Phenylethanolamine n-methyltransferase (PNMT) inhibitors to regulate adrenaline responses

Abstract

Disclosed herein are phenylethanolamine n-methyltransferase (PNMT) inhibitors to regulate adrenaline responses. The inhibitors exhibit high specificity for PNMT over other DNA and protein methyltransferases. Also disclosed are pharmaceutical compositions of the inhibitors and methods of treating various diseases associated with associated with dysregulated epinephrine production.

Description

PHENYLETHANOLAMINE N-METHYLTRANSFERASE (PNMT) INHIBITORS TOREGULATE ADRENALINE RESPONSESTECHNICAL FIELD

[0001] Disclosed herein are inhibitors of phenylethanolamine N-methyltransferase and their use in treating various diseases and conditions.BACKGROUND

[0002] Epinephrine is a hormone, synthesized in the adrenal medulla, which is released into the blood stream in response to stress and produces profound physiological changes which serve to prepare the animal to cope with the stressor situation. For example, epinephrine produces anxiety, an increase in blood pressure, acceleration of heart rate and increase in cardiac output. These changes are detrimental in individuals with certain disease conditions such as angina pectoris, myocardial infarction and anxiety neuroses.

[0003] Phenylethanolamine N-methyltransferase (PNMT) catalyzes the final step in the biosynthesis of epinephrine, that is the transfer of a methyl group from S-adenosylmethionine to norepinephrine to produce epinephrine. Studies suggest that PNMT may play a role in human disease states including hypertension, myocardial infarction, Alzheimer’s, and Parkinson’s diseases. Catecholamines may also be involved in post-traumatic stress disorder (PTSD). PNMT- knockout mice (Pnmt-KO) induced with PTSD showed a decrease in stress-related behaviors compared to wild-type mice. However, when administered with Epinephrine, an increase in stress behavior was observed, suggesting that Epinephrine may be involved in the persistence of traumatic memories in PTSD, conceivably by enhancing the expression of Nr4a2 and Nr4a3 genes in the hippocampus. Nr4a is a subfamily of orphan nuclear receptors genes which encode for transcription factors Nr4al, Nr4a2, and Nr4a3, which have been implicated in contextual fear memory formation.

[0004] Thus, there is a need to develop new agents for treating diseases or conditions associated with overproduction of epinephrine or where epinephrine production is detrimental.SUMMARY

[0005] The compounds disclosed herein inhibit phenylethanolamine N-methyltransferase and thus reduce the formation of epinephrine. These compounds mimic the geometry and electrostatic properties of an enzymatic transition state in chemically stable structures and exhibit high affinity to bind their cognate active sites for inhibition of methyltransferase enzymes.

[0006] An aspect of the patent document provides a compound of Formula I and pharmaceutically acceptable salts thereof. The compound is generally represented as follows:Formula IWherein:L is a linker selected from the group consisting of 3-6 membered heterocylic, 6- membered aryl, 6-membered heteroaryl, -(CH2)aC(0)NRa-, -C(O)NRa-, - (CH2)aC(O)NRa(CH2)b-, -(CH2)2+a-, -(CH2)aO(CH2CH2O)c-, -(CH2)2+aS, - (CH2)aheterocyclyl-, -(CH2)aC(O)-, -(CH2)aNRa-, -CRa=N-NRa-, -CRa=N-O-, -CRa=N- NRb-CO-, -N=N-CO-, -S-S-, and any combination thereof, wherein a, b, and c are each an integer selected from 0 to 8;R1, R2, R3, and R4in each instance is independently selected from the group consisting of H, Ci-4alkyl, OCl-4alkyl, NRaRb, haloCi-4alkyl, halogen, OH, CN, hydroxyl-alkyl, dihydroxyl-alkyl, C(O)ORa, C(O)NRaRa, C(O)Ci-ealkyl, phenyl and 5- or 6-membered hereroaryl, wherein the phenyl and 5- or 6-membered hereroaryl are optionally substituted with one or more substituents selected from the group consisting of Ci-4alkyl, OCi-4alkyl, NRaRb, halo Ci-4alkyl, halogen, OH, and CN, wherein at least one of R1and R2is not H;Raand Rbin each instance are independently H or Ci-4alkyl;R5is H, Ci-4alkyl, C(O)ORa, or C(O)Ci-6alkyl;R6is H or Ci-4alkyl;R7in each instance is independently H or Ci-4alkyl;R8in each instance is independently H, Ci-4alkyl, C(O)ORa, C(O)NRaRa, or C(O)Ci- ealkyl;Ring A is a 5- or 6-membered hetercyclyl; n is an integer of 0 to 5; provided that the compound is notr its stereoisomer.

[0007] In some embodiments, the A ring is 6-membered. In some embodiments, L is a C3- salkylene. In some embodiments, L is a C4alkylene. In some embodiments, R1and R2are independently selected from the group consisting of halogen, Ci-4alkyl, OCl-4alkyl, haloCi-4alkyl, and CN. In some embodiments, R1and R2are each independently halogen or Ci-4alkyl. In some embodiments, R1and R2are each chlorine. In some embodiments, R3and R4are each H. In some embodiments, R5is H. In some embodiments, R5is H, R7is H. In some embodiments, R5is H, R6is H, R7is H.

[0008] In some embodiments, the compound

[0009] Also provided are pharmaceutical compositions comprising a therapeutically effective amount of a compound of Formula I disclosed herein or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

[0010] Another aspect provides a method for treating a disease or condition. The method includes administering to a subject in need thereof a compound of formula I, a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof.

[0011] Another aspect provides a method for inhibiting phenylethanolamine N- methyltransferase. The method includes contacting a cell with an effective amount of thecompound of formula I or a pharmaceutically acceptable salt thereof or a pharmaceutical composition thereof.DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 illustrates the synthesis of a compound of Formula I.

[0013] Figure 2 illustrates the synthesis of a compound of Formula II.DETAILED DESCRIPTION

[0014] Various embodiments of this patent document disclose compounds as inhibitors of Phenylethanolamine N-m ethyl transferase (PNMT). The inhibitors exhibit high specificity for PNMT over other targets such as DNA and protein methyltransferases and a2-adrenoceptor. Because multiple diseases including for post-traumatic stress disorder (PTSD), hypertension, myocardial infarction, Alzheimer’s disease, Parkinson’s disease, and neuropathic pain are associated with overproduction of epinephrine production, the compounds disclosed herein possess great therapeutic utilities for treating diseases associated with imbalance in PNMT catalyzed conversion of norepinephrine to epinephrine.

[0015] While the following text may reference or exemplify specific embodiments of a compound, substituent, or use thereof, it is not intended to limit the scope of the compound, substituent or its use to such particular references or examples. Various modifications may be made by those skilled in the art, in view of scientific and practical considerations, such as replacement of a substituent or treatment of other diseases.

[0016] The articles "a" and "an" as used herein refer to "one or more" or "at least one," unless otherwise indicated. That is, reference to any element or component of an embodiment by the indefinite article "a" or "an" does not exclude the possibility that more than one element or component is present.

[0017] The term "alkyl" refers to a hydrocarbon or a hydrocarbon chain which may be either straight-chained or branched. The term "Ci-6 alkyl" refers to alkyl groups having 1, 2, 3, 4, 5 or 6 carbon atoms. Non-limiting examples include groups such as CH3, (CH2)2CHs, CH2CH(CH3)CH3, and the like. Similarly, the term "C2-5 alkyl" refers to alkyl groups having 2, 3, 4 or 5 carbon atoms.

[0018] The term “alkylene” refers to a divalent hydrocarbon or a hydrocarbon chain which may be either straight-chained or branched. Non-limiting examples include groups such as CH2,(CH2)2CH2, CH2CH(CH3)CH2, and the like. A Ci-3alkylene includes alkylenes with 1, 2 or 3 carbons such as CH2, (CH2)2, CHCH3,(CH2)3, and CH(CH3)CH2.

[0019] The term "cycloalkyl" refers to saturated and partially unsaturated cyclic hydrocarbon groups having 3 to 12 ring carbons, for example 3 to 8 carbons, and as a further example 3 to 6 carbons, wherein the cycloalkyl group additionally is optionally substituted. Examples of cycloalkyl groups include, without limitation, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, and cyclooctyl.

[0020] The term "aryl" group refers to a Ce-14 aromatic moiety comprising one to three aromatic rings, which is optionally substituted. Examples of aryl groups include, without limitation, phenyl, naphthyl, anthracenyl, fluorenyl, and dihydrobenzofuranyl.

[0021] The term “haloalkyl” refers to a Ce-ioalkyl chain, straight or branched, in which one or more hydrogen has been replaced by a halogen. Non-limiting examples of haloalkyls include CHF2, CFH2, CF3, CH2CHF2, CH2CH2C1, CH2CF3, and CH2CH2F. In some embodiments, the alkyl in haloalkyl has 1, 2, 3 or 4 carbons.

[0022] The term “heteroalkyl” refers to a Ce-ioalkyl group, straight or branched, wherein one or more carbon atoms in the chain are replaced by one or more heteroatoms selected from the group consisting of O, S, N and NRm. In some embodiments, the alkyl in heteroalkyl has 1 to 10 carbons. In some embodiments, the alkyl in heteroalkyl has 2, 3, 4 or more than 2 carbons.

[0023] The term “hydroxy alkyl” refers to a Ce-ioalkyl chain, straight or branched, wherein a carbon is substituted with a hydroxyl group. The carbon the hydroxyl is attached to is a primary carbon or secondary carbon. In some embodiments, the alkyl in hydroxylalkyl has 2, 3, 4 or more than 2 carbons.

[0024] The term “dihydroxyalkyl” refers to a C2-ioalkyl chain, straight or branched, wherein two carbons are each substituted with a hydroxyl group. In some embodiments, the alkyl in dihydroxylalkyl has 2, 3, 4 or more than 2 carbons.

[0025] The term “heterocyclyl” or “heterocyclic” group is a ring structure having from about 3 to about 12 atoms, for example 4 to 8 atoms, wherein one or more atoms are selected from the group consisting of N, O, and S, the remainder of the ring atoms being carbon. The heterocyclyl may be a monocyclic, a bicyclic, a spirocyclic or a bridged ring system. Examples of heterocyclic groups include, without limitation, epoxy, azetidinyl, aziridinyl, azocanyl, azepanyl, diazepanyl, dihydrofuranyl, tetrahydrofuranyl, tetrahydropyranyl, oxazepanyl, pyrrolidinyl, pyrrolidinonyl, piperidinyl, piperazinyl, imidazolidinyl, thiazolidinyl, thiooxazepanyl, dithianyl, trithianyl,dioxolanyl, oxazolidinyl, oxazolidinonyl, decahydroquinolinyl, piperidonyl, 4-piperidinonyl, thiomorpholinyl, thiomorpholinyl 1,1 dioxide, morpholinyl, oxazepanyl, azabicyclohexanes, azabicycloheptanes and oxa azabiocycloheptanes. Specifically excluded from the scope of this term are compounds having adjacent annular O and / or S atoms.

[0026] The term “heteroaryl” refers to groups having 5 to 14 ring atoms, preferably 5, 6, 9, or 10 ring atoms; having 6, 10, or 147t electrons shared in a cyclic array; and having, in addition to carbon atoms, from one to three heteroatoms per ring selected from the group consisting of N, O, and S. Examples of heteroaryl groups include acridinyl, azocinyl, benzimidazolyl, benzofuranyl, benzothiofuranyl, benzothiophenyl, benzoxazolyl, benzthiazolyl, benztriazolyl, benztetrazolyl, benzisoxazolyl, benzisothiazolyl, benzimidazolinyl, carbazolyl, 4aH-carbazolyl, carbolinyl, chromanyl, chromenyl, cinnolinyl, furanyl, furazanyl, imidazolinyl, imidazolyl, 1H- indazolyl, indolenyl, indolinyl, indolizinyl, indolyl, 3H-indolyl, isobenzofuranyl, isochromanyl, isoindazolyl, isoindolinyl, isoindolyl, isoquinolinyl, isothiazolyl, isoxazolyl, methylenedioxyphenyl, naphthyridinyl, octahydroisoquinolinyl, oxadiazolyl, 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, oxazolidinyl, oxazolyl, oxazolidinyl, pyrimidinyl, phenanthridinyl, phenanthrolinyl, phenazinyl, phenothiazinyl, phenoxathiinyl, phenoxazinyl, phthalazinyl, piperonyl, pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl, pyridooxazole, pyridoimidazole, pyridothiazole, pyridinyl, pyridyl, pyrimidinyl, pyrrolinyl, 2H-pyrrolyl, pyrrolyl, quinazolinyl, quinolinyl, 4H-quinolizinyl, quinoxalinyl, quinuclidinyl, tetrahydroisoquinolinyl, tetrahydroquinolinyl, tetrazolyl, 6H-1,2,5- thiadiazinyl, 1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl, 1,3,4-thiadiazolyl, thianthrenyl, thiazolyl, thienyl, thi enothiazolyl, thienooxazolyl, thienoimidazolyl, thiophenyl, triazinyl, 1,2,3-triazolyl, 1,2,4-triazolyl, 1,2,5-triazolyl, 1,3,4-triazolyl, and xanthenyl.

[0027] The term “halogen” refers to F, Cl, Br or I.

[0028] The term “subject” refers to humans or animals including for example sheep, horses, cattle, pigs, dogs, cats, rats, mice, birds, and reptiles. Preferably, the subject is a human or other mammal.

[0029] The term “effective amount” or “therapeutically effective amount” of a compound is an amount that is sufficient to ameliorate, or in some manner reduce a symptom or stop or reverse progression of a condition, or negatively modulate or inhibit activity. Such amount may be administered as a single dosage or may be administered according to a regimen, whereby it is effective.

[0030] The term “pharmaceutically acceptable” refers to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues, organs, and / or bodily fluids of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit / risk ratio.

[0031] The term “pharmaceutically acceptable carrier” refers to a chemical compound that facilitates the delivery or incorporation of a compound or therapeutic agent into cells or tissues.

[0032] The term “pharmaceutically acceptable salts” means salts of compounds of the present invention which are pharmaceutically acceptable, as defined above, and which possess the desired pharmacological activity. Non-limiting examples of such salts include acid addition salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, and phosphoric acid; or with organic acids such as 1,2-ethanedi sulfonic acid, 2-hydroxy ethanesulfonic acid, 2-naphthalenesulfonic acid, 3 -phenylpropionic acid, 4,4'-methylenebis(3-hydroxy- 2-ene- 1 -carboxylic acid), 4-methylbicyclo[2.2.2]oct-2-ene- 1 -carboxylic acid, acetic acid, aliphatic mono- and dicarboxylic acids, aliphatic sulfuric acids, aromatic sulfuric acids, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, carbonic acid, cinnamic acid, citric acid, cyclopentanepropionic acid, ethanesulfonic acid, fumaric acid, glucoheptonic acid, gluconic acid, glutamic acid, glycolic acid, heptanoic acid, hexanoic acid, hydroxynaphthoic acid, lactic acid, laurylsulfuric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, muconic acid, o-(4-hydroxybenzoyl)benzoic acid, oxalic acid, / ?-chlorobenzenesulfonic acid, phenyl -substituted alkanoic acids, propionic acid, -toluenesulfonic acid, pyruvic acid, salicylic acid, stearic acid, succinic acid, tannic acid, tartaric acid, tertiarybutyl acetic acid, and trimethylacetic acid. Pharmaceutically acceptable salts also include base addition salts which may be formed when acidic protons present are capable of reacting with inorganic or organic bases. Acceptable inorganic bases include sodium hydroxide, sodium carbonate, potassium hydroxide, aluminum hydroxide and calcium hydroxide. Nonlimiting examples of acceptable organic bases include ethanolamine, diethanolamine, ethylenediamine, triethanolamine, tromethamine, and / ' / -methylglucamine. It should be recognized that the particular anion or cation forming a part of any salt of this invention is not critical, so long as the salt, as a whole, is pharmacologically acceptable. Additional examples of pharmaceutically acceptable salts and their methods of preparation and use are presented in Handbook of Pharmaceutical Salts: Properties, and Use (P. H. Stahl & C. G. Wermuth eds., Verlag Helvetica Chimica Acta, 2002).

[0033] The term “pharmaceutical composition” refers to a mixture of a compound disclosed herein with other chemical components, such as diluents or additional carriers. The pharmaceutical composition facilitates administration of the compound to an organism. Multiple techniques of administering a pharmaceutical composition exist in the art including, but not limited to, oral, injection, aerosol, parenteral, intranasal, sublingual, inhalational, and topical administration. In some embodiments, pharmaceutically acceptable salts of the compounds disclosed herein are provided.

[0034] The term "treating" or "treatment" of any disease or condition refers, in some embodiments, to ameliorating the disease or disorder (i.e., arresting or reducing the development of the disease or at least one of the clinical signs and symptoms thereof). In some embodiments "treating" or "treatment" refers to ameliorating at least one physical parameter, which may not be discernible by the subject. In some embodiments, "treating" or "treatment" refers to modulating the disease or disorder, either physically, (e.g., stabilization of a discernible symptom), physiologically, (e.g., stabilization of a physical parameter), or both. In some embodiments, "treating" or "treatment" refers to delaying the onset of the disease or disorder, or even preventing the same. “Prophylactic treatment” is to be construed as any mode of treatment that is used to prevent progression of the disease or is used for precautionary purpose for persons at risk of developing the condition.

[0035] Phenylethanolamine Mm ethyl transferase (PNMT) catalyzes the 5-adenosyl-L- methionine (SAM)-dependent methylation of norepinephrine to form epinephrine. Epinephrine is implicated in the regulation of blood pressure, respiration, Alzheimer’s disease, and post-traumatic stress disorder (PTSD). Transition state (TS) analogues bind their target enzymes orders of magnitude more tightly than their substrates.

[0036] Kinetic isotope effect studies indicate a transition state (TS) for PNMT proceeding through an early dissociative SN2 mechanism. The rate limiting step is the transfer of the methyl group from 5-adenosyl-L-methionine (SAM) to norepinephrine. The bisubstrate nature of the reaction catalyzed by PNMT led to the development of norepinephrine or SAM substrate analogues as inhibitors. Most norepinephrine mimics that compete with the binding of norepinephrine exhibit problems of specificity and permeability, prohibiting their further development. SAM analogues have been developed as methyltransferase inhibitors, but the wide range of SAM-based methyltransferases poses a challenge to develop selective agents.

[0037] Transition state analogues (TSA) mimic the geometry and electrostatic properties of an enzymatic transition state in chemically stable structures and have the potential to bind their cognate active sites orders of magnitude more tightly than substrate analogues. TS analogues are specific for their target protein and have the potential to minimize off-target interactions.

[0038] Compounds

[0039] An aspect of the disclosure provides a compound of Formula X a pharmaceutically acceptable salt thereof as a transition state PNMT inhibitor, which is represented by the structure below:

[0040] When M is M-l, Formula X is represented by Formula I below.L is a linker selected from the group consisting of 3-6 membered heterocylic, 6- membered aryl, 6-membered heteroaryl, -(CH2)aC(0)NRa-, -C(O)NRa-, - (CH2)aC(O)NRa(CH2)b-, -(CH2)2+a-, -(CH2)aO(CH2CH2O)c-, -(CH2)2+aS, - (CH2)aheterocyclyl-, -(CH2)aC(O)-, -(CH2)aNRa-, -CRa=N-NRa-, -CRa=N-O-, -CRa=N- NRb-CO-, -N=N-CO-, -S-S-, and any combination thereof, wherein a, b, and c are each an integer selected from 0 to 8, all subunits (e.g. 0, 1, 2, 3, 4, 5, 6, 7, 8) included;R1, R2, R3, and R4in each instance is independently selected from the group consisting of H, Ci-4alkyl, OCl-4alkyl, NRaRb, haloCi-4alkyl, halogen, OH, CN, C(O)ORa, C(O)N(Ra)2, C(O)Ci-ealkyl, phenyl and 5- or 6-membered hereroaryl, wherein the phenyl and 5- or 6- membered hereroaryl are optionally substituted with one or more substituents selected from the group consisting of Ci-4alkyl, OCi-4alkyl, NRaRb, halo Ci-4alkyl, halogen, OH, and CN, wherein at least one of R1and R2is not H;Raand Rbin each instance are independently H or Ci-4alkyl;R5is H, Ci-4alkyl, C(O)ORa, or C(O)Ci-6alkyl;R6is H or Ci-4alkyl;R7in each instance is independently H or Ci-4alkyl;R8in each instance is independently H, Ci-4alkyl, C(O)ORa, C(O)NRaRa, or C(O)Ci- ealkyl;Ring A is a 5- or 6-membered hetercyclyl; and n is an integer of 0 to 5.

[0041] In some embodiments, n is 1, 2 or 3.

[0042] In some embodiments, the compound is notIn some embodiments, the compound is notr its stereoisomer.

[0043] In instances where the compound contains one or more chiral centers, each of them can be R or S configuration.

[0044] Ring A can be a 4, 5, or 6 membered ring. In some embodiments, A is a 6-membered ring. In some embodiments, the compound is represented by Formula I-a.

[0045] Linker L can be a carbon chain, wherein each carbon may be replaced with a heteroatom or group (e.g. O, S, NH, or NCi-4alkyl) or substituted with a group such as NHCi-4alkyl, NH2, Ci-4alkyl, OCi-4alkyl, OH, halogen, and SH). In some embodiments, the linker is a C3- salkylene, which is optionally replaced or substituted with one or more aforementioned atoms or groups. In some embodiments, the linker is C4alkylene (CH2CH2CH2CH2), which is optionally substituted as described above.

[0046] In some embodiments, compound is represented by Formula I-bm wherein L is C3- salkylene, and is preferably C4alkylene (CH2CH2CH2CH2). In some embodiments, R3and R4are both H.Formula I-b

[0047] In some embodiments, the compound of Formula I is represented by Formula I-c.

[0048] None limiting examples of I-c include the following (n=0, 1, or 2)

[0049] In some embodiments, R1and R2are independently selected from the group consisting of halogen, Ci-4alkyl, OCl-4alkyl, haloCi-4alkyl, and CN. In some embodiments, R1and R2are each independently halogen or Ci-4alkyl. In some embodiments, R1and R2are each chlorine. In some embodiments, R3and R4are each H.

[0050] In some embodiments, R5is H. In some embodiments, R5is H, R7is H. In some embodiments, R5is H, R6is H, R7is H.

[0051] In some embodiments, the compound is represented by the formula below. Other stereocenters of the compound are independently in an R or S configuration. In some emodiments, the carbon marked with an asterisk has an R configuration. In some emodiments, the carbon marked with an asterisk has an S configuration. In some embodiments, the compound is a mixture of two diasteromers where one has R configuration in the asterisk carbon and the other has S configuration.

[0052] When M is M-2, Formula X is represented by Formula II below.Formula IIL1and L2are each a Ci-salkylene linker;R1, R2, and R3in each instance are independently selected from the group consisting of H, Ci-4alkyl, OCl-4alkyl, NRaRb, haloCi-4alkyl, halogen, OH, CN, hydroxyl-alkyl, dihydroxyl-alkyl, C(O)ORa, C(O)NRaRa, C(O)Ci-ealkyl, phenyl and 5- or 6-memberedhereroaryl, wherein the phenyl and 5- or 6-membered hereroaryl are optionally substituted with one or more substituents selected from the group consisting of Ci-4alkyl, OCi-4alkyl, NRaRb, halo Ci-4alkyl, halogen, OH, and CN, wherein at least one of R1and R2is not H;Raand Rbin each instance are independently H or Ci-4alkyl;R4and R5are independently H, Ci-4alkyl, C(O)ORa, or C(O)Ci-ealkyl;R6is H or Ci-4alkyl;R7in each instance is independently H or Ci-4alkyl;R8in each instance is independently H, Ci-4alkyl, C(O)ORa, C(O)NRaRa, or C(O)Ci- ealkyl; and n is an integer of 0 to 5.

[0053] In instances where the compound contain one or more chiral centers, each of them can be R or S configuration.

[0054] In some embodiments, L1and L2are each a Ci-2alkylene linker. In some embodiments, L1and L2are each a methylene linker.

[0055] In some embodiments, R1, R2and R3in each instance are independently selected from the group consisting of H, Ci-4alkyl, and halogen. In some embodiments, R1, R2and R3are each hydrogen.

[0056] In some embodiments, R4and R5are independently H or Ci-4alkyl. In some embodiments, R4and R5are hydrogen.

[0057] In some embodiments, R6is H. In some embodiments, R7in each instance is independently H or Ci-4alkyl. In some embodiments, R8in each instance is independently H or Ci- 4alkyl. In some embodiments, R6, R7andR8are each hydrogen.

[0058] In some embodiments, the compound iswherein m is 1, 2 3 or 4.

[0059] Pharmaceutical Composition and Kit

[0060] Another aspect of the patent specification provides a pharmaceutical composition comprising a compound of Formula I or II or a pharmaceutically acceptable salt thereof disclosed herein and a pharmaceutically acceptable carrier, excipient, or diluent. Compounds described in this patent specification may be formulated by any method well known in the art and may be prepared for administration by any route, including, without limitation, parenteral, peroral, sublingual, buccal, intrathecal, transdermal, topical, subcutaneous, intramuscular, intraperitoneal, intranasal, intratracheal, or intrarectal.

[0061] Nonlimiting examples of pharmaceutically acceptable carriers include physiologically acceptable surface active agents, glidants, plasticizers, diluents, excipients, smoothing agents, suspension agents, complexing agents, film forming substances, and coating assistants. Preservatives, stabilizers, dyes, sweeteners, fragrances, flavoring agents, and the like may be provided in the pharmaceutical composition. For example, sodium benzoate, ascorbic acid and esters of p-hydroxybenzoic acid may be added as preservatives. In addition, antioxidants and suspending agents may be used. In various embodiments, alcohols, esters, sulfated aliphatic alcohols, and the like may be used as surface active agents. Suitable exemplary binders include crystalline cellulose, sucrose, D-mannitol, dextrin, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, and the like. Suitable exemplary disintegrants include starch, carboxymethylcellulose, calcium carboxymethylcellulose, croscarmellose sodium, sodium carboxymethylstarch, and the like. Suitable exemplary solvents or dispersion media include water, alcohol (for example, ethanol), polyols (for example, glycerol, propylene glycol, and polyethylene glycol, sesame oil, corn oil, and the like), and suitable mixtures thereof that are physiologically compatible. Suitable exemplary solubilizing agents include polyethylene glycol, propylene glycol, D-mannitol, benzylbenzoate, cyclodextrins, ethanol, trisaminomethane, cholesterol, triethanolamine, sodium carbonate, sodium citrate, and the like. Suitable exemplary suspending agents include surfactants such as stearyltriethanolamine, sodium laurylsulfate, laurylaminopropionic acid, lecithin, benzalkonium chloride, benzethonium chloride, glycerin monostearate, coconut oil, olive oil, sesame oil, peanut oil, soya and the like; and hydrophilic polymers such as polyvinyl alcohol, polyvinylpyrrolidone, sodium carboxymethylcellulose, methylcellulose, hydroxymethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, and the like. Suitable exemplary isotonic agent includes sodium chloride, glycerin, D-mannose, and the like. Suitable exemplary buffer agents include buffer solutions of salts, such as phosphate, acetates, carbonates, and citrates. Suitable exemplary soothing agentsinclude benzyl alcohol, and the like. Suitable exemplary antiseptic substances include paraoxybenzoic acid esters, benzethonium chloride, benzalkonium chloride, chlorobutanol, benzyl alcohol, phenethyl alcohol, dehydroacetic acid, sorbic acid, and the like. Suitable exemplary antioxidants include sulfite salts, ascorbic acid, and the like. Suitable exemplary sealers include, but are not limited to HPMC (or hypromellose), HPC, PEG and combinations thereof. Suitable exemplary lubricants include magnesium stearate, calcium stearate, talc, colloidal silica, hardened oil and the like.

[0062] In further exemplary embodiments for solid preparations, carriers or excipients include diluents, lubricants, binders, and disintegrants. In exemplary embodiments for liquid preparations, carriers include solvents, solubilizing agents, suspending agents, isotonic agents, buffer agents, soothing agents, and the like. Acceptable additional carriers or diluents for therapeutic use and the general procedures for the preparation of pharmaceutical compositions are well known in the pharmaceutical art, and are described, for example, in Remington’s Pharmaceutical Sciences, 18th Ed., Mack Publishing Co., Easton, PA (1990), which is incorporated herein by reference in its entirety.

[0063] The compound of Formula I or II may also be in a pharmaceutically acceptable salt form. Examples of such salts include, but are not limited to acid addition salts formed with inorganic acids (for example, hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid, and the like), and salts formed with organic acids such as acetic acid, oxalic acid, tartaric acid, succinic acid, malic acid, ascorbic acid, benzoic acid, tannic acid, pamoic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acid, naphthalenedisulfonic acid, and polygalacturonic acid. The compounds can also be administered as pharmaceutically acceptable quaternary salts known by those skilled in the art, which specifically include the quaternary ammonium salt, wherein the counterion includes, for example, chloride, bromide, iodide, -O-alkyl, toluenesulfonate, methyl sulfonate, sulfonate, phosphate, or carboxylate (such as benzoate, succinate, acetate, glycolate, maleate, malate, citrate, tartrate, ascorbate, benzoate, cinnamoate, mandeloate, benzyloate, and diphenylacetate).

[0064] A related aspect provides a kit, which includes a compound of Formula I or II or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof and an instruction for treating or preventing certain diseases or conditions. In some embodiments, the kit further includes an additional secondary therapeutic agent.

[0065] Nonlimiteing examples of secondary agents include atypical antipsychotics, mood stabilizers, benzodiazepines, buspirone, stimulants, norepinephrine reuptake inhibitors, augmentation strategies (such as lithium, thyroid hormone, or atypical antipsychotics), gabapentin, pregabalin, bupropion, aripiprazole, ziprasidone, quetiapine, risperidone, olanzapine, lamotrigine, valproate, clonazepam, lorazepam, alprazolam, triazolam, zolpidem, zaleplon, eszopiclone, mirtazapine, trazodone, vilazodone, vortioxetine, agomelatine, modafinil, armodafmil, memantine, topiramate, riluzole, tramadol, opioids (in select cases), dextromethorphan, memantine, omega-3 fatty acids, n-acetylcysteine, s-adenosylmethionine (same), 1-methylfolate, 1- tryptophan, st. John's wort, lithium orotate, dehydroepiandrosterone (dhea), naltrexone, and paliperidone.

[0066] In some embodiments, the secondary agent is a selective serotonin reuptake inhibitor. Nonlimiting examples of selective serotonin reuptake inhibitors include Fluoxetine, Sertraline, Escitalopram, Paroxetine, Citalopram, Fluvoxamine, Vilazodone, Vortioxetine, Desvenlafaxine, Levomilnacipran, Alaproclate, Ifoxetine, Citalopram hydrobromide, Paroxetine mesylate, Fluvoxamine maleate, Levomilnacipran hydrochloride, Escitalopram oxalate, Vilazodone hydrochloride, Zimelidine, Edivoxetine, Fluoxetine hydrochloride, Dapoxetine, Femoxetine, Indalpine, Etoperidone, Zimelidine dihydrochloride, Vortioxetine hydrobromide, PRC-050, Zimelidine hydrochloride, Sertraline hydrochloride, Fluoxetine hydrochloride hemihydrate, Fluvoxamine maleate hemihydrate, Fluvoxamine hydrochloride, Citalopram hydrobromide monohydrate, Fluoxetine hydrochloride, Paroxetine hydrochloride hemihydrate, Paroxetine hydrochloride, Fluoxetine hydrochloride hydrate, Sertraline hydrochloride hydrate, Levomilnacipran hydrochloride hydrate, Fluoxetine hydrochloride monohydrate, Citalopram hydrochloride, Paroxetine hydrochloride monohydrate, Escitalopram oxalate hydrate, Escitalopram oxalate monohydrate, Fluoxetine hydrochloride dihydrate, Vilazodone hydrochloride hydrate, Fluoxetine hydrochloride anhydrous, Sertraline hydrochloride monohydrate, Paroxetine hydrochloride anhydrous, Sertraline hydrochloride dihydrate, Citalopram hydrobromide hydrate, Escitalopram oxalate dihydrate, Levomilnacipran hydrochloride dihydrate, Fluvoxamine hydrochloride monohydrate, Paroxetine hydrochloride dihydrate, Citalopram hydrochloride hydrate, Escitalopram oxalate anhydrous, Vilazodone hydrochloride dihydrate, Fluoxetine hydrochloride trihydrate, and Sertraline hydrochloride hydrate.

[0067] In some embodiments, the secondary agent is a serotonin and norepinephrine reuptake inhibitor. Nonlimiting examples of serotonin and norepinephrine reuptake inhibitorsinclude Venlafaxine, Duloxetine, Desvenlafaxine, Levomilnacipran, Milnacipran, Atomoxetine, Reboxetine, Maprotiline, Nefazodone, Mirtazapine, Amoxapine, Bupropion, Trazodone, Viloxazine, Sibutramine, Meptazinol, Seproxetine, Indalpine, PRC200-SS, Tesofensine, Talopram, Nisoxetine, Cyclizine, Sertraline, Clemastine, Clomipramine, Levomilnacipran, Methoxetamine, Venlafaxine XR, Vilazodone, Desipramine, Duloxetine HC1, Pridopidine, Agomelatine, Duloxetine hydrochloride, Pirlindole, PNU-99761, NS-2359, Tapentadol, Paroxetine, Bicifadine, Flibanserin, Bupropion hydrobromide, Esreboxetine, IFO-300, PNU- 50,194A, Sibutramine hydrochloride monohydrate, Etoperidone, GW-353162, ORG-13008, VMAT2 inhibitors, Radafaxine, Tenilapine, F-2695, Zimelidine, Indatraline, Dasotraline, Viloxazine hydrochloride, Epelsiban, Tianeptine, and Nisoxetine hydrochloride.

[0068] Method of Treating Diseases

[0069] Another aspect of the patent specification provides for methods for treating a disease or condition via inhibition of phenylethanolamine N-methyltransferase. The method includes administering to a subject in need thereof the compound of formula I or II, a pharmaceutically acceptable salt thereof, or a corresponding pharmaceutical composition disclosed herein. The compound or a pharmaceutically acceptable salt thereof or a pharmaceutical composition thereof can be administered prophylactically and after the occurrence of a symptom. The method may include administering a second agent described above.

[0070] Phenylethanolamine N-methyltransferase (PNMT) catalyzes the conversion of norepinephrine (NEpi) to epinephrine (Epi) as the final step in the catecholamine biosynthetic pathway. In humans, expression of PNMT occurs largely in the adrenal gland where epinephrine acts as a hormone for adrenergic receptors. Epinephrine in the human central nervous system (CNS) has been associated with cardiovascular homeostasis, regulation of circadian cycle, and adrenergic receptor activation. Studies suggest that PNMT may also play a role in human disease states or conditions including hyperadrenergic condition, hypertension, myocardial infarction, Alzheimer’s, and Parkinson’s diseases. Catecholamines may also be involved in post-traumatic stress disorder (PTSD). PNMT-knockout mice (Pnmt-KO) induced with PTSD showed a decrease in stress-related behaviors compared to wild-type mice. However, when administered with Epi, an increase in stress behavior was observed, suggesting that Epi may be involved in the persistence of traumatic memories in PTSD, conceivably by enhancing the expression of Nr4a2 and Nr4a3 genes in the hippocampus. Nr4a is a subfamily of orphan nuclear receptors genes which encode for transcription factors Nr4al, Nr4a2, and Nr4a3, which have been implicated in contextual fearmemory formation. Thus, there is a plausible mechanism suggesting a driving role of Epi production as the key to memory persistence in PTSD patient experiences.

[0071] Other diseases or conditions associated with dysregulation, dysfunction or overproduction of epinephrine and can be treated with the compounds disclosed herein include for example post-traumatic stress disorder (PTSD), hypertension, myocardial infarction, Alzheimer’s disease, Parkinson’s disease, neuropathic pain, major depressive disorder, obsessive-compulsive disorder (OCD), panic disorder, and bulimia nervosa, social anxiety disorder, generalized anxiety disorder, pheochromocytoma, paroxysmal tachycardia, panic disorder, generalized anxiety disorder, hyperthyroidism, graves' disease, essential tremor, postural orthostatic tachycardia syndrome (pots), carcinoid syndrome, neuroblastoma, medullary thyroid carcinoma, men 2a and 2b syndromes, multiple endocrine neoplasia type 1 (menl), sympathetic ophthalmia, takotsubo cardiomyopathy (broken heart syndrome), catecholaminergic polymorphic ventricular tachycardia (cpvt), hyperadrenergic orthostatic hypotension, systemic mastocytosis, neuroendocrine tumors, orthostatic intolerance, factitious disorder, labile hypertension, erythromelalgia, hyperhidrosis, von hippel-lindau disease, hyperkinetic movement disorders, carotid body tumor, autonomic dysreflexia, carcinoid heart disease, cyclic vomiting syndrome, obsessive-compulsive disorder, premenstrual dysphoric disorder, bipolar depression, persistent depressive disorder (dysthymia), eating disorders (such as binge eating disorder), body dysmorphic disorder, trichotillomania (hairpulling disorder), hoarding disorder, premature ejaculation, sleep disorders (such as insomnia), fibromyalgia (for pain management), chronic fatigue syndrome, irritable bowel syndrome, pain disorders (such as chronic pain or migraines), interstitial cystitis, dermatological conditions (such as pruritus or chronic urticaria), alcohol use disorder (as part of a comprehensive treatment plan), nicotine dependence, raynaud's phenomenon, premature ejaculation, borderline personality disorder, selective mutism, hot flashes associated with menopause, postmenopausal osteoporosis, neuropathic pain associated with spinal cord injury, raynaud's phenomenon, chronic fatigue syndrome, and irritable bowel syndrome.

[0072] Hyperadrenergic condition refers to a state characterized by excessive activity or increased release of adrenergic neurotransmitters. In some embodiments, the disease or condition to be treated with the compound or pharmaceutically salt thereof disclosed herein is hyperadrenergic condition or state, which includes for example, pheochromocytoma, paroxysmal sympathetic hyperactivity (psh), hyperthyroidism, anxiety disorders (such as generalized anxiety disorder, panic disorder, post-traumatic stress disorder), orthostatic intolerance (including postural orthostatic tachycardia syndrome - pots), essential tremor, hyperhidrosis, neuroblastoma, acutestress reaction, hyperadrenergic postural orthostatic tachycardia syndrome (hyperpots), hyperadrenergic dysautonomia, primary orthostatic tremor, neurocirculatory asthenia (da costa's syndrome), migraine (in some cases), restless legs syndrome, chronic fatigue syndrome, fibromyalgia, irritable bowel syndrome, raynaud's phenomenon, takotsubo cardiomyopathy (broken heart syndrome), panic attacks, postural orthostatic tachycardia syndrome associated with ehlers-danlos syndrome, chronic pain syndromes, primary hyperhidrosis, hyperkinetic movement disorders (such as restless legs syndrome), stress-induced cardiomyopathy, excessive sweating, vasovagal syncope, orthostatic syncope, and hypertensive crisis.

[0073] Another aspect of the patent document discloses a method of inhibiting phenylethanolamine N-m ethyltransferase (PNMT). The method includes contacting the PNMT with a compound or the pharmaceutically acceptable salt thereof disclosed herein.

[0074] As further illustrated in the examples, the compounds dislocsed herein exhibit excellent binding affinity to the catalytic site of PNMT. Meanwhile, the compounds demonstrate high specificity for PNMT relative to other targets including for example, SAM-based protein arginine or lysine methyltransferases, DNA methyltransferases including DNMT1, DNMT3a, nicotinamide methyltransferases, protein N-terminal methyltransferases and a2-adrenoceptor.

[0075] In some embodiments, the compound exhibits a speficity for PNMT over the above targets or a different target by more than 10-fold, more than 50-fold, more than 100-fold, more than 200-fold, more than 500-fold, more than 1000-fold, more than 2000-fold, more than 5000-fold, more than 10,000-fold, more than 12,000-fold, more than 15,000-fold, more than 20,000-fold, more than 50,000-fold, or more than 80,000-fold.

[0076] Administration Regimen

[0077] The compound of Formula I or II, or a pharmaceutically acceptable salt thereof or a pharmaceutically composition thereof for the methods or kit described herein described herein may be administered to the subject by any suitable means. Non-limiting examples of methods of administration include, among others, (a) administration though oral pathways, which administration includes administration in capsule, tablet, granule, spray, syrup, or other such forms; (b) administration through non-oral pathways such as rectal, vaginal, intraurethral, intraocular, intranasal, or intraauricular, which administration includes administration as an aqueous suspension, an oily preparation or the like or as a drip, spray, suppository, salve, ointment or the like; (c) administration via injection, subcutaneously, intraperitoneally, intravenously, intramuscularly, intradermally, intraorbitally, intracapsularly, intraspinally, intrasternally, or thelike, including infusion pump delivery; as well as (d) administration topically; as deemed appropriate by those of skill in the art for bringing the active compound into contact with living tissue.

[0078] Advantageously, the compound of Formula I or II, or a pharmaceutically acceptable salt thereof or a pharmaceutically composition thereof for administrations described above are prepared into dosage forms in a unit dose suited to fit a dose of the active ingredients. Such dosage forms in a unit dose include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc.

[0079] In exemplary embodiments of the pharmaceutical composition of the compound of Formula I, or a pharmaceutically acceptable salt thereof for oral administration, the composition can be a tablet, coated tablet, capsule, caplet, cachet, lozenges, gel capsule, hard gelatin capsule, soft gelatin capsule, troche, dragee, dispersion, powder, granule, pill, liquid, an aqueous or nonaqueous liquid suspension, an oil-in-liquid or oil-in-water emulsion, including sustained release formulations that are known in the art. For pediatric and geriatric applications, suspensions, syrups and chewable tablets are especially suitable.

[0080] The therapeutically effective amount (dosage) of the compound of Formula I or II, or a pharmaceutically acceptable salt thereof required will depend on the route of administration, the species (human or animal), and the physical characteristics of the particular subject or patient being treated. The dose can be tailored to achieve a desired effect, but will depend on such factors as weight, diet, concurrent medication and other factors which those skilled in the medical arts will recognize. More specifically, a therapeutically effective amount means an amount of compound effective to prevent, alleviate or ameliorate symptoms of disease or prolong the survival of the patient or animal being treated. Determination of a therapeutically effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.

[0081] In non-human animal studies, applications of potential products are commenced at higher dosage levels, with dosage being decreased until the desired effect is no longer achieved or adverse side effects disappear. The dosage may range broadly, depending upon the desired effects and the therapeutic indication. Typically, dosages may be about 10 pg / kg to about 100 mg / kg body weight, preferably about 100 pg / kg to about 10 mg / kg body weight. Alternatively, dosages may be based and calculated upon the surface area of the animal, as understood by those of skill in the art.

[0082] The exact formulation, route of administration and dosage for the pharmaceutical compositions can be chosen by the individual physician in view of the patient’s condition, (see e.g., Fingl et al. 1975, in “The Pharmacological Basis of Therapeutics”, which is hereby incorporated herein by reference in its entirety, with particular reference to Ch. 1, p. 1). In some embodiments, the dose range of the compound of Formula I or a pharmaceutically acceptable salt thereof administered to the subject or patient can be from about 0.5 to about 1000 mg / kg of their body weight. The dosage may be a single one or a series of two or more given in the course of one or more days, as is needed by the patient. In instances where human dosages for compounds have been established for at least some conditions, those same dosages, or dosages that are about 0.1% to about 500%, more preferably about 25% to about 250% of the established human dosage may be used.

[0083] It should be noted that the attending physician would know how to and when to terminate, interrupt, or adjust administration due to side-effects, toxicity or organ dysfunctions. Conversely, the attending physician would also know to adjust treatment to higher levels if the clinical response was not adequate (precluding toxicity). The magnitude of an administrated dose in the management of the disorder of interest will vary with the severity of the condition to be treated and to the route of administration. The severity of the condition may, for example, be evaluated, in part, by standard prognostic evaluation methods. Further, the dose and perhaps dose frequency will also vary according to the age, body weight, and response of the individual patient. A program comparable to that discussed above may also be used in veterinary medicine.

[0084] Although the exact dosage will be determined on a drug-by-drug basis, in most cases, some generalizations regarding the dosage can be made. The daily dosage regimen for an adult human patient may be, for example, a peroral dose of about 0.01 mg to 2000 mg of the active ingredient, preferably from about 0.01 mg to about 500 mg. In other embodiments, an intravenous, subcutaneous, or intramuscular dose of the active ingredient of about 0.01 mg to about 100 mg, preferably about 0.01 mg to about 60 mg is used. In cases of administration of a pharmaceutically acceptable salt, dosages may be calculated as the freebase. In some embodiments, the composition is administered 1 to 4 times per day. Alternatively, a compound of Formula I or a pharmaceutically acceptable salt thereof may be administered by continuous intravenous infusion, preferably at a dose of up to about 1000 mg per day. As will be understood by those of skill in the art, in certain situations it may be necessary to administer a compound of Formula I or II or a pharmaceutically acceptable salt thereof disclosed herein in amounts that exceed, or even far exceed, the abovestated, preferred dosage range in order to effectively and aggressively treat particularly intractablediseases or conditions. In some embodiments, a compound of Formula I or a pharmaceutically acceptable salt thereof will be administered for a period of continuous therapy, for example for a week or more, or for months or years.

[0085] In some embodiments, a compound of Formula I or II or a pharmaceutically acceptable salt thereof is formulated into a dosage form for release for a period of 1 to 12, typically 3 to 12 hours, more typically 6-12 hours after administration. In some embodiments, the oral pharmaceutical compositions described herein may be administered in single or divided doses, from one to four times a day. The oral dosage forms may be conveniently presented in unit dosage forms and prepared by any methods well known to those skilled in the art of pharmacy.

[0086] A compound of Formula I or II or a pharmaceutically acceptable salt thereof can be evaluated for efficacy and toxicity using known methods. For example, the toxicology of the compound may be established by determining in vitro toxicity towards a cell line, such as a mammalian, and preferably human, cell line. The results of such studies are often predictive of toxicity in animals, such as mammals, or more specifically, humans. Alternatively, the toxicity may be determined in an animal model (such as mice, rats, rabbits, or monkeys) using known methods. The efficacy of a particular compound may be established using several recognized methods, such as in vitro methods, animal models, or human clinical trials. Recognized in vitro models exist for nearly every class of condition. Similarly, acceptable animal models may be used to establish the efficacy of chemicals to treat such conditions. When selecting a model to determine efficacy, the skilled artisan can be guided by the state of the art to choose an appropriate model, dose, and route of administration, and dosing regime. Of course, human clinical trials can also be used to determine the efficacy of a compound of Formula I or a pharmaceutically acceptable salt thereof in humans.

[0087] A compound of Formula I or II or a pharmaceutically acceptable salt thereof may, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the active ingredient. The pack may for example comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration. The pack or dispenser may also be accompanied with a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the drug for human or veterinary administration. Such notice, for example, may be the labeling approved by the U.S. Food and Drug Administration for prescription drugs, or the approved product insert. Compositions comprising a compound of Formula I or a pharmaceutically acceptable salt thereofformulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition.

[0088] The following non-limiting examples serve to further illustrate the embodiments of the present disclosure.

[0089] Examples

[0090] Materials and assays

[0091] Isothermal titration calorimetry (ITC) experiments. A MicroCAL PEAQ-ITC instrument (Malvern Panalytical) was used to measure the thermodynamic parameters of PNMT binding to inhibitor 4. The PNMT was stored in a buffer containing 50 mM potassium phosphate (pH 8.0), 1 mM EDTA, 0.5 mM tris (2-carboxyethyl) phosphine hydrochloride (TCEP), and 15% glycerol. A stock solution of the desired inhibitor was prepared in D2O and the concentration was measured using 'H NMR spectroscopy. Volume of the NMR sample was measured precisely before it was freeze dried. The dry sample was resuspended in identical volume of enzyme storage buffer prior to the ITC experiment. This extra step helps eliminate the heat exchange resulting from buffer mismatch. Concentration of the protein was determined using nanodrop (Xaso, e = 44.92 crrr'mM-1). The titration solutions were prepared by dilution of stock solutions into the enzyme storage buffer. In a typical ITC experiment, 300 pL of protein was placed in the cell and varying concentrations of the inhibitor solution was taken by the syringe. An initial 0.4 pL injection was followed by 18 injections of 1.5 pL at 25 °C at 150 s intervals. The reference power was 10 kcal / s and the stirring rate was 750 rpm. Control ITC experiments involved the titration of ligand into buffer solution, buffer to buffer, and the working solution of PNMT to buffer to account for heat of dilution and mixing. These titrations were subtracted from the experimental results before the data were analyzed.

[0092] Cell culture. HEK293T cells were maintained in complete medium comprising of Gibco high glucose DMEM with L-Glutamine (Thermofisher, Waltham, MA) supplemented with 10% Gibco heat inactivated Fetal Bovine Serum (FBS) (Thermofisher, Waltham, MA). Cells were cultured at 37 °C in 5% CO2 atmosphere. Cells were subcultured at a 1 :3 ratio every other day to maintain a monolayer of cells. Cells were detached with 2 mL of 0.025% (w / v) EDTA free trypsin (Gibco, Thermofisher, Waltham, MA) per 10 cm dish for 3 minutes at room temperature. The reaction was stopped by adding 3 volumes of complete medium and cells were recovered by centrifugation at 300 g for 2 minutes at room temperature. Nexelcom Cellometer was used to count cells using trypan blue to assess cell density and viability.

[0093] Transient transfection and PNMT overexpression. The lentiviral vector pLVfExp]- EGFP:T2A:Puro-EFlA>3xFLAG / PNMT was synthesized by VectorBuilder (Chicago, IL). This was designed to express EGFP and puromycin resistance genes separated by the T2A self-cleaving peptide sequence. 3x Flag-tagged PNMT was cloned into this vector. The plasmid was produced by transformation into E. coli DH5a and purified using Qiagen midi prep kit (Qiagen). Cells were transfected using the calcium phosphate transfection protocol in a 96-well tissue culture treated plate. Briefly, DNA calculated to 1 pg final in each well was mixed with 62 pL 2M CaCh, sterile water was added to a total volume of 500 pL. 500 pL of 2x HEPES buffered saline (HBS) was added to this mixture in 100 pL increments with mixing. The DNA mixture was incubated for 30 minutes at room temperature after which the mixture was added directly to the wells containing approximately 50,000 cells per well. Cells were transfected at 70 - 90% confluency. The plate was incubated at 37 °C, 5% CO2 for 7 hours after which fresh media was added and cultured under the same conditions overnight. Cells were monitored for PNMT overexpression by western blotting using anti-flag primary antibody (Abeam) and a goat anti-mouse horse radish peroxidase (HRP) conjugated secondary antibody (BD Biosciences). Alpha tubulin (a-tubulin) was used as a loading control using anti- a-tubulin antibody (1 : 10000) (R&D Systems).

[0094] Cellular inhibition of PNMT. Inhibition assay was performed approximately 24 hours after transfection in 96-well tissue culture treated plates. Assay was performed in a 50 pL volume in phosphate buffered saline (PBS) for 4 hours at 37 °C, 5% CO2. The linear production of epinephrine in this system was monitored by varying norepinephrine concentrations from 0 - 150 pM with equimolar amounts of SAM. For the inhibition assay, 50 pM each of norepinephrine and SAM was added to each well. Inhibitor 4 concentration was varied from 0 - 10 pM. Controls were set up using the known PNMT inhibitor, SK&F 64139, and a non-transfected cell in the presence of inhibitor, norepinephrine, and SAM. Non-transfected cells incubated with norepinephrine and SAM were used to determine background epinephrine levels which was subtracted from all values.

[0095] Inhibition was assayed by using the norepinephrine / epinephrine ELISA kit from Abnova following the manufacturers protocol. Briefly, cells were lysed using Pierce IP lysis buffer with Halt protease inhibitor cocktail (Thermofisher, MA) following the manufacturer’s protocol. 10 pL of cell lysate was analyzed for epinephrine levels using ELISA. IC50 values were determined by plotting inhibitor concentration against epinephrine levels using the 4-parameter equation in GraphPad Prism. All inhibition assays were performed three times in duplicates.

[0096] General binding assay to ADRA2a receptor: To 100 pL of binding assay buffer containing 50 mM HEPES pH 7.4, 150 mM NaCl, 5 mM MgCh, 1 mM CaCl2, and 0.2% BSA was added 16 nM of [3H]-RX821002. 1 unit of membrane lysate containing ADRA2a (5 pL, 4.13 nM ADRA2a protein) was added, followed by cold epinephrine (chase) or inhibitor 4’ or inhibitor 4. Samples were adjusted to 140 pL, mixed and incubated at room temperature for 5 minutes before spinning through a 10 kDa MWCO filter at 15,000 g for 5 minutes and 30-40 pL of filtrate was analyzed by liquid scintillation counting.

[0097] PNMT crystallization and soaking with the diastereomeric mixture of inhibitor 4. Purified PNMT contained strongly bound SAH. Initial X-ray crystallography experiments with PNMT produced SAH-bound structures. To obtain structures containing only inhibitor 4, we first crystallized PNMT in complex with SAH. Subsequently, the SAH was replaced with inhibitor 4 through time course soaking of the crystals with inhibitor 4. Crystallization of PNMT was achieved by using hanging drop vapor diffusion at 22 °C. The 10 mg / mL protein was screened with previously known crystallization conditions. Crystallization trials were done in 24-well VDXm hanging drop plates (Hampton Research). Crystallization drops contained 1 pL of PNMT and 1 pL of well solution. The volume of the well solution was 1 mL. Diffraction quality crystals were obtained in two weeks in a crystallization condition containing 100 mM sodium cacodylate pH 5.5, 170 mM LiCl, and 18 % PEG 6000. These crystals were washed in mother liquor and incubated in soaking buffer containing 100 mM sodium cacodylate pH 5.5, 170 mM LiCl, 20 % PEG 6000, and 1.0 mM inhibitor 4 for different time points. After soaking, crystals were cryoprotected with 20% ethylene glycol and flash frozen for diffraction data collection.

[0098] Structure determination and refinement. Crystal structures of PNMT in complex with TSA inhibitor 4 was solved by molecular replacement using PHASER program. Chain-A of wild-type PNMT (PDB ID: 4MIK) structure was used as the initial search model. The model obtained from PHASER was manually built and completed using the program COOT. Structure refinement was performed with PHENIX-REFINE, using standard protocols for the NCS refinement.58Inhibitor molecule was deleted from the model to initiate the refinement. After the addition of water molecules, inhibitor molecule was fitted into its electron density.

[0099] Structure analysis. Crystal structures of PNMT in complex with SK&F 64139; (PDB ID: 1 YZ3, chain: B), inhibitor 4’ (PDB ID: 6WS1, chain: B) and inhibitor 4 (PDB ID: 7TX2, chain: B) were used in the structural comparison. Structural superimpositions were done using SSM protocol of COOT and the geometry analyses of the final model was achieved using MolProbity. Additional structural analyses, including the calculation of the B-factor profiles wasaccomplished using BAVERAGE of the CCP4 suite. Structural figures were generated with the molecular graphics program PyMOL. For PNMT -inhibitor 4 complex structure (PDB ID: 7TX2), subunit-B was used for all the structural analyses and comparisons.

[0100] Synthesis

[0101] Synthesis of TS analogue inhibitor 4. Intermediate 1 was converted to fragment B in one step through a Pd-catalyzed hydroformylation reaction. Fragment A and fragment C were synthesized as previously described in literature (Journal of the American Chemical Society 2020, 142, 14222 - 14233). Assembly of the final molecule was achieved by two consecutive reductive amination reactions aided by titanium iso-propoxide. Satisfactory results were obtained when the first coupling occurred between the amine group of fragment C and the aldehyde moiety of fragment B to yield compound 2. This was followed by a second reductive amination with fragment A to yield compound 3. A global deprotection step involved trifluoroacetic acid and anisole in water to give TS analogue inhibitor 4 as a diastereomeric mixture.

[0102] Inhibition Assay. Inhibition constants of the PNMT catalyzed reaction were measured using a coupled-enzyme assay. Briefly, deamination of SAH by TM0936 yields 5- inosyl-L-homocysteine (SIH) and the change in absorbance is monitored at 263 nm. Commonly, the assay mixture contains norepinephrine, inhibitor 4, excess TM0936 (5-methylthioadenosine / S- adenosylhomocysteine deaminase [EC:3.5.4.31; 3.5.4.28]), and PNMT in a phosphate buffer. Upon initiation with SAM at 30 °C, the UV absorbance was monitored at 263 nm (detailed protocol in Supporting Information). Since the concentration of inhibitor was less than ten times the concentration of enzyme, the results were fitted to the Morrison equation. In this manner, the Ki value of the diastereomeric mixture of inhibitor 4 was calculated to be 1.2 ± 0.4 nM. TSA inhibitor 4 is an order of magnitude more potent than TSA inhibitor 4’. An increased number of favorable interactions in the norepinephrine binding pocket could account for more favorable binding. However, addition of a methylene bridge also imposes an entropic penalty, cancelling some of the enthalpic improvements gained through active site interactions. The thermodynamic properties of binding by isothermal titration calorimetry (ITC) was investigated.

[0103] Isothermal Titration Calorimetry (ITC) Studies. Binding of TSA inhibitor 4 (as a diastereomeric mixture) to PNMT was consistent with a two-site binding model with a negative cooperative sequential mechanism. Human PNMT is a homodimer and two active sites are accessible to the ligand. Inhibitor 4 binding is driven by large entropic (-TAS) values: -9.7 kcal / mol at the first active site; and -14.7 kcal / mol at the second active site of the homodimer. These largeentropic contributions are accompanied by a small favorable enthalpic contribution of -1.6 kcal / mol at the first active site and an unfavorable contribution of 4 kcal / mol at the second active site. Entropic contributions are attributed to the dynamic components, water exclusion, or hydrophobic differences while enthalpic contributions are attributed to the formation of hydrogen bond or ionic interactions between the ligand and active site residues. In ITC analysis, inhibitor 4 binds to the first site of the PNMT homodimer with a KA of 4.9 nM. Binding at the second site gives a KA of 15 nM, approximately 3-fold less than the first site, a negative cooperative interaction. The longer linker in inhibitor 4 provides a 10-fold improvement in binding when compared to an analog having a shorter linker, suggesting that 4 is a closer mimic of the TS. This increased binding affinity results from improved thermodynamic parameters with increased favorable enthalpy and corresponding entropic compensation.

[0104] Table 1. Thermodynamic constants for binding ofTS analogue inhibitor 4 to PNMT

[0105] Cellular Activity of the Diastereomeric Mixture of Inhibitor 4. The cellular activity of PNMT and its expression often uses rat pheochromocytoma cells, PC12, or the mouse pheochromocytoma cell line (MPC). It was observed that hPheol failed to produce detectable levels of PNMT protein even upon differentiation induced with dexamethasone. PNMT was overexpressed in HEK293T cells using a lentiviral vector expressing GFP in addition to flag- tagged PNMT. Western blots of transfected cell lysates and immunofluorescence for GFP expression confirmed overexpression of PNMT. HEK293T cells do not produce significant norepinephrine, hence exogenous norepinephrine and SAM were provided in the assay culture medium. The linear increase in epinephrine formation with increasing norepinephrine and SAM concentrations in intact cultured HEK293T cells overexpressing PNMT confirmed the expression and PNMT catalytic activity in this system. Addition of TSA inhibitor 4 to cells in the presence of norepinephrine and SAM inhibited the activity of PNMT in a concentration dependent manner with an ICso value of 81 ± 10 nM. Similarly, a positive control experiment with SK&F 64139, gave an ICso value of 15 ± 2 nM. Despite studies on the inhibition of PNMT in PC12 cells and animal models, no other reports of dose titration with SK&F 64139 in a cell-based assay were found that supports its known cellular availability. The cell permeability of inhibitor 4 suggests that it can act as a candidate for physiological PNMT inhibition. Although SK&F 64139 shows a lower cellularICso, it is non-selective by blocking both adrenaline biosynthesis and the biological activity of a.2- adrenoceptors, thereby complicating the interpretation of biological results when tested in vivo.

[0106] Specificity of Bisubstrate SAM-Methyltransferase Inhibitors. The enzyme specificity of inhibitor 4 can be compared to SAM bisubstrate inhibitor analogues for nicotinamide N-m ethyltransferase (NNMT) and protein TV-terminal methyltransferase 1 (NTMT1) reported earlier. Compound LL320 and the chemically related NAH-C4-GPKRIA have been reported to be bisubstrate analogue inhibitors of NNMT and NTMT1, respectively. LL320 was reported to be a 1.6 nM inhibitor for NNMT. As LL320 contains elements of SAM methyl donor and aromatic acceptor group, it was also screened against PNMT, where the apparent ICso was > 100 pM. Therefore, compound LL320 shows a > 62,000 preference for NNMT relative to PNMT, despite the common elements. Inhibitor NAH-C4-GPKRIA was reported to be a 130 pM inhibitor for NTMT1 and showed a > 3000-fold selectivity when compared to euchromatic histone-lysine N- methyltransferase (G9a) domain-containing protein 7 (SETD7), NNMT, protein arginine methyltransferase 1 (PRMT1), PRMT3, Trypanosoma brucei PRMT7, or 5-adenosyl homocysteine hydrolase (SAHH). These reports suggest the inhibitor scaffolds of SAM-based methyltransferases are highly specific for their targets.TSA Inhibitor 4 LL320 NAH-C4-GPKRIA

[0107] Inhibitor 4 and its analog with a 3-carbon linker (inhibitor 4’) were tested against DNA methyltransferases, DNMT1 and DNMT3b. Inhibitor 4’ gave ICso values of 35 pM and 19 pM for DNMT1 and DNMT3b respectively, demonstrating a specificity of 2,900 and 1,600 respectively for PNMT relative to the DNA methyltransferases. Inhibitor 4 gave ICso values of 61 pM and 17 pM for DNMT1 and DNMT3b, demonstrating a specificity of 51,000 and 14,000 respectively for PNMT relative to the DNA methyltransferases.

[0108] Inhibitors 4 and 4’ were also tested for their effects on protein methyltransferase function in cultured cells. HT-29 colorectal cells expressing protein arginine and protein lysine methyltransferases (PRMTs and PKMTs) were treated with inhibitor 4’ or inhibitor 4 for 6 daysbefore the soluble protein was extracted and blotted for protein symmetric dimethyl arginine (SDMA), asymmetric dimethylarginine (ADMA), and trimethyl lysine (Mes-lys e) post- translational methylations. Positive controls included PRMT5 -specific inhibitor GSK3326595 (SDMA) and Type I PRMT5 (PRMT1 / 4) specific inhibitor MS023 (ADMA), Depletion of protein methylation marks occurs on a biological scale of hours to days. Cells treated with GSK3326595 showed no band corresponding to SDMA while cells treated with MS023 showed no evidence of AD A after 6 days treatment. Western Blot analysis showed no significant change in total SDMA, ADMA, and Mes-lysine in samples treated with 20 pM inhibitor 4’ or inhibitor 4, indicating that neither of these molecules influenced protein methylation. TSA inhibitors 4’ and 4 are highly specific for their target with a specificity of > 1,600-fold for PNMT over protein methyltransferases.

[0109] Binding of Inhibitors 4’ and 4 to Adrenergic Receptors. Previously described inhibitors of PNMT showed significant affinity for a2-adrenoceptors to complicate the interpretation of biological outcomes. The specificity of TSA inhibitors 4’ and 4 for PNMT was compared to their binding to human a.2 \ adrenergic receptor (ADRA2a). A membrane preparation of ADRA2a was mixed with [3H]-RX821002, a selective a2-adrenoceptor antagonist with a binding affinity of 1.6 nM, approximately 1000-fold tighter than the binding affinity of ADRA2a for epinephrine (1 pM). Inhibitors 4’ and 4 were used as unlabeled competitors in the binding assay at 160 pM to ADRA2a receptor. Variable concentrations of epinephrine, inhibitors 4’ and 4 were used to estimate binding affinity. At 1.6 mM of inhibitor 4, less ligand was displaced from ADRA2a than with epinephrine.

[0110] The Kd value of epinephrine for the ADRA2a receptor was calculated to be 10.0 ± 0.2 pM, approximately 10-fold higher than previously reported. The Kd value of inhibitors 4’ and 4 for the ADRA2a receptor was calculated to be 14.6 ± 1.1 pM and 14.3 ± 0.2 pM, respectively. These experiments demonstrate that TSA inhibitors 4’ and 4 bind to PNMT orders of magnitude tighter than to the ADRA2a receptor. With a Kd of 1.2 nM for PNMT, inhibitor 4 binds its target enzyme 12,000 times tighter than the ADRA2a receptor.

[0111] Structure of PNMT with the Diastereomeric Mixture of Inhibitor 4. X-ray crystallography experiments with purified PNMT without added ligands produced a structure showing SAH bound to both catalytic sites. The PNMT complex with inhibitor 4 was obtained by soaking experiments. Crystals of the wild-type PNMT-SAH complex were soaked in a crystallization buffer containing 1 mM inhibitor 4. PNMT crystals soaked with inhibitor 4 for two hours yielded a structure with two subunits in the asymmetric unit, one bound with inhibitor 4 andthe second bound with SAH. Crystals soaked for 12 hours produced structures with inhibitor 4 in both subunits. The structure of PNMT in complex with inhibitor 4 and SAH was determined at 2.10 A resolution in the space group P4s2i2. The structure of PNMT in complex with inhibitor 4 in both subunits was determined at a 2.43 A resolution, in the space group P4s2i2. The electron density for SAH and inhibitor 4 can be seen clearly in the corresponding binding pocket of both structures. Comparing structures with first-generation inhibitor 4’ and inhibitor 4, reveals substantial protein conformational changes between the PNMT-SAH and PNMT-inhibitor 4’ structures, but not between the PNMT-SAH and PNMT-inhibitor 4 structures. The accessible surface area of the PNMT dimer interface is 11481 A2 by PISA analysis. A Ca-superposition of the SAH-bound subunit with the inhibitor 4-bound subunit shows an RMSD of 0.144 A, demonstrating no significant structural difference between the two chains. The PNMT monomer contains an a / 0 domain with 7-stranded mixed 0-sheets (01 -07) flanked on either side by 4-helices. The binding site for norepinephrine and SAM are buried and located at the edge of the 0-sheet. Both binding sites are tightly covered by surrounding motifs.

[0112] Catalytic Site Interaction and Structural Comparison. Binding of inhibitor 4 at the catalytic site of PNMT fills both the SAM and norepinephrine binding. The N-13 and N-15 atoms of inhibitor 4 form hydrogen bonds with the peptide nitrogen of Vai 159 and carboxylate of Aspl58, respectively. The 0-39 of inhibitor 4 is in hydrogen bond interaction with the carboxylate of AsplOl and carboxamide of Asnl06, whereas 0-40 is hydrogen bonded to the carboxylate of AsplOl . The carboxyl group in inhibitor 4 is hydrogen bonded to three tyrosine residues including the hydroxyl groups of Tyr35, Tyr40, and Tyr85. The N-23 of inhibitor 4 is hydrogen bonded to peptide carbonyls of Gly79, Ser80, and Alal81. The adenine base is sandwiched between the side chains of Phel02 and Vall87 providing strong hydrophobic interactions. Tyr27 and Phe30 residues create a hydrophobic pocket facilitating inhibitor binding. The dichloro-THIQ moiety of inhibitor 4 is bound in the norepinephrine binding site where a pocket is formed by Tyr35, Val53, Lys57, Phel82, Glu219, Tyr222, Met258, Val269, and Val272. The aromatic ring in the norepinephrine site is in a TI-TI stacking interaction with Phel82. The binding of dichloro-THIQ is stabilized by the hydrophobic interaction in the binding pocket. The N-33 atom of dichloro-THIQ is in strong hydrogen bonding interaction with the carboxylate of Glu219.

[0113] Structure of the PNMT-inhibitor 4 complex was compared with the SK&F 64139, and inhibitor 4’-bound PNMT structures. The SAM mimic of inhibitor 4’ and 4 binds in the same conformation in the cofactor (SAM) binding pocket. Unlike the inhibitor 4’ -bound structure, binding of inhibitor 4 does not induce conformational changes in the Glu218-Val234 and His261-Lys270 loops to cause any rearrangement of the substrate binding pocket. These loops have the same conformation as the SK&F 64139-bound structure. With inhibitor 4’ bound, conformational changes are observed in residues Asn39 (1.2A), Glu219 (1.7A), Tyr222 (1.2 A), and Asp267 (1.5 A). However, in the inhibitor 4-bound structure, these residues have the same conformation as in the SK&F 64139- or SAH-bound PNMT complexes. The side chain of Arg44 is disordered in the inhibitor 4’ -bound complex but not in the inhibitor 4-bound structure. When compared to the SK&F 64139-bound structure (PDB ID: 1 YZ3), the dichloro-THIQ moiety in inhibitor 4 is bound to the substrate (norepinephrine) or SK&F 64139 binding pocket. The N-2 atom of SK&F 64139 is hydrogen bonded to carboxylate of Glu219. A similar interaction is observed with the N-33 atom of inhibitor 4 in the substrate binding pocket. Unlike the inhibitor 4’-PNMT complex, the other interactions of SK&F 64139 with PNMT active site residues are similar in the inhibitor 4-bound structure.

[0114] All references cited herein are incorporated by reference in their entireties.

[0115] It will be appreciated by persons skilled in the art that the invention described herein is not limited to what has been particularly shown and described. Rather, the scope of the invention is defined by the claims which follow. It should further be understood that the above description is only representative of illustrative examples of embodiments. The description has not attempted to exhaustively enumerate all possible variations. The alternate embodiments may not have been presented for a specific substituent of the compound, or a step of the method, and may result from a different combination of described substituent or step, or that other undescribed alternate embodiments may be available for a compound or method, is not to be considered a disclaimer of those alternate embodiments. It will be appreciated that many of those un-described embodiments are within the literal scope of the following claims, and others are equivalent.

Claims

Claims1. A compound or a pharmaceutically acceptable salt thereof, wherein the compound is represented by Formula I,Formula IWherein:L is a linker selected from the group consisting of 3-6 membered heterocylic, 6- membered aryl, 6-membered heteroaryl, -(CH2)aC(0)NRa-, -C(O)NRa-, - (CH2)aC(O)NRa(CH2)b-, -(CH2)2+a-, -(CH2)aO(CH2CH2O)c-, -(CH2)2+aS, - (CH2)aheterocyclyl-, -(CH2)aC(O)-, -(CH2)aNRa-, -CRa=N-NRa-, -CRa=N-O-, -CRa=N- NRb-CO-, -N=N-CO-, -S-S-, and any combination thereof, wherein a, b, and c are each an integer selected from 0 to 8;R1, R2, R3, and R4in each instance is independently selected from the group consisting of H, Ci-4alkyl, OCl-4alkyl, NRaRb, haloCi-4alkyl, halogen, OH, CN, hydroxyl-alkyl, dihydroxyl-alkyl, C(O)ORa, C(O)NRaRa, C(O)Ci-ealkyl, phenyl and 5- or 6-membered hereroaryl, wherein the phenyl and 5- or 6-membered hereroaryl are optionally substituted with one or more substituents selected from the group consisting of Ci-4alkyl, OCi-4alkyl, NRaRb, halo Ci-4alkyl, halogen, OH, and CN, wherein at least one of R1and R2is not H;Raand Rbin each instance are independently H or Ci-4alkyl;R5is H, Ci-4alkyl, C(O)ORa, or C(O)Ci-6alkyl;R6is H or Ci-4alkyl;R7in each instance is independently H or Ci-4alkyl;R8in each instance is independently H, Ci-4alkyl, C(O)ORa, C(O)NRaRa, or C(O)Ci- ealkyl;Ring A is a 5- or 6-membered hetercyclyl;n is an integer of 0 to 5; provided that the compound is notr its stereoisomer.

2. The compound or the pharmaceutically acceptable salt thereof of claim 1, wherein the A ring is 6-membered.

3. The compound or the pharmaceutically acceptable salt thereof of claim 1,Formula I-a wherein the compound is represented by Formula I-a.

4. The compound or the pharmaceutically acceptable salt thereof of claim 1, wherein L is a Cs-salkylene.

5. The compound or the pharmaceutically acceptable salt thereof of claim 1, wherein L is a C4alkylene.

6. The compound or the pharmaceutically acceptable salt thereof of claim 1, wherein the compound is represented by Formula I-bFormula I-b wherein L is a Cs-salkylene.

7. The compound or the pharmaceutically acceptable salt thereof of claim 1, R1and R2are independently selected from the group consisting of halogen, Ci-4alkyl, OCl-4alkyl, haloCi-4alkyl, and CN.

8. The compound or the pharmaceutically acceptable salt thereof of claim 1, wherein R1and R2are each independently halogen or Ci-4alkyl.

9. The compound or the pharmaceutically acceptable salt thereof of claim 1, wherein R1and R2are each chlorine.

10. The compound or the pharmaceutically acceptable salt thereof of claim 1, wherein R3and R4are each H.

11. The compound or the pharmaceutically acceptable salt thereof of claim 1, wherein R5is H.

12. The compound or the pharmaceutically acceptable salt thereof of claim 1, wherein R5is H, R7is H.

13. The compound or the pharmaceutically acceptable salt thereof of claim 1, wherein R5is H, R6is H, R7is H.

14. The compound or the pharmaceutically acceptable salt thereof of claim 6, wherein the compound is15. A pharmaceutical composition comprising a therapeutically effective amount of the compound or the pharmaceutically acceptable salt thereof of claim 1.

16. A method of treating a central nervous disease in a subject, comprising administering to the subject a therapeutically effective amount of the compound or the pharmaceutically acceptable salt thereof of claim 1.

17. The method of claim 16, wherein the disease is selected from the group consisting of post-traumatic stress disorder (PTSD), hypertension, myocardial infarction, Alzheimer’s disease, Parkinson’s disease, neuropathic pain, and hyperadrenergic condition,18. The method of claim 16, wherein the disease is PTSD.

19. The method of claim 16, further comprising administering to the subject an agent selected from the group consisting of SSRI (selective serotonin reuptake inhibitor), and SNRI (the serotonin and norepinephrine reuptake inhibitor).

20. The method of claim 16, wherein the compound or the pharmaceutically acceptable salt thereof is administered prophylactically.

21. A method of inhibiting phenylethanolamine N-methyltransferase (PNMT), comprising contacting the PNMT with the compound or the pharmaceutically acceptable salt thereof of claim 1.

22. The method of claim 21, wherein the PNMT is inhibited selectively over one or more targets selected from the group consisting of SAM-based protein arginine or lysine methyltransferases, DNA methyltransferases, nicotinamide methyltransferases, protein N- terminal methyltransferases and a2-adrenoceptor.

23. The method of claim 21, wherein the PNMT is inhibited in vivo.

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