Potent and selective human neuronal nitric oxide synthase inhibitors

Abstract

Disclosed are neuronal nitric oxide synthase (nNOS) inhibitors and methods of using the same in treating a disease or disorder associated with nNOS activity, such as a neurological disease or disorder, or melanoma.

Description

[0001] POTENT AND SELECTIVE HUMAN NEURONAL NITRIC OXIDE SYNTHASE INHIBITORS CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U. S. provisional application serial no. 63 / 746,815 filed January 17, 2025. The contents of which are incorporated by reference herein in their entirety.

[0002] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0003] This invention was made with government support under grant number GM131788 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0004] BACKGROUND OF THE INVENTION

[0005] In mammals, there are three nitric oxide synthase (NOS) isoforms: neuronal NOS (nNOS), inducible NOS (iNOS), and endothelial NOS (eNOS).1Each isoform plays a vital role in human physiology by producing nitric oxide (NO), a crucial secondary cell-signaling molecule, through the NADPH and molecular oxygen dependent oxidation of L-arginine to L-citrulline.2'3Nitric oxide participates in the regulation of essential processes such as neurotransmission,4immune response,5smooth muscle relaxation,6and vasodilation.7Neuronal NOS is responsible for producing NO in the central nervous system, which is essential for neuronal communication.8'10Endothelial NOS generates NO to help lower blood pressure by relaxing smooth muscle.11Inducible NOS produces cytotoxic NO, supporting the immune response in fighting pathogens and microorganisms.12

[0006] The pivotal role of NO in the development of neurological disorders in the nervous system is well-known.13-15While NO produced at normal physiological levels by nNOS is crucial for neuronal communication,16the overproduction of NO gives rise to highly reactive compounds like peroxynitrite (ONOO⁻) in the presence of superoxide.17'19Excess peroxynitrite can lead to the disruption of protein function via nitration and / or nitrosylation.20nNOS is implicated in a range of neurodegenerative conditions, including Alzheimer's disease (AD),21Parkinson's disease (PD),22Huntington's disease (HD),23amyotrophic lateral sclerosis (ALS),24chronic headaches,25and neuronal damage during stroke.26NO also has been shown to cause

[0007] Page 1

[0008] QB\702581.02752\100011397.1degeneration of skin melanocytes leading to melanoma.27Therefore, nNOS is a promising target for treating these various neurological disorders.

[0009] Targeting nNOS shows promise in preventing and treating neuronal damage in animal models.28,38However, non-selective inhibition of nNOS could result in hypertension and cardiovascular problems by affecting eNOS,29or the immune response by impacting iNOS.30Therefore, precise targeting of nNOS over other isoforms is preferable for the effective treatment of neurological disorders.1,31-32Owing to very similar active sites, achieving selective inhibition of one NOS isoform over others presents a significant challenge.33'39

[0010] There remains a need for the development of NOS inhibitors that retain high potency, selectivity, and blood-brain barrier permeability.

[0011] BRIEF SUMMARY OF THE INVENTION

[0012] Disclosed herein are neuronal nitric oxide synthase (nNOS) inhibitors and methods of using the same in the treatment of diseases or disorders.

[0013] One aspect of the present disclosure provides a compound of formula (I), or a pharmaceutically acceptable salt, hydrate, or solvate thereof.

[0014] 'n

[0015]

[0016] (I)

[0017] wherein

[0018] X is CR3, orN,

[0019] each

[0020]

[0021] is independently H, halo, cyano, or haloalkyl,

[0022] n is 0, 1, or 2,

[0023] m is 1 or 2, and

[0024] (i) R1is H or alkyl and R2is alkyl or haloalky l, or

[0025] (ii) R1and R2together with the nitrogen atom they are attached to form a heterocycloalkyl substituted with 0-2 halo.

[0026] Another aspect of the present disclosure provides a pharmaceutical composition comprising the compound disclosed herein and a pharmaceutically acceptable excipient, carrier, or diluent.

[0027] Page 2

[0028] QB\702581.02752\100011397.1Another aspect of the present disclosure provides a method for treating a disease or disorder associated with neuronal nitric oxide synthase (nNOS) activity in a subject in need thereof, the method comprising administering an effective amount of the compound disclosed herein, or a pharmaceutically acceptable salt, hydrate, or solvate thereof; or the pharmaceutical composition disclosed herein to the subject.

[0029] BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Non-limiting embodiments of the present invention will be described by way of example with reference to the accompanying figures, which are schematic and are not intended to be drawn to scale. In the figures, each identical or nearly identical component illustrated is typically represented by a single numeral. For purposes of clarity, not every component is labeled in every figure, nor is every component of each embodiment of the invention shown where illustration is not necessary to allow those of ordinary skill in the art to understand the invention.

[0031] Figure 1 shows compound 11 bound to (A) mNOS, (B) hnNOS, and (C) heNOS. In all following structural figures, major H-bonds that secure inhibitor binding are depicted as dashed lines and the bond distances are labeled in A. Heme propionates (A and D) are labeled.

[0032] Figure 2 shows compound 12 bound to (A) hnNOS and (B) heNOS.

[0033] Figure 3 shows compound 14 bound to (A) mNOS and (B) hnNOS.

[0034] Figure 4 shows compound 16 bound to (A) mNOS, (B) hnNOS, and (C) heNOS. Figure 5 shows compound 18 bound to (A) mNOS, (B) hnNOS, and (C) heNOS. Figure 6 shows compound 19 bound to (A) mNOS and (B) heNOS.

[0035] Figure 7 shows compound 20 bound to (A) mNOS and (B) hnNOS.

[0036] Figure 8 shows compound 23 bound to (A) mNOS. (B) hnNOS, and (C) heNOS. Figure 9 shows compound 13 bound to (A) mNOS, (B) hnNOS, and (C) heNOS. Figure 10 shows compound 15 bound to (A) mNOS and (B) heNOS.

[0037] Figure 11 shows compound 17 bound to heNOS.

[0038] Figure 12 shows compound 21 bound to (A) mNOS, (B) hnNOS, and (C) heNOS. Figure 13 shows compound 22 bound to (A) hnNOS and (B) heNOS.

[0039] Figure 14 shows the1H NMR spectrum of compound 42 (solvent: CDCh).

[0040] Figure 15 shows the13C NMR spectrum of compound 42 (solvent: CDCh).

[0041] Figure 16 shows the DEPT-13513C NMR spectrum of compound 20 (solvent: CDCh).

[0042] Page 3

[0043] QB\702581.02752\100011397.1DETAILED DESCRIPTION OF THE INVENTION

[0044] Disclosed herein are compounds and derivative thereof for use as neuronal nitric oxide synthase (nNOS) inhibitors. The compounds and compositions disclosed herein may be used in methods of treating diseases or disorders, such as a neurological disease or disorder, or melanoma

[0045] Chemical Entities

[0046] The term "alkyl" refers to a straight-chain, branched, or cyclized alkyl radical in all of its isomeric forms, such as a straight or branched group of 1-12, 1-10, or 1-6 carbon atoms, referred to herein as Ci-Ci2-alkyl, Ci-Cio-alkyl, and Ci-Cs-alkyl, respectively.

[0047] The term "alkylene" refers to a diradical of straight-chain or branched alkyl group (e.g., a diradical of straight-chain or branched C1-C12 alkyl group). Exemplary alkylene groups include, but are not limited to -CH2-, -CH2CH2-, -CH2CH2CH2-, -CH(CH3)CH2-, -CH2CH(CH3)CH2-, -CH(CH2CH3)CH2-, and the like.

[0048] The term "alkenyl" refers to an unsaturated straight or branched hydrocarbon having at least one carbon-carbon double bond, such as a straight or branched group of 2-12, 2-10, or 2-6 carbon atoms, referred to herein as C2-C 12- alkenyl, C2-Cio-alkenyl, and C2-Ce-alkenyl, respectively.

[0049] The terms "alkoxy" or "alkoxyl" refers to an alkyl group, as defined above, having an oxygen radical attached thereto. Representative alkoxy groups include methoxy, ethoxy, tertbutoxy and the like.

[0050] The term "cycloalkyl" refers to a monovalent saturated or partially saturated cyclic, bicyclic, or bridged cyclic (e.g., adamantyl) hydrocarbon group of 3-12, 3-8, 4-8, or 4-6 carbons, referred to herein, e.g., as " C4-8-cycloalkyl," derived from a cycloalkane. Unless specified otherwise, the cycloalkyl group is not substituted, i.e., it is unsubstituted.

[0051] The term "heterocycloalkyl" (or "heterocyclyl") refers to a monovalent saturated or partially saturated cyclic, bicyclic, or bridged cyclic hydrocarbon group of 3-12, 3-8, 4-8, or 4-6 carbons in which at least one carbon of the cycloalkane is replaced with a heteroatom such as, for example, N, O, and / or S.

[0052] The term "halo" refers to a halogen atom or halogen radical (e.g., -F, -Cl, -Br, or -I). The term "haloalkyl" refers to an alkyl group that is substituted with at least one halogen. For example, -CH2F, -CHF2, -CF3, -CH2CF3, -CF2CF3, and the like.

[0053] The term "cyano" refers to -CN.

[0054] Page 4

[0055] QB\702581.02752\100011397.1The term "aryl" refers to a carbocyclic aromatic group. The term "aryl" includes monocyclic ring systems, and polycyclic ring systems having two or more carbocyclic rings in which two or more carbons are common to two adjoining rings (the rings are "fused rings") wherein at least one of the rings is aromatic and, e.g., the other ring(s) may be cycloalkyls, cycloalkenyls, cycloalkynyls, and / or ary ls. Unless specified otherwise, the aryl ring is unsubstituted. In certain embodiments, the aryl group is a 6-10 membered ring structure. Representative aryl groups include phenyl, naphthy l, anthracenyl, 1,3-benzodioxolyl and the like.

[0056] The substituents on the aryl (e.g., phenyl) rings in the compounds of the disclosure may be at ortho-, meta-, or para- positions.

[0057] The term "heteroaryl" refers to an aromatic 5- to 10-membered ring structure, alternatively 6- to 10-membered rings, whose ring structures include one to four heteroatoms, such as nitrogen, oxygen, and sulfur. The number of ring atoms in the heteroaryl group can be specified using Cx-Cx nomenclature where x is an integer specifying the number of ring atoms. For example, a C3-C7 heteroaryl group refers to an aromatic 3- to 7-membered ring structure containing one to four heteroatoms, such as nitrogen, oxygen, and sulfur. The term "heteroaryl" includes monocyclic ring systems, and polycyclic ring systems having two or more heterocyclic rings in which two or more carbon or heteroatom are common to two adjoining rings (the rings are "fused rings") wherein at least one of the rings is a heterocyclic aromatic group and. e.g., the other ring(s) may be cycloalkyls, cycloalkenyls, cycloalkynyls. and / or aryls. Representative heteroaryl groups include pyridinyl, quinolinyl, furanyl, thionyl, indolyl, and the like.

[0058] The term “optionally substituted'’ refers to one or more carbon atoms in the group being independently substituted with one or more functional groups described herein.

[0059] The compounds of the disclosure may contain one or more chiral centers and / or double bonds and, therefore, exist as stereoisomers, such as geometric isomers, enantiomers or diastereomers. The term "stereoisomers" when used herein consist of all geometric isomers, enantiomers or diastereomers. These compounds may be designated by the symbols " R" or " S’," or "+" or depending on the configuration of substituents around the stereogenic carbon atom and or the optical rotation observed. The present invention encompasses various stereoisomers of these compounds and mixtures thereof. Stereoisomers include enantiomers and diastereomers. Mixtures of enantiomers or diastereomers may be designated (±)" in nomenclature, but the skilled artisan will recognize that a structure may denote a chiral center Page 5

[0060] QB\702581.02752\100011397.1implicitly. It is understood that graphical depictions of chemical structures, e.g., generic chemical structures, encompass all stereoisomeric forms of the specified compounds, unless indicated otherwise. Also contemplated herein are compositions comprising, consisting essentially of, or consisting of an enantiopure compound, which composition may comprise, consist essentially of, or consist of at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of a single enantiomer of a given compound (e.g., at least about 99% of an R enantiomer of a given compound).

[0061] As used herein, "salt" refers to acid addition salts and basic addition salts. It may also refer to those salts that may be prepared in situ during the final isolation and purification of the compounds of the invention.

[0062] Examples of acid addition salts include, but are not limited to acetate, adipate, alginate, citrate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, camphorate, camphorsulfonate, digluconate, glycerophosphate, hemisulfate, heptanoate, hexanoate, fumarate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethansulfonate (isothionate), lactate, malate, maleate, methanesulfonate, nicotinate, 2-naphthalenesulfonate, oxalate, palmitate, pectinate, persulfate, 3 -phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, phosphate, glutamate, bicarbonate, p-toluenesulfonate and undecanoate. Also, the basic nitrogen-containing groups may be quatemized with such agents as lower alkyl halides such as, but not limited to, methyl, ethyl, propyl, and butyl chlorides, bromides and iodides; dialkyl sulfates like dimethyl, diethyl, dibutyl and diamyl sulfates; long chain halides such as, but not limited to, decyl, lauryl, myristyl and stearyl chlorides, bromides and iodides; aryl alky I halides like benzyl and phenethyl bromides and others. Water or oil-soluble or dispersible products are thereby obtained. Examples of acids which may be employed to form pharmaceutically acceptable acid addition salts include such inorganic acids as hydrochloric acid, hydrobromic acid, sulfuric acid, and phosphoric acid and such organic acids as acetic acid, fumaric acid, maleic acid, 4-methylbenzenesulfonic acid, succinic acid, and citric acid.

[0063] Basic addition salts may be prepared in situ during the final isolation and purification of compounds of this invention by reacting a carboxylic acid-containing moiety with a suitable base such as, but not limited to, the hydroxide, carbonate or bicarbonate of a pharmaceutically acceptable metal cation or with ammonia or an organic primary, secondary or tertiary amine. Pharmaceutically acceptable salts include, but are not limited to, cations based on alkali metals or alkaline earth metals such as, but not limited to, lithium, sodium, potassium, calcium, magnesium and aluminum salts and the like and nontoxic quaternary ammonia and amine Page 6

[0064] QB\702581.02752\100011397.1cations including ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, diethylamine, ethylamine and the like. Other examples of organic amines useful for the formation of base addition salts include ethylenediamine, ethanolamine, diethanolamine, piperidine, piperazine and the like.

[0065] Compounds described herein may exist in unsolvated as well as solvated forms, including hydrated forms, such as hemi-hydrates. In general, the solvated forms, with pharmaceutically acceptable solvents such as water and ethanol among others are equivalent to the unsolvated forms for the purposes of the invention.

[0066] Compounds

[0067] One aspect of the present disclosure provides a compound of formula (I), or a pharmaceutically acceptable salt, hydrate, or solvate thereof.

[0068] 'n

[0069]

[0070] (I)

[0071] wherein

[0072] X is CR3, orN,

[0073] each

[0074]

[0075] is independently H, halo, cyano, or haloalkyl,

[0076] n is 0, 1, or 2,

[0077] m is 1 or 2, and

[0078] (i) R1is H or alkyl, and R2is alky l or haloalkyl, or

[0079] (ii) R1and R2together with the nitrogen atom they are attached to form a heterocycloalkyl substituted with 0-2 halo.

[0080] In some embodiments, the compound has a formula of (la).

[0081]

[0082] QB\702581.02752\100011397.1In some embodiments, m is 1.

[0083] In some embodiments, m is 2.

[0084] In some embodiments, each R3is independently halo.

[0085] In some embodiments, R3is fluoro.

[0086] In some embodiments, R3is selected from H and halo. For example, R3may be selected from H and fluoro, or from H and chloro.

[0087] In some embodiments, R3is selected from H and haloalkyl. For example, R3may be selected from H and -CF3,

[0088] In some embodiments, R3is selected from H and cyano.

[0089] In some embodiments, R1is H and R2is alkyl. For example, R1may be H and R2may be methyl.

[0090] In some embodiments, R1is H and R2is haloalkyl. For example, R1may be H and R2may be -CH2CH2F.

[0091] In some embodiments, R1is alkyl and R2is alkyl. For example, R1may be methyl and R2may be methyl.

[0092] In some embodiments, R1and R2together with the nitrogen atom they are attached to form a 4- or 6-membered heterocycloalkyl substituted with 2 halo. In some such embodiments,

[0093] R1and R2together with the nitrogen atom they are attached to form

[0094]

[0095] FOr

[0096]

[0097] F

[0098] In some embodiments, the compound has a formula of (lb).

[0099]

[0100] (Ib)

[0101] In some embodiments of formula (Ib), m is 1.

[0102] In some embodiments of formula (Ib). R1is H and R2is alkyl. For example, R1may be H and R2may be methyl.

[0103] Page 8

[0104] QB\702581.02752\100011397.1In some embodiments, the compound is

[0105]

[0106] Page 9

[0107] QB\702581.02752\100011397.1

[0108]

[0109] or a pharmaceutically acceptable salt, hydrate, or solvate thereof.

[0110] In some embodiments, the compound is

[0111]

[0112] or a pharmaceutically acceptable salt, hydrate, or solvate thereof.

[0113] In some embodiments, HC1 salts of the disclosed compounds are provided, such as a dihydrochloride salt of the compound described herein.

[0114] The disclosed compounds may be formulated as pharmaceutical compositions comprising any of the compounds disclosed herein and a pharmaceutically acceptable excipient, carrier, or diluent.

[0115] The disclosed compounds and compositions comprising the same may exhibit one or more biological activities. In some embodiments, the disclosed compounds inhibit the activity of neuronal nitric oxide synthase (nNOS). The NOS inhibitory activity of the compounds disclosed herein may be measured using a hemoglobin (Hb) NO capture assay. In some embodiments, the disclosed compounds have an inhibition constant (Ki) for human nNOS which is less than about of 100 nM, 90 nM, 80 nM, 70 nM, 60 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 5 nM, 1 nM, 0.5 nM. 0.1 nM or lower. The inhibition constants (Ki) may be

[0116] Page 10

[0117] QB\702581.02752\100011397.1calculated from the IC50 values of the dose-response curves using the Cheng-Prusoff equation, according to methods described herein.

[0118] In some embodiments, the disclosed compound adopts the same binding mode to rnNOS (rat) and hnNOS (human). In some embodiments, the ratio of the Ki values of the inhibitors for human and rat nNOS (the hn / rn ratio) is from about 0.5 to about 1.8, from about 0.6 to about 1.5, from about 0.7 to about 1.4. from about 0.8 to about 1.3, from about 0.9 to about 1.2, or about 1.0.

[0119] In some embodiments, the disclosed compound is selective for human nNOS over human inducible NOS (iNOS). The isoform selectivity is determined by comparing the Ki values of human nNOS with human iNOS. In some embodiments, the disclosed compounds exhibit a selectivity for human nNOS over human iNOS (hn / hi) which is at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or higher. In some embodiments, the NOS inhibited by the disclosed compounds is a neuronal NOS which is inhibited selectively versus iNOS.

[0120] In some embodiments, the disclosed compound is selective for human nNOS over human endothelial NOS (eNOS). The isoform selectivity is determined by comparing the Ki values of human nNOS with human eNOS. In some embodiments, the disclosed compounds exhibit a selectivity for human nNOS over human eNOS (hn / he) which is at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, or higher. In some embodiments, the NOS inhibited by the disclosed compounds is a neuronal NOS which is inhibited selectively versus eNOS.

[0121] The disclosed NOS inhibitors may penetrate the blood-brain barrier (BBB). To improve BBB permeability several factors come into play may have one or more of the following properties: lower molecular weight (MW <500 Da, e.g., 350-400 Da), lower topological polar surface area (TPSA <76 A2, e.g.. 25-60 A2), higher lipophilicity (Clog P <5). a low number of hydrogen bond donors (HBD), both OH and NH2(HBD ≤ 5; e.g., 3), a reduced number of rotatable bonds (RB) (RB < 8; e.g., 4-5), a low number of hydrogen bond acceptors (HBA), both O andN atoms (HBA <10, e.g., 7), reduced pKa of basic amino groups (pKa 7.5−10.5, e.g., 8.4), and reduced P-glycoprotein (P-gp) efflux (ER <2).34In some instances, the disclosed compounds have 2, 3, 4, 5. 6, 7, or all 8 of these properties. Notably, increasing the number of rotatable bonds (NRB) may have a detrimental effect on the permeation rate. Furthermore, reducing the number of rotatable bonds not only enhances permeability but also improves oral bioavailability.46

[0122] Page 11

[0123] QB\702581.02752\100011397.1In some embodiments, the disclosed compounds exhibit an effective permeability (Pe) for the blood brain barrier of at least about 5.0 x 10’6cm / s, 6.0 x 10'6cm / s, 7.0 x 10’6cm / s, 8.0 x IO’6cm / s, 9.0 x 10'6cm / s, 10.0 x 10’6cm / s, 11.0 x 10’6cm / s, 12.0 x IO’6cm / s, 13.0 x 10'6cm / s, 14.0 x 10'6cm / s, 15.0 x 10'6cm / s, 16.0 x 10'6cm / s, 17.0 x 10'6cm / s, 18.0 x 10'6cm / s, 19.0 x 10'6cm / s, or 20.0 x 10'6cm / s. In some embodiments, the blood-brain barrier penetration may be estimated using the PAMPA-BBB assay, according to methods described herein. Pharmaceutical Compositions

[0124] Another aspect of the present disclosure provides a pharmaceutical composition comprising the compound disclosed herein and a pharmaceutically acceptable excipient, carrier, or diluent.

[0125] The compounds employed in the compositions and methods disclosed herein may be administered as pharmaceutical compositions and, therefore, pharmaceutical compositions incorporating the compounds are considered to be embodiments of the compositions disclosed herein. Such compositions may take any physical form which is pharmaceutically acceptable; illustratively, they can be orally administered pharmaceutical compositions. Such pharmaceutical compositions contain an effective amount of a disclosed compound, which effective amount is related to the daily dose of the compound to be administered. Each dosage unit may contain the daily dose of a given compound or each dosage unit may contain a fraction of the daily dose, such as one-half or one-third of the dose. The amount of each compound to be contained in each dosage unit can depend, in part, on the identity of the particular compound chosen for the therapy and other factors, such as the indication for which it is given. The pharmaceutical compositions disclosed herein may be formulated so as to provide quick, sustained, or delayed release of the active ingredient after administration to the patient by employing well known procedures.

[0126] In some embodiments, the compounds disclosed herein may be formulated as pharmaceutical compositions that include: (a) a therapeutically effective amount of one or more compounds as disclosed herein; and (b) one or more pharmaceutically acceptable carriers, excipients, or diluents. The pharmaceutical composition may include the compound in a range of about 0.1 to 2000 mg (preferably about 0.5 to 500 mg, and more preferably about 1 to 100 mg). The pharmaceutical composition may be administered to provide the compound at a daily dose of about 0.1 to about 1000 mg / kg body weight (preferably about 0.5 to about 500 mg / kg body weight, more preferably about 50 to about 100 mg / kg body weight). In some embodiments, after the pharmaceutical composition is administered to a subject (e.g., after Page 12

[0127] QB\702581.02752\100011397.1about 1, 2, 3, 4, 5, or 6 hours post-administration), the concentration of the compound at the site of action may be within a concentration range bounded by end-points selected from 0.001 nM, 0.005 nM, 0.01 nM, 0.5 nM, 0.1 nM, 1.0 nM, 10 nM, and 100 nM (e.g, 0.1 nM - 1.0 nM).

[0128] It is understood by those skilled in the art that dosage amount will vary with the activity of a particular inhibitor compound, disease state, route of administration, duration of treatment, and like factors well-known in the medical and pharmaceutical arts. In general, a suitable dose will be an amount which is the lowest dose effective to produce a therapeutic or prophylactic effect. If desired, an effective dose of such a compound, pharmaceutically acceptable salt thereof, or related composition may be administered in two or more sub-doses, administered separately over an appropriate period of time.

[0129] The compounds for use according to the methods of disclosed herein may be administered as a single compound or a combination of compounds. For example, a compound that inhibits neuronal nitric oxide synthase may be administered as a single compound or in combination with another compound that inhibits neuronal nitric oxide synthase or that has a different pharmacological activity. In some embodiments, one or more additional therapeutic agents may be administered with the disclosed compounds or with pharmaceutical compositions comprising the disclosed compounds, where the additional therapeutic agent is administered prior to, concurrently with, or after administering the disclosed compounds or the pharmaceutical compositions comprising the disclosed compounds. In some embodiments, the disclosed pharmaceutical composition is formulated to comprise the disclosed compounds and further to comprise one or more additional therapeutic agents, for example, one or more additional therapeutic agents for treating diseases and disorders.

[0130] As indicated above, pharmaceutically acceptable salts of the compounds are contemplated and also may be utilized in the disclosed methods. The term 'pharmaceutically acceptable salt” as used herein, refers to salts of the compounds, which are substantially nontoxic to living organisms. Typical pharmaceutically acceptable salts include those salts prepared by reaction of the compounds as disclosed herein w ith a pharmaceutically acceptable mineral or organic acid or an organic or inorganic base. Such salts are known as acid addition and base addition salts. It will be appreciated by the skilled reader that most or all of the compounds as disclosed herein are capable of forming salts and that the salt forms of pharmaceuticals are commonly used, often because they are more readily cry stallized and purified than are the free acids or bases. In some embodiments, HC1 salts of the disclosed compounds are provided.

[0131] Page 13

[0132] QB\702581.02752\100011397.1Acids commonly employed to form acid addition salts may include inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, and the like, and organic acids such as p-toluenesulfonic, methanesulfonic acid, oxalic acid, p-bromophenylsulfonic acid, carbonic acid, succinic acid, citric acid, benzoic acid, acetic acid, and the like. Examples of suitable pharmaceutically acceptable salts may include the sulfate, pyrosulfate, bisulfate, sulfite, bisulfate, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, bromide, iodide, acetate, propionate, decanoate, caprylate, acrylate, formate, hydrochloride, dihydrochloride, isobutyrate, caproate, heptanoate, propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleat-, butyne-.1,4-dioate, hexyne-l,6-dioate, benzoate, chlorobenzoate, methylbenzoate, hydroxybenzoate, methoxybenzoate, phthalate, xylenesulfonate, phenyl acetate, phenylpropionate, phenylbutyrate, citrate, lactate, a-hydroxybutyrate, glycolate, tartrate, methanesulfonate, propanesulfonate, naphthalene- 1 -sulfonate, naphthal ene-2-sulfonate, mandelate, and the like.

[0133] Base addition salts include those derived from inorganic bases, such as ammonium or alkali or alkaline earth metal hydroxides, carbonates, bicarbonates, and the like. Bases useful in preparing such salts include sodium hydroxide, potassium hydroxide, ammonium hydroxide, potassium carbonate, sodium carbonate, sodium bicarbonate, potassium bicarbonate, calcium hydroxide, calcium carbonate, and the like.

[0134] The particular counter-ion forming a part of any salt of a compound disclosed herein may not be critical to the activity of the compound, so long as the salt as a whole is pharmacologically acceptable and as long as the counterion does not contribute undesired qualities to the salt as a whole. Undesired qualities may include undesirably solubility or toxicity.

[0135] Pharmaceutically acceptable esters and amides of the compounds can also be employed in the compositions and methods disclosed herein. Examples of suitable esters include alkyl, aryl, and aralkyl esters, such as methyl esters, ethyl esters, propyl esters, dodecyl esters, benzy l esters, and the like. Examples of suitable amides include unsubstituted amides, monosubstituted amides, and disubstituted amides, such as methyl amide, dimethyl amide, methyl ethyl amide, and the like.

[0136] In addition, the methods disclosed herein may be practiced using solvate forms of the compounds or salts, esters, and / or amides, thereof. Solvate forms may include ethanol solvates, hydrates, and the like.

[0137] Page 14

[0138] QB\702581.02752\100011397.1Compositions can be formulated in a unit dosage form, each dosage containing from about 1 to about 500 mg of each compound individually or in a single unit dosage form, such as from about 5 to about 300 mg, from about 10 to about 100 mg, and / or about 25 mg. The term “unit dosage form” refers to a physically discrete unit suitable as unitary dosages for a patient, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical carrier, diluent, or excipient.

[0139] The pharmaceutical composition may include the compound in a range of about 0.1 to 2000 mg. In some embodiments, the pharmaceutical composition may include the compound in a range of from about 0.5 to 500 mg. In some embodiments, the pharmaceutical composition may include the compound in a range of from about 1 to 100 mg. The pharmaceutical composition may be administered to provide the compound at a daily dose of about 0.1 to about 1000 mg / kg body weight. In some embodiments, the pharmaceutical composition may be administered to provide the compound at a daily dose of about 0.5 to about 500 mg / kg body¬ weight. In some embodiments, the pharmaceutical composition may be administered to provide the compound at a daily dose of about 50 to about 100 mg / kg body weight. A typical daily dose may contain from about 0.01 mg / kg to about 100 mg / kg (such as from about 0.05 mg / kg to about 50 mg / kg and / or from about 0.1 mg / kg to about 25 mg / kg) of each compound used in the present method of treatment. In some embodiments, after the pharmaceutical composition is administered to a subject (e.g, after about 1, 2. 3, 4, 5. or 6 hours post-administration), the concentration of the compound at the site of action may be within a concentration range bounded by end-points selected from 0.001 nM, 0.005 nM, 0.01 nM, 0.5 nM, 0.1 nM, 1.0 nM, 10 nM, and 100 nM (e.g., 0.1 nM - 1.0 nM).

[0140] The compounds utilized in the methods disclosed herein may be formulated as a pharmaceutical composition that includes a carrier. For example, the carrier may be selected from the group consisting of proteins, carbohydrates, sugar, talc, magnesium stearate, cellulose, calcium carbonate, and starch-gelatin paste.

[0141] The compounds utilized in the methods disclosed herein may be formulated as a pharmaceutical composition that includes one or more binding agents, filling agents, lubricating agents, suspending agents, sweeteners, flavoring agents, preservatives, buffers, wetting agents, disintegrants, and effervescent agents. Filling agents may include lactose monohydrate, lactose anhydrous, and various starches; examples of binding agents are various celluloses and cross-linked polyvinylpyrrolidone, microcrystalline cellulose, such as Avicel®

[0142] Page 15

[0143] QB\702581.02752\100011397.1PH101 and Avicel® PH102, microcrystalline cellulose, and silicified microcrystalline cellulose (ProSolv SMCC™). Suitable lubricants, including agents that act on the flowability of the powder to be compressed, may include colloidal silicon dioxide, such as Aerosil®200, talc, stearic acid, magnesium stearate, calcium stearate, and silica gel. Examples of sweeteners may include any natural or artificial sweetener, such as sucrose, xylitol, sodium saccharin, cyclamate, aspartame, and acsulfame. Examples of flavoring agents are Magnasweet® (trademark of MAFCO), bubble gum flavor, and fruit flavors, and the like. Examples of preservatives may include potassium sorbate, methylparaben, propylparaben, benzoic acid and its salts, other esters of parahydroxybenzoic acid such as butylparaben, alcohols such as ethyl or benzyl alcohol, phenolic compounds such as phenol, or quaternary compounds such as benzalkonium chloride.

[0144] Suitable diluents may include pharmaceutically acceptable inert fillers, such as microcrystalline cellulose, lactose, dibasic calcium phosphate, saccharides, and mixtures of any of the foregoing. Examples of diluents include microcrystalline cellulose, such as Avicel® PH101 and Avicel® PH102; lactose such as lactose monohydrate, lactose anhydrous, and Pharmatose® DCL21; dibasic calcium phosphate such as Emcompress®; mannitol; starch; sorbitol; sucrose; and glucose.

[0145] Suitable disintegrants include lightly crosslinked polyvinyl pyrrolidone, com starch, potato starch, maize starch, and modified starches, croscarmellose sodium, cross-povidone, sodium starch glycolate. and mixtures thereof.

[0146] Examples of effervescent agents are effervescent couples such as an organic acid and a carbonate or bicarbonate. Suitable organic acids include, for example, citric, tartaric, malic, fumaric, adipic, succinic, and alginic acids and anhydrides and acid salts. Suitable carbonates and bicarbonates include, for example, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, magnesium carbonate, sodium glycine carbonate, L-lysine carbonate, and arginine carbonate. Alternatively, only the sodium bicarbonate component of the effervescent couple may be present.

[0147] The compounds utilized in the methods disclosed herein may be administered in conventional dosage forms prepared by combining the active ingredient with standard pharmaceutical carriers or diluents according to conventional procedures well known in the art. These procedures may involve mixing, granulating and compressing or dissolving the ingredients as appropriate to the desired preparation.

[0148] Page 16

[0149] QB\702581.02752\100011397.1Pharmaceutical compositions comprising the compounds may be adapted for administration by any appropriate route, for example by the oral (including buccal or sublingual), rectal, nasal, topical (including buccal, sublingual or transdermal), vaginal or parenteral (including subcutaneous, intramuscular, intravenous or intradermal) route. Such formulations may be prepared by any method known in the art of pharmacy, for example by bringing into association the active ingredient with the carrier(s) or excipient(s).

[0150] Pharmaceutical compositions adapted for oral administration may be presented as discrete units such as capsules or tablets; powders or granules; solutions or suspensions in aqueous or non-aqueous liquids; edible foams or whips; or oil-in-water liquid emulsions or water-in-oil liquid emulsions.

[0151] Pharmaceutical compositions adapted for transdermal administration may be presented as discrete patches intended to remain in intimate contact with the epidermis of the recipient for a prolonged period of time. For example, the active ingredient may be delivered from the patch by iontophoresis.

[0152] Pharmaceutical compositions adapted for topical administration may be formulated as ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, impregnated dressings, sprays, aerosols or oils and may contain appropriate conventional additives such as preservatives, solvents to assist drug penetration and emollients in ointments and creams.

[0153] For applications to the eye or other external tissues, for example the mouth and skin, the pharmaceutical compositions are in some embodiments applied as a topical ointment or cream. When formulated in an ointment, the compound may be employed with either a paraffinic or a water-miscible ointment base. Alternatively, the compound may be formulated in a cream with an oil-in-water cream base or a water-in-oil base. Pharmaceutical compositions adapted for topical administration to the eye include eye drops where the active ingredient is dissolved or suspended in a suitable earner, especially an aqueous solvent.

[0154] Pharmaceutical compositions adapted for topical administration in the mouth include lozenges, pastilles and mouth washes.

[0155] Pharmaceutical compositions adapted for rectal administration may be presented as suppositories or enemas.

[0156] Pharmaceutical compositions adapted for nasal administration where the carrier is a solid include a coarse pow der having a particle size (e.g., in the range 20 to 500 microns) which is administered in the manner in which snuff is taken (i. e., by rapid inhalation through the nasal passage from a container of the powder held close up to the nose). Suitable formulations where Page 17

[0157] QB\702581.02752\100011397.1the carrier is a liquid, for administration as a nasal spray or as nasal drops, include aqueous or oil solutions of the active ingredient.

[0158] Pharmaceutical compositions adapted for administration by inhalation include fine particle dusts or mists which may be generated by means of various types of metered dose pressurized aerosols, nebulizers or insufflators.

[0159] Pharmaceutical compositions adapted for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or spray formulations.

[0160] Pharmaceutical compositions adapted for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient: and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets.

[0161] Oral administration is an illustrative route of administering the compounds employed in the compositions and methods disclosed herein. Other illustrative routes of administration include transdermal, percutaneous, intravenous, intramuscular, intranasal, buccal, intrathecal, intracerebral, or intrarectal routes. The route of administration may be varied in any way, limited by the physical properties of the compounds being employed and the convenience of the subj ect and the caregiver.

[0162] Tablets and capsules for oral administration may be in unit dose presentation form, and may contain conventional excipients such as binding agents, for example syrup, acacia, gelatin, sorbitol, tragacanth, or polyvinylpyrrolidone; fillers, for example lactose, sugar, maize-starch, calcium phosphate, sorbitol or glycine; tableting lubricants, for example magnesium stearate, talc, polyethylene glycol or silica; disintegrants, for example potato starch; or acceptable wetting agents such as sodium lauryl sulphate. The tablets may be coated according to methods well known in normal pharmaceutical practice. Oral liquid preparations may be in the form of, for example, aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, or may be presented as a dry product for reconstitution with water or other suitable vehicle before use. Such liquid preparations may contain conventional additives, such as suspending agents, for example sorbitol, methyl cellulose, glucose syrup, gelatin, hydroxyethyl cellulose,

[0163] Page 18

[0164] QB\702581.02752\100011397.1carboxymethyl cellulose, aluminium stearate gel or hydrogenated edible fats, emulsifying agents, for example lecithin, sorbitan monooleate, or acacia; non-aqueous vehicles (which may include edible oils), for example almond oil, oily esters such as glycerine, propylene glycol, or ethyl alcohol; preservatives, for example methyl or propyl p-hydroxybenzoate or sorbic acid, and, if desired, conventional flavoring or coloring agents.

[0165] Methods of preparing pharmaceutical formulations or compositions include the step of bringing a disclosed compound into association with a carrier and, optionally, one or more additional adjuvants or ingredients. For example, standard pharmaceutical formulation techniques can be employed, such as those described in Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA.

[0166] Regardless of composition or formulation, those skilled in the art will recognize various avenues for medicament administration, together with corresponding factors and parameters to be considered in rendering such a medicament suitable for administration.

[0167] As one skilled in the art will appreciate, suitable formulations include those that are suitable for more than one route of administration. For example, the formulation can be one that is suitable for both intrathecal and intracerebral administration. Alternatively, suitable formulations include those that are suitable for only one route of administration as well as those that are suitable for one or more routes of administration, but not suitable for one or more other routes of administration. For example, the formulation can be one that is suitable for oral, transdermal, percutaneous, intravenous, intramuscular, intranasal, buccal, and / or intrathecal administration but not suitable for intracerebral administration.

[0168] Methods

[0169] The disclosed compounds and pharmaceutical compositions comprising the disclosed compounds may be administered in methods of treating a disease or a disorder in a subject in need thereof, such as in methods of treating a disease or disorder associated with neuronal nitric oxide synthase activity.

[0170] Another aspect of the present disclosure provides a method of treating a disease or disorder associated with neuronal nitric oxide synthase (nNOS) activity in a subject in need thereof, the method comprising administering an effective amount of the compound disclosed herein, or a pharmaceutically acceptable salt, hydrate, or solvate thereof; or the pharmaceutical composition disclosed herein to the subject.

[0171] As used herein, the terms "treating” or “to treat’" each mean to alleviate symptoms, eliminate the causation of resultant symptoms either on a temporary or permanent basis, and / or Page 19

[0172] QB\702581.02752\100011397.1to prevent or slow the appearance or to reverse the progression or severity of resultant symptoms of the named disease or disorder. As such, the methods disclosed herein encompass both therapeutic and prophylactic administration.

[0173] A “subject in need thereof’ as utilized herein refers to a subject in need of prevention and / or treatment for a disease or disorder associated with nNOS activity. The term “subject” may be used interchangeably with the terms “individual” and “patient” and includes human and non-human mammalian subjects. In some embodiments, the treated subject may be a mammalian subject. Although the methods disclosed herein are particularly intended for the treatment of humans, other mammals are included. By way of non-limiting examples, mammalian subjects include monkeys, equines, cattle, canines, felines, mice, rats and pigs.

[0174] As used herein, the term "disorder" refers to a condition in which there is a disturbance of normal functioning. A "disease" is any abnormal condition of the body or mind that causes discomfort, dysfunction, or distress to the person affected or those in contact with the person. Sometimes the term is used broadly to include injuries, disabilities, syndromes, symptoms, deviant behaviors, and atypical variations of structure and function, while in other contexts these may be considered distinguishable categories. It should be noted that the terms "disease", "disorder", "condition" and "illness", are equally used herein.

[0175] In some embodiments, the disease or disorder is a neurological disease or disorder, or melanoma. Neurological diseases or disorders include, but are not limited to. Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), chronic headache, or neuronal damage during stroke.

[0176] In some embodiments of the disclosed treatment methods, the subject may be administered a dose of a compound as low as 1.25 mg, 2.5 mg, 5 mg, 7.5 mg, 10 mg, 12.5 mg, 15 mg, 17.5 mg, 20 mg, 22.5 mg, 25 mg, 27.5 mg, 30 mg, 32.5 mg, 35 mg, 37.5 mg, 40 mg, 42.5 mg, 45 mg, 47.5 mg, 50 mg, 52.5 mg, 55 mg, 57.5 mg, 60 mg, 62.5 mg, 65 mg, 67.5 mg, 70 mg, 72.5 mg, 75 mg, 77.5 mg, 80 mg, 82.5 mg, 85 mg, 87.5 mg, 90 mg, 100 mg. 200 mg, 500 mg, 1000 mg, or 2000 mg once daily, twice daily, three times daily, four times daily, once weekly, twice weekly, or three times per week in order to treat the disease or disorder in the subject. In some embodiments, the subject may be administered a dose of a compound as high as 1.25 mg, 2.5 mg, 5 mg, 7.5 mg, 10 mg, 12.5 mg, 15 mg, 17.5 mg, 20 mg, 22.5 mg, 25 mg, 27.5 mg, 30 mg, 32.5 mg, 35 mg, 37.5 mg, 40 mg, 42.5 mg, 45 mg, 47.5 mg, 50 mg, 52.5 mg, 55 mg, 57.5 mg, 60 mg, 62.5 mg, 65 mg, 67.5 mg, 70 mg, 72.5 mg, 75 mg, 77.5 mg, 80 mg, 82.5 mg, 85 mg, 87.5 mg, 90 mg, 100 mg. 200 mg, 500 mg. 1000 mg, or 2000 mg, once daily,

[0177] Page 20

[0178] QB\702581.02752\100011397.1twice daily, three times daily, four times daily, once weekly, twice weekly, or three times per week in order to treat the disease or disorder in the subject. Minimal and / or maximal doses of the compounds may include doses falling within dose ranges having as end-points any of these disclosed doses (e.g., 2.5 mg - 200 mg).

[0179] In some embodiments of the disclosed treatment methods, a minimal dose level of a compound for achieving therapy in the disclosed methods of treatment may be at least about 10. 20. 30. 40. 50, 60, 70, 80, 90, 100, 150. 200, 250. 300, 350, 400, 450, 500. 550, 600, 650.

[0180] 700, 750, 800, 850, 900, 950, 1000, 1200, 1400, 1600, 1800, 1900, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 15000, or 20000 ng / kg body weight of the subject. In some embodiments, a maximal dose level of a compound for achieving therapy in the disclosed methods of treatment may not exceed about 10. 20. 30. 40. 50. 60. 70. 80. 90. 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1200, 1400, 1600, 1800, 1900, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 15000, or 20000 ng / kg body weight of the subject. Minimal and / or maximal dose levels of the compounds for achieving therapy in the disclosed methods of treatment may include dose levels falling within ranges having as end-points any of these disclosed dose levels (e.g., 500 – 2000 ng / kg body weight of the subject).

[0181] As used herein the term “effective amount'’ refers to the amount or dose of the compound or composition, upon single or multiple dose administration to the subject, which provides the desired effect in the subject under diagnosis or treatment. The disclosed methods may include administering an effective amount of the disclosed compounds (e.g., as present in a pharmaceutical composition) for treating a disease or disorder associated with neuronal nitric oxide synthase activity.

[0182] An effective amount can be readily determined by the attending diagnostician, as one skilled in the art, by the use of known techniques and by observing results obtained under analogous circumstances. In determining the effective amount or dose of compound administered, a number of factors can be considered by the attending diagnostician, such as: the species of the subject; its size, age, and general health; the degree of involvement or the severity of the disease or disorder involved; the response of the individual subject; the particular compound administered; the mode of administration; the bioavailability characteristics of the preparation administered; the dose regimen selected; the use of concomitant medication; and other relevant circumstances.

[0183] Page 21

[0184] QB\702581.02752\100011397.1The compounds and compositions disclosed herein may be administered in methods of treatment as known in the art. Accordingly, various such compounds and compositions can be administered in conjunction with such a method in any suitable way. For example, administration may comprise oral, intravenous, intraarterial, intramuscular, subcutaneous, intraperitoneal, parenteral, transdermal, intravaginal, intranasal, mucosal, sublingual, topical, rectal or subcutaneous administration, or any combination thereof.

[0185] Miscellaneous

[0186] Unless otherwise specified or indicated by context, the terms “a”, “an”, and “the” mean “one or more.” For example, “a molecule” should be interpreted to mean “one or more molecules.”

[0187] As used herein, “about”, “approximately,” “substantially,” and “significantly” will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which they are used. If there are uses of the term which are not clear to persons of ordinary skill in the art given the context in which it is used, “about” and “approximately” will mean plus or minus <10% of the particular term and “substantially” and “significantly” will mean plus or minus >10% of the particular term.

[0188] As used herein, the terms “include” and “including” have the same meaning as the terms “comprise” and “comprising.” The terms “comprise” and “comprising” should be interpreted as being “open” transitional terms that permit the inclusion of additional components further to those components recited in the claims. The terms “consist” and “consisting of’ should be interpreted as being “closed” transitional terms that do not permit the inclusion additional components other than the components recited in the claims. The term “consisting essentially of’ should be interpreted to be partially closed and allowing the inclusion only of additional components that do not fundamentally alter the nature of the claimed subject matter.

[0189] The phrase “such as” should be interpreted as “for example, including.” Moreover the use of any and all exemplary language, including but not limited to “such as”, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed.

[0190] Furthermore, in those instances where a convention analogous to “at least one of A, B and C, etc.” is used, in general such a construction is intended in the sense of one having ordinary skill in the art would understand the convention (e.g., “a system having at least one of A, B and C” would include but not be limited to systems that have A alone, B alone. C alone, A and B together, A and C together, B and C together, and / or A, B, and C together.). It will be Page 22

[0191] QB\702581.02752\100011397.1further understood by those within the art that virtually any disjunctive word and / or phrase presenting two or more alternative terms, whether in the description or figures, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase “A or B” will be understood to include the possibilities of “A” or ‘B or “A and B.”

[0192] All language such as “up to." “at least,” “greater than,” “less than,” and the like, include the number recited and refer to ranges which can subsequently be broken down into ranges and subranges. A range includes each individual member. Thus, for example, a group having 1-3 members refers to groups having 1, 2, or 3 members. Similarly, a group having 6 members refers to groups having 1, 2, 3, 4, or 6 members, and so forth.

[0193] The modal verb “may” refers to the preferred use or selection of one or more options or choices among the several described embodiments or features contained within the same. Where no options or choices are disclosed regarding a particular embodiment or feature contained in the same, the modal verb “may” refers to an affirmative act regarding how to make or use and aspect of a described embodiment or feature contained in the same, or a definitive decision to use a specific skill regarding a described embodiment or feature contained in the same. In this latter context, the modal verb “may” has the same meaning and connotation as the auxiliary verb “can.”

[0194] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary' language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0195] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

[0196] Preferred aspects of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred aspects may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect a person having ordinary skill in the art to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and Page 23

[0197] QB\702581.02752\100011397.1equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherw ise clearly contradicted by context.

[0198] EXAMPLES

[0199] Example 1

[0200] In this study, we have developed a series of compact, biaryl NOS inhibitors, aiming to achieve good membrane permeability, improve synthetic accessibility while maintaining potency and isoform selectivity by reducing the number of rotatable bonds (NRB). Previous reports indicated that the inclusion of a 2-aminopyridine scaffold is vital in the key binding interactions of nNOS inhibitors with Glu-592 and Glu-597 at the active sites of rat nNOS and human nNOS, respectively.33'37,52Furthermore, the addition of a lipophilic methyl group at the 4-position of the 2-aminopyridine enhanced both potency and selectivity.53Based on the above experimental observations and recent X-ray crystallographic analyses, we have synthesized inhibitors with diverse structures. Moreover, we also have developed a fast and efficient synthetic method using the Suzuki-Miyaura cross-coupling reaction, allowing for quick access to these inhibitors together with excellent yields.

[0201] RESULTS AND DISCUSSION

[0202] Chemical Synthesis of the nNOS Inhibitors

[0203] The synthetic route to inhibitors 11-14 and 17 is described in Scheme 1. We developed a highly efficient, short synthetic procedure, enabling rapid access to NOS inhibitors using Suzuki-Miyaura cross-coupling reactions. This method has the advantage over classical Suzuki-Miyaura cross-coupling reactions as it not only enables the creation of aryl boronate esters (borylation) in situ but also facilitates efficient second cross-coupling reactions with halo coupling components in a one-pot approach.54'57Initially, we focused on synthesizing the aryl linker with an amine tail fragment starting from commercially available benzyl bromide (24, 27, and 30) with / -butyl-N-methy Icarbamate via SN2 substitution using NaH at 0 °C,58which provided intermediates of 25, 28, and 31 in excellent yields. The in situ borylation of species 26, 29, and 32 was achieved with bis(pinacolato)diboron (B2Pin2) using PdCl2(dppf) at 100 °C, followed by the Suzuki-Miyaura cross-coupling reactions with 6-bromo-4-methylpyridin-2-amine. This yielded the corresponding Boc-protected inhibitors, which, upon hydrolysis with 3 M HC1 in CH3OH,59gave the final NOS inhibitors (11-12 and 14). Similarly, 13 and 17 were

[0204] Page 24

[0205] QB\702581.02752\100011397.1prepared using methylamine hydrochloride34, 60instead of tert-butyl-N-methylcarbamate, followed by A-Boc protection61from commercially available benzyl bromide (33 and 37).

[0206] Scheme 1. Synthetic Route to 11-14, and 17"

[0207] 34, R₁ = CF₃ 35, R₁ = CF₃ 36, R₁ = CF₃ R₁ 13, R₁ = CF₃ R, 38, R, = CN 39, R, = CN 40, R-j = ON 17, R, = CN “Reagents and conditions: (a) / e / 7-butyl-.'V-methylcarbamate. NaH, DMF, 0 °C, 30 min, then 24, 27, and 30, 0-25 °C, 1-24 h; (b) CH3NH2«HC1, K2CO3. CH3CN, 25 °C, 24 h; (c) BOC2O. Et3N, CH2C12, 25 °C, 8-24 h; (d) B2Pin2, PdCl2(dppf), KOAc, 1,4-dioxane (0.2 M), 100 °C, 1- 8 h, and then 6-bromo-4-methylpyridin-2-amine, aq. K2CO3 (1.8 M). 120 °C, 8-24 h; (e) 3 M HC1 in CH3OH, 25 °C, 1-24 h.

[0208]

[0209] “Reagents and conditions: (a) (CH3)2NH«HC1, K2CO3, CH3CN, 25 °C, 24 h; (b) B2Pin2, PdCl2(dppf), KOAc, 1,4-dioxane (0.2 M), 100 °C, 1-8 h, and then 6-bromo-4-methylpyridin- 2-amine, aq. K2CO3 (1.8 M), 120 °C, 8-24 h; (c) 3 M HC1 in CH3OH, 25 °C, 1-24 h.

[0210] Compound 15 was synthesized using a similar method as compounds 11-14 and 17 (Scheme 1). It was prepared by combining 30 with dimethylamine hydrochloride at 25 °C, resulting in the formation of the desired aryl linker with a dimethylamine tail (41).34, 60The borylated species was then generated in situ through a Suzuki-Miyaura cross-coupling reaction of 41 with bis(pinacolato)diboron. This species undergoes further cross-coupling with 6- bromo-4-methylpyridin-2-amine to produce the freebase (42) of final compound 15 in a one- pot process. Finally, the freebase was converted to its HC1 salt by acidification with 3 M HC1 in methanol (Scheme 2).59

[0211] Page 25

[0212] QB\702581.02752\100011397.1Scheme 3. Synthetic Route to 16

[0213]

[0214] •2HCI " Reagents and conditions: (a) CH3NH2 in EtOH (33 wt. %), CH3OH, 25 °C. 8 h, and then NaBEk, 25 °C, 2 h; (b) BOC2O, Et3N, CH2CI2, 25 °C, 24 h; (c) B2Pin2, PdCl2(dppf), KO Ac, 1,4-dioxane (0.2 M), 100 °C, 1-8 h, and then 6-bromo-4-methylpyridin-2-amine, aq. K2CO3 (1.8 M), 120 °C, 8-24 h; (d) 3 M HC1 in CH3OH, 25 °C, 1-24 h.

[0215] The pyridine-containing linker with a methylamine tail (45) fragment was successfully obtained from commercially available 5-bromonicotinaldehyde (43) through reductive amination62with methylamine in a stepwise manner, followed by A-Boc-protection.61Then, 45 underwent a one-pot borylation-Suzuki-Miyaura cross-coupling reaction with 6-bromo-4-methylpyridin-2-amine to produce the freebase (46) of compound 16. This was further converted to its HCl salt by acidic work-up with 3 M HC1 in methanol (Scheme 3).59

[0216] The difluoro aryl-containing linker was synthesized with different amine tails (secondary and tertiary amines) such as in 48, 51, 53, 56, and 58, through a simple SN2 substitution of benzyl bromide 47 with appropriate amines (Scheme 4).34’60This was followed by A-Boc-protection61(only for secondary’ amines) and a one-pot borylation-Suzuki-Miyaura cross-coupling reaction with 6-bromo-4-methylpyridin-2-amine to produce the final desired compounds (18-22). Finally, these compounds were converted to their HC1 salts by acidification yvith 3 M HC1 in methanol.59

[0217] Page 26

[0218] QB\702581.02752\100011397.1Scheme 4. Synthetic Route to 18-22a

[0219] aReagents and conditions: (a) Appropriate amine hydrochloride, K2CO3, CH3CN, 25 °C, 24 h; (b) Boc2O, EtsN, CH2CI2, 25 °C, 24 h; (c) B2Pin2, PdCl2(dppf), KO Ac, 1,4-dioxane (0.2 M), 100 °C, 1-8 h, and then 6-bromo-4-methylpyridin-2-amine, aq. K2CO3 (1.8 M), 120 °C, 8-24 h; (d) 3 M HC1 in CH3OH, 25 °C, 1-24 h.

[0220] Scheme 5.aSynthetic Route to 23a

[0221]

[0222] “Reagents and conditions: (a) BH3»SMe2, THF, 0 °C to 25 °C, 24 h; (b) CBn, PPI13, 0 °C to 25 °C, 28 h, CH2CI2; (c) KCN, (nBu)4NBr, CH2C12: H2O (1:1) (0.18 M), 24 h; (d) BH3*SMe2. THF, 0 °C to 25 °C, 30 h; (e) BOC2O, EtsN, CH2CI2, 25 °C, 24 h; (1) NaH, DMF, 0 °C, 30 min, and then CH3I, 0-25 °C, 4 h; (g) B2Pin2, PdCl2(dppf), KO Ac, 1,4-dioxane (0.2 M), 100 °C, 1-8 h, and then 6-bromo-4-methylpyridin-2-amine, aq. K2CO3 (1.8 M), 120 °C, 8-24 h; (h) 3 M HC1 in CH3OH, 25 °C, 1-24 h.

[0223] Page 27

[0224] QB\702581.02752\100011397.1The synthetic route to inhibitor 23 is described in Scheme 5. First, 6333was obtained from commercially available carboxylic acid 60 by converting it to benzyd alcohol 61 through reduction with BH3’SMe2. Then, 47 was prepared by the Appel reaction63of 61, followed by cyanation (62) of the corresponding benzyl bromide 47 and reduction with BHs’SMe2 to achieve the desired one-carbon extension of the chain and amine installation (63). Compound 63 then was subjected to A-Boc protection (64),61followed by methylation to give 65 in excellent yields. Finally, 65 was converted to 66 via a one-pot borylation-Suzuki-Miyaura cross-coupling reaction with 6-bromo-4-methylpyridin-2-amine, which, upon hydrolysis with 3 M HC1 in methanol,59yielded the final compound 23 in excellent yield.

[0225] Biochemical Characterization of nNOS Inhibitors

[0226] All nNOS inhibitors (11-23) were converted to their hydrochloride (HC1) salt before biochemical assays. The potency and selectivity of the new NOS inhibitors were determined using the NO hemoglobin (Hb) capture assay as previously reported,64with L-arginine (a NO donor) and a NOS inhibitor (inhibitor of NO formation) as positive and negative controls, respectively.65The results are summarized in Table 1. All compounds were assayed with purified rat nNOS to test inhibitory activity ( 7i) as an initial screen. The most promising inhibitors were selected for further assays with isoforms of human NOS enzymes (hnNOS, hiNOS, and heNOS) to evaluate potency and isoform selectivity. All inhibitors were chosen for evaluation using additional assays, including the PAMPA-BBB assay to assess cell membrane permeability. The ratio, hnNOS / rat nNOS (hn / m), is defined as the ratio of the Ki values of the inhibitors for human and rat nNOS. The hn / m ratio is important for evaluating the potential translation of these inhibitors from preclinical to clinical studies. Isoform selectivity for human nNOS over human iNOS (hn / hi) and eNOS (hn / he) was obtained by comparing the Ki values of human nNOS with human iNOS and human eNOS, respectively.

[0227] Table 1. List of K values and selectivity of 11-23

[0228] K (nM)“ Selectivity* Compound rat Human human human

[0229] hn / rn hn / hi hn / he nNOS nNOS iNOS eNOS

[0230] 11 ’2HC1 40 38 1416 4916 1.0 37 129 12 -211( 1 47 65 3152 51486 1.4 48 792 13 »2HC1 815 1152 122978 320417 1.4 107 278 14 «2HC1 109 95 20244 104206 0.9 213 1097 15 ’211( 1 941 1365 132222 NT 1.5 97 NT

[0231]

[0232] Page 28

[0233] QB\702581.02752\100011397.116 »2HC1 62 82 3956 28815 1.3 48 351 17 «2HC1 298 358 16356 115457 1.2 46 323 18 «2HC1 40 30 2240 24632 0.8 75 821 19 »2HC1 697 698 23333 35717 1.0 33 51 2O «2HC1 98 116 5898 27797 1.2 51 240 21 «2HC1 4172 8448 142356 130243 2.0 17 15 22 *2HC1 3975 3566 245956 125501 0.9 69 35 23 «2HC1 63 47 8342 12455 0.7 177 265

[0234]

[0235] aKi values were calculated from the IC50 values of the corresponding dose-response curves using the Cheng-Prusoff equation. For each compound, 8 to 11 concentrations were tested, and the IC50 value was calculated from an average of at least two duplicates. All standard errors were less than 10%.bSelectivity values were determined by calculating the ratios of respective K values. The ratio hn / m is desired to be as close to 1.0 as possible to avoid significant differences between rat and human dosages for clinical studies. For hn / hi and hn / he ratios, higher values are favorable. NT = not tested.

[0236] Assessment of Permeability of nNOS Inhibitors

[0237] All nNOS inhibitors (11-23) were selected and tested for the ability to permeate cell membranes using the PAMPA-BBB assay, which serves as a model for blood-brain permeability. Permeability (Pe) values were compared with reported Pevalues of five commercial drugs.33’6,66The results of this comparison are summarized in Table 2.

[0238] Table 2. Effective permeability (Pe) of five commercial drugs and novel nNOS inhibitors in the PAMPA-BBB assay

[0239] Reported PeDetermined Pe

[0240] Compounds Prediction6(106cm s1)" (IO-6cm s

[0241] (±)- Verapamil 16 22.6± 0.76 CNS (+) Desipramine 12 22.8 ± 0.93 CNS (+) Chlorpromazine 6.5 10.6 ± 0.94 CNS (+) Dopamine 0.2 0.13 ± 0.14 CNS (-) Theophylline 0.12 0.08 ± 0.04 CNS (-) 11 *2HC1 6.5 ± 0.24 CNS (+) 12 »2HC1 11.2 ± 0.73 CNS (+) 13 »2HC1 17.4 ± 0.28 CNS (+)

[0242]

[0243] Page 29

[0244] QB\702581.02752\100011397.114 *2HC1 15.3 ± 0.19 CNS (+) 15 »2HC1 21.5 ± 0.69 CNS (+) 16 »2HC1 3.6 ± 0.21 CNS (+ / -) 17 *2HC1 8.2 ± 0.36 CNS (+) 18 «2HC1 10.7 ± 0.18 CNS (+) 19 »2HC1 20.0 ± 0.23 CNS (+) 20 • 2HC1 22.5 ± 0.45 CNS (+) 21 »2HC1 24.5 ± 1.02 CNS (+) 22 • 2HC1 18.2 ± 0.36 CNS (+) 23 • 2HC1 12.0 ± 0.49 CNS (+)

[0245]

[0246] aReported effective permeability (Pe) values from the literature.hEffective permeability values were obtained in our in-house conditions. All assays were performed over 17 h at a concentration of 200 / / M and tested in a triplicate for each compound.cCNS (+) = high BBB permeation. CNS (-) = low BBB permeation. CNS (+ / -) = BBB permeation uncertain. Note that the “predictions” for the five literature-based compounds listed have been experimentally tested.

[0247] An inhibitor possessing fewer rotatable bonds is anticipated to have enhanced capacity to permeate membranes due to diminished flexibility. Removing a carbon chain between the 2-aminopyridine head and the aryl linker segment in the simplified version of inhibitors 11-23 did not significantly impact the ability to pass through membranes, except for compound 16.

[0248] For the simplified versions of inhibitors, 11-23, the phenyl linker (11) showed moderate permeability (Pe = 6.5 × 10⁻⁶ cm s⁻¹). By introducing halogen atoms such as fluorine or chlorine into the aryl linker (12-15) and fluorine atoms into the phenyl linker as well as the amine tail (18-23), the permeability of NOS inhibitors was significantly enhanced. Replacing hydrogen with a fluorine atom (12) not only improved heNOS selectivity but also doubled permeability (Pe = 11.2 × 10⁻⁶ cm s⁻¹) compared to 11. The trifluoromethyl substituted phenyl linker showed nearly three times higher permeability (Pe = 17.4 × 10⁻⁶ cm s⁻¹) in the PAMPA-BBB assay compared to 11. Inhibitors containing chloro-substituents at the phenyl linkers with methylamine (14) and Ar. A’-dimethylamine (15) tail functionalities exhibited excellent permeabilities (Pe = 15.3 × 10⁻⁶ cm s⁻¹ and 21.5 × 10⁻⁶ cm s⁻¹, respectively). Notably, compound 21, with a fluorinated azetidine as the tail, displayed the highest permeability (Pe = 24.5 × 10⁻⁶ cm s⁻¹), while the pyridyl ring with methylamine as the tail (16) showed the lowest permeability Page 30

[0249] QB\702581.02752\100011397.1(P e = 3.6 x 10'6cm s'1) in this series. Also, a cyano-substituted phenyl (17, Pe= 8.2 x 10'6cm s'1) linker was a better permeant than the phenyl linker (11), whereas a pyridine-substituted linker (16, Pe= 3.6 x 10'6cm s'1) was a poorer permeant than the phenyl linker (11) in this experiment. The presence of fluorinated alkyl substituents (20-22) at the tail significantly improved permeability presumably through enhanced hydrophobicity.

[0250] Effects compact structures (11-17)

[0251] Biaryl molecules without any carbon chain between the aminopyridine and bridging phenyl ring or any carbon chain between the phenyl ring and tail segments (11-17) result in compact inhibitors with fewer rotatable bonds. Figure 1 shows the crystal structure of 11 bound to rnNOS (A) and hnNOS (B). As with many other NOS inhibitors, the aminopyridine of 11 hydrogen bonds with the conserved active site Glu (Glu592 in rnNOS or Glu597 in hnNOS). Since the bridging phenyl ring directly connects to the aminopyridine, the planes of the two aromatic rings must twist away from each other by 55-60° to avoid steric clashes. The tail secondary amine forms an H-bond with heme propionate A without disturbing the water molecule between H4B and propionate A. Surprisingly, the bridging phenyl ring of 11 bound to heNOS (Figure 1, panel C) adopts a totally different orientation. A major contributing factor for this difference is the presence of a O' anion in the active site of heNOS. If the linker phenyl group adopted the same orientation as in hnNOS, there would be unfavorable clashes with the Cl' anion. However, the tail amine of 11 reorients relative to its position in hnNOS to interact with the Cl' anion in heNOS. This tail amine Cl' interaction could explain why 11 binds more tightly to heNOS relative to other inhibitors bound to heNOS (Table 1).

[0252] Effects of adding bulky substituents on the bridging phenyl ring (12-17) Replacing hydrogen with a fluorine atom on the bridging phenyl ring in 11 to give 12 is advantageous in enhancing selectivity over heNOS by ~6-fold (hn / he = 792) compared to 11 (hn / he = 129) because 12 binds to heNOS 10-fold more weakly than 11. 11 and 12 bind very much the same to hnNOS and mNOS (Figure 2) and as a result exhibit very similar potency (Table 1). In heNOS, however, the bridging phenyl of 12 reorients relative to 11 such that the tail amine H-bonds with heme propionate A rather than the Cl' anion. In addition, the fluorine atom is close to heme propionate D which is electrostatically unfavorable. This may account for why 12 exhibits a 10-fold drop in potency compared to 11.

[0253] Similarly, to enhance the selectivity over heNOS, we doubled the steric bulk at the bridging phenyl ring by substituting a fluorine atom with a trifluoromethyl group (13) or a chlorine atom (14). 13 exhibits poor potency and selectivity and is thus an ineffective nNOS Page 31

[0254] QB\702581.02752\100011397.1inhibitor. The detailed structure-activity analysis for this compound is provided in ESI. Replacement of fluorine with chlorine at the bridging phenyl ring in 14 improved selectivity by 1.4-fold (hn / he = 1097), but the potency also decreased by 1.5-fold (Ki (hnNOS) = 95 nM) compared to compound 12.

[0255] Crystal structures show (Figure 3) that the orientation of the bridging phenyl ring in 14 flips from that observed for 12 in hnNOS (Figure 2, panel A) so that the chlorine atom points toward propionate D. The tail amine is between the active site Glu and propionate A with a bridging water molecule between the tail amine and active site Asp. This difference between 12 and 14 contributes to a modest drop in potency. Unfortunately, determination of a heNOS-14 structure was not successful.

[0256] Additionally, changing the steric bulk from a secondary to a tertiary amine at the tail, as seen in compound 15, resulted in a 13-fold decrease in potency (Ki (hnNos) = 1365 nM) compared to compound 14. Keeping a secondary amine tail while using 4-cyanophenyl (17) as the linker reduced both potency and isoform selectivity7as compared to 12. A structural discussion of 15 and 17 that rationalizes the effect of the methyl group is provided in the ESI.

[0257] When the bridging phenyl ring is replaced by a pyridyl ring in 16, the potency for nNOS is maintained. In both the mNOS and hnNOS structures, 16 adopts a similar binding mode with the pyridine ring N atom pointing upward aw ay from the heme plane and H-bonds w ith a water molecule (Figure 4). A unique feature in these tw o structures is the position of propionate D which bends toward propionate A, thus enabling the tail amine to form H-bonds with both propionates. With heNOS, 16 does not result in the movement of either heme propionate, and as such the tail amine interacts with only propionate A. However, the pyridine N atom interacts with the Cl’ ion. This latter interaction may compensate for the one missing H-bond between the tail amine and heme propionate in heNOS-16 compared to hnNOS-16, which is one possible reason for the modest selectivity of 16 (hn / he =351).

[0258] Effects of enhancing the lipophilicity of nNOS inhibitors (18-19)

[0259] We next focused on increasing lipophilicity to improve membrane permeability. This was achieved by adding more fluorine atoms to the bridging phenyl ring and by modifying the amine tail to include aliphatic groups. While adding one fluorine atom to compound 12 to give compound 18 does not improve membrane permeability, there is a modest 2.2-fold (Ki (hnNOS) = 30 nM) potency increase in hnNOS and a slight increase in selectivity over heNOS (hn / he = 821, Table 1).

[0260] Page 32

[0261] QB\702581.02752\100011397.1The binding mode of 18 to mNOS and to hnNOS is shown in Figure 5. Both fluorine atoms of the difluorobenzene ring point upward toward a Gin residue (Gln478 in mNOS or Gln483 in hnNOS) for possible H-bonding interactions. The tail amine H-bonds with both heme propionates. 18 is the best inhibitor in this series of compounds (Table 1) owing to the tail amine H-bonding with both heme propionates and the favorable electrostatic interactions between inhibitor fluorine atoms and the active site Gin.

[0262] The difluorobenzene ring in the heNOS-18 adopts the same orientation as in the heNOS-12 complex that places the fluorine atoms close to propionate D which is electrostatically unfavorable. However, the tail amine H-bonds with Glu361 and is 3.7 A from the Cl" anion (Figure 5, panel C). These favorable interactions are one possible reason why 18 is a slightly better inhibitor than 12 of heNOS (Table 1). Even so, 18 is the most selective inhibitor because 18 binds so tightly to hnNOS (Table 1).

[0263] The secondary amine in 18 was replaced by a tertiary amine to give 19. The crystal structure (Figure 6, panel A) shows that the binding mode of 19 in mNOS is the same as that for 18. However, unlike with 18 where the secondary amine has two protons that form favorable interactions with both propionates. 19 has only one proton. As a result, the less favorable electrostatic stabilization of the tail amine in 19 compared to 18 is most likely why 19 is a less potent inhibitor (Table 1). In heNOS, 19 binds much the same as 18 except the tertiary amine electron density is very weak indicating weak interactions with the heme propionates. As a result, 19 loses substantial potency relative to 18 (Table 1).

[0264] Effects of modulating amine basicity of nNOS inhibitors (20-22)

[0265] Addition of an electron withdrawing fluorine atom to the substituent attached to the tail amine as in 20, resulted in a slight decrease in inhibitory activity (Table 1). We also found that the addition of an electron-donating methyl (19) or an electron-withdrawing fluorinated cyclic alkyl species (21-22) to the secondary amine in 18 are not as effective as compound 18 (Table 1).

[0266] Figure 7 shows 20 bound to mNOS and hnNOS. The mNOS-20 structure is very similar to the mNOS-19 structure. Propionate D moves up enabling the tail amine to be situated within H-bonding distance to both propionates. In contrast, propionate D in hnNOS-20 does not move thereby weakening interactions with the tail amine. The subtle difference in K values of 20 in mNOS versus hnNOS, reflects this difference in the H-bonding interactions observed between inhibitor-bound mNOS and hnNOS structures. Attempts to obtain the heNOS-20 structure were unsuccessful.

[0267] Page 33

[0268] QB\702581.02752\100011397.1Finally, addition of the electron-withdrawing fluorinated cyclic alkyl species (21-22) to 18 to create a bulky tertiary amine decreased both potency and selectivity. The structureactivity interpretations of these inhibitors are provided in ESI.

[0269] Effects of Lengthening the Tail of Inhibitor 18 in 23

[0270] Since the tail amine of 18 interacts with the propionates, we next explored the possibility that lengthening the tail amine might optimize interactions with the propionates. The addition of one more carbon through homologation did give an inhibitor (23) with good potency (Ki (hnNOS) = 47 nM) but moderate selectivity (hn / he = 265 and hn / hi = 177, Table 1).

[0271] The structural underpinning for this loss of selectivity7is that the binding mode of 23 bound to mNOS. hnNOS, and heNOS is nearly identical (Figure 8). The difluorobenzene ring fluorine atoms point upward, one of which can H-bond with a Gin residue (Gln478 in mNOS, Gln483 in hnNOS, and Gln247 in heNOS). Recall that in the heNOS-18 complex, the difluorobenzene ring is oriented quite differently (Figure 5) and as such there is no favorable electrostatic interaction between the fluorine atoms and Gln247. This additional electrostatic stabilization in heNOS-23 relative to heNOS-18 is, in part, why 23 exhibits slightly better potency in heNOS than 18 as well as reduced selectivity7(Table 1). The reason the difluorobenzene in 23 orients differently than 18 in heNOS involves a complex balance of favorable and unfavorable interactions. While the orientation of the difluorobenzene in 23 allows favorable interactions between a fluorine atom and Gln247, there are unfavorable electrostatic interactions with the Cl’ anion. However, this is compensated for by the longer tail amine which can hydrogen bond with propionate D. This type of compensation is not possible with 18 owing to the shorter tail amine.

[0272] CONCLUSIONS

[0273] An especially challenging problem in nNOS inhibitor design is the requirement that nNOS inhibitors have positively charged groups that interact with the active site Glu. This, of course, raises problems with blood-brain barrier penetration. This study was undertaken to obtain selectivity and potency while increasing membrane permeability'. Another important goal was to streamline the synthesis and increase yields so that it would be possible to examine many inhibitors. This was achieved by employing the powerful Suzuki-Miyaura crosscoupling. This series of biaryl inhibitors with short carbon linker between the aromatic rings also led to a desirable feature, that is, the inhibitor binding mode to mNOS and hnNOS is the same, which makes the translation of pre-clinic and clinic studies more straightforward. Of the many compounds characterized in this study, inhibitor 18 meets the criteria of simplicity, ease Page 34

[0274] QB\702581.02752\100011397.1of synthesis, good potency toward hnNOS (Ki = 30 nM), selectivity (821-fold over heNOS) and membrane permeability (Pe= 10.7 x 10'6cm s’1). Our current efforts also have provided three important lessons.

[0275] First, adding fluorine atoms to the linker phenyl group to 11 both improves, as expected, membrane permeability but also selectivity. Inhibitor 11 that has no fluorine atoms has good potency toward hnNOS but poor selectivity over heNOS and poor membrane permeability. Adding one fluorine atom as in 12 or two fluorine atoms as in 18 gives potency similar to 11 toward hnNOS but greatly improved membrane permeability and selectivity. Increase in selectivity of 12 and 18 is due to a decrease in potency toward heNOS of 12 and 18 relative to 11 (Table 1).

[0276] Second is the unexpected role that the Cl’ anion plays in the selectivity of 12 and 18.

[0277] Owing to the presence of the Cl’ anion in heNOS, the fluorinated phenyl ring in 12 and 18 cannot adopt a favorable orientation as in hnNOS since this would result in an electrostatic clash between fluorine and the Cl’ anion (Figures 2 and 5). The orientation that the phenyl ring does adopt in heNOS results in an electrostatic clash with heme propionate D (Figures 2 and 5). Given that there is no favorable orientation of the fluorinated phenyl ring in heNOS, the potency of 12 and 18 decrease relative to 11. An additional structural insight is why heNOS binds Cl’ but not hnNOS. In heNOS Asn366 directly interacts with the Cl’ anion while this residue is Asp366 in hnNOS and, therefore, hnNOS cannot bind Cl’ (Figure 2). This new insight could prove useful in the design of new and selective hnNOS inhibitors.

[0278] A third important lesson is that the tail amine should be a secondary7amine. The secondary7tail amine (11-12, 14, 16, 18, 20, and 23) exhibited higher potency and isoform selectivity7, except for compound 13 or 17, where the poor binding affinity is due to a bulky -CFs or a lengthy -CN group attached to the phenyl ring, than the tertiary amines (15, 19, 21, 22). The tertiary amine has only one proton while the secondary amine has two protons and is thus able to H-bond with both heme propionates.

[0279] In summary, we have been able to improve the pharmacophore properties of selective NOS inhibitors without a significant compromise in potency while improving membrane permeability. In addition, crystal structures have provided important insights on what is required for maintaining high potency and selectivity in the simplified inhibitors described in this study. Most unexpected, but potentially important in future inhibitor design, is the unique Cl’ binding site in heNOS.

[0280] EXPERIMENTAL SECTION

[0281] Page 35

[0282] QB\702581.02752\100011397.1All reactions described were carried out under an argon atmosphere unless otherwise specified. Reagents and anhydrous solvents, such as THF, 1.4-dioxane, CH2CI2. CH3OH, EtsN, CH3CN, and DMF, were purchased from several chemical industries including Ambeed, Oakwood, Combi-blocks, Sigma- Aldrich, and Astatech. Metal catalysts, particularly [1,1’-Bis(diphenylphosphino)ferrocene]dichloropalladium(II), were acquired from Sigma- Aldrich. Specific compounds like l-bromo-3-(bromomethyl)benzene (24), l-bromo-3-(bromomethyl)-5 -fluorobenzene (27), and l-bromo-3-(bromomethyl)-5-chlorobenzene (30) were obtained from Combi-blocks, while l-bromo-3-(bromomethyl)-5-(trifluoromethyl)benzene (33) and 3-bromo-5-(bromomethyl)benzonitrile (37) were purchased from Ambeed. Suzuki-Miyaura cross-coupling reactions were conducted using Chemglass pressure vessels. All reactions were monitored by thin layer chromatography (TLC) using Merck TLC silica gel 60 F254; 0.25 mm glass plates. Visualization was done with ultraviolet light (254 nm) and / or KMnO4 or phosphomolybdic acid stain.JH and13C NMR spectra were recorded on a Bruker Avance III NMR spectrometer in CDCI3, CD3OD, or DMSO-d6. Chemical shifts are reported in parts per million, and multiplicities are indicated by s = singlet, d = doublet, t = triplet, q = quartet, sep = septet, dd = doublet of doublet, dt = doublet of triplet, m = multiplet, and br = broad resonance. Coupling constants “J” were reported in hertz. High-resolution mass spectral (HRMS) data were obtained on an Agilent 6210 LC-TOF spectrometer in the positive-ion mode using electrospray ionization (ESI) with an Agilent G1312A HPLC pump and an Agilent G1367B autoinjector at the Integrated Molecular Structure Education and Research Center (IMSERC), Northwestern University. Flash column chromatography was performed on an Agilent 971-FP automated flash purification system with a Varian column station and various SiliCycle cartridges (4-80 g, 40−63 μm, 60 Å). All compounds used in the biological testing were >95% pure by HPLC analysis. The synthetic procedures for all intermediates are discussed in Supporting Information.

[0283] General Procedure A: Boc-protected amine alkylation.

[0284] Compounds 25, 28, and 31 were prepared following a reported procedure with modifications.58A 100 mL two-necked flask with a stir bar was filled with sodium hydride (1.5 equiv, 60% dispersion in mineral oil) and dry DMF under an argon atmosphere. The mixture was cooled to 0 °C in an ice bath and stirred for 5 min. Following this, a solution of tert-butyl methylcarbamate (1 equiv) in DMF was slowly added dropwdse over 10 min, leading to a rapid evolution of hydrogen gas. The resulting mixture was stirred for 30 min at 0 °C and then for 10 min at room temperature. Afterward, the reaction mixture was again cooled to 0 °C in an Page 36

[0285] QB\702581.02752\100011397.1ice bath, and benzy l bromide (24, 27, and 30, 1 equiv) was added dropwise over 5 min. The reaction mixture was allowed to warm up to room temperature and continued to be stirred for 1-24 hours. A color change from grey to pale yellow to deep yellow was observed after the addition of benzyl bromide with an exothermic reaction. Upon completion of the reaction, as indicated by TLC, the reaction mixture was carefully quenched with water. The resulting mixture was then extracted twice with Et2O. The combined organic layers were washed with water, dried over Na2SO4, filtered, and concentrated under vacuum. The crude product was further purified by flash column chromatography on silica gel, eluting with a mixture of hexanes for 10 min and followed by hexanes / EtOAc (90:10, v:v) for 15 min to yield the products (25, 28, and 31).

[0286] General Procedure B: Amine alkylation.

[0287] Compounds 34, 38, 41, 48, 51, 53, 56, and 58 were prepared following a reported procedure with modifications.34 60A 100 mL two-necked flask with a stir bar was filled with an appropriate amine hydrochloride (1.0 equiv), K2CO3 (1.2 equiv), and anhydrous CHsCN at 25 °C under an argon atmosphere. Then, a solution of the appropriate benzyl bromide (1.0 equiv) in anhydrous CH3CN was added to the stirred suspension. The mixture was left to stir at 25 °C for 24 h. After the reaction was completed, as indicated by TLC, the reaction mixture was diluted with water and then transferred to a separatory funnel and extracted several times with CH2CI2. The combined organic layers were washed with brine, dried over Na2SO4, filtered, and concentrated under vacuum with a rotary evaporator to obtain the crude product. The crude residue was purified by flash column chromatography using hexane / EtOAc (50:50, v / v) for 10 min, followed by the solvent mixture CH2Cl2 / CH3OH / Et3N (85:10:5, v / v) for 15 min, resulting in the isolation of compounds 34, 38, 41, 48, 51, 53, 56, and 58.

[0288] General Procedure C: N-Boc protection.

[0289] Compounds 35, 39, 45, 49, 54, and 64 were prepared using a previously reported procedure with modifications.61A 200 mL two-necked flask with a stir bar was filled with the appropriate amine (1 equiv) and CH2CI2 under an argon atmosphere. Then, a solution of di-tert-butyl dicarbonate (1.2 equiv) in CH2CI2 and triethylamine (2.0 equiv) was added and stirred at 25 °C for 24 h. Once the reaction was complete, as indicated by TLC, the reaction mixture was diluted with water and then extracted twice with CH2CI2. The combined organic layers were washed with brine, dried over Na2SO4, filtered, and concentrated under vacuum using a rotary evaporator to obtain the crude product. The crude product was further purified

[0290] Page 37

[0291] QB\702581.02752\100011397.1by flash column chromatography on silica gel, eluting with hexanes for 10 min, followed by hexanes / EtOAc (70:30, v:v) for 15 min to yield the products (35, 39, 45, 49, 54, and 64).

[0292] General Procedure D: Suzuki-Miyaura cross-coupling reaction.

[0293] Compounds 26, 29, 32, 36, 40, 42, 46, 50, 52, 55, 57, 59, and 66 were synthesized using a previously reported method with some modifications.54-57A mixture of bromoaryl linker with amine tail (1.0 equiv), PdCb(dppf) (10 mol%), bis(pinacolato)diboron (1.2 equiv), and KO Ac (3 equiv) was added to an oven-dried pressure vessel. The vessel was then sealed, evacuated, and backfilled with argon 3 times. After adding 1,4-dioxane (0.2 M, degassed), the mixture was heated to 100 °C for 1-8 hours. Upon complete borylation conversion (as indicated by TLC), aqueous K2CO3 (3 equiv, 1.8 M, degassed) was added, followed by 6-bromo-4-methylpyridin-2-amine (1 equiv) under argon. The resulting mixture was further heated from 100 °C to 120 °C for 1-24 h. After completion of the reaction (indicated by TLC), the reaction mixture was cooled, diluted with water, and then extracted twice with 2 x 50 mL of Et2O or EtOAc. The combined organic layers were dried over Na2SO4, filtered, and concentrated under vacuum. The crude product was purified by flash column chromatography on silica gel, eluting with hexanes for 10 min followed by hexanes / EtOAc (50:50 to 0: 100, v:v) for 15 min to yield the products (26, 29, 32, 36, 40, 42, 46, 50, 52, 55, 57, 59, and 66). In some cases, the byproduct pinacol w as contaminated with the product and w as removed by azeotropic distillation (Tbath 60-70 °C / 20-5 mbar) with 50% aqueous methanol under reduced pressure using a rotary evaporator.68-69

[0294] General Procedure E: N-Boc deprotection.

[0295] The N-Boc deprotection was achieved using a previously reported method with some modifications.59A stirred solution of N-Boc protected NOS inhibitor (26, 29, 32, 36, 40, 46, 50, 55, and 66) in methanol was mixed with an excess of hydrogen chloride solution (3 M in methanol) at 25 °C and stirred at this temperature for 1-24 hours. After confirming the completion of the reaction based on LCMS, the reaction mixture was evaporated under reduced pressure to dryness to give the desired dihydrochloride salt of inhibitors 11-14, 16-18, 20, and 23.

[0296] General Procedure F: HCl salt formation.

[0297] The freebase of the inhibitors (42, 52, 57, and 59) was transformed into their dihydrochloride salts33by treating them with an excess of hydrogen chloride solution (3.0 M in CH3OH) at 25 °C under an argon atmosphere. The resulting mixture was stirred for 8 h, and

[0298] Page 38

[0299] QB\702581.02752\100011397.1then the solvent was evaporated under reduced pressure using a rotary evaporator, resulting in the dihydrochloride salt of compounds 15, 19, 21-22.

[0300] General Procedure G: Stepwise reductive amination.

[0301] The stepwise reductive amination was achieved by a slight modification of the reported procedure.62A 100 mL two-necked flask with a stir bar was filled with 5-bromonicotinaldehyde (1 equiv) and methanol under an argon atmosphere. Then, a solution of methylamine in ethanol (33 wt. %) (4 equiv) was added to the mixture and continued the stirring at 25 °C for 8 h. After that, solid sodium borohydride (2.0 equiv) was added in several portions over 30 min at 25 °C with stirring and continued the reaction for 2 h. (Caution: a brisk effervescence was observed while adding the sodium borohydride to the reaction mixture). After completion of the reaction, as indicated by the TLC, the reaction mixture was quenched with saturated aqueous Na2CO3 carefully dropwise, and then extracted the organic layer several times with CH2CI2. The combined organic layers were washed with brine, dried over Na2SO4, filtered, and concentrated under vacuum using a rotary evaporator to obtain the crude product. The crude residue was then purified by flash column chromatography using hexane / EtOAc (50:50, v / v) for 10 mm, followed by the solvent mixture CH2Cl2 / CH3OH / Et3N (85:10:5, v / v) for 15 min, resulting in the isolation of compound 44.

[0302] 4-Methyl-6-(3-((methylamino)methyl)phenyl)pyridin-2-amine dihydrochloride (11)

[0303]

[0304] Compound 11 was synthesized according to general procedure E using 26 (520 mg, 1.59 mmol) with excess hydrogen chloride (8 mL). Compound 11 was isolated as a white powder (380 mg, 1.27 mmol, 80%). H NMR (500 MHz, CD3OD): 58.04 (t, J= 1.8 Hz, 1 H), 7.91 (dt,.7= 7.5, 1.7 Hz, 1 H), 7.77 - 7.66 (m, 2 H), 7.14 (d, J= 1.3 Hz, 1 H), 6.84 (t, J= 1.2 Hz, 1 H), 4.33 (s, 2 H), 2.78 (s, 3 H), 2.48 (s, 3 H).13C NMR (126 MHz, CD3OD): 5 159.1, 156.5, 146.6. 134.1, 133.6, 131.5, 130.2, 129.4, 114.4, 112.1, 53.1, 33.3, 22.1. HRMS (ESI) Calcd for C14H18N3 [(M+H)+]: 228.1495, found: 228.1492.

[0305] 6-(3-Fluoro-5-((methylamino)methyl)phenyl)-4-methylpyridin-2-anune dihydrochloride (12)

[0306] Page 39

[0307] QB\702581.02752\100011397.1

[0308]

[0309] Compound 12 was synthesized according to general procedures D-E, using 28 (1.70 g, 5.35 mmol), PdCl2(dppf) (391 mg, 0.535 mmol), bis(pinacolato)diboron (1.63 g, 6.42 mmol), KOAc (1.57 g, 16.0 mmol), and 6-bromo-4-methylpyridin-2-amine (1.00 g, 5.35 mmol), and K2CO3 (8.91 rnL, 16.0 mmol, 1.8 M in water). This resulted in a mixture of '-Boc protected amine (29) and a freebase of inhibitor 12 in an almost 1: 1 ratio. The freebase was obtained as a yellow semi-solid (500 mg, 2.04 mmol, 38%), and the A-Boc protected amine (29) was obtained as a yellow- viscous oil (780 mg, 2.26 mmol, 42%). The NMR analysis of the freebase is provided below. 1H NMR (500 MHz, CDCl3): 5 7.68 - 7.62 (m, 1 H), 7.53 (ddd, J= 10.0, 2.6, 1.6 Hz, 1 H), 7.04 (ddd, J = 9.2. 2.5, 1.5 Hz, 1 H), 6.93 (dd, J= 1.2, 0.7 Hz, 1 H), 6.31 (t, J= 1.0 Hz, 1 H), 4.44 (bs, 2 H, 1 x NH2), 3.80 (s, 2 H), 2.47 (s, 3 H), 2.29 (s, 3 H).13C NMR (126 MHz, CDCh): 5 163.4 (d, JC-F = 245.7 Hz), 158.5, 154.7 (d, JC-F = 2.5 Hz), 149.6, 143.0 (d, JC-F = 7.6 Hz). 142.1 (d, JC-F = 8.8 Hz), 122.1 (d, JC-F = 1.3 Hz), 115.0 (d, JC-F = 21.4 Hz), 112.8, 112.4 (d, JC-F = 22.7 Hz), 108.2, 55.8 (d, JC-F = 1.3 Hz), 36.2, 21.3. HRMS (ESI) Calcd for C14H17FN3 [(M+H)+]: 246.1401, found: 246.1432. The freebase and A-Boc protected amine (29) were combined and transformed into dihydrochloride salt with excess hydrogen chloride following the general procedure E.

[0310] 4-Methyl-6-(3-((methylamino)methyl)-5-(trifhioromethyl)phenyl)pyridin-2-amine dihydrochloride (13)

[0311]

[0312] Compound 13 was synthesized according to general procedure E using 36 (900 mg, 2.28 mmol) with excess hydrogen chloride (15 mL). Compound 13 was isolated as a paleyellow solid (704 mg, 1.91 mmol, 84%). H NMR (500 MHz, CD3OD): 5 8.39 (s, 1 H), 8.26 (s, 1 H), 8.12 (s, 1 H), 7.25 (s, 1 H), 6.92 (s, 1 H), 4.45 (s, 2 H), 2.82 (s, 3 H), 2.50 (s, 3 H).13C Page 40

[0313] QB\702581.02752\100011397.1NMR (126 MHz, CD3OD): 5 159.1, 156.5, 144.8, 135.5, 135.0, 134.2, 133.5 (q, JC-F = 32.8 Hz), 130.2 (q, JC-F = 2.6 Hz), 126.3 (q. JC-F = 3.8 Hz), 124.8 (q, JC-F = 272.2 Hz), 115.2, 113.0, 52.5, 33.6, 22.1. HRMS (ESI) Calcd for C15H17F3N3 [(M+H)+]: 296.1369, found: 296.1374.

[0314] 6-(3-Chloro-5-((metliylandno)methyl)phenyl)-4-methylpyridin-2-an ne dihydrochloride (14)

[0315]

[0316] Compound 14 was synthesized according to general procedure E using 32 (390 mg, 1.08 mmol) with excess hydrogen chloride (6 mL) solution. Compound 14 was isolated as white powder (334 mg, 1.00 mmol, 93%). 'H NMR (500 MHz, CD3OD): 57.98 (p, J= 1.7 Hz, 2 H). 7.80 (t. J = 1.7 Hz. 1 H), 7.16 (d. J= 1.4 Hz. 1 H).6.87 (t. J= 1.2 Hz. 1 H). 4.33 (s. 2 H), 2.79 (s, 3 H), 2.48 (s, 3 H).13C NMR (126 MHz, CD3OD): 5 159.1, 156.4, 145.0, 137.1, 136.0, 135.8, 133.3, 129.4, 128.7, 114.9, 112.8, 52.4, 33.4, 22.1. HRMS (ESI) Calcd for C14H17CIN3 [(M+H)+]: 262.1106, found: 262.1099.

[0317] 6-(3-Chloro-5-((dimethylamino)methyl)phenyl)-4-inethylpyridin-2-amine dihydrochloride (15)

[0318]

[0319] Compound 15 was synthesized according to general procedures D-E, using 41 (748 mg, 3.01 mmol), PdC12(dppf) (220 mg, 0.301 mmol), bis(pinacolato)diboron (917 mg, 3.61 mmol), KOAc (886 mg, 9.02 mmol), 6-bromo-4-methylpyridin-2-amine (563 mg, 3.01 mmol), and K2CO3 (5.01 mL, 9.02 mmol, 1.8 M in water). The freebase of 15 was obtained as a yellow oil (564 mg, 2.05 mmol, 68%). 'H NMR (500 MHz, CDCh): 6 7.83 (t, J= 1.8 Hz, 1 H), 7.72 (d, J= 1.5 Hz. 1 H), 7.31 (t, J= 1.8 Hz, 1 H), 6.92 (s, 1 H), 6.30 (t, J= 1.0 Hz. 1 H). 4.50 (bs. 2 H, 1 x NH2), 3.45 (s, 2 H), 2.28 (s, 3 H), 2.26 (s, 6 H).13C NMR (126 MHz, CDCh): 5 158.5, 154.5, 149.6, 141.5, 141.2, 134.6, 129.0, 125.9, 125.6, 112.8, 108.2, 64.0, 45.6, 21.3. HRMS Page 41

[0320] QB\702581.02752\100011397.1(ESI) Calcd for C15H19CIN3 [(M+H)+]: 276.1262, found: 276.1270. The freebase of (15) was transformed into the dihydrochloride salt with excess hydrogen chloride following the general procedure E.

[0321] 4-Methyl-5'-((methylamino)methyl)-[2,3 '-bipyridin]-6-amine dihydrochloride (16)

[0322]

[0323] Compound 16 was synthesized according to general procedure E using 46 (890 mg, 2.71 mmol) with excess hydrogen chloride (15 mL). Compound 16 was isolated as a brown solid (653 mg, 2.17 mmol. 80%). 'H NMR (500 MHz, CD3OD): 59.36 (s, 1 H), 9.15 (s, 1 H), 9.07 (s, 1 H), 7.33 (s, 1 H), 6.97 (s, 1 H), 4.56 (s, 2 H), 2.86 (s, 3 H), 2.51 (s, 3 H).13C NMR (126 MHz, CD3OD): 8 159.0, 156.7, 148.7, 145.7, 144.5, 141.3, 132.0, 131.9, 116.3, 114.2, 49.9, 33.8, 22.2. HRMS (ESI) Calcd for C13H17N4 [(M+H)4]: 229.1448, found: 229.1453.

[0324] 3-(6-Amino-4-methylpyridin-2-yl)-5-((methylamino)methyl)benzonitrile dihydrochioride (17)

[0325]

[0326] Compound 17 was synthesized according to general procedure E using 40 (1.10 g, 3.12 mmol) with excess hydrogen chloride (15 mL). Compound 17 was isolated as a brown solid (953 mg, 2.93 mmol, 94%).

[0327]

[0328] NMR (500 MHz, CD3OD): 88.40 (s, 1 H), 8.33 (s, 1 H), 8.13 (s, 1 H), 7.23 (s, 1 H), 6.91 (s, 1 H), 4.42 (s, 2 H), 2.81 (s, 3 H), 2.49 (s, 3 H).13C NMR (126 MHz, CD3OD): 8 159.1, 156.5, 144.3, 136.7, 135.6, 135.1, 134.7, 133.0, 118.2, 115.5, 115.2, 113.2, 52.2, 33.6. 22.1. HRMS (ESI) Calcd for C15H17N4 [(M+H)4]: 253.1448, found: 253.1438.

[0329] 6-(2,3-Difluoro-5-((methylamino)methyl)phenyl)-4-methylpyridin-2-amine dihydrochioride (18)

[0330] Page 42

[0331] QB\702581.02752\100011397.1

[0332]

[0333] Compound 18 was synthesized according to general procedure E using 50 (700 mg, 1.93 mmol) with excess hydrogen chloride (12 mL). Compound 18 was isolated as white solid (604 mg, 1.79 mmol, 93%). 'H NMR (500 MHz, CD3OD): 8 7.78 - 7.68 (m, 2 H), 7.06 (s, 1 H), 6.92 (d, J= 1.7 Hz, 1 H), 4.30 (s, 2 H), 2.78 (s, 3 H), 2.49 (s, 3 H).13C NMR (126 MHz, CD3OD): 8 158.9, 156.3, 151.9 (dd, Jc-v = 250.7, 12.6 Hz), 149.7 (dd, JC-F = 257.0, 13.9 Hz), 140.2, 130.6 (dd. JC-F = 7.6, 5.0 Hz), 128.7 (d, JC-F = 2.5 Hz). 124.1 (d. JC-F = 10.1 Hz), 122.7 (d, JC-F = 17.6 Hz), 117.2 (d, JC-F = 2.5 Hz), 113.5, 52.1, 33.3, 22.1. HRMS (ESI) Calcd for C14H16F2N3 [(M+H)+]: 264.1307, found: 264.1312.

[0334] 6-(5-((dimethylamino)methyl)-2,3-difluorophenyl)-4-methylpyridin-2-amine dihydrochloride (19)

[0335]

[0336] Compound 19 was synthesized according to general procedure F using 52 (454 mg, 1.64 mmol) with excess hydrogen chloride (10 mL). Compound 19 was isolated as a brown solid (527 mg, 1.50 mmol, 92%). H NMR (500 MHz, CDsOD): 87.80 (ddd, J= 8.7, 6.9, 2.9 Hz, 2 H), 7.09 (d, J= 1.3 Hz, 1 H), 6.92 (t, J= 1.2 Hz, 1 H), 4.45 (s, 2 H), 2.93 (s, 6 H), 2.48 (s, 3 H).13C NMR (126 MHz, CD3OD): 8 158.9, 156.3, 152.0 (dd. JC-F = 250.7, 12.6 Hz), 150.3 (dd, JC-F = 258.3, 15.1 Hz), 140.0 (d, JC-F = 2.5 Hz), 130.0 (d, JC-F = 2.5 Hz), 129.1 (dd, JC-F = 7.6, 5.0 Hz), 124.2 (d, JC-F = 10.1 Hz), 123.7 (d, JC-F = 18.9 Hz), 117.3 (d, JC-F = 2.5 Hz), 113.5, 60.4, 43.2, 22.1. HRMS (ESI) Calcd for C15H18F2N3 [(M+H)+]: 278.1463, found: 278.1471.

[0337] 6-(2,3-Difluoro-5-(((2-fluoroethyl)amino)methyl)phenyl)-4-methylpyridin-2-amine dihydrochloride (20)

[0338] Page 43

[0339] QB\702581.02752\100011397.1

[0340]

[0341] Compound 20 was synthesized according to general procedure E using 55 (780 mg, 1.97 mmol) with excess hydrogen chloride (10 mL). Compound 20 was isolated as an off-white solid (676 mg, 1.84 mmol, 93%). H NMR (500 MHz, CD3OD): 57.78 (dd, J= 10.9, 6.2 Hz, 2 H), 7.07 (s, 1 H), 6.92 (s, 1 H), 4.78 (t, J= 4.6 Hz, 1 H), 4.40 (s, 2 H), 3.53 (t, J= 4.6 Hz, 1 H), 3.48 (t, J = 4.6 Hz, 1 H), 2.49 (s, 3 H).13C NMR (126 MHz, CD3OD): 5 158.9, 156.4, 151.9 (dd, JC-F = 250.7, 12.6 Hz), 150.0 (dd, JC-F = 257.0, 15.1 Hz), 140.1 (d, JC-F = 2.5 Hz), 130.3 (dd, JC-F = 6.3, 5.0 Hz), 129.0 (d, JC-F = 3.8 Hz), 124.0 (d, JC-F = 10.1 Hz), 123.0 (d. JC-F = 18.9 Hz), 117.2 (d, JC-F = 2.5 Hz), 113.5, 80.3 (d, JC-F = 168.8 Hz), 51.0, 48.9 (d, JC-F = 22.6 Hz, merged with c / - meth anol reference peaks), 22.1. HRMS (ESI) Calcd for C15H17F3N3 [(M+H)+]: 296.1369. found: 296.1380.

[0342] 6-(5-((3,3-Difluoroazetidin-l-yl)methyl)-2,3-difluorophenyl)-4-methylpyridin-2-amine dihydrochloride (21)

[0343]

[0344] Compound 21 was synthesized according to general procedure F using 57 (605 mg, 1.86 mmol) with excess hydrogen chloride (10 mL). This produced a mixture consisting of the desired HC1 salt (21) and its freebase form in an approximately 1.5:1 ratio, respectively. The mixture was obtained as a pale-yellow solid (385 mg, 0.97 mmol, 52%). The NMR analysis of this mixture is provided below. 'H NMR (500 MHz, CD3OD): 57.80 (tt, J= 8.2, 6.8, 2.7 Hz, 4 H), 7.16 - 7.03 (m, 2 H), 6.92 (t, J= 1.2 Hz, 2 H), 4.81 (t, J= 10.9 Hz, 4 H), 4.64 (s, 2 H, N-CH2), 4.47 (s, 2 H. N-CH2), 4.11 (t, J = 13.4 Hz. 2 H). 3.90 (t. J = 15.3 Hz, 2 H), 2.49 (s, 6 H).

[0345] 13C NMR (126 MHz, CD3OD): 5 158.9, 158.8, 156.4, 156.3, 153.1-148.9 (m, 4xC), 140.1 (d, JC-F = 2.5 Hz, IxC), 139.9 (d, JC-F = 3.8 Hz, IxC), 129.7-129.6 (m, IxC), 129.4 (d, JC-F = 2.5 Hz, IxCH), 129.3-129.2 (m, IxC), 129.0 (d, JC-F = 3.8 Hz, IxCH), 124.3 (d, JC-F = 10.1 Hz,

[0346] Page 44

[0347] QB\702581.02752\100011397.1IxC), 124.0 (d, JC-F = 10.1 Hz, IxC), 123.3 (d, JC-F = 18.9 Hz, IxCH), 123.0 (d, JC-F = 18.9 Hz. IxCH), 119.6 (t. JC-F = 247.0 Hz. 1XCF2), 117.2 (d, JC-F = 3.8 Hz. IxCH), 117.1 (d, JC-F = 2.5 Hz, IxCH), 114.4 (1, JC-F = 270.9 Hz, 1XCF2), 113.5 (d, JC-F = 2.5 Hz, 2xCH), 65.8 (t, JC-F = 30.2 Hz, 2XCH2), 58.3, 51.5, 43.6 (t, JC-F = 30.2 Hz, 2xCH2), 22.1, 22.0. HRMS (ESI) Calcd for Ci6Hi6F2N3 [(M+H)+]: 326.1275, found: 326.1257.

[0348] 6-(5-((4,4-Difluoropiperidin-l-yl)methyl)-2,3-difluorophenyl)-4-niethylpyridin-2-amine dihydrochloride (22)

[0349]

[0350] Compound 22 was synthesized according to general procedure F using 59 (823 mg, 2.33 mmol) with excess hydrogen chloride (15 mL). Compound 22 was isolated as an off-white solid (755 mg, 1.77 mmol, 76%). H NMR (500 MHz, CD3OD): 5 8.00 - 7.76 (m, 2 H), 7.14 (s, 1 H), 6.92 (s, 1 H), 4.52 (s, 2 H), 3.86 - 3.52 (m, 2 H), 3.50 - 3.28 (m, 2 H), 2.60 - 2.30 (m, 7 H, 1XCH3and 2xCH2).13C NMR (126 MHz, CD3OD): 5 158.8, 156.4, 151.9 (dd, JC-F = 250.7, 12.6 Hz), 150.1 (dd, JC-F = 257.0, 13.9 Hz), 140.0 (d. JC-F = 2.5 Hz), 130.2 (d, C-F = 3.8 Hz), 128.4, 124.1 (d, JC-F = 17.6 Hz), 124.0, 120.3 (t, JC-F = 243.2 Hz), 117.3 (d, JC-F = 3.8 Hz), 113.4, 59.3, 50.7 (t, JC-F = 5.7 Hz), 32.1 (t, JC-F = 26.5 Hz), 22.1. HRMS (ESI) Calcd for CI8H2OF2N3 [(M+H)4]: 354.1588, found: 354.1590.

[0351] 6-(2,3-Difluoro-5-(2-(methylamino)ethyl)phenyl)-4-methylpyridin-2-amine dihydrochioride (23)

[0352]

[0353] Compound 23 was synthesized according to general procedure E using 66 (198 mg, 0.525 mmol) with excess hydrogen chloride (5 mL). Compound 23 was isolated as yellow solid (171 mg, 0.488 mmol, 93%). 'H NMR (500 MHz, CD3OD): 57.72 (dd, J = 11.3, 6.6 Hz, 2 H), 7.24 (s, 1 H), 7.09 (s, 1 H), 3.40 - 3.37 (m, 2 H), 3.15 - 3.12 (m, 2 H), 2.77 (s, 3 H), 2.48 (s, 3

[0354] Page 45

[0355] QB\702581.02752\100011397.1H).13C NMR (126 MHz, CD3OD): 5 158.8, 156.3, 152.0 (dd, JC-F = 249.5, 12.6 Hz), 148.3 (dd, C-F = 253.3, 13.9 Hz), 140.4, 136.0 (dd, JC-F = 6.3. 5.0 Hz), 127.1, 123.4 (d, J = 10.1 Hz), 121.7 (d, J= 18.9 Hz), 117.1 (d, J= 3.8 Hz), 113.2, 50.8, 34.0, 32.4, 22.2. HRMS (ESI) Calcd for C15H18F2N3 [(M+H)+]: 278.1463, found: 278.1470.

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[0391] (33) Vasu, D.; Do, H. T.; Li, H.; Hardy, C. D.; Awasthi, A.; Poulos, T. L.; Silverman, R. B., Potent, Selective, and Membrane Permeable 2-Amino-4-Substituted Pyridine-Based Neuronal Nitric Oxide Synthase Inhibitors, J. Med. Chem. 2023, 66, 9934-9953.

[0392] (34) Vasu, D.; Li, H.; Hardy, C. D.; Poulos, T. L.; Silverman, R. B. 2-Aminopyridines with a Shortened Amino Sidechain as Potent, Selective, and Highly Permeable Human Neuronal Nitric Oxide Synthase Inhibitors, Bioorg. Med. Chem. 2022, 69, 116878.

[0393] (35) Do, H. T.; Li, H.; Chreifi, G.; Poulos. T. L.; Silverman, R. B. Optimization of Blood-Brain Barrier Permeability with Potent and Selective Human Neuronal Nitric Oxide Synthase Inhibitors Having a 2-Aminopyridine Scaffold. J. Med. Chem. 2019, 62, 2690-2707.

[0394] (36) Do, H. T.; Wang, H.-Y.; Li, H.; Chreifi, G.; Poulos, T. L.; Silverman, R. B. Improvement of Cell Permeability of Human Neuronal Nitric Oxide Synthase Inhibitors Using

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[0397] (37) Kang, S.: Li, H.; Tang, W.; Martasek, P.; Roman, L. J.; Poulos, T. L.; Silverman, R. B. 2-Aminopyridines with a truncated side chain to improve human neuronal nitric oxide synthase inhibitory potency and selectivity'. J. Med. Chem. 2015, 58, 5548-5560.

[0398] (38) Mukherjee, P.; Cinelli, M. A.; Kang, S.; Silverman, R. B. Development of nitric oxide synthase inhibitors for neurodegeneration and neuropathic pain. Chem. Soc. Rev. 2014, 43, 6814-6838.

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[0400] (40) Rankovic, Z. CNS drug design: balancing physicochemical properties for optimal brain exposure. J. Med. Chem. 2015, 58, 2584-2608.

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[0404] (44) Misra, A.; Ganesh, S.; Shahiwala, A.; Shah, S. P. Drug delivery' to the central nervous system: a review, J Pharm Pharm Sci. 2003, 6. 252-73.

[0405] (45) Wager, T. T.; Chandrasekaran, R. Y.; Hou, X.; Troutman, M. D.; Verhoest, P. R.; Villalobos, A.; Will, Y. Defining Desirable Central Nervous System Drug Space through the Alignment of Molecular Properties, in Vitro ADME, and Safety Attributes. ACS Chem. Neurosci., 2010, 1. 420-434.

[0406] (46) Veber, D. F.; Johnson, S. R.; Cheng, H.-Y.; Smith, B. R.; Ward, K. W.; Kopple, K. D. Molecular Properties That Influence the Oral Bioavailability of Drug Candidates. J. Med. Chem. 2002, 45, 2615-2623.

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[0408] QB\702581.02752\100011397.1(47) Kang, S.; Tang, W.; Li, H.; Chreifi, G.; Martasek, P.; Roman, L. J.; Poulos, T. L.; Silverman, R. B. Nitric Oxide Synthase Inhibitors That Interact with Both Heme Propionate and Tetrahydrobiopterin Show High Isoform Selectivity. J. Med. Chem. 2014, 57, 4382-4396.

[0409] (48) Cinelli, M. A.; Li, H.; Chreifi, G.; Poulos, T. L.; Silverman, R. B. Nitrile in the hole: discovery of a small auxiliary pocket in neuronal nitric oxide synthase leading to the development of potent and selective 2-aminoquinoline inhibitors. J. Med. Chem. 2017, 60, 3958-3978.

[0410] (49). Labby, K. J.; Xue, F.; Kraus, J. M.; Ji, H.; Mataka, J.; Li, H.; Martasek, P.; Roman, L. J.; Poulos, T. L.; Silverman, R. B. Intramolecular hydrogen bonding: A potential strategy for more bioavailable inhibitors of neuronal nitric oxide synthase. Bioorg Med Chem. 2012, 20, 2435-2443.

[0411] (50) Xue, F.; Li, H.; Delker, S. L.; Fang, J.; Martasek, P.; Roman, L. J.; Poulos, T. L.; Silverman, R. B. Potent, highly selective, and orally bioavailable gem-difluorinated monocationic inhibitors of neuronal nitric oxide synthase. J. Am. Chem. Soc. 2010, 132, 14229-14238.

[0412] (51) Silverman, R. B.; Lawton, G. R.; Ranaivo, H. R.; Chico, L. K; Seo, J.; Watterson, D. M. Effect of potential amine prodrugs of selective neuronal nitric oxide synthase inhibitors on blood-brain barrier penetration. Bioorg. Med. Chem. 2009, 17, 7593-7605.

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[0417] (56) Molander, G. A.; Trice, S. L. J.; Tschaen, B. A modified procedure for the palladium catalyzed borylation / Suzuki-Miyaura cross-coupling of aryl and heteroar l halides utilizing bis-boronic acid, Tetrahedron, 2015, 71, 5758-5764.

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[0419] QB\702581.02752\100011397.1(57) Bui, M. H. T. et al, - Pyrazolo-Pyrimidin-Amino-Cycloalkyl Compounds and Their Therapeutic Uses, WO2019236631.

[0420] (58) Hussain, N.; Kim, B. -S.; Walsh, P. J. Palladium-Catalyzed C(sp3)-H Arylation of A-Boc Benzylalkylamines via a Deprotonative Cross-Coupling Process, Chem. Eur. J. 2015, 21, 11010 - 11013.

[0421] (59) Chappie. T. A. etal. Imidazo-triazine derivatives as pdelO inhibitors, WO2014 / 177977 Al.

[0422] (60) Porzio, A. D.; Galli, U.; Amato, J; Zizza, P.; Iachettini, S.; Iaccarino, N.; Marzano, S.; Santoro, F.; Brancaccio, D.; Carotenuto, A.; Tito, S. D.; Biroccio, A.; Pagano, B.; Tron, G. C.; Randazzo, A. Synthesis and Characterization of Bis-Triazolyl-Pyridine Derivatives as Noncanonical DNA-Interacting Compounds. Int. J. Mol. Sci. 2021. 22, 11959.

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[0025] Nitric-oxide synthase assays. Methods Enzymol.

[0427] 1.904, 233, 250-258.

[0428] (65) Csonka, C.; Pah, T.; Bencsik, P.; Gorbe, A.; Ferdinandy, P.; Csont, T. Measurement of NO in biological samples. Br. J. Pharmacol., 2015, 172, 1620-1632.

[0429] (66) Di, L.; Kems. E. H.; Fan, K.; McConnell. O. J.; Carter, G. T. High throughput artificial membrane permeability assay for blood- brain barrier. Eur. J. Med. Chem. 2003, 38, 223-232.

[0430] (67) Xue, F.; Li, H.; Delker, S. L.; Fang, J.; Martasek, P; Roman, L. J. Poulos, T. L.; Silverman, R. B. Potent. Highly Selective, and Orally Bioavailable Gem-Difluorinated Monocationic Inhibitors of Neuronal Nitric Oxide Synthase, J. Am. Chem. Soc. 2010, 132, 14229-14238.

[0431] (68) Bagutski, V.; Ros, A.; Aggarwal, V. K. Improved method for the conversion of pinacolboronic esters into trifluoroborate salts: facile synthesis of chiral secondary and tertiary trifluoroborates, Tetrahedron., 2009, 65, 9956-9960.

[0432] Page 51

[0433] QB\702581.02752\100011397.1(69) Freitas, J. J. R.; Freitas, Q. P. S. B.; Andrade, S. R. C. P.; Freitas, J. C. R.; Oliveira, R. A.; Menezes, P. H. Efficient method for propargylation of aldehydes promoted by allenylboron compounds under microwave irradiation, BeilsteinJ Org. Chem. 2020, 16, 168— 174.

[0434] Example 2

[0435] 1. NOS Enzyme Inhibition Assay

[0436] The NOS inhibitory activity of 11-23 was measured using the hemoglobin (Hb) NO capture assay following a protocol described previously.1'2The production of NO was monitored by a rapid oxidation of oxyhemoglobin (oxyHb) to methemoglobin (metHb) by NO.1Purified recombinant full-length NOSs, including rat nNOS (mNOS),3'4human nNOS (hnNOS),5human iNOS (hiNOS),6and human eNOS (heNOS),5 were used in activity assays. These proteins were expressed in Escherichia coli and purified as described previously.3'6Activity assays were performed in 100 mM HEPES (4-(2-hydroxyethyl)-l -piperazineethanesulfonic acid) buffer with 10% glycerol (pH 7.4-7.5) at 37 °C in the presence of 10 pM L-Arg, 10 pM H4B, 100 pM NADPH, 0.83 mM CaC12, 320 units / mL calmodulin, and 3 pM human oxyhemoglobin. A concentration of L-Arg of 10 pM was used as it does not cause NOS uncoupling and is close to the Km values for all three NOS isoforms so that competitive inhibitors can be detected effectively. In the case of iNOS. CaC12 and calmodulin were omitted and replaced by HEPES buffer (100 mM, 10 % glycerol, pH 7.4-7.5) because iNOS activation is calcium-independent. The assay was performed in 96-well plates using a Biotek Gen5 microplate reader, with NOS enzymes and hemoglobin dispensed automatically by the plate reader. NO production was kinetically monitored at 401 nm for 6 min. The inhibition constants (Ki) for all NOSs were calculated from the ICso values of the dose-response curves using the Cheng-Prusoff equation,7

[0437] SCgg

[0438] K ~;- ty 'sty

[0439] where 'm is the Michaelis constant: Km (hnNOS) = 1.6 pM; im (mNOS) = 1.3 pM; 'm (hiNOS) = 8 pM; 'm ( eNos) = 3.9 pM.8 Dose-response curves were constructed from ten to eleven test concentrations (10 pM to 10 nM), and ICso values were calculated by nonlinear regression using GraphPad Prism software. The calculated standard deviations from dose-response curves of the assays were less than 10% with all NOSs.

[0440] Page 52

[0441] QB\702581.02752\100011397.12. PAMPA-BBB Assay

[0442] Blood-brain barrier penetration was estimated using the PAMPA-BBB assay, in which a porcine brain lipid (PBL) was used as an artificial membrane.9'11The five commercial drugs, (±)-verapamil, desipramine, chlorpromazine, dopamine, and theophylline; phosphate buffer saline (PBS, 10 rnM); DMSO (for biology), and dodecane (analytical standard) were purchased from Sigma-Aldrich. The porcine brain lipid (PBL) was obtained from Avanti Polar Lipids (100 mg, powder, catalogue no. 141101P). The donor plate used in the assay is a 96-well filter plate with a hydrophobic polyvinylidene fluoride (PVDF) membrane (pore size 0.45 pm, nonsterile, catalogue no. MAIPNTR10), and the acceptor plate is a 96-well transport receiver plate (catalogue no. MATRNPS50), both from Millipore Sigma. A 96-well UV plate with a flat bottom obtained from Greiner Bio- One was used for UV measurements (catalogue no.

[0443] 655801). Test compounds were first dissolved in DMSO to make a 10 mM stock solution. Then 40 pL of stock solution was diluted with 1960 pL of 10 mM PBS buffer (pH 7.5) to generate a final concentration of 200 pM (2% DMSO). The acceptor plate was filled with 250 pL of 10 mM PBS (2% DMSO, pH 7.5). The donor plate was first coated with 4 pL of PBL (20 mg / mL in dodecane), then 250 pL of a test compound (200 pM) was added to the donor plate. Each test compound was measured in triplicate. The donor plate was then carefully placed on top of the acceptor plate to make a “sandwich’', which was incubated at 25 °C for 17 h in a saturated humidity atmosphere with an orbital agitation at 100 rpm. During this time, compounds diffuse from the donor plate to the acceptor plate. After incubation, 150 pL of test solution was taken from each well from both donor and acceptor plates and transferred to the UV plate for measurement. The concentration of a compound in each donor and acceptor well was determined by using a standard curve, which was built from its UV absorbance at / .max of various concentrations (1-200 pM). The effective permeability’ (Pe) was calculated using the following equation,12

[0444] „ 2.3Q3 Vx-Vp / (VA*VO) \ / CA(Q \

[0445] (W9” \(1 - Hj VD / \ Cf»(OV

[0446]

[0447] where Peis the effective permeability (cm s '); VA and VD are the volume of the acceptor and donor wells (0.25 cm3), respectively; CA (t) is the concentration of the acceptor well at time t; CD(0) and CD(t) are the concentrations of the donor well at to and t, respectively; A is the filter well area (0.21 cm2); t is the incubation time (s); Tss is the time to reach a steady state (usually very short compared with the incubation time); and R is the retention membrane factor, which was calculated using the following equation:

[0448] Page 53

[0449] QB\702581.02752\100011397.1V& CA(O

[0450] rs - 1 - • -

[0451]

[0452] Cfi(O) VQ Cp(O)

[0453] Pe is reported as an average of triplicates with a standard deviation.

[0454] 3. Preparation of NOS heme domain samples for crystallography

[0455] In our earlier work, the heme domains of mNOS, hnNOS, and heNOS for crystallography were generated by trypsin digestion of full-length proteins as described.4-5More recently we have expressed just the heme domains of mNOS and hnNOS. These heme domain preparations were used in structures described in the current work (see section 4).

[0456] To generate the expression construct for the rat nNOS heme domain, amino acids Arg299 to Trp716 of rat nNOS were amplified from the mNOS expression construct, pCWori-mNOS,4which includes an R349A mutation. A thrombin-cleavage site was engineered into the reverse primer to create a thrombin-cleavable C-terminal His6 tag. The resulting PCR product was cloned into the Ndel and Xhol sites of pET22b. Similarly, amino acids Cys302 to Lys722 from human nNOS were cloned into pET22b by amplification of this region from the construct pCWori-hnNOS EID (which includes both PDZ and heme domains),5which also contains R354A and G356D mutations. The PCR product was then cloned into the Ndel and Xhol sites of pET22b. The design of primers was reported previously.13

[0457] Expression of the mNOS and hnNOS heme domain constructs was carried out in BL21 / DE3 cells. Cell grow th and Ni column purification were described in detail previously.13 Peak fractions from the Ni column eluate were pooled and concentrated using an AmiconUltra centrifugal filter unit (Millipore, 30 kD cutoff). One round of buffer exchange was carried out using 150 mM NaCl. 20 mM Tris, 10% glycerol pH 8.5, and the sample was concentrated to < 3 mL. This sample was digested with thrombin (MP Biomedicals) using a ratio of 30 U thrombin per mg of protein and incubated overnight at 4 °C. The thrombin-digested sample was passed over aNi column in Ni column buffer (as above except also including 0.25 mM PMSF and 20 mM imidazole) to remove uncleaved protein and His tags. The flow-through was collected and concentrated to < 1.5 mL. The sample was then loaded onto an S-200 column and run as reported previously.13Fractions from the S-200 column were pooled based on spectral ratio (280 nm / 405 nm) and concentrated to > 8 mg / mL for crystallization.

[0458] To study the new Zn2+binding site observed in many mNOS structures, 14 we mutated one of the Zn2+binding ligands. His692, to a Phe to disrupt Zn2+binding. This mutation was engineered into the full-length mNOS R349A expression construct by site-directed mutagenesis using PrimerSTAR Max DNA polymerase (Takara). The full-length mNOS Page 54

[0459] QB\702581.02752\100011397.1R349A / H692F was expressed and purified as previously described for wild-type nN0S4 which was then used to generate the heme domain by trypsin digestion as previously described for wild-type nNOS.4In this study, this heme domain sample of mNOS H692F was used only in one structure, mNOS R349A / H692F-11 (Table 3). Because of the large distance between His692 and the active site of mNOS, the mutant is expected to behave like the wild-ty pe protein with regard to inhibitor binding.

[0460] 4. Inhibitor Complex Crystal Preparation

[0461] The sitting drop vapor diffusion method was used to grow crystals at 4 °C for the heme domains of mNOS R349A (8 mg / mL containing 20 mM histidine), hnNOS R354A / G357D (10 mg / mL), and wild-type heNOS (10 mg / mL). The crystal growth conditions were as described previously.4-5Seeding techniques were applied for all cases often at a reduced protein concentration to improve the size and quality of crystals. As described previously,4the final step of cryo-soakings for both mNOS and hnNOS crystals were carried out in HEPES buffer (pH 7.5); for heNOS, Bis-Tris buffer at pH 7.5 was used. Crystals were further soaked with 5-10 mM inhibitor for 2-4 h at 4 °C before being flash cooled with liquid nitrogen and stored until data collection.

[0462] 5. X-ray Diffraction Data Collection, Data Processing, and Structural Refinement The cry ogenic (100 K) X-ray diffraction data were collected remotely at the Stanford Synchrotron Radiation Lightsource (SSRL) or the Advanced Light Source (ALS) through data collection control software Blu-Ice15and a crystal-mounting robot. Although the crystal growth conditions were not changed, the mNOS and hnNOS crystals produced with the cloned heme domain proteins showed different symmetry from crystals produced from the heme domain proteins generated by trypsin cleavage of their respective full-length NOS.4-5The mNOS crystals changed from P212121 space group4to C222i, with only one subunit per asymmetric unit. The hnNOS crystals changed from P2i2i2i to P2i space group even though the [3 angle shows almost no deviation from 90.0° with two dimers in the asymmetric unit.16Owing to the random choices of the asymmetric unit by the program the cell dimensions vary by the rearrangement of the three cell edges. In this study, all the mNOS and hnNOS structures were determined using the subcloned and expressed heme domain protein. All the heNOS crystals were in the P2i space group rather than orthorhombic P2i2i2i reported previously^ with a angle less than 1° off 90°. Therefore, a molecular replacement calculation with PHASER-MR17was needed initially to solve the structure. In the P2i space group, there are two heNOS dimers in the asymmetric unit.

[0463] Page 55

[0464] QB\702581.02752\100011397.1Whenever duplicate data sets were available, merging and scaling the two together was attempted. For the fine-sliced pixel array data the higher redundancy of merged data from 2 crystals often lead to better scaling statistics and were thus used in the following structure refinements. The number of crystals used for the refinement of each structure are listed in Table 3. Raw CCD data frames were indexed, integrated, and scaled using iM0SFLM,18 but the pixel array data were preferably processed with XDS19and scaled with Aimless.20The binding of inhibitors was detected by initial difference Fourier maps calculated with REFMAC.21The inhibitor molecules were then modeled in Coot22and refined using REFMAC and then PHENIX.23Disordering in portions of inhibitors bound in the NOS active sites was often observed, sometimes resulting in poor density quality. However, partial structural features were usually still visible if the contour level of the sigma A weighted 2m|Fo| - D|Fc| map was dropped to 0.5o, which enabled the building of reasonable models into disordered regions. Water molecules were added in PHENIX and checked visually in Coot. The TLS24protocol was implemented in the PHENIX refinements with each subunit as one TLS group. The omit Fo - Fc density’ maps were calculated by the Polder map routine in PHENIX by removing the bound inhibitors in the map calculation.23The refined structures were validated through the validation facility in wwPDB before the final deposition to the Protein Data Bank. The crystallographic data collection and refinement statistics are reported in Table 3, which includes the PDB codes for all structures in this study.

[0465] 6. Compounds 13, 15, 17, 21, and 22, Figures 9-13

[0466] Compound 13 has a bulky -CF3 substitution on the bridging phenyl ring and shows poor potency and selectivity compared to 12. As shown in Figure 9, in both mNOS and hnNOS 13 share the same binding mode. The bridging phenyl ring of 13 has an orientation flipped from what was observed for 12 that places the trifluoromethyl group against the propionate D forcing the latter into a downward conformation. The fluorine atoms are in van der Waals distances from a Tyr residue (Tyr706 in mNOS or Tyr711 in hnNOS), a Met residue (Met570 in mNOS or Met575 in hnNOS), and the propionate D. This phenyl ring orientation brings the tail amine to a H-bonding distance to the active site Glu (Glu592 in mNOS or Glu597 in hnNOS). Compound 13 bound to heNOS (Figure 9, panel C) has similar positions for the two aromatic rings and the trifluoromethyl group, but the tail amine shows some ambiguity among the 4 subunits in the structure as to whether it is H-bonded to propionate A or to a nearby Cl" anion. Although 13 does not show any uncertainty in its binding to NOS, the crowding

[0467] Page 56

[0468] QB\702581.02752\100011397.1associated with the bulky trifluoromethyl group may be the main reason for its poor binding affinity to all three NOSs.

[0469] The only change from 14 to 15 is the placement of a secondary amine with a tertiary amine. The structure of mNOS-15 (Figure 10, panel A) indicates that both positions of the aminopyridine and the chlorophenyl rings remain the same in 14 and 15 in mNOS. Propionate D is still pushed into a downward conformation, however, the exact amine N atom position changes substantially. In mNOS, the tertiary amine in 15 only makes van der Waals contacts with propionate A without the H-bond with a water molecule observed for 14. In the heNOS-15 structure (Figure 10, panel B), the chlorine atom approaches closer to Met339 than it does to Met570 in the mNOS-15 structure, allowing the tail amine to make a H-bond to propionate A directly.

[0470] The lengthy -CN group on the bridging phenyl ring in 17 makes it difficult to fit into the active site of nNOS. Good density could be observed for both aminopyridine and phenyl rings, but there is no indication where the tail amine and -CN group go. Surprisingly, only the structure of heNOS-17 has a complete density for 17 (Figure 11). The -CN group sticks into the propionate D position forcing it into a downward conformation. The tail amine interacts with the C1‘ anion. The O' anion may stabilize the binding of 17 in heNOS while without the Cl" in nNOS, the orientation of the phenyl ring is disordered. Overall, 17 is a poor inhibitor in this series.

[0471] Compound 21 has a fluorinated azetidine as the tail tertiary amine. A similar binding mode is found in the structures of mNOS, hnNOS, and heNOS (Figure 12). The difluorobenzene ring has its fluorine atoms pointing upw ard. One of the fluorine atoms H-bonds with a Gin residue (Gln478 in mNOS, Gln483 in hnNOS, and Gln247 in heNOS), a water molecule in mNOS and hnNOS or the Cl" anion in heNOS. The water molecule (or the Cl" anion) is further H-bonded to a Tyr residue (Tyr588 in mNOS, Tyr593 in hnNOS, and Tyr457 in heNOS). The tail azetidine N atom is not facing to the heme propionates thus there is no H-bonding interaction. The lack of H-bonding with the propionates may be the main reason for its poor potency since for many inhibitors in this series, one or more H-bonds from the tail secondary amine to the propionates are part of the structural basis for good potency.

[0472] Compound 22 has a tail fluorinated piperidine and its N atom serves as a tertiary amine. As show n in Figure 13, the binding mode of 22 in hnNOS is similar to that of 21, that is, the bridging difluorobenzene ring has its fluorine atoms pointing upward with one of the fluorine atoms H-bonding to Gln483 and a water molecule. The tail piperidine N atom faces away from Page 57

[0473] QB\702581.02752\100011397.1the heme propionate. One of the fluorine atoms off the piperidine ring approaches Asn574 for a H-bond. However, in the heNOS-22 structure, the bridging difluorobenzene ring flips from the orientation observed in hnNOS-22 with its fluorine atoms pointing toward heme, and pushes propionate D in a downward conformation. This bridging ring orientation brings the tail piperidine ring N atom toward propionate A for a weak H-bond. One of the fluorine atoms is within H-bonding distance of Arg365. This binding mode of 22 in heNOS is unique owing to the bulky piperidine tail and is quite different than with a simple tertiary amine as that in 19. The bulky cyclic fluorinated tertiary amine introduced in 21 and 22 improves membrane permeability (Table 2), but unfortunately, results in poor binding especially to nNOS by losing the H-bond from the tail amine to heme propionates (Table 1).

[0474] Page 58

[0475] QB\702581.02752\100011397.17. Table 3. Crystallographic data collection and refinement statistics Data set0ir. NOb R349A raNOS R349A-I3 HJNOS R349A-14 ffiNOS R349A-15

[0476] H692F-II

[0477] Data collectioa

[0478] PD8 code 9C¥1 9CVJ 9CVK 5> CVL Space group C222t <.222; C2221 C222i Cell dimensions

[0479] a, b, £■ (A) 4S S: • r;1640 49.0114.21640 4S.7.1113 163.1 48,£ 114 1 164.4 RestshOicn (A) 2.03 (2.08-2 S3) 1.79 (1 83-179) 190(1.94-1 99} 192 (1 97-192) & SKgS 0.141 (4729) 0.077 (8.431) 0.100(6.546) 0.190 (8.523)

[0480] 0.068 (2.128) 034 (3.630) •3.034 (2.832) 0.059 (2.776) 0,157(5.194) 0.085 (9.200) 0.106(7.146) 0,1.90 (3.976) CC122 0996 (0.344) 0.999 (0.411) 0.998 (0.322) 0.997(0.449) < I 7 C'l ' 5.9 (1.35)£8.8 (1.20)c8.3 (1.33)'’ 6.6 (1.2?)sNo. unique rejections 29446 (2153) 43102 (2567) 35691 (2254) 35583 (2367) Cotapletesiess (%) 98.4(989) 986 (99.2} 99.6(98.0) 99.9 (999) Redmidancy 55 (58) 5.9 (61) £.1 (6.1) 11 2 {10.71 No. crystals 1 1 1 2 Wilson B factor (A) 46.3 37. S 399 40.0 Refiuemsat

[0481] Resolstioti (A) 2.03 1.79 1.35 1.92 No. reflections used 27594 39550 3367? 35139 WW 023W297 0.218 / 6.263 S219 / 0.274 0256 / 0299 No. atoms, Protein 3339 3408 3388 3384 Ligaadioa £2 86 83 S4 Water 30 42 42 41 R tn.s. de\73torss

[0482] Bond lengths (A) 0.008 0.008 0.006 0.907 Bond angles (*) 1.08 094 ISO 0.95

[0483]

[0484] Page 59

[0485] QB\702581.02752\100011397.1Data se mNOS R349A-16 nsNQS R349A-1S inNOS &349A-19 stiNOS R349A-2® Data I'oltec tion

[0486] PDB code 9CVM 9CVN 9CVO 9CVP Space group C222-. C2225C222i C222j Cell dimeussms

[0487] «. b. c (A) 48..5 113.0162 £ 49.0113.81344 48.8 113.0 163.2 4881134164.0 Resolution (A) l.£0 (1.84-1 80) 1.85 (1.89-1.85) 2.08(2.14-2.08) L97 (202- 1.^7)

[0488] C 086 (S 713) 0.090 (6526) 0198 (5342) 0.116(7.128) 0027 (2.907) 0.032 (3.087) 0.067 (2,530) 0.052 (3.155) XxiSflSJf}® C.090 (066) 0.096 (7.248) 0-209 (5.930) 0.128(7.810) CCP2 0999 (0.714) 0.993 (0.375) 0.999 (0.641) 0.999 (0.231) ■; 1 -ii - 9.2 (1.83)c& S (1 04)16.3 (1.35):9,0(1.23) No, unique reflections 41948 (2427) 39793 (2297) 37540 (2347) 32396 (2232) CotnpleteaeBS (%) 190.0(100.0) 99.3 (93 7) 98.5 (97. S) 99.2 (99.2) Redsifidascy 11.2 (9.0) 6.7 (5.2) 8,6 (5.5) 5.8 (6.0) No. crystals 2 1 t 1 Wifcos B factor (A) 2S 5 48.2 44.2 40.7 Refinemeiit

[0489] Rescltsiai (A) 1.80 1.85 2.08 1 97 No refleciiciis used 39£04 37505 37290 36418 0216 / 0256 S.210256 0222 / 02S5 0228®.288 No. atoms, Protein 3344 3384 3337 3344 Ligaadfen 82 90 84 82 Water 411 46 39 39 R.m.s. deviations

[0490] Bond lengths (A) a.oos •9.00S 0.007 0.908 Betid angles (s) 1 02 1 04 0.5t£ l t

[0491]

[0492] Page 60

[0493] QB\702581.02752\100011397.1Data set0mNOSK349A-2l iiiNOS R349 A-23 hiiNQS R354A hiiNQS R354A G557D-I1 G357D-12 Data cofecfioa

[0494] PDB cede 9CVQ 9CVR 9CVS

[0495] Space group C2221P2j P21Cell dmiesistorss

[0496] a. A c (A) 48.6 1132 163.2 484 112.8 1626 52.4 118.81651 52.5 11S.1 165.1 3 C) 90.1 96.05 ResduticKi (A) 1.95 (2.00-1.90) 1.87 (1.91-1.78) 1.®) (1.83-1.80) 2,00(2.03-2.00)

[0497] 0.157 (9.162) 0.143 (8.244) 0.149 (5.049) 0.229 (4.178) 0.847 (2.297) 9045 (2.528) 0.065 (2.329) 0.086 (2.234) 0.164 (9.590) 0.150 (8.637) 0.163 (5.580) 0.245 (4.760) CC 1 / 2 0999 (0499) 9996 (0756) 6996 (0309) 0.992 (0297) <?7 n7 / - 15.3 ( 1.74} <• 9.7 (1.30);5.7 (0.68F 6.0 (1.09) ■ No. unique refiectiaus 32337 (2225) 37012 (2376) 184082 (8637) 133861 (6265) Completeness (%) 972 (97.1) 995 (99.8) 98 S (945) 9S 1 (91.4) Redundancy 12.0 (11.5) 11.4 (11.0) 6.2: (5.6) 7.8 (4.5) No. crystals? 2 I 1 Wilson B factor (A) 42.9 38.7 33.4 33. £ Refinesient

[0498] Resolnhon (A) 1.95 1.37 1.80 2.00 No. reflections used 31959 36486 168247 133249 0232 / 0269 $.232 / 0.260 0.180 / 0.222 0.184 / 0.231 No. atoms, Proteus 3381 3324 13620 13681 Lsgand / ion 84 85 340 278 Water 52 50 1129 846 R.m.s devitrfions

[0499] Bond lengths ( A) 0.007 0.006 0.007 0.007 Bond angles (a) 1 16 093 0.91 0.92

[0500]

[0501] Page 61

[0502] QB\702581.02752\100011397.1Data set* haNOS B354A hsNOS R354A hnNOS R354A hiiNQS R354A G357D-13 G357D-14 G357D-16 G357D-18 Data cc< Bectioia

[0503] PDB code 9CVU 9CVW 9CVX 9CVY Space group?2j P2i P21

[0504] Cell dimenskm

[0505] a. &. c (A) 117.3 51.5 163.2 52.3 113 3 165 2 51.8 1165 163 3 522 1644 1186 p o 90.9 90.18 90.03 90.0 Resolution (A) 1 94 (1 97-1 94) 1 87 (1 90-1 87} 1 30 (1 83-1.80) 2 39 (2 44-239)

[0506] 8 183 (4015) 0 165 (1 764) 9 121 (2 110) 0 390 (3 516) 0690 (2 139) D 059 (0 946) 0 062 (1 291) 0 195 (2 008) 0.204 (4 575) 0.176 (2 009) 0.136 (2.486) 0438 (4076) CC 2 / 2 0.991 (0.158) 0.997 (0.557) 0 994 (0.274) 0.953 (0.114) 3.6 (1.0Q)':10.4 (1.38):4.3 (0.99)c3.0 (1.19) 4 No. unique rejections 145729 (6664) 164286 (7073) 178635 (8638) 78289 (382:9) Conipletersess (%) 996 (91 9) 989 (869) 99 9 (98 2) 99 1 (71 6) Redundaticy 48 (39) 79 (4 6) 48 (3 4) 4.8 (3 6) crvsisls 1 1 1 1 Wilson B factor (A) 38.4 31.4 30.4 41 1 Refiiiemeiit

[0507] Resolution (A) 1.94 1.87 1 80 2.39 No. reflections used 145573 164023 178452 73944 r> '?3 &

[0508] '2‘-fees 0.189 / 0.233 0.170 / 0.208 0.17443.216 0.209(0276 No atoms, Protest 13602 13615 13602 13587 Ligaad / iosi 368 344 352 354 Water 854 1280 1244 434 R m s deviations

[0509] Bond lengths {A} 0.007 0.007 0.006 0.009 Bond angles (°) 0 97 091 092 1 12

[0510]

[0511] Page 62

[0512] QB\702581.02752400011397.1Dai J wrThnNOS R3S4A htosOS R354A hnNOS R354A haNOS R354A G357D-20 G357D-21 G357D-22 G357D-25 Data tjUecfion

[0513] PDB code 9CVZ 9CW0 9CW3 9CW1 Space gic iip F-i P2; P2j P2-. Cell dimensions

[0514] a, b, c (A) 118.8 52.2 164.5 116.4 51.8 163.1 118.5 52.1 164.8 51.5 116.1 163.0

[0515] 90.0 50.0 90.0 90.04 Resshshon (A) 2 18 (222-2.18) 2.10 (2.14-2.10) 2 19 (2.23-2 19) 1 90 (1 93 -190)

[0516] 8.2:78 (4.419) 0.212 (2.072) 0.556(9.221) 0.118 (1.506) 0 140 (2.260) 0.131 (1.256) 0.181 (3.292) 0077(0.981) 0.311 (4.984) 0.249 (2.428) 0.585 (9.814) 0.142 (1.804) CC 1 / 2 0986 (0 127) 0.972 (0.294) 0.985 (0.205) 0988 (0.341) ■■■■ i '<?> / ' 3.0 (1.42)c4.1 (1 10) - 5.2 (1.08)c4.5 (1.54)" No. unique reileciions 10508S (5105) 144915 (5469) 104426 (5066) 158418 (7402) Completeness V / H) 99.1 (99.0) 98.6 (97.0) 99.9 (98. S) 98.8(99.8) Redundsncy 48(48) 37 (3.7) 102(75) 3.3 (3.3) No. crystals I 2 1 Wilson B factor (A] 43.1 28.1 38.1 27.3 RefEHemeiti

[0517] Resolntion (A) 2 13 2.10 2.19 1.98 No. reBevtions used 91063 114453 10430G 149322 0.289 / 0.277 0.195 / 0.236 0.193 / 0.251 0.175^.220 No atoms, Protein 13641 13597 13601 13680 Ligand / ion 362 376 378 358 Water 347 1078 670 1263 RJH. S. deviations

[0518] Braid lengths (A) 0.008 0.007 0.008 0.007 Bead angles (“) 1.04 1.10 0.95 0.95

[0519]

[0520] Page 63

[0521] QB\702581.02752\100011397.1Date heNOS-11 heNCS-12 heNOS-13 l-eN0S-I5 Data c oflectioa

[0522] PDB code 9CW2 9CW4 9CW5 9CW6 Space group P2: P2:- P2; P21Cell dimensions

[0523] t'i, b, c (A) 5S.9 151.4107.5 592 153.0 108.8 59.2 151.9 107.8 59.5 153.3 108.9 e> 90.6 90.5 90.7 90.8 Rssdutkxi (A) 2,10 (3.14-2.10) 2.00 (3.03-2.00) 1.88 (1.91-1.88) 215 (2.19-2.15)

[0524] 0.183 (2.553) 0.203 (5079) 0.086(1.407) 0.194 (3.429) 0.094 (1.294) 0698 (2.526) 0052 (0919) 0099 {1 769) ■^MSBKSJS 0.206 (2.849) 0.226 (5.693) 0.101 (1,591) 0.219 (3.871) CCl / > 0.991 (0.275) 0988 (0.403) 0997 (0400) 0.5B8 (0197) I ■ > 3.8 (1.16) < 4.0 AB? 6.9 (1.19) < 4.5 {i.53){No unique reflections 109241 (5387) 121391 (5847) 154010 (7585) 104103 (5172) Coiapfeteiiegs {%) 99.7 (99.6) 93.3 (90.7) 99.9 (99.0) 99.9 (999) Redundancy 4.5 (4.7) 4,9 (46) 37 (3.1) 4.6 (4.6) No. cry sials 1 1 1 1 Wilson B factor {A} 34.0 38.2 32.2 41.5 Refinement

[0525] Resohstsoa (A) 2.10 2.00 188 2.15 No reflections used 107719 111.903 153838 99987 Rwatk / / £&«* 0.217 / 0.277 0.223 / 0.284 0.187 / 0228 0.208 / 0.266 No, atoms, Praietn 12841 12925 12835 12844 Ligasd / ion 524 426 522 514 Wafer 488 350 807 302 Rm s deviations

[0526] Bond lengths (A) 0.009 0.009 0.009:0005 Bond angles (°) 1 05 096 095 1.06

[0527]

[0528] Page 64

[0529] QB\702581.02752\100011397.1Data set13heNOS-16 heNOS-17 heNOS-18 heNOS-19 Data ecSeeti&u

[0530] PDB cotte 9CW7 9CW8 9CW9 9CWD Space group P2v P2i P2i P2j Cell dunenstons

[0531] a, £>, c (A) 60.0 152.4 108.7 592 152.0 1077 593 152.7 108.7 59.4.152.6 108.3 p f) 90. S 90.6 90.6 90.7 ReBolutiosi (A) 1.83 (1.86-1.83) 1.92 (1.95-1.92) 2.12 < 2 16-2 12) 1.92 (1 95-1.92)

[0532] 0.100 (1.902) 0.081 (1.314) 0.277 (1.884) 0.128 (1.838) 0.052 (1.039) 0.054 (0.922) 0.096 (0.933) 0066 (0936) 0.113 (2.177) 0.098 (1.614) 8.294 (2.109) 0.144 (2.068) CC 1 / 2 0.996 (0.321) 0.995 (0.334) 0.991 (0.339) 0.998 (0.329)

[0533] S.1 (1.26)17.1 (1.24)e5.6 (1.41)c6.2 (1.23)cNo unique reflections 168643 (8187) 142999 (6924) 108982 (5319) 144080 (7090) Completeness (%) 98.6 (90) 98.8 (97.2) 99.8 (98.8) 98.4(98.7) Re&inda&cy 4.6 (4.2) 3.2 (2.9) 90 (4.9) 4.5 (4.7) No. crystals I 1 7 1 Wilsoa B factor (A) 31.9 348 35.1 35.1 Refin«naeiit

[0534] Resolution (A) 183 1 92 2 12 1 92 No. reflections used 168577 142341 100875 143947 0.189 / 0.228 0.186 / 0.231 0.215 / 0.256 0.238 / 0.252 No. atoms, Protein 12829 12859 12844 12841 LigatidAou 504 508 514 530 Water 981 770 571 686 Rm s deviations

[0535] Bond lengths (A) 0. Q09 0009 0.008 0.010 Bond angles («) 099 1 0'2 1 00 1 02

[0536]

[0537] Page 65

[0538] QB\702581.027521100011397.1Data seP heNOS-21 heNOS-22 heNOS-23 Data coltectism

[0539] PDB cade 9CW1 9CWJ 9CWK Space group P21P21P21Cell dmiensians

[0540] a, &, e (A) 59.4 152.8 108 3 58.9 151.8 107.9 61.7 152.3 109.2 90.8 90.8 90.8 9904 Resotuticsi (A) 1.73 (1.76-1.73} 1.90 (1.93-1.90) 1.90 (1.93-1.90)

[0541] 0.080(2.122) 0 140(1.985) 0.134 (0.506) 0.041 (1.150) 0.069 (0.996) 0.085 (0.352) 8.090 (2.424) 0.156(2.231) 0.159 (0.620) CW 0.998 (0 322) 0.996 (0.456) 0.954 (0.567) •. If 10.4(1 21}L6.0 (1.11)c4.2 (1.77}rNo. unique refleoicsis 197817 (9145) 147478 (7174) 155555 (7498) Cwiipleteaess (%) 986 (91.9) 99.1 (98.0) 98.5 (95. S) Redundaacy 4.7 (42) 4.6 (4.2) 3.2 (2.9) No. crystals 1 1 1 Wilson B factor (A) 29.3 23.3 23.7

[0542] Refiaenwirt

[0543] Resoiistiosi (A) 1.73 1.90 1.90 No. reSecti&ius used 197735 147134 155352 0.194^.222 0 196 / 0.23S 0 182 / 0.217 No. atoms.. Protean 12838 12S44 12843 LtgRaciioH 542 550 494 Water 987 743 1421 R.m.s deviations

[0544] Bond lengths (A) 0.007 0.009 0.009 Bond angles (>yi 1.16 0.97 0.99

[0545]

[0546] bRfree was calculated with the 5% of reflection set aside throughout the refinement. The set of reflections for the Rfreecalculation were kept the same for all data sets according to those used in the data of the starting model (7TS9 for mNOS in C222i, 7TS7 for hnNOS in P2i, and 5UO8 for the heNOS in P2i).

[0547] cThe <1 ' ol> value in the highest resolution shell calculated by Xtriage and reported in the wwPDB X-ray Structure Validation Report.

[0548] Page 66

[0549] QB\702581.02752\100011397.18. Experimental procedures

[0550] The benzyl bromides such as l-bromo-3-(bromomethyl)benzene (24), l-bromo-3-(bromomethyl)-5-fluorobenzene (27), l-bromo-3-(bromomethyl)-5-chlorobenzene (30), 1-bromo-3-(bromomethyl)-5-(trifluoromethyl)benzene (33), and 3-bromo-5-(bromomethyl)benzonitrile (37) were purchased directly from the commercial sources.

[0551] / e / L Butyl (3-bromobenzyl)(methyl)carbamate (25)2628

[0552] 25

[0553]

[0554] Compound 25 (Scheme 1) was synthesized according to general procedure A using 24 (8.50 g, 34.00 mmol. 1.0 equiv), sodium hydride (2.04 g, 51.00 mmol, 1.5 equiv, 60% dispersion in mineral oil), and tert-butyl methylcarbamate (4.46 g, 34.00, 1.0 equiv). Compound 25 was isolated as a yellow-viscous oil (10.00 g, 33.3 mmol, 98%). The NMR spectral data was identical to those reported previously.27LRMS (ESI) Calcd for C9H10BrNO2[(MH- / Bu)+]: 242.99, found: 243.45.

[0555] / c / 7- butyl (3-(6-amino-4-methylpyridin-2-yl)benzyl)(methyl)carbamate (26)

[0556]

[0557] Compound 26 (Scheme 1) was synthesized according to general procedures D, using 25 (481 mg, 1.60 mmol, 1.0 equiv), PdCb(dppl) (117 mg, 0.160 mmol. 10 mol%), bis(pinacolato)diboron (489 mg, 1.93 mmol, 1.2 equiv), KO Ac (472 mg, 4.81 mmol, 3.0 equiv), and 6-bromo-4-methylpyridin-2-amine (300 mg, 1.60 mmol, 1.0 equiv), and K2CO3 (2.67 ml, 4.81 mmol, 1.8 M in water, 3.0 equiv). This resulted in the formation of coupled product 26 as pale-yellow viscous oil (420 mg, 1.28 mmol, 80%). LRMS (ESI) Calcd for C15H17N3O2[(MH-rBu) ]: 271.32, found: 271.72. The product was taken to the next step without further characterization.

[0558] tert-butyl (3-bromo-5-fluorobenzyl)(methyl)carbamate (28)26-28

[0559] Page 67

[0560] QB\702581.02752\100011397.1

[0561]

[0562] Compound 28 (Scheme 1) was synthesized according to general procedure A using 27 (5.00 g, 18.66 mmol, 1.0 equiv), sodium hydride (1.12 g, 27.99 mmol, 1.5 equiv, 60% dispersion in mineral oil), and tert-butyl methylcarbamate (2.45 g, 18.66, 1.0 equiv). Compound 28 was isolated as a yellow oil (4.18 g, 13.12 mmol, 70%).XH NMR (500 MHz, CDCh, shows mixture of rotamers): 57.17 - 7.13 (m, 2 H), 6.89 - 6.86 (m, 1 H), (4.40 + 4.35 (s, 2 H)), (2.87 + 2.81 (s, 3 H)), (1.49 + 1.45 (s, 9 H)).13C NMR (126 MHz, CDCh. shows mixture of rotamers): 6 162.9 (d, JC-F= 250.7 Hz), (156.2 + 155.6 (s, IxC)). 142.6 (bs, IxC), 126.3 (d, JC-F = 35.3 Hz), 122.8 (d, Jo F= 8.8 Hz), 118.0 (d, JC-F = 25.2 Hz), 113.3 (dd, JC-F = 46.6, 21.4 Hz), (80.4 + 80.3 (s, IxC)), (52.1 + 51.4 (s, 1XACH2), 34.4, 28.5.

[0563] tert- butyl (3-bromo-5-chlorobenzyl)(methyl)carbamate (31)2628

[0564]

[0565] Compound 31 (Scheme 1) was synthesized according to general procedure A using 30 (2.00 g, 7.03 mmol, 1.0 equiv). sodium hydride (0.42 g, 10.55 mmol, 1.5 equiv, 60% dispersion in mineral oil), and tert-butyl methylcarbamate (0.92 g, 7.033 mmol, 1.0 equiv). Compound 31 was isolated as a yellow solid (1.29 g, 3.85 mmol, 55%). 'H NMR (500 MHz, CDCh, shows mixture of rotamers): 57.41 (s, 1 H), 7.25 (s, 1 H), 7.14 (s, 1 H), (4.38 + 4.34 (s, 1 x ACH2. 2 H)), (2.87 + 2.80 (s, 1 x 7VCH3, 3 H)), (1.49 + 1.45 (s. 1 x / VCH3. 9 H)).13C NMR (126 MHz, CDCh. shows mixture of rotamers): 5 (156.2 + 155.6 (s, 1 x C, shows two singlets due to restricted rotation)), 142.1, 135.4, 130.3, (129.0 + 128.6 (s, 1 x CH)), (126.5 + 126.2 (s, 1 x CH), 123.0, (80.5 + 80.3 (s, 1 x C)), (52.0 + 51.3 (s, 1 x 7VCH2)), (34.5 + 34.4 (s, 1 x ACH3)), 28.5.

[0566] tert-butyl (3-(6-amino-4-methylpyridin-2-yl)-5-chlorobenzyl)(methyl)carbamate (32)

[0567] Page 68

[0568] QB\702581.02752\100011397.1

[0569]

[0570] Compound 32 (Scheme 1) was synthesized according to general procedures D, using 31 (500 mg, 1.49 mmol, 1.0 equiv), PdCl2(dppf) (109 mg, 0.15 mmol, 10 mol%), bis(pinacolato)diboron (455 mg, 1.79 mmol, 1.2 equiv), KO Ac (440 mg, 4.48 mmol, 3.0 equiv), and 6-bromo-4-methylpyridin-2-amine (279 mg, 1.49 mmol, 1.0 equiv), and K2CO3 (2.49 mL, 4.48 mmol, 1.8 M in water, 3.0 equiv). This resulted in the formation of coupled product 32 as pale-yellow viscous oil (390 mg, 1.08 mmol, 72%). LRMS (ESI) Calcd for C15H17ClN3O2[(MH-tBu)+]: 306.10, found: 306.15. The product was taken to the next step without further characterization.

[0571] l-(3-bromo-5-(trifluoromethyl)phenyl)-N-methylmethanainine (34)2930

[0572] Brx

[0573] N

[0574] H

[0575] 34

[0576]

[0577] CF

[0578] Compound 34 (Scheme 1) was synthesized according to general procedure B using 33 (2.00 g, 6.29 mmol, 1.0 equiv), methylamine hydrochloride (0.43 g, 6.29 mmol, 1.0 equiv), and K2CO3 (1.04 g, 7.56 mmol, 1.2 equiv). Compound 34 was isolated as a pale-yellow oil (1.38 g, 5.16 mmol, 82%). 'H NMR (500 MHz, CDCh): 5 7.71 - 7.66 (m, 1 H), 7.64 (d, J = 1.9 Hz, 1 H), 7.55 - 7.49 (m, 1 H), 3.79 (s, 2 H), 2.45 (s, 3 H).13C NMR (126 MHz, CDCh): 5 143.7, 134.6 (d, JC-F = 1.3 Hz), 132.5 (q, JC-F = 32.8 Hz), 127.1 (q, JC-F = 3.8 Hz), 123.7 (q, Jc F = 3.8 HZ), 123.3 (q, JC-F= 273.4 Hz), 122.8, 55.1, 36.2.

[0579] / eH-butyl (3-bromo-5-(trifluoromethyl)benzyl)(methyl)carbamate (35)31

[0580]

[0581] Compound 35 (Scheme 1) was synthesized according to general procedure C using 34 (1.86 g, 6.95 mmol, 1.0 equiv), BOC2O (1.82 g, 8.34 mmol, 1.2 equiv), and EtsN (1.94 mL, 18.89 mmol, 2.0 equiv). Compound 35 was isolated as a yellow7oil (2.40 g, 6.52 mmol, 94%).

[0582] Page 69

[0583] QB\702581.02752\100011397.1'H NMR (500 MHz, CDCk, shows mixture of rotamers): 5 7.66 (s, 1 H), 7.55 (s, 1 H). 7.40 (s, 1 H), 4.43 (s. 2 H), (2.88 + 2.83 (s. 3 H)), (1.47 + 1.46 (s, 9 H)).13C NMR (126 MHz, CDCh, shows mixture of rotamers): 5 (156.2 + 155.5 (s, 1 x C)), 141.8, (133.9 + 133.7 (s, 1 x CH)), 132.7 (q, JC-F = 32.8 Hz), 127.5 (q, JC-F = 3.8 Hz), 123.2 (q, JC-F = 273.4 Hz), 123.1, 122.9, 80.5, (52.2 + 51.5 (s, 1 x NCH2)), 34.6, 28.5. LRMS (ESI) Calcd for C10H9BrF3NO2[(MH-tBu)+]: 310.98, found: 310.98.

[0584] rc / 7-butyl (trifluoromethyl)benzyl)(methyl)carbamate (36)

[0585]

[0586] Compound 36 (Scheme 1) was synthesized according to general procedures D, using 35 (1.00 g, 2.71 mmol, 1.0 equiv), PdCl2(dppf) (198 mg, 0.27 mmol, 10 mol%), bis(pinacolato)diboron (826 mg, 3.25 mmol, 1.2 equiv), KOAc (798 mg, 8.31 mmol, 3.0 equiv), and 6-bromo-4-methylpyridin-2-amine (507 mg, 2.71 mmol, 1.0 equiv), and K2CO3 (4.52 ml, 8.13 mmol, 1.8 M in water, 3.0 equiv). This resulted in the formation of coupled product 36 as a brown viscous oil (0.90 g, 2.28 mmol, 84%). LRMS (ESI) Calcd for C20H25F3N3O2[(M+H)+]: 396.19, found: 396.29. The product was taken to the next step without further characterization.

[0587] 3-bromo-5-((methylamino)methyl)benzonitrile (38)2930

[0588]

[0589] Compound 38 (Scheme 1) was synthesized according to general procedure B using 37 (1.00 g, 3.64 mmol, 1.0 equiv), methylamine hydrochloride (0.25 g, 3.64 mmol, 1.0 equiv), and K2CO3 (0.60 g, 4.36 mmol, 1.2 equiv). Compound 38 was isolated as ayellow oil (655 mg, 2.91 mmol, 80%).

[0590]

[0591] NMR (500 MHz, CDCh): 57.74 (d, J = 1.8 Hz, 1 H), 7.67 (d, J = 1.5 Hz, 1 H), 7.58 (s, 1 H), 3.77 (s, 2 H), 2.44 (s, 3 H).13C NMR (126 MHz, CDCh): 5 144.1, 135.8, 133.3, 130.4, 123.0, 117.6, 114.2, 54.7, 36.1.

[0592] rc / 7-butyl (3-bromo-5-cyanobenzyl)(inethyl)carbamate (39)31

[0593] Page 70

[0594] QB\702581.02752\100011397.1

[0595]

[0596] Compound 39 (Scheme 1) was synthesized according to general procedure C using 38 (0.79 g, 3.41 mmol, 1.0 equiv). BOC2O (0.92 g. 4.21 mmol, 1.2 equiv). and EtsN (0.98 mL, 7.02 mmol, 2.0 equiv). Compound 39 was isolated as a pale-yellow oil (0.700 g, 2.15 mmol, 61%). iH NMR (500 MHz, CDCh, shows mixture of retainers): 87.69 (s, 1 H), 7.61 (s, 1 H), 7.44 (s, 1 H), 4.42 (s, 2 H), (2.87 + 2.84 (s, 3 H)), 1.52 (s, 9 H). LRMS (ESI) Calcd for C10H10BrN2O2[(MH-tBu)+]: 268.99, found: 269.08.

[0597] / c / 7-butyl (3-(6-amino-4-methylpyridin-2-yl)-5-cyanobenzyl)(methyl)carbamate (40)

[0598]

[0599] Compound 40 (Scheme 1) was synthesized according to general procedures D, using 39 (1.14 g, 3.51 mmol. 1.0 equiv), PdCb(dppf) (256 mg, 0.351 mmol, 10 mol%), bis(pinacolato)diboron (1.07 g, 4.21 mmol, 1.2 equiv), KOAc (1.03 g, 10.52 mmol. 3.0 equiv), and 6-bromo-4-methylpyridin-2-amine (656 mg, 3.51 mmol, 1.0 equiv), and K2CO3 (5.84 mL, 10.52 mmol, 1.8 M in water, 3.0 equiv). This resulted in the formation of coupled product 40 as a brown viscous oil (0.89 g, 2.52 mmol, 72%). LRMS (ESI) Calcd for C20H25N4O2[(M+H)+]: 353.20. found: 353.34. The product was taken to the next step without further characterization.

[0600] l-(3-bromo-5-chlorophenyl)-N, N-dimethylmethanamine (41)29-30

[0601]

[0602] Compound 41 (Scheme 2) was synthesized according to general procedure B using 30 (2.00 g, 7.03 mmol, 1.0 equiv), ethylamine hydrochloride (0.57 g, 7.03 mmol, 1.0 equiv), and

[0603] Page 71

[0604] QB\702581.02752\100011397.1K2CO3 (1.17 g. 8.44 mmol, 1.2 equiv). Compound 41 was isolated as a brown oil (1.54 g, 6.19 mmol, 88%). 'H NMR (500 MHz, CDCh): 87.39 (t, J = 1.9 Hz, 1H). 7.36 (d, J = 1.7 Hz. 1H), 7.25 (t, J = 1.7 Hz, 1H), 3.35 (s, 2H), 2.23 (s, 6H).13C NMR (126 MHz, CDCh): 8 143.03, 135.04, 130.22, 130.10, 127.84, 122.70, 63.35, 45.53.

[0605] l-(5-bromopyridin-3-yl)-N-methylmethanamine (44)

[0606]

[0607] Compound 44 (Scheme 3) was synthesized according to general procedure G using 43 (2.00 g, 10.75 mmol, 1.0 equiv), methylamine (4.06 g. 43.00 mmol. 4.0 equiv) in EtOH (33 wt.%), NaBEU (0.81 g, 21.5 mmol. 2.0 equiv). Compound 44 was isolated as a yellow oil (1.35 g, 6.73 mmol, 63%). 'H NMR (500 MHz, CDCh): 5 8.57 (d, J = 2.2 Hz, 1 H), 8.46 (d, J = 1.7 Hz, 1 H), 7.85 (t, J = 2.0 Hz, 1 H), 3.75 (s, 2 H), 2.45 (s, 3 H).13C NMR (126 MHz, CDCh): 5 149.7, 147.9, 138.5, 137.6, 121.0, 52.8, 36.2. LRMS (ESI) Calcd for C7H10BrN2[(M+H)+]: 201.00. found: 202.07.

[0608] tert-butyl ((5-bromopyridin-3-yI)methyl)(methyl)carbamate (45)31

[0609] Bae

[0610]

[0611] Compound 45 (Scheme 3) was synthesized according to general procedure C using 44 (1.35 g, 6.73 mmol, 1.0 equiv), BOC2O (1.76 g, 8.08 mmol, 1.2 equiv), and EtsN (1.88 mL, 13.47 mmol, 2.0 equiv). Compound 45 was isolated as a pale-yellow solid (1.80 g, 5.98 mmol, 89%). 'H NMR (500 MHz, CDCh, shows mixture of rotamers): 8 8.57 (s, 1 H), 8.40 (s, 1 H), 7.71 (s, 1 H), 4.39 (s, 2 H), (2.86 + 2.82 (s, 3 H)), 1.46 (s, 9 H).

[0612] tert-butyl ((6-amino-4-methyl-[2,3'-bipyridin]-5'-yl)methyl)(methyl)carbamate (46)

[0613] Page 72

[0614] QB\702581.02752\100011397.1

[0615]

[0616] Compound 46 (Scheme 3) was synthesized according to general procedures D, using 45 (1.00 g, 3.32 mmol, 1.0 equiv), PdCl2(dppf) (243 mg, 0.33 mmol, 10 mol%), bis(pinacolato)diboron (1.02 g, 3.98 mmol, 1.2 equiv), KOAc (0.98 g, 9.96 mmol, 3.0 equiv), and 6-bromo-4-methylpyridin-2-amine (621 mg, 3.32 mmol, 1.0 equiv), and K2CO3 (5.53 mL, 9.96 mmol, 1.8 M in water, 3.0 equiv). This resulted in the formation of coupled product 46 as a brown viscous oil (0.89 g, 2.71 mmol, 82%). 'H NMR (500 MHz, CDCh, shows mixture of rotamers): 5 9.01 (d, J = 2.0 Hz, 1 H), 8.46 (s, 1H), 8.12 (s, 1 H), 6.91 (s, 1 H), 6.32 (s, 1 H), 4.54 (s, 2 H), 4.47 (bs, 2 H, NH2), (2.88 + 2.81 (s, 3 H)), 2.27 (s, 3 H), 1.48 (s, 9 H). LRMS (ESI) Calcd for C18H25N4O2[(M+H)+]: 329.20, found: 329.32.

[0617] l-(3-bromo-4,5-difluorophenyl)-N-methylmethanamine (48)2930

[0618] x-x x

[0619] 1 J H

[0620] rT p 48

[0621]

[0622] Compound 48 (Scheme 4) was synthesized according to general procedure B using 47 (2.00 g, 7.00 mmol, 1.0 equiv), methylamine hydrochloride (0.47 g, 7.00 mmol, 1.0 equiv), and K2CO3 (1.16 g, 8.39 mmol, 1.2 equiv). Compound 48 was isolated as a pale-yellow oil (1.45 g, 6.16 mmol, 88%).XH NMR (500 MHz, CDCh): 57.30 (dt, J = 5.7, 2.0 Hz, 1 H), 7.13 (ddd, J = 10.5, 6.9, 2.1 Hz, 1 H), 3.70 (s, 2 H), 2.43 (s, 3 H).13C NMR (126 MHz, CDCl3): δ 150.7 (dd, JC-F= 250.0, 13.9 Hz), 147.0 (dd, JC-F= 248.2, 15.1 Hz), 137.7 (t, JC-F= 5.0 Hz), 127.6 (d, JC-F= 3.8 Hz), 116.2 (d, JC-F= 17.6 Hz), 110.2 (d, JC-F= 17.6 Hz), 54.6 (d, JC-F= 2.5 Hz), 35.9.

[0623] rt-butyl (3-bromo-4,5-difluorobenzyl)(methyl)carbamate (49)31

[0624]

[0625] Page 73

[0626] QB\702581.02752\100011397.1Compound 49 (Scheme 4) was synthesized according to general procedure C using 48 (0.73 g, 3.08 mmol, 1.0 equiv). BOC2O (0.85 g. 3.69 mmol, 1.2 equiv). and EI3N (0.86 mL, 6.15 mmol, 2.0 equiv). Compound 49 was isolated as a yellow viscous oil (0.80 g, 2.38 mmol, 77%). 'H NMR (500 MHz, CDCI3, shows mixture of rotamers): 8 7.20 - 7.16 (m, 1 H), 7.06 - 6.92 (m, 1 H), (4.36 + 4.32 (s, 2 H)), (2.86 + 2.80 (s, 3 H)), (1.49 + 1.46 (s, 9 H).

[0627] ZerZ-butyl (3-(6-amino-4-methylpyridin-2-yl)-4,5-difluorobenzyl)(methyl)carbamate (50 )31

[0628]

[0629] Compound 50 (Scheme 4) was synthesized according to general procedures D, using 49 (0.80 g, 2.38 mmol, 1.0 equiv), PdC12(dppf) (174 mg, 0.24 mmol, 10 mol%), bis(pinacolato)diboron (725 mg, 2.86 mmol. 1.2 equiv), KOAc (0.70 g, 7.14 mmol. 3.0 equiv), and 6-bromo-4-methylpyridin-2-amine (445 mg, 2.38 mmol, 1.0 equiv), and K2CO3 (4.11 mL, 7.14 mmol, 1.8 M in water, 3.0 equiv). This resulted in the formation of coupled product 50 as a yellow oil (0.70 g, 1.93 mmol, 81%). The product was taken to the next step without further characterization.

[0630] l-(3-bromo-4,5-difluorophenyl)-N, N-dimethylmethanamine (51)29-30’39

[0631]

[0632] Compound 51 (Scheme 4) was synthesized according to general procedure B using 47 (2.00 g, 7.00 mmol, 1.0 equiv), ethylamine hydrochloride (0.57 g, 7.00 mmol, 1.0 equiv), and K2CO3 (1.16 g, 8.39 mmol, 1.2 equiv). Compound 51 was isolated as a yellowish-brown oil (1.42 g, 5.66 mmol, 81%).1H NMR (500 MHz, CDCl3): δ 7.33 (dt, J = 5.5, 1.9 Hz, 1 H), 7.19 (ddd, J = 10.4, 6.9, 2.0 Hz, 1 H), 3.44 (s, 2 H), 2.30 (s, 6 H).13C NMR (126 MHz, CDCl3): 8 150.7 (dd, JC-F = 252.0, 13.9 Hz), 147.3 (dd, JC-F = 248.2, 13.9 Hz), 135.6, 128.6 (d, JC-F= 3.8 Hz), 117.2 (d, JC-F = 17.6 Hz), 110.3 (d, JC-F = 17.6 Hz), 62.6, 45.1. LRMS (ESI) Calcd for C9H10BrF2N [(M+H)+]: 249.00, found: 249.25.

[0633] Page 74

[0634] QB\702581.02752\100011397.16-(5-((dimethylamino)methyl)-2,3-difluorophenyl)-4-methylpyridin-2-amine (52)

[0635]

[0636] Compound 52 (Scheme 4) was synthesized according to general procedures D, using 49 (0.50 g, 2.00 mmol, 1.0 equiv), PdCl2(dppf) (146 mg, 0.20 mmol, 10 mol%), bis(pinacolato)diboron (0.61 g, 2.40 mmol, 1.2 equiv), KOAc (0.70 g, 6.00 mmol, 3.0 equiv), and 6-bromo-4-methylpyridin-2-amine (374 mg, 2.00 mmol, 1.0 equiv), and K2CO3 (3.33 mL, 6.00 mmol, 1.8 M in water, 3.0 equiv). This resulted in the formation of coupled product 52 as a pale-yellow solid (0.45 g, 1.64 mmol, 82%). 'H NMR (500 MHz. CDCh): 57.56 - 7.49 (m.

[0637] 1 H), 7.17 (ddd, J = 10.8, 7.2, 2.3 Hz, 1 H), 6.99 - 6.89 (m, 1 H), 6.33-6.32 (m, 1 H), 4.53 (s, 2 H), 3.40 (s, 2 H), 2.29 (s, 3 H), 2.24 (s, 6 H).1?C NMR (126 MHz, CDCh): 5 158.6, 151.0 (dd, JC-F= 248.2, 13.9 Hz), 150.6, 149.4, 147.7 (dd, JC-F = 250.7, 13.9 Hz), 135.5 (t, C-F= 5.0 Hz), 129.3 (d, JC F= 8.8 HZ), 125.6 (t. JC-F = 1.9 HZ). 117.1 (d. JC-F = 17.6 HZ), 116.6 (d, JC-F = 7.6 Hz), 108.5, 63.5 (d, JC-F = 1.3 Hz), 45.5, 21.3.

[0638] N-(3-bromo-4,5-difluorobenzyl)-2-fluoroethan-l-amine (53)2930

[0639]

[0640] Compound 53 (Scheme 4) was synthesized according to general procedure B using 47 (2.39 g, 8.35 mmol, 1.0 equiv), 2-fluoroethylamine hydrochloride (0.83 g, 8.35 mmol, 1.0 equiv), and K2CO3 (1.38 g, 10.01 mmol. 1.2 equiv). Compound 53 was isolated as a paleyellow oil (1.90 g, 7.09 mmol, 85%). H NMR (500 MHz, CDCh): 57.31 (dt, J = 5.6, 1.9 Hz, 1 H), 7.19 - 7.11 (m, 1 H), 4.67 - 4.56 (m, 1 H), 4.56 - 4.45 (m, 1 H), 3.78 (s, 2 H), 2.96 -2.89 (m, 1 H), 2.89 - 2.82 (m, 1 H), 1.72 (s, 1 H, NH).13C NMR (126 MHz, CDCh): 5 150.8 (dd, JC-F = 252.0, 13.9 Hz), 147.0 (dd, JC-F = 248.2, 13.9 Hz), 137.8 (dd, JC-F = 6.3. 5.0 Hz), 127.5 (d, JC-F = 2.5 Hz), 116.1 (d, J = 17.6 Hz), 110.2 (d, JC-F= 17.6 Hz). 83.6 (d, JC-F= 165.1 Hz), 52.2 (d, J = 1.3 Hz), 49.0 (d, JC-F= 18.9 Hz).

[0641] ter / -butyl (3-bromo-4,5-difluorobenzyl)(2-fluoroethyl)carbamate (54)31

[0642] Page 75

[0643] QB\702581.02752\100011397.1

[0644]

[0645] Compound 54 (Scheme 4) was synthesized according to general procedure C using 53 (0.97 g, 3.63 mmol, 1.0 equiv), BOC2O (0.83 g, 3.81 mmol, 1.05 equiv), and EtsN (1.01 mL, 7.25 mmol, 2.0 equiv). Compound 57 was isolated as a pale-yellow semisolid (1.10 g, 2.99 mmol, 82%).1H NMR (500 MHz, CDCI3, shows mixture of rotamers): 57.21 - 7.16 (m, 1 H), 7.06 - 6.98 (m, 1 H), 4.64 - 4.53 (m, 2 H), (4.46 + 4.43 (s, 2 H)), 3.58 - 3.32 (m, 2 H), (1.49 + 1.43 (s, 9 H).

[0646] tert-butyl 3-(6-amino-4-methylpyridin-2-yl)-4,5-difluorobenzyl)(2-fluoroethyl)carbamate (55)

[0647]

[0648] Compound 55 (Scheme 4) was synthesized according to general procedures D, using 54 (0.86 g, 2.34 mmol, 1.0 equiv), PdC12(dppf) (171 mg, 0.23 mmol, 10 mol%), bis(pinacolato)diboron (0.71 g, 2.81 mmol, 1.2 equiv), KOAc (0.69 g, 7.01 mmol, 3.0 equiv), and 6-bromo-4-methylpyridin-2-amine (437 mg, 2.34 mmol, 1.0 equiv), and K2CO3 (3.90 mL, 7.01 mmol, 1.8 M in water, 3.0 equiv). This resulted in the formation of coupled product 55 as a pale-yellow oil (786 mg, 1.99 mmol, 85%). The product was taken to the next step without further characterization.

[0649] l-(3-bromo-4,5-difluorobenzyl)-3,3-difluoroazetidine (56)2930

[0650]

[0651] Compound 56 (Scheme 4) was synthesized according to general procedure B using 47 (1.67 g, 5.83 mmol, 1.0 equiv), 3,3-difluoroazetidine hydrochloride (0.76 g, 5.83 mmol, 1.0 equiv), and K2CO3 (0.97 g, 6.995 mmol, 1.2 equiv). Compound 56 was isolated as a yellow oil Page 76

[0652] QB\702581.02752\100011397.1(1.40 g, 4.70 mmol, 81%). ’H NMR (500 MHz, CDCh): 57.28 (dt, J = 5.5, 1.9 Hz, 1 H), 7.12 (ddd, J = 10.4, 6.9, 2.1 Hz. 1 H), 3.67 (s. 2 H). 3.60 (t. J = 10.0 Hz, 4 H).13C NMR (126 MHz, CDCh): 8 150.8 (dd, JC-F = 252.3, 13.9 Hz), 147.3 (dd, JC-F = 249.5, 15.1 Hz), 135.2 (t, JC-F = 5.0 Hz), 127.7 (d, JC-F = 3.8 Hz), 116.3 (t, JC-F = 275.9 Hz), 116.2 (d, JC-F = 17.6 Hz), 110.5 (d, JC-F = 17.6 Hz), 64.7 (t, JC-F = 23.3 Hz), 61.5.

[0653] 6-(5-((3,3-difluoroazetidin-l-yl)methyl)-2,3-difluorophenyl)-4-methylpyridin-2-amine (57)

[0654]

[0655] Compound 57 (Scheme 4) was synthesized according to general procedures D, using 56 (0.70 g, 2.35 mmol, 1.0 equiv), PdCl2(dppf) (172 mg, 0.23 mmol, 10 mol%), bis(pinacolato)diboron (0.72 g, 2.81 mmol, 1.2 equiv), KOAc (0.69 g, 7.01 mmol, 3.0 equiv), and 6-bromo-4-methylpyridin-2-amine (439 mg, 2.35 mmol, 1.0 equiv), and K2CO3 (3.91 mL, 7.04 mmol, 1.8 M in water, 3.0 equiv). This resulted in the formation of coupled product 57 as a yellow solid (605 mg, 1.86 mmol, 79%). The product was taken to the next step without further characterization.

[0656] l-(3-bromo-4,5-difluorobenzyl)-4,4-difluoropiperidine (58)2930

[0657]

[0658] Compound 58 (Scheme 4) was synthesized according to general procedure B using 47 (1.82 g, 6.35 mmol, 1.0 equiv), 4,4-difluoropiperidine hydrochloride (1.0 g, 6.35 mmol, 1.0 equiv), and K2CO3 (1.05 g, 7.62 mmol, 1.2 equiv). Compound 58 was isolated as a yellow oil (1.66 g, 5.08 mmol, 80%). 'H NMR (500 MHz, CDCh): 67.28 (dt, J = 5.7, 2.0 Hz, 1 H), 7.14 (ddd, J = 10.5, 6.9, 2.0 Hz, 1 H), 3.46 (s, 2 H), 2.53 (t, J = 5.7 Hz. 4 H), 2.00 (ddd, J = 19.7, 11.9. 5.7 Hz. 4 H).13C NMR (126 MHz, CDCh): 6 150.8 (dd. JC-F = 253.3, 13.9 Hz), 147.1 (dd, JC-F = 249.5, 13.9 Hz), 136.3 (t, JC-F = 5.0 Hz), 128.0 (d, JC-F = 2.5 Hz), 121.98 (t, JC-F =

[0659] Page 77

[0660] QB\702581.02752\100011397.1241.9 Hz), 116.6 (d, JC-F = 17.6 Hz), 110.2 (d, JC-F = 17.6 HZ), 60.7 (d, JC-F = 1.3 Hz), 50.0 (t, JC-F = 5.7 Hz), 34.1 (t, JC-F = 23.3 Hz).

[0661] 6-(5-((4,4-difluoropiperidin-l-yl)methyl)-2,3-difluorophenyl)-4-methylpyridin-2-amine (59)

[0662]

[0663] Compound 59 (Scheme 4) was synthesized according to general procedures D, using 58 (1.00 g, 3.07 mmol, 1.0 equiv), PdCl2(dppf) (224 mg, 0.31 mmol, 10 mol%), bis(pinacolato)diboron (0.93 g, 3.68 mmol, 1.2 equiv), KOAc (0.90 g, 9.20 mmol, 3.0 equiv), and 6-bromo-4-methylpyridin-2-amine (574 mg, 3.07 mmol, 1.0 equiv), and K2CO3 (5.11 mL, 9.20 mmol, 1.8 M in water, 3.0 equiv). This resulted in the formation of coupled product 59 as a yellow viscous oil (823 mg, 2.33 mmol, 76%). ’H NMR (500 MHz, CDCh): 5 7.55 (dt, J = 6.3, 1.9 Hz, 1 H), 7.21 (ddd, J = 10.8, 7.2, 2.2 Hz, 1 H), 6.98 - 6.93 (m, 1 H), 6.36 (t, J = 1.0 Hz. 1 H). 4.60 (bs, 2 H, NH2), 3.53 (s, 2 H), 2.55 (t, J = 5.5 Hz, 4 H), 2.31 (s, 3 H), 2.09 - 1.91 (m, 4 H).13C NMR (126 MHz, CDCh): 5 158.4, 151.1 (dd, JC-F = 248.2, 13.9 Hz), 149.9, 149.8, 147.8 (dd, JC-F = 252.0, 13.9 Hz), 134.9 (t, JC-F = 5.0 Hz), 129.1 (d, JC-F = 8.8 Hz), 125.4 (dd, JC-F = 2.5, 1.3 HZ), 122.2 (t, JC-F = 242.0 Hz), 117.02 (d, JC-F = 17.6 HZ), 116.7 (d, JC-F = 7.6 Hz), 108.8, 61.2 (d, JC-F = 2.5 Hz), 50.0 (t, JC-F = 5.7 Hz). 34.1 (t. JC-F = 23.3 Hz), 21.4.

[0664] (3-bromo-4,5-difluorophenyl)methanol (61)

[0665]

[0666] F

[0667] Compound 61 (Scheme 5) was prepared following a reported procedure with slight modification.39-41To a stirred solution of 3-bromo-4,5-difluorobenzoic acid (60, 5.00 g, 21.09 mmol, 1 equiv) in THF (100 mL) was added BH3*SMe2 (3.00 mL. 31.65 mmol. 1.5 equiv) dropwise at 0 °C for 10 minutes under an argon atmosphere. After the addition, the resulting heterogeneous mixture was gradually warmed to 25 °C and stirred for 24 hours. After completion, as indicated by TLC, the reaction mixture was poured slowly over saturated Page 78

[0668] QB\702581.02752\100011397.1sodium bicarbonate and extracted it with Et2O (2 x 100 mL). The combined organic layers were washed with brine, dried over Na2SO4, filtered, and concentrated under reduced pressure. This resulted in the formation of alcohol 61 as a pale-yellow oil (4.47 mg, 20.02 mmol, 95%). The NMR spectral data matched those reported previously.39

[0669] l-bromo-5-(bromomethyl)-2,3-difluorobenzene (47)

[0670]

[0671] Compound 47 (Scheme 5) was prepared following a reported procedure with slight modification39-42In a 200 mL two-necked flask containing a stir bar, (3-bromo-4,5-difluorophenyl)methanol (61, 3.00 g, 13.45 mmol, 1.0 equiv), carbon tetrabromide (4.59 g, 17.49 mmol, 1.3 equiv), and 50 mL of anhydrous CH2CI2 were placed at 0 °C under an argon atmosphere. Next, a solution of triphenylphosphine (4.59 g, 17.49 mmol. 1.3 equiv) in 20 mL of CH2CI2 was added dropwise via a dropping funnel over 30 minutes with vigorous stirring. After the addition of triphenylphosphine at 0 °C, the reaction mixture was allowed to warm to 25 °C. The colorless solution turned to a pale-orange color and was stirred for an additional 24 hours at room temperature. Once the reaction was complete, as indicated by TLC, the reaction mixture was concentrated, and the resulting crude product was treated with excess hexanes (200 mL). The white precipitate was filtered, and the filtrate was concentrated under reduced pressure. The crude residue was purified by flash column chromatography using hexanes / EtOAc (80:20, v / v) to yield compound 47 as a yellow oil (3.42 g, 11.96 mmol, 89%). The NMR spectral data matched those reported previously.39

[0672] 2-(3-bromo-4,5-difluorophenyl)acetonitrile (62)

[0673]

[0674] Compound 62 (Scheme 5) was prepared following a reported procedure with slight modifications.39-43A 250 mL round bottom flask was charged with 47 (3.42 g, 11.96 mmol, 1 equiv), potassium cyanide (0.94 g, 14.35 mmol), 18-crown-6 (10 mg, Cat.,) and tetrabutylammonium bromide (386 mg, 1.20 mmol) under an argon atmosphere. To this, a mixture of CH2CI2 and water in a 1: 1 ratio (60 mL, 0.2 M) was added, and the resulting biphasic Page 79

[0675] QB\702581.02752\100011397.1mixture was stirred vigorously for 24 h at room temperature. After completion, as indicated by TLC. the organic phase was carefully separated from the aqueous phase and collected in a conical flask (Caution: care must be taken while handling this reaction due to cyanide poisoning. Appropriate safety protocol must be followed and recommended). The aqueous phase was extracted with CH2CI2 (2 x 30 rnL). The combined organic layers were washed with brine, dried over Na2SO4, filtered, and concentrated under reduced pressure giving compound 62 as a brown solid (2.34 g, 10.0 mmol, 84%). The crude was taken to the next step without further purification. The waste phases (aqueous and brine) were carefully treated with excess bleach (sodium hypochlorite) at pH 11-14, and the resulting mixture was stirred for 24 at room temperature before disposal into a cyanide waste container.

[0676] 2-(3-bromo-4,5-difluorophenyl)ethan- 1 -amine (63)

[0677] Brx

[0678] 63

[0679] F

[0680]

[0681] Compound 63 (Scheme 5) was prepared following a reported procedure with slight modifications.39,44To a solution of 62 (2.30 g, 9.91 mmol, 1.0 equiv) in THF (50 mL, 0.2 M) was added BH3*SMe2 (2.82 mL, 29.74 mmol, 3.0 equiv) dropwise at 0 °C for 10 min under an argon atmosphere. After the addition, the resulting mixture was gradually warmed to 25 °C and stirred for 30 h. After completion of the reaction, as indicated by TLC, the reaction mixture was carefully quenched with CH3OH (30 rnL) and then 6 M HC1 in water (10 mL) at 0 °C and concentrated under reduced pressure. The crude ammonium salt was then dissolved in water (50 mL) and washed with EtOAc (2 x 30 mL). The aqueous layer was neutralized with 10% NaOH (pH 11). extracted with EtOAc (2 x 50 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure. The crude product was further purified by flash column chromatography, eluting with hexanes / EtOAc (50:50, v / v) for 10 min to remove any nonpolar residues and followed by the solvent mixture (CH2Cl2 / CH3OH / Et3N, 70:20:10, v / v) for another 15 min to afford 63 a yellow oil (1.71 g, 7.24 mmol, 73%). LRMS (ESI) Calcd for C8H9BrF2N [(M+H)+]: 235.99, found: 236.89. The NMR spectral data matched those reported previously.39tert-butyl (3-bromo-4,5-difluorophenethyl)carbamate (64)

[0682] Page 80

[0683] QB\702581.02752\100011397.1''BOG

[0684]

[0685] Compound 64 (Scheme 5) was synthesized according to general procedure C using 63 (1.00 g, 4.24 mmol, 1.0 equiv), BOC2O (1.11 g, 5.08 mmol, 1.2 equiv), and EtsN (1.18 mL, 8.47 mmol, 2.0 equiv). Compound 64 w as isolated as a pale-yellow' solid (0.90 g, 2.57 mmol, 61%).1H NMR (500 MHz, CDCl3): δ 7.15 (dt, J = 5.6, 1.9 Hz, 1 H), 6.96 (ddd, J = 10.5, 6.8, 2.1 Hz, 1 H), 4.56 (bs, 1 H, NH), 3.35 – 3.31 (m, 2 H), 2.76 – 2.73 (m, 2 H), 1.43 (s, 9 H).

[0686] tert- butyl (3-bromo-4,5-difluorophenethyl)(methyI)carbamate (65)

[0687] 65

[0688]

[0689] A 100 mL two-necked flask with a stir bar was filled with sodium hydride (71.4 mg, 1.79 mmol, 3.0 equiv. 60% dispersion in mineral oil) and dry DMF (2 mL) under an argon atmosphere. The mixture was cooled to 0 °C in an ice bath and stirred for 5 minutes. Following this, a solution of 64 (200 mg, 0.59 mmol, 1.0 equiv) in DMF (1 mL) was slowly added dropwise over 10 minutes, leading to a rapid evolution of hydrogen gas. The resulting mixture was stirred for 30 minutes at 0 °C and then for 10 minutes at room temperature. Afterward, the reaction mixture was again cooled to 0 °C in an ice bath, and methyl iodide (74 pL, 1.19 mmol, 2.0 equiv) was added dropwise over 5 minutes. The reaction mixture was allowed to warm up to room temperature and continued to be stirred for 24 hours. A color change from brown to dark brown to pale-brown was observed after the addition of methyl iodide. Upon completion of the reaction as indicated by TLC. the reaction mixture was carefully quenched with water. The resulting mixture was then extracted twice with EtOAc (2 x 50 mL). The combined organic layers were washed with water, dried over Na2SO4, filtered, and concentrated under vacuum. The crude product was further purified by flash column chromatography on silica gel, eluting with hexanes for 10 min and followed by hexanes / EtOAc (80:20, v / v) for 15 min to yield the product as yellow oil (143 mg, 0.41 mmol, 69%). LRMS (ESI) Calcd for C14H19BrF2NO2[(M+H)+]: 350.06, found: 350.39. The product was taken to the next step without further characterization.

[0690] Page 81

[0691] QB\702581.02752\100011397.1tert-butyl (3-(6-amino-4-methylpyridin-2-yl)-4,5-difluorophenethyl)(methyl)carbamate (66)

[0692]

[0693] Compound 66 (Scheme 5) was synthesized according to general procedures D, using 65 (0.20 g, 0.57 mmol, 1.0 equiv), PdCl2(dppf) (41.8 mg, 0.06 mmol, 10 mol%), bis(pinacolato)diboron (174 mg, 0.69 mmol, 1.2 equiv), KOAc (0.17 g, 1.71 mmol, 3.0 equiv), and 6-bromo-4-methylpyridin-2-amine (106.8 mg, 0.57 mmol, 1.0 equiv), and K2CO3 (0.95 mL, 1.71 mmol, 1.8 M in water, 3.0 equiv). This resulted in the formation of coupled product 66 as a pale-yellow solid (177 mg, 0.47 mmol, 82%). LRMS (ESI) Calcd for C20H26F2N3O2[(M+H)+]: 378.20, found: 378.32. The product was taken to the next step without further characterization.

[0694] 11. References

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[0697] (3) Roman, L. J.; Sheta, E. A.; Martasek, P.; Gross, S. S.; Liu, Q.; Masters, B. S. High-level expression of functional rat neuronal nitric oxide synthase in Escherichia coli. Proc. Natl. Acad. Sci. U. S. A. 1.905, 92, 8428-8432.

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[0743] QB\702581.02752\100011397.1

Claims

CLAIMSI / We claim:

1. A compound of formula (I), or a pharmaceutically acceptable salt, hydrate, or solvate thereof,whereinX is CR3, orN,each R3is independently H, halo, cyano, or haloalkyl,n is 0, 1, or 2,m is 1 or 2, and(i) R1is H or alkyl and R2is alkyl or haloalkyl, or(ii) R1and R2together with the nitrogen atom they are attached to form a heterocycloalkyl substituted with 0-2 halo.

2. The compound of claim 1 having a formula of (la).R2R3(la)3. The compound of claim 1 or 2, wherein m is 1.

4. The compound of any one of claims 1-3. wherein each R3is independently halo.

5. The compound of claim 4, wherein R3is fluoro.Page 87QB\702581.02752\100011397.

16. The compound of any one of claims 1-3. wherein R3is selected from H and halo.

7. The compound of any one of claims 1-6, wherein R1is H and R2is alkyl.

8. The compound of any one of claims 1-6, wherein R1is H and R2is haloalkyl.

9. The compound of any one of claims 1-6. wherein R1is alkyl and R2is alkyl.

10. The compound of any one of claims 1-6, wherein R1and R2together with the nitrogen atom they are attached to form a 4- or 6-membered heterocycloalkyl substituted with 2 halo.

11. The compound of claim 1 having a formula of (lb).R2(lb)12. The compound of claim 11, wherein m is 1.

13. The compound of claim 11, wherein R1is H and R2is alkyl.

14. The compound of claim 1, or a pharmaceutically acceptable salt, hydrate, or solvate thereof, wherein the compound isPage 88QB\702581.02752\100011397.1QB\702581.02752\100011397.

115. The compound of claim 1, or a pharmaceutically acceptable salt, hydrate, or solvate thereof, wherein the compound isF16. The compound of any one of claims 1-15, wherein the compound adopts the same binding mode to mNOS and hnNOS.

17. The compound of any one of claims 1-16, wherein the compound is selective for human nNOS over human endothelial NOS (eNOS).

18. The compound of any one of claims 1-17, wherein the compound is selective for human nNOS over human inducible NOS (iNOS).

19. The compound of any one of claims 1-18, wherein the compound has a Ki value of human nNOS that is less than 100 nM, calculated using the Cheng-Prusoff equation.Page 90QB\702581.02752\100011397.

120. The compound of any one of claims 1-19, wherein the effective permeability (Pe) for the blood brain barrier of the compound is at least 5.0 x 10’6cm s’1.

21. A pharmaceutical composition comprising the compound according to any one of claims 1-20 and a pharmaceutically acceptable excipient, carrier, or diluent.

22. A method for treating a disease or disorder associated with neuronal nitric oxide synthase (nNOS) activity in a subject in need thereof, the method comprising administering an effective amount of the compound of any one of claims 1-20, or a pharmaceutically acceptable salt, hydrate, or solvate thereof; or the pharmaceutical composition of claim 21 to the subject.

23. The method of claim 22, wherein the disease or disorder is a neurological disease or disorder, or melanoma.

24. The method of claim 23, wherein the neurological disease or disorder is Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), chronic headache, or neuronal damage during stroke.

25. The method of claim 23. wherein the disease or disorder is melanoma.Page 91QB\702581.02752\100011397.1

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