma Aldrich; St. Louis, MO, USA), 1% antibiotic-antimycotic solution (Mediatech Inc.; Tewksbury, MA, USA)], and grown for 48 h until > -90% confluent.
• DMEM 4.5 g/L glucose, L-glutamine, sodium pyruvate
• FBS Sigma Aldrich; St. Louis, MO, USA
• 1% antibiotic-antimycotic solution Mediatech Inc.; Tewksbury, MA, USA
• 3T3-L1 pre-adipocytes were cultured with standard culture media (DMEM, 4.5 g/L glucose, L- glutamine, sodium pyruvate, 10% FBS, 1% antibiotic-antimycotic solution) and grown for 48 h before initiating differentiation using the manufacturer's suggested protocol and modified from previous published work. Briefly, standard culture media was supplemented with scheduled addition of adipogenic agents [3-isobutyl- 1 methyl xanthine (IBMX), Sigma Aldrich; MO, USA), dexamethasone (Sigma Aldrich; MO, USA), insulin (Gibco Life Technologies Inc.; Grand Island, NY,
• the 1-MNA NNMT reaction product was quantified from peak area ratios using AB Sciex Analyst and MultiQuant 2.1 software and the parent precursor and Q3 masses set to m/z 137.1 and 94.1, respectively. Fragment ions at m/z of 92.1 and 77.9 were additionally used for the detection and confirmation of 1-MNA, respectively. Processing of undifferentiated 3T3-L1 pre-adipocytes (day 0) and differentiated adipocytes (day 10) were optimized for recovery and reproducibility of 1- MNA levels across cultured batches of 3T3-L1 cells (-passages 7-8) and the 1-MNA levels were compared between the pre-adipocytes and adipocytes.
• MRM multiple reaction monitoring
• NNMT inhibitor As an internal standard (IS) to extract cellular metabolites.
• Adherent cells were scrapped, then centrifuged at 4°C and 13000 g for 15 min, and the resulting supernatants processed using established protocols. Intracellular levels of 1-MNA and as well as the IS were determined from LC/MS/MS peak areas. Data were subsequently normalized to the IS peak area and transformed as % control values for cross-sample comparisons. The above procedure was repeated with inhibitor concentrations spanning 0.3 - 60 ⁇ to determine the effective concentration (EC50) required to inhibit 50% N MT activity in cultured adipocytes. Choice of inhibitor concentrations and time period was chosen based on the results from the MTT studies.
• NNMT inhibitors were screened in biochemical assays for activity against three structurally similar methyltransferases, including catechol-O-methyltransferase (COMT), DNA (cytosine-5)-methyltransferase 1 (DNMT1), and protein arginine methyltransferase 3 (PRMT3). Additional biochemical assays were used to test the ability of compounds to inhibit nicotinamide phosphoribosyl transferase (NAMPT) and NAD+-dependent protein deacetylase sirtuin 1 (SIRTl), two enzymes in the NAD+ biosynthesis/salvage pathway.
• NAMPT nicotinamide phosphoribosyl transferase
• SIRTl NAD+-dependent protein deacetylase sirtuin 1
• IC50 values were calculated from dose-response curves established with 10 concentrations of a half-log dilution series.
• established enzyme specific inhibitors were included as positive controls for enzyme function and assay reproducibility.
• IC50 values were determined by nonlinear least- squares fitting of a 4-parameter dose-response curve to collected data points (Graphpad Prism 7.0; La Jolla, CA, USA). [000240] 5.5(a). D MT1 activity assay. A radiometric assay was performed by RBC using 100 ⁇ 5 nM SAH as an inhibitor positive control.
• the analogues, 1,8- diMQ and 5-amino-lMQ were tested at concentrations from 200 ⁇ - 10 nM and 600 ⁇ - 10 nM, respectively. Reactions were performed with 0.001 mg/ml DNA substrate Poly(dl-dC), 1 ⁇ radiolabelled S-adenosyl-L-[methyl-3H] methionine
• SAM SAM co-substrate
• human D MT1 enzyme Activity was monitored via quantification of radiolabeled reaction product DNA 5-[methyl-3H]- cytosine.
• PRMT3 activity assay A radiometric assay was performed by RBC using 100 ⁇ -5 nM SAH as an inhibitor positive control. The analogues, 1,8- diMQ and 5-amino-lMQ were tested at concentrations from 200 ⁇ - 10 nM and 600 ⁇ - 10 nM, respectively. Reactions were performed with 5 ⁇ histone H3 (histone L-arginine) substrate, 1 ⁇ radiolabeled S-adenosyl-L-[methyl-3H] methionine (SAM) co-substrate, and recombinant human PRMT3 enzyme. Activity was monitored via quantification of radiolabeled reaction product histone [methyl-
• COMT activity assay A radiometric assay was performed by RBC using 1 ⁇ - 50 pM tolcapone as an inhibitor positive control. The analogues, 1,8-diMQ and 5-amino- 1MQ were tested at concentrations from 200 ⁇ - 10 nM and 600 ⁇ - 10 nM, respectively. Reactions were performed with 0.5 ⁇ catechol substrate COMT-SOl, 1 ⁇ radiolabelled S-adenosyl-L-[methyl-3H] methionine (SAM) co-substrate, and recombinant human COMT enzyme. Activity was monitored via quantification of methylated catechol reaction product (guaiacol [methyl-3H]).
• SAM S-adenosyl-L-[methyl-3H] methionine
• NAMPT activity assay A fluorometric assay was performed by RBC using 1 ⁇ - 50 pM FK866 as an inhibitor positive control. The analogue 5- amino-lMQ was tested at concentrations from 600 ⁇ - 30 nM. Reactions were performed with 2 ⁇ nicotinamide and 30 ⁇ phosphoribosyl pyrophosphate (PRPP) in the presence of ImM ATP and recombinant human NAMPT enzyme. Activity was monitored using fluorescence detection and quantification of the nicotinamide mononucleotide (NMN) reaction product.
• NPN nicotinamide mononucleotide
• SIRT-1 activity assay A fluorometric assay was performed by RBC using 100 ⁇ -5 nM suramin sodium as an inhibitor positive control. The analogue 5-amino-lMQ was tested at concentrations from 600 ⁇ - 30 nM. Reactions were performed with 50 ⁇ RHKKAc, a fluorogenic peptide substrate from p53 residues 379-382, 500 ⁇ ⁇ + co- substrate, and recombinant human SIRT-1 (NAD+-dependent) enzyme. Activity was monitored by the formation of a fluorescent product (coumarin) generated by a two-step coupled reaction that involved deacetylation of substrate by SIRT-1 followed by secondary release of the fluorophore.
• coumarin coumarin
• mice were continued to be fedHFD (Open Source Diets formula D12451 from Research Diets Inc.; New Brunswick, NJ, USA), containing 45% energy from fat. Water was available ad libitum.
• HFD Open Source Diets formula D12451 from Research Diets Inc.; New Brunswick, NJ, USA
• mice in the vehicle cohort received three subcutaneous (SC) saline (1 ml/kg) injections/day (-0930, 1330, 1730 h) and mice in the treatment cohort received three SC injections of the NNMT inhibitor 5-amino-lMQ at a dose of 20 mg/kg/injection for a total dose of -34 mg/kg/day of the parent compound (calculated according to free weight) for 11 days.
• SC subcutaneous
• mice in the treatment cohort received three SC injections of the NNMT inhibitor 5-amino-lMQ at a dose of 20 mg/kg/injection for a total dose of -34 mg/kg/day of the parent compound (calculated according to free weight) for 11 days.
• the dose chosen was based on an initial dose escalation study (ranging from
• Body weight and food intake were measured every other day.
• mice were subjected to a 4 h fast period, then deeply anesthetized using isoflurane and trunk blood was collected by decapitation. Plasma was separated from every sample and the samples were submitted to Texas A&M Veterinary Medical Diagnostic Laboratory (TVMDL; College Station, TX, USA) for plasma lipid-panel measurements (total cholesterol and triglycerides).
• TVMDL Texas A&M Veterinary Medical Diagnostic Laboratory
• Triglycerides values were not included for analysis since the measurements were confounded by sample hemolysis that interfered with the triglyceride reagent in the assay.
• Epididymal fat pads epididymal white adipose tissue; EWAT
• EWAT epididymal white adipose tissue
• NNMT inhibitors display high membrane permeability.
• ND Not determined;
• Quinoiine is ars NNMT substrste[25]
• Differentiated 3T3-L1 adipocytes provide a relevant cell- based system to validate NNMT inhibitor mechanism-of-action.
• differentiated 3T3-L1 adipocytes could be utilized as a cell-based system for mechanism-of-action and phenotypic characterization of NNMT inhibitors, we measured the expression levels of NNMT and used LC/MS/MS to assess the levels of NNMT reaction product 1-MNA in fully differentiated adipocytes (day 9-10 post-differentiation) and undifferentiated pre- adipocytes (day 0).
• NNMT protein expression was found to be ⁇ 37-fold higher in the adipocytes (day 9) vs pre- adipocyte (P ⁇ 0.0001).
• 1-MNA levels normalized to total cellular protein were ⁇ 7.5-fold higher in adipocytes compared to pre- adipocytes (P ⁇ 0.05, pre-adipocytes vs. adipocytes), suggesting relatively higher activity of the NNMT enzyme in the fully differentiated adipocytes.
• NNMT inhibition using 5-amino- 1MQ (30 ⁇ concentration) in both the pre-adipocytes (P ⁇ 0.01, treated pre- adipocytes vs. untreated controls) and the adipocytes (P ⁇ 0.05, treated adipocytes vs. untreated controls) resulted in significant reduction in the intracellular levels of 1-MNA.
• NNMT inhibitors decrease production of 1-MNA in differentiated adipocytes.
• the relative effectiveness of NNMT inhibitors to lower 1-MNA levels in the differentiated adipocytes were compared at a single concentration of 10 ⁇ (concentration well below the cytotoxic concentration range for NNMT inhibitors).
• Dunnett's posthoc tests revealed that all membrane-permeable NNMT inhibitors tested significantly decreased 1-MNA levels in the adipocytes relative to control (5-amino-lMQ, P ⁇ 0.001; 3-amino-6-fluoro-lMQ, P ⁇ 0.01; and
• NNMT inhibition increases intracellular concentrations of NAD+ and SAM in differentiated adipocytes.
• Figure 1A outlines the major elements of the mammalian NAD+ salvage pathway using NA as the starting substrate. Since the NNMT inhibitor 5-amino-lMQ significantly reduced intracellular 1-MNA concentrations, we hypothesized that NNMT inhibition in adipocytes would increase intracellular concentrations of the co-substrates NA and SAM and shunt more NA into the NAD+ salvage cycle.
• NNMT inhibitors are selective and do not impact related methyltransferases or enzymes in the NAD+ salvage pathway.
• the selectivity of NNMT inhibitors was confirmed by testing against a panel of structurally similar methyltransferases and two enzymes in the NAD+ salvage pathway (NAMPT and SIRT1; Figures 1A and 7). Concentrations of 1,8-diMQ and 5-amino-lMQ ranging from 10 nM to 200 or 600 ⁇ , respectively, did not inhibit DNMT1 or PRMT3.
• Sigmoidal dose-response curves and reliable estimates of IC50 values based on non-linear least-squares fitting to the available data could not be obtained since no significant inhibition of DNMT1 and PRMT3 was observed at the tested NNMT inhibitor concentrations (Table 6). Additionally, 1,8-diMQ and 5-amino-lMQ showed little inhibition of COMT at maximal tested concentrations of 200 ⁇ (20% inhibition) and 600 ⁇ (10% inhibition), respectively, although no clear trend of concentration-dependent inhibition was observed. As was noted for D MT1 and PRMT3, sigmoidal dose-response curves and reliable estimates of IC50 values could not be obtained since no significant inhibition was observed at the tested N MT inhibitor concentrations.
• 5-amino-lMQ did not inhibit SIRT1 concentrations ranging from 10 nM - 300 ⁇ , and minor reduction in SIRT1 activity was observed with 600 ⁇ 5- amino-lMQ.
• sigmoidal dose-response curves and reliable estimates (i.e., R2 > 0.8) of IC50 values could not be obtained since no significant inhibition was observed with the tested concentrations of 5-amino-lMQ.
• NNMT inhibitor caused weight loss and reduced adipose tissue mass in DIO mice. Since in vitro studies showed 5-amino-lMQ to have high cell permeability, enzyme selectivity, and cell culture efficacy, a sub-chronic (11- day) proof-of-concept in vivo study was conducted to test the effect of NNMT inhibition on obesity in HFD fed mice. Three times daily systemic (SC) treatment of DIO mice with 20 mg/kg of 5-amino- 1MQ produced a progressive loss of body weight over the treatment period compared to controls ( Figure 8A).
• SC systemic
• cholesterol levels in the NNMT inhibitor-treated DIO mice were similar to cholesterol levels reported by the vendor for age-matched normal chow-fed C57B1/6 mice (www.jax.org/jax-mice-and-services/find-and-order-jax- mice/most- popular-jax-mice-strains/dio-b6).
• NNMT inhibition suppresses lipogenesis in 3T3-L1 cells.
• lipid accumulation was determined in adipocytes following treatment of 3T3-L1 cells with the NNMT inhibitor in media containing adipogenic factors.

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