Potent human neuronal nitric oxide synthase inhibitors

Abstract

To provide 2-aminopyridine derivative compounds for use as inhibitors of nitric oxide synthase.SOLUTION: Disclosed are 2-aminopyridine derivative compounds for use as inhibitors of nitric oxide synthase (NOS). In particular, the field of the present invention relates to 2-aminopyridine derivative compounds for use as inhibitors of neuronal nitric oxide synthase (nNOS), which are formulated as pharmaceutical compositions for treating nNOS-associated diseases and disorders such as Alzheimer's disease, Parkinson's disease, and Huntington's disease, as well as amyotrophic lateral sclerosis, cerebral palsy, stroke / ischemic brain injury, and migraine headaches.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] Statement on Federally Sponsored Research and Development This invention was made with government support under Grant No. GM049725 awarded by the National Institutes of Health (NIH). The U.S. government has certain rights in this invention.

Background Art

[0002] Background The field of the present invention relates to 2-aminopyridine derivative compounds for use as inhibitors of nitric oxide synthase. In particular, the field of the present invention relates to 2-aminopyridine derivative compounds for use as inhibitors of nitric oxide synthase formulated as pharmaceutical compositions for the treatment of neurological diseases or disorders including, but not limited to, Alzheimer's disease, Parkinson's disease, and Huntington's disease, amyotrophic lateral sclerosis, cerebral palsy, stroke / ischemic brain injury, and migraine.

[0003] In some aspects, the disclosed subject matter relates to methods of treating neurological diseases and disorders, particularly neurodegenerative diseases. Neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and Huntington's disease, which are generally known, are characterized by the gradual degeneration and death of neurons in the central nervous system (CNS) that cause problems with a patient's muscle movement and mental function. Despite an urgent medical need, comprehensive treatment of these diseases remains very limited. 1,2 One of the most difficult challenges in CNS drug discovery is the effective delivery of therapeutic agents to the human brain, mainly due to the presence of the blood-brain barrier (BBB) located mainly at the boundary between blood vessels and brain tissue. 3 The BBB is composed of a layer of endothelial cells with tight junctions that prevent access of external toxins, thereby protecting the brain and maintaining its optimal physiological environment. However, this cell layer also limits access of valuable therapeutic agents to the brain. 4The major route for CNS drugs to cross the BBB is passive diffusion through its lipid membrane. In addition to the tight junctions of endothelial cells, high expression levels of efflux transporters on the BBB, particularly P-glycoprotein (P-gp), greatly contribute to the limited brain exposure of CNS drugs. 5 Consequently, establishing strategies that include a combination of increasing passive permeability and reducing P-gp-mediated efflux is required in CNS drug discovery. 3,6,7 。

[0004] Neuronal nitric oxide synthase (nNOS) has been identified as a promising therapeutic target in the development of novel treatments for neurodegenerative diseases. 8-10 In the brain, nitric oxide (NO) produced by nNOS participates in neuronal signaling. 11 However, excessive production of intracellular NO is harmful. In particular, excessive NO formed by overactivated nNOS in the CNS can cause excessive nitration and nitrosylation of proteins, which leads to their misfolding and aggregation. 12 In addition, the reaction of superoxide anion with NO generates peroxynitrite, a powerful oxidant that damages DNA and causes lipid peroxidation. These processes lead to neuronal cell death and dysfunction in neuronal signaling. 13,14 Therefore, limiting NO production by inhibiting nNOS is beginning to emerge as an essential approach that may protect neurons and cure certain degenerative diseases. 15,16 。

[0005] nNOS is a homodimeric enzyme, and each monomer contains one C-terminal reductase domain and one N-terminal oxygenase domain. The C-terminal reductase domain contains nicotinamide adenine dinucleotide phosphate (NADPH), flavin adenine dinucleotide (FAD), and flavin mononucleotide (FMN), whereas the N-terminal oxygenase domain contains non-catalytic zinc, tetrahydrobiopterin (H4B), and heme. These two domains are linked to each other by a calmodulin domain. When dimerization occurs, the electron flow from the reductase domain to the oxygenase domain is promoted, through which L-Arg is oxidized to L-Cit and NO is released. 15、17 Propelling a molecule that competes with the L-Arg binding at the active site of this enzyme is one of the basic approaches for inhibiting nNOS. 16 The challenge of this task is related not only to the efficacy of nNOS inhibitors but also to their binding selectivity to nNOS over two isoforms, eNOS and iNOS, which share very similar structural features with it. 18,19 Since eNOS inhibition can cause circulatory insufficiency while iNOS inhibition can trigger immune system disruption, it is necessary to avoid excessive inhibition of both eNOS and iNOS. 20 .

[0006] In recent years, the inventors' efforts to achieve nNOS inhibitors with excellent efficacy and high isoform activity have led to a promising molecular class possessing a 2-aminopyridine scaffold. Using this molecular scaffold, the inventors obtained nNOS inhibitors that showed excellent activity at concentrations in the <30 nM range. 15,21,22 However, the first generation of nNOS inhibitors possessing a 2-aminopyridine scaffold showed expected poor permeation through the BBB, as revealed by very low Caco-2 permeability. 23。Recently, the present inventors have been able to improve the cell membrane permeability of 2-aminopyridine nNOS inhibitors while retaining their high inhibitory activity against nNOS. The inventors first identified the lead compound (1, Figure 1), which showed excellent efficacy and selectivity against human nNO (K i hnNOS = 30 nM; hnNOS / heNOS = 2799), and also showed an excretion ratio (ER) of 5.9 in the Caco-2 assay 24 。To progress towards CNS drug discovery, the cell membrane permeability of 2-aminopyridine nNOS inhibitors must be further improved to accompany an ER < 2.5, which is required for a promising CNS(+) agent 7,25 。

Summary of the Invention

[0007] Overview 2-Aminopyridine compounds, pharmaceutical compositions, and methods for treating diseases or disorders associated with nitric oxide synthase (NOS) activity are disclosed. Diseases and disorders treatable by the compounds, pharmaceutical compositions, and methods of the present disclosure can include neurological diseases or disorders. Neurological diseases or disorders treatable by the disclosed 2-aminopyridine compounds include, but are not limited to, neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and Huntington's disease, as well as amyotrophic lateral sclerosis, cerebral palsy, stroke / ischemic brain injury, and migraine headache

[0008] The disclosed compounds include derivatives of 2-aminopyridine. The disclosed compounds can have the following formula:

[0009]

Chemical Formula

[0010] In the formula,

[0011]

Chemical Formula

[0012] represents a single bond, double bond, or triple bond; Y is a substituted aryl (e.g., substituted phenyl) or substituted heteroaryl (e.g., substituted quinolinyl such as substituted quinolin-3-yl), and Y is, at one or more ring positions, substituted by a substituent having the formula -X-R a wherein X is selected from C1-C6-alkyl, C1-C6-alkenyl, and C1-C6-alkynyl; R a is amino, alkylamino (e.g., methylamino), dialkylamino (e.g., dimethylamino), or a 4- to 6-membered heterocycle containing at least one nitrogen atom and whose heterocycle is optionally substituted at one or more positions by alkyl (e.g., methyl), alkoxy (e.g., ethoxy), or halo (e.g., fluoro); and, Y is optionally substituted at one or more ring positions by halo (e.g., 2,3-difluoro-phenyl). Also contemplated are salts of the disclosed compounds, including pharmaceutically acceptable salts of the disclosed compounds. Solvates of the disclosed compounds are also contemplated.

[0013] Specifically, the disclosed compounds can have the formula (I):

[0014]

Chemical formula

[0015] wherein R 2 , R 3 , R 4 and R 6 are each independently H or halogen (e.g., fluoro).

[0016] The disclosed compounds can be formulated as pharmaceutical compositions containing the compound or a pharmaceutically acceptable salt thereof in a pharmaceutically acceptable carrier for use in a method of treatment for a subject in need thereof. In some embodiments, the disclosed compounds and pharmaceutical compositions can be utilized to treat a disease or disorder associated with nitric oxide synthase activity. In particular, the disclosed compounds and pharmaceutical compositions can be utilized to inhibit nitric oxide synthase in a subject in need thereof and to treat a disease or disorder associated with nitric oxide synthase activity. In some embodiments, the disclosed compounds and pharmaceutical compositions can be utilized to treat a neurological disease or disorder in a subject in need thereof. In particular, the disclosed compounds and pharmaceutical compositions can be utilized to treat Alzheimer's disease, Parkinson's disease and / or Huntington's disease, amyotrophic lateral sclerosis (ALS), cerebral palsy, and migraine.

Brief Description of the Drawings

[0017]

Figure 1

Mode for Carrying Out the Invention

[0018] Detailed Description The present invention will be described using several definitions throughout this specification as set forth below.

[0019] Definitions

[0020] The disclosed subject matter may be further described using the following definitions and terms. The definitions and terms used herein are for the purpose of merely describing particular embodiments and are not intended to be limiting.

[0021] As used in this specification and the claims, the singular forms “a,” “an,” and “the” include the plural unless the context clearly dictates otherwise. For example, the term “a substituent” should be construed to mean “one or more substituents” unless the context clearly dictates otherwise.

[0022] As used herein, “about,” “approximately,” “substantially,” and “significantly” are understood by those of ordinary skill in the art and will vary to some extent depending on the context in which they are used. Where there is ambiguity in the use of terms not clear to those of ordinary skill in the art in the context in which they are used, “about” and “approximately” shall mean plus or minus up to 10% of the particular term, and “substantially” and “significantly” shall mean plus or minus more than 10% of the particular term.

[0023] As used in this specification, the terms “comprising” and “including” have the same meaning as the terms “containing” and “having.” The terms “containing” and “having” should be construed as open-ended conjunctions that allow for the inclusion of additional elements beyond those recited in the claims. The terms “consisting of” and “consisting essentially of” should be construed as closed-ended conjunctions that do not allow for the inclusion of additional elements beyond those recited in the claims. The term “consisting essentially of” should be construed as being partially restrictive and allowing for the inclusion of additional elements only if they do not materially change the nature of the claimed subject matter.

[0024] The phrase "etc." should be interpreted as "including, for example". Further, the use of any and all illustrative phrases, including but not limited to "etc.", is merely intended to clarify the present invention better and, unless otherwise claimed, does not result in a limitation of the scope of the present invention.

[0025] Furthermore, in cases where a convention similar to "at least 1 of A, B, and C etc." is used, generally, such a construction is intended to mean that those skilled in the art will understand the convention (for example, "a system having at least 1 of A, B, and C" includes, but is not limited to, a system having only A, only B, only C, A and B together, A and C together, B and C together, and / or A, B, and C together). Further, it will be understood by those skilled in the art that in either the description or the drawings, virtually any adjacent words and / or adjacent phrases representing two or more alternative terms are intended to contemplate the possibility of including one of those terms, any of those terms, or both of those terms. For example, the phrase "A or B" will be understood to include the possibilities of "A" or "B" or "A and B".

[0026] All words such as "up to", "at least", "greater than", "less than", and the like include the recited numerical value and refer to a range that can then be subdivided into ranges and sub-ranges. The range includes each individual numerical value. Thus, for example, a group having members from 1 to 3 refers to a group having members 1, 2, or 3. Similarly, a group having 6 members refers to a group having members 1, 2, 3, 4, or 6, etc.

[0027] The modal verb "may" refers to the preferred use and selection of one or more options or alternatives among some of the described embodiments or features included therein. If an option or alternative is not specified with respect to a particular embodiment or feature included therein, the modal verb "may" refers to positive measures regarding how to make or use the described embodiment or feature included therein and aspects, or refers to a definitive determination regarding the use of a particular technique with respect to the described embodiment or feature included therein. In this latter context, the modal verb "may" has the same meaning and implication as the auxiliary verb "can".

[0028] As used herein, "subject in need thereof" refers to a subject in need of treatment for a disease or disorder associated with neuronal nitric oxide synthase (nNOS) activity and / or nitric oxide (NO) level, such as a disease or disorder in which an elevated NO level is undesirable. The term "subject" may be used interchangeably with the terms "individual" and "patient", and includes subjects that are human and non-human mammals.

[0029] Diseases and disorders associated with nNOS activity may include, but are not limited to, neurological diseases and disorders. Neurological diseases and disorders may include, but are not limited to, neurodegenerative diseases and disorders such as Alzheimer's disease, Parkinson's disease, and Huntington's disease, as well as amyotrophic lateral sclerosis, cerebral palsy, stroke / ischemic brain injury, and migraine headache.

[0030] The disclosed compounds may be used, without limitation, to modulate enzymatic activity including nNOS activity. The term "modulate" should be understood broadly to include "inhibit" enzymatic activity and / or otherwise modulate enzymatic activity.

[0031] New Chemical Entities

[0032] Novel chemical entities are disclosed herein and may be described using terms known in the art and defined herein.

[0033] As used herein, the term "alkyl" refers to a saturated, straight or branched hydrocarbon, e.g., C1-C 12 alkyl, C1-C 10 -alkyl, and C1-C6-alkyl, each referring to a straight or branched group of 1 to 12, 1 to 10, or 1 to 6 carbon atoms, respectively.

[0034] The term "alkylene" refers to the diradical of an alkyl group. An exemplary alkylene group is -CH2CH2-.

[0035] The term "haloalkyl" refers to an alkyl group substituted by at least one halogen, e.g., -CH2F, -CHF2, -CF3, -CH2CF3, -CF2CF3, etc.

[0036] As used herein, the term "heteroalkyl" refers to an "alkyl" group in which at least one carbon atom is replaced by a heteroatom (e.g., an O, N, or S atom). One type of heteroalkyl group is an "alkoxyl" group.

[0037] As used herein, the term "alkenyl" refers to an unsaturated, straight or branched hydrocarbon having at least one carbon-carbon double bond, e.g., C2-C 12 -alkenyl, C2-C 10 -alkenyl, and C2-C6-alkenyl, each referring to a straight or branched group of 2 to 12, 2 to 10, or 2 to 6 carbon atoms, respectively. "Cycloalkene" is a compound having a ring structure (e.g., of 3 or more carbon atoms) and containing at least one double bond.

[0038] As used herein, the term "alkynyl" refers to an unsaturated, straight or branched hydrocarbon having at least one carbon-carbon triple bond, e.g., C2-C 12 -alkynyl, C2-C 10-alkynyl, and C2-C6-alkynyl, each refers to a linear or branched group having 2 to 12, 2 to 10, or 2 to 6 carbon atoms, respectively.

[0039] The term "cycloalkyl" refers to, for example, a monovalent saturated cyclic, bicyclic, or bridged cyclic (e.g., adamantyl) hydrocarbon group of 3 to 12, 3 to 8, 4 to 8, or 4 to 6 carbons, such as "C 4-8 -cycloalkyl" as referred to herein. Unless otherwise specified, the cycloalkyl group is optionally substituted at one or more ring positions, for example, by alkanoyl, alkoxy, alkyl, haloalkyl, alkenyl, alkynyl, amide, amidino, amino, aryl, arylalkyl, azide, carbamate, carbonate, carboxy, cyano, cycloalkyl, ester, ether, formyl, halogen, haloalkyl, heteroaryl, heterocyclyl, hydroxyl, imino, ketone, nitro, phosphate, phosphonato, phosphinato, sulfate, sulfide, sulfonamide, sulfonyl, or thiocarbonyl, etc. In some embodiments, the cycloalkyl group is unsubstituted, i.e., it is non-substituted.

[0040] The term "cycloalkylene" refers to the diradical of a cycloalkyl group.

[0041] The term "partially unsaturated carbocyclic" refers to a monovalent cyclic hydrocarbon in which at least one ring of the carbocyclic is not aromatic and contains at least one double bond between ring atoms. The partially unsaturated carbocyclic may be characterized according to the number of ring carbon atoms. For example, the partially unsaturated carbocyclic can contain 5 to 14, 5 to 12, 5 to 8, or 5 to 6 ring carbon atoms, and accordingly, is referred to as a 5- to 14-, 5- to 12-, 5- to 8-, or 5- to 6-membered partially unsaturated carbocyclic, respectively. The partially unsaturated carbocyclic can be in the form of a monocyclic carbocyclic, bicyclic carbocyclic, tricyclic carbocyclic, bridged carbocyclic, spirocyclic carbocyclic, or other carbocyclic system. Illustrative partially unsaturated carbocyclic groups include cycloalkenyl groups and bicyclic carbocyclic groups that are partially unsaturated. Unless otherwise specified, the partially unsaturated carbocyclic group is optionally substituted at one or more ring positions, for example, by alkanoyl, alkoxy, alkyl, haloalkyl, alkenyl, alkynyl, amide, amidino, amino, aryl, arylalkyl, azide, carbamate, carbonate, carboxy, cyano, cycloalkyl, ester, ether, formyl, halogen, haloalkyl, heteroaryl, heterocyclyl, hydroxyl, imino, ketone, nitro, phosphate, phosphonato, phosphinato, sulfate, sulfide, sulfonamide, sulfonyl, or thiocarbonyl, etc. In some embodiments, the partially unsaturated carbocyclic is unsubstituted, i.e., it is non-substituted.

[0042] The term "aryl" is approved in the art and refers to a carbocyclic aromatic group. Representative aryl groups include phenyl, naphthyl, anthracenyl, etc. The term "aryl" includes polycyclic ring systems having two or more carbocyclic rings where two or more carbons are shared with two adjacent rings (these rings are "fused rings"), where at least one of these rings is aromatic, and where, for example, the other ring(s) can be cycloalkyl, cycloalkenyl, cycloalkynyl, and / or aryl. Unless otherwise specified, this aromatic ring can be substituted at one or more ring positions, for example, by halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl, amino, nitro, sulfhydryl, imino, amide, carboxylic acid, -C(O)alkyl, -CO2alkyl, carbonyl, carboxyl, alkylthio, sulfonyl, sulfonamide, sulfonamide, ketone, aldehyde, ester, heterocyclyl, aryl or heteroaryl moieties, -CF3, -CN, etc. In some embodiments, the aromatic ring is substituted at one or more ring positions by halogen, alkyl, hydroxyl, or alkoxyl. In some other embodiments, the aromatic ring is unsubstituted, i.e., it is non-substituted. In some embodiments, the aryl group has a 6- to 10-membered ring structure.

[0043] The terms "heterocyclyl" and "heterocyclic group" are approved in the art and refer to saturated, partially unsaturated, or aromatic 3- to 10-membered ring structures, or 3- to 7-membered rings, whose ring structure contains 1 to 4 heteroatoms, such as nitrogen, oxygen, and sulfur, etc. The number of ring atoms of the heterocyclic group can be specified using the 5 Cx-Cx nomenclature, where x is an integer specifying the number of ring atoms. For example, a C3-C7 heterocyclic group refers to a saturated or partially unsaturated 3- to 7-membered ring structure containing 1 to 4 heteroatoms, such as nitrogen, oxygen, and sulfur. The definition "C3-C7" indicates that the heterocycle contains any heteroatoms occupying ring atom positions and a total of 3 to 7 ring atoms.

[0044] The terms "amine" and "amino" are approved in the art and refer to both unsubstituted and substituted amines, where the substituents may include, for example, alkyl, cycloalkyl, heterocyclyl, alkenyl, and aryl.

[0045] The term "alkoxyl" or "alkoxy" is approved in the art and refers to an alkyl group as defined above having an oxygen radical attached thereto. Representative alkoxyl groups include methoxy, ethoxy, tert-butoxy, and the like.

[0046] "Ether" is two hydrocarbons covalently linked by oxygen. Thus, the substituents of alkyl that make it an ether may be alkoxyl or similar thereto and may be represented by one of, for example, -O-alkyl, -O-alkenyl, -O-alkynyl, and the like.

[0047] The term "carbonyl" as used herein refers to the radical -C(O)-.

[0048] The term "carboxy" or "carboxyl" as used herein refers to the radical -COOH or its corresponding salt, such as -COONa and the like.

[0049] The term "amide" or "amide" or "carboxamide" as used herein refers to a radical of the form -R 1 C(O)N(R 2 )-, -R 1 C(O)N(R 2 )R 3 -, -C(O)NR 2 R 3 , or -C(O)NH2, where R 1 , R 2 and R 3Each is independently alkoxy, alkyl, alkenyl, alkynyl, amide, amino, aryl, arylalkyl, carbamate, cycloalkyl, ester, ether, formyl, halogen, haloalkyl, heteroaryl, heterocyclyl, hydrogen, hydroxyl, ketone, or nitro.

[0050] The compounds of the present disclosure can contain one or more chiral centers and / or double bonds and thus exist as stereoisomers, such as geometric isomers, enantiomers or diastereomers. As used herein, the term "stereoisomer" encompasses all geometric isomers, enantiomers or diastereomers. These compounds can be designated by the symbols "R" or "S" depending on the arrangement of the substituents around the stereogenic carbon atoms. The present invention encompasses various stereoisomers of these compounds and mixtures thereof. Stereoisomers include enantiomers and diastereomers. A mixture of enantiomers or diastereomers is designated "(±)" in the nomenclature, but those skilled in the art will recognize that the structure may implicitly represent chiral centers. It is understood that chemical structures, for example, illustrations of general chemical structures, encompass all stereoisomeric forms of the specified compound unless otherwise specified.

[0051] Pharmaceutical Compositions

[0052] The compounds utilized in the compositions and methods disclosed herein can be administered as pharmaceutical compositions, and thus pharmaceutical compositions incorporating such compounds are considered embodiments of the compositions disclosed herein. Such compositions can take any physically acceptable form as a medicament; specifically, these can be pharmaceutical compositions that can be administered orally. Such pharmaceutical compositions contain an effective amount of the disclosed compound, and this effective amount is related to the daily dose of the compound to be administered. Each dosage unit can contain the daily dose of a given compound, or each dosage unit can contain a fraction of the daily dose, such as 1 / 2 or 1 / 3 of the daily dose. The amount of each compound to be included in each dosage unit is determined in part by the identity of the particular compound selected for therapy and by other factors such as the indication for which it is given. The pharmaceutical compositions disclosed herein can be formulated, by utilizing well-known procedures, to provide immediate release, sustained release, or delayed release of the active ingredient after administration to a patient.

[0053] The compounds for use in accordance with the methods disclosed herein can be administered as a single compound or as a combination of compounds. For example, a compound that inhibits neuronal nitric oxide synthase can be administered as a single compound or in combination with another compound that inhibits neuronal nitric oxide synthase or has a different pharmacological activity.

[0054] As shown above, pharmaceutically acceptable salts of the present compounds are contemplated and may be utilized in the methods also disclosed herein. As used herein, the term "pharmaceutically acceptable salt" refers to salts of the present compounds which are substantially non-toxic to a living organism. Representative pharmaceutically acceptable salts include those salts prepared by reaction of the compounds disclosed herein with a pharmaceutically acceptable mineral or organic acid or organic or inorganic base. Such salts are known as acid addition salts and base addition salts. It will be understood by those skilled in the art that most or all of the compounds disclosed herein are capable of forming salts, and that the salt form of pharmaceuticals is commonly used because they are often more readily crystallized and purified than the free acid or free base.

[0055] Acids commonly used to form acid addition salts include inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, etc., and organic acids such as p-toluenesulfonic acid, methanesulfonic acid, oxalic acid, p-bromophenylsulfonic acid, carbonic acid, succinic acid, citric acid, benzoic acid, acetic acid, etc. Examples of suitable pharmaceutically acceptable salts can include sulfates, pyrosulfates, bisulfates, sulfites, bisulfites, phosphates, monohydrogen phosphates, dihydrogen phosphates, metaphosphates, pyrophosphates, bromides, iodides, acetates, propionates, decanoates, caprylates, acrylates, formates, hydrochlorides, dihydrochlorides, isobutyrates, caproates, heptanoates, propionates, oxalates, malonates, succinates, suberates, sebacates, fumarates, maleates, butyne-1,4-dioates, hexyne-1,6-dioates, benzoates, chlorobenzoates, methylbenzoates, hydroxybenzoates, methoxybenzoates, phthalates, xylenesulfonates, phenylacetates, phenylpropionates, phenylbutyrates, citrates, lactates, α-hydroxybutyrates, glycolates, tartrates, methanesulfonates, propanesulfonates, naphthalene-1-sulfonates, naphthalene-2-sulfonates, mandelates, etc.

[0056] The base addition salts include those derived from inorganic bases such as ammonium or hydroxides, carbonates, bicarbonates of alkali or alkaline earth metals, etc. Bases useful for preparing such salts include sodium hydroxide, potassium hydroxide, ammonium hydroxide, potassium carbonate, sodium carbonate, sodium bicarbonate, potassium bicarbonate, calcium hydroxide, calcium carbonate, etc.

[0057] The specific counterions that form part of any salt of the compounds disclosed herein are generally not critical to the activity of the compounds as long as the salts are pharmaceutically acceptable and the counterions generally do not contribute undesirable qualities to the salts. Undesirable qualities can include undesirable solubility or toxicity.

[0058] Pharmaceutically acceptable esters and amides of the compounds can also be utilized in the compositions and methods disclosed herein. Examples of suitable esters include alkyl, aryl, and aralkyl esters such as methyl ester, ethyl ester, propyl ester, dodecyl ester, benzyl ester, etc. Examples of suitable amides include unsubstituted amides, mono-substituted amides, and di-substituted amides such as methyl amide, dimethyl amide, methyl ethyl amide, etc.

[0059] In addition, the methods disclosed herein can be practiced using solvate forms of the compounds or their salts, esters, and / or amides. Solvate forms can include ethanol solvate, hydrate, etc.

[0060] This pharmaceutical composition can be used in a method for treating a disease or disorder associated with neuronal nitric oxide synthase activity. As used herein, the terms "treating" or "treatment" each mean alleviating symptoms, eliminating the cause of symptoms that occur either temporarily or permanently, and / or preventing or delaying the onset of symptoms resulting from the named disease or disorder or reversing its progression or severity. Accordingly, the methods disclosed herein encompass both therapeutic and prophylactic administration.

[0061] As used herein, the term "effective amount" refers to the amount or dosage of the compound upon administration of a single or repeated dose to a subject that provides the desired effect to the subject being diagnosed or treated. The disclosed methods can include administering an effective amount of the disclosed compound (such as as present in a pharmaceutical composition) for treating a disease or disorder associated with neuronal nitric oxide synthase activity.

[0062] The effective amount can be readily determined by a diagnostician acting as a person skilled in the art, using known techniques and by observing results obtained in similar circumstances. In determining the effective amount or dosage of the compound to be administered, numerous factors can be considered by the diagnostician in charge, such as: the subject's race; their build, age, and general state of health; the degree or severity of involvement of the disease or disorder involved; the individual subject's response; the particular compound being administered; the mode of administration; the bioavailability characteristics of the preparation being administered; the dosing regimen selected; the use of concomitant medications; and other related circumstances.

[0063] A representative daily dose can 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.

[0064] The composition can be formulated into unit dosage forms, and each dosage contains about 1 to about 500 mg, such as about 5 to about 300 mg, about 10 to about 100 mg, and / or about 25 mg of each compound, either individually or in a single unit dosage form. The term "unit dosage form" refers to physically discrete units suitable as unit dosages for a patient, and each unit contains a predetermined amount of the active substance calculated to produce the desired therapeutic effect, together with a suitable pharmaceutical carrier, diluent, or excipient.

[0065] Oral administration is an exemplary route of administration for the compounds utilized in the compositions and methods disclosed herein. Other exemplary routes of administration include transdermal, subcutaneous, intravenous, intramuscular, intranasal, intraoral, intrathecal, intracerebral, or rectal routes. The route of administration may be varied in any manner limited by the physical characteristics of the compound utilized and the convenience of the subject and caregiver.

[0066] As will be understood by those skilled in the art, suitable formulations include those suitable for two or more routes of administration. For example, the formulation can be suitable for both intrathecal and intracerebral administration. Alternatively, suitable formulations include those suitable for only one route of administration, as well as those 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 suitable for oral, transdermal, subcutaneous, intravenous, intramuscular, intranasal, intraoral, and / or intrathecal administration but not suitable for intracerebral administration.

[0067] The inert ingredients of this pharmaceutical composition and the form of the preparation are common ones. The usual preparation methods used in pharmaceutical science may be used here. All the usual types of compositions, including tablets, chewable tablets, capsules, solutions, parenteral solutions, nasal sprays or powders, troches, suppositories, transdermal patches, and suspensions, may be used. Generally, the composition contains the compound in a total amount of about 0.5% to about 50% depending on the desired dosage and the type of composition used. However, the amount of the compound is best defined as an "effective amount", that is, the amount of the compound that provides the desired dosage for a patient in need of such treatment. The activity of the compounds utilized in the compositions and methods disclosed herein is not considered to be significantly dependent on the nature of the composition, and thus the present composition can be selected and formulated primarily or simply for convenience and economy.

[0068] Capsules are prepared by mixing the compound with a suitable diluent and filling an appropriate amount of this mixture into capsules. Usual diluents include inert powdered substances (such as starch), powdered cellulose (especially crystalline and microcrystalline cellulose), sugars (such as fructose, mannitol, and sucrose), cereal flours, and similar edible powders.

[0069] Tablets are prepared by direct compression, wet granulation, or dry granulation. These preparations usually incorporate (in addition to the compound) diluents, binders, lubricants, and disintegrants. Representative diluents include, for example, various types of starch, lactose, mannitol, kaolin, calcium phosphate or calcium sulfate, inorganic salts (such as sodium chloride), and powdered sugar. Powdered cellulose derivatives can also be used. Representative tablet binders include substances such as starch, gelatin, and sugars (such as lactose, fructose, glucose, etc.). Natural and synthetic rubbers, including gum acacia, alginate, methylcellulose, polyvinylpyrrolidine, etc., can also be used. Polyethylene glycol, ethylcellulose, and waxes can also act as binders.

[0070] The tablets can be coated with saccharides, for example, as flavor enhancers and sealants. These compounds may also be formulated as chewable tablets by using a large amount of bitter correctors, such as mannitol, in the formulation. For example, an immediate-dissolving tablet-like formulation can also be utilized to ensure that the patient consumes the dosage form and to avoid the difficulty that some patients may have in swallowing solid substances.

[0071] Lubricants can be used in the formulation of tablets to protect the tablets from sticking in the tablet press and to punch them out. Lubricants can be selected from slippery solids such as talc, magnesium stearate, calcium stearate, stearic acid, and hardened vegetable oils.

[0072] Tablets can also contain disintegrants. Disintegrants are substances that swell when wet, break up the tablets, and release the present compounds. These include starch, clay, cellulose, algin, and rubber. As further examples, corn and potato starch, methylcellulose, agar, bentonite, wood cellulose, powdered natural sponge, cation exchange resin, alginic acid, guar gum, citrus pulp, sodium lauryl sulfate, and carboxymethyl cellulose can be used.

[0073] The composition can be formulated as an enteric-coated agent, for example, to protect the active ingredient from the strongly acidic contents of the stomach. Such a formulation can be made by coating a solid dosage form with a polymeric film that is insoluble in an acidic environment and soluble in a basic environment. Illustrative films include cellulose acetate phthalate, polyvinyl acetate phthalate, hydroxypropyl methylcellulose phthalate, and hydroxypropyl methylcellulose acetate succinate.

[0074] Transdermal patches can also be used to deliver the present compound. The transdermal patch includes a resinous composition in which the present compound is dissolved or partially dissolved; and a film that protects the composition and holds the resinous composition in contact with the skin. Additionally, more complex patch compositions can also be used, such as those having a membrane penetrated by a plurality of pores through which the drug is pumped by osmotic action.

[0075] As will also be understood by those skilled in the art, the present formulation can be prepared with materials (such as active excipients, carriers (such as cyclodextrin), diluents, etc.) having properties (such as purity) that make the formulation suitable for administration to humans. Alternatively, the present formulation can be prepared with materials having a purity and / or other properties that make the formulation suitable for administration to non-human subjects but not suitable for administration to humans.

[0076] 2-Aminopyridine-Derived Compounds and Their Use

[0077] A 2-aminopyridine-derived compound, a pharmaceutical composition containing the compound, and a method of using the compound and the pharmaceutical composition for treating diseases and disorders related to neuronal nitric oxide synthase activity are disclosed herein. The disclosed compound can include a derivative of a 2-aminopyridine compound having the following formula or a salt thereof:

[0078]

Chemical formula

[0079] In the formula,

[0080]

Chemical formula

[0081] represents a single bond, a double bond, or a triple bond; Y is a substituted aryl (e.g., substituted phenyl) or a substituted heteroaryl (e.g., substituted quinolinyl such as substituted quinolin-3-yl), and Y is, at one or more ring positions, of the formula -X-R a substituted by a substituent having; X is selected from C1-C6-alkyl, C1-C6-alkenyl, and C1-C6-alkynyl; R a is amino, alkylamino (e.g., methylamino), dialkylamino (e.g., dimethylamino), or contains at least one nitrogen atom, and its heterocycle is optionally substituted at one or more positions by alkyl (e.g., methyl), alkoxy (e.g., ethoxy), or halo (e.g., fluoro), and is selected from 4- to 6-membered heterocycles; and, Y is optionally substituted at one or more ring positions by halo (e.g., 2,3-difluoro-phenyl). Salts of the disclosed compounds are also contemplated herein. Solvates of the disclosed compounds are also contemplated.

[0082] In some embodiments, the disclosed compounds can optionally be pyrrolidinyl (e.g., pyrrolidin-2-yl) substituted at one or more positions by alkyl (e.g., N-methyl-pyrrolidin-2-yl or 4-methyl-pyrrolidin-2-yl) or alkoxy (e.g., 4-ethoxy-pyrrolidin-2-yl) or halo (e.g., 4-fluoro-pyrrolidin-2-yl), azetidinyl (e.g., azetidin-2-yl) substituted at one or more positions by alkyl (e.g., N-methyl-azetidin-2-yl), and morpholinyl (e.g., morpholin-3-yl) substituted at one or more positions by alkyl (e.g., N-methyl-morpholin-3-yl) selected R a can have.

[0083] In some embodiments, the disclosed compounds can have formula (I):

[0084]

Chemical formula

[0085] Wherein, R 2 , R 3 , R 4 , and R 6 are each independently H or halogen (e.g., fluoro).

[0086] In some embodiments, R 3 is halogen (e.g., fluoro).

[0087] In some embodiments, R 2 is halogen (e.g., fluoro).

[0088] In some embodiments, R 2 is halogen (e.g., fluoro), and R 3 is halogen (e.g., fluoro).

[0089] In some embodiments, X is methyl, ethyl, or propyl.

[0090] In some embodiments, R a is dimethylamino.

[0091] In some embodiments, the disclosed compound can have the following formula:

[0092]

Chemical formula

[0093] Wherein, R 2 , R 3 , R 4 , and R 6 are each independently H or halogen (e.g., fluoro).

[0094] Specifically, the compounds disclosed herein can include compounds having a formula selected from the following:

[0095]

Chem.

[0096]

Chem.

[0097]

Chem.

[0098]

Chem.

[0099]

Chem.

[0100]

Chem.

[0101]

Chem.

[0102]

Chem.

[0103]

Chem.

[0104]

Chem.

[0105]

Chem.

[0106]

Chem.

[0107]

Chem.

[0108]

Chem.

[0109]

Chem.

[0110]

Chem.

[0111] Salts of the disclosed compounds are contemplated herein. In particular, pharmaceutically acceptable salts of the disclosed compounds are contemplated.

[0112] The disclosed compounds, their salts, and / or their hydrates can be formulated as pharmaceutical compositions containing the compound, its salt, and / or its hydrate in a pharmaceutically acceptable carrier. The pharmaceutical composition can be formulated for treating a disease or disorder associated with nitric oxide synthase activity.

[0113] In some embodiments, the disclosed compounds and pharmaceutical compositions can be used for the treatment or prevention of a disease or disorder associated with nitric oxide synthase (NOS) in a subject, and the method includes administering the compound and / or the pharmaceutical composition to the subject. In these methods, an amount of the compound sufficient to inhibit NOS activity can be administered to the subject.

[0114] In some embodiments, the disclosed compounds and pharmaceutical compositions can be used to treat or prevent a neurodegenerative disease or disorder in a subject in need thereof. In these methods, a subject can be administered an amount of the compound sufficient to inhibit NOS activity. In these methods, the subject can have a neurodegenerative disease or disorder selected from, but not limited to, Alzheimer's disease, Huntington's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), cerebral palsy, and migraine.

[0115] In some embodiments, the disclosed compounds and pharmaceutical compositions can be used to inhibit NOS in a subject in need thereof. In these methods, a subject can be administered an amount of the compound sufficient to inhibit NOS activity.

[0116] In some embodiments, the NOS inhibited by the disclosed compounds is neuronal NOS. In some embodiments, the disclosed compounds have a K i for human nNOS that is less than about 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.

[0117] In some embodiments, the NOS inhibited by the disclosed compounds is neuronal NOS that is selectively inhibited against iNOS. In some embodiments, the compound exhibits a selectivity (n / i) of nNOS over iNOS of at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or higher.

[0118] In some embodiments, the NOS inhibited by the disclosed compounds is neuronal NOS, which is selectively inhibited with respect to eNOS. In some embodiments, the compound exhibits an nNOS to eNOS selectivity (n / e) of at least about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, or higher.

[0119] In some embodiments, the NOS inhibited by the disclosed compounds is neuronal NOS present in the brain of a subject in need thereof. In these methods, the disclosed compounds preferably have an effective permeability (P e ) of at least about 5×10 -6 cm / s, 6×10 -6 cm / s, 7×10 -6 cm / s, 8×10 -6 cm / s, 9×10 -6 cm / s, 10×10 -6 cm / s, 11×10 -6 cm / s, 12×10 -6 cm / s, 13×10 -6 cm / s, 14×10 -6 cm / s, 15×10 -6 cm / s, 16×10 -6 cm / s, 17×10 -6 cm / s, 18×10 -6 cm / s, 19×10 -6 cm / s, or 20×10 -6 cm / s.

[0120] Exemplary Embodiments The following embodiments are illustrative and are not to be construed as limiting the scope of the claimed subject matter.

[0121] Embodiment 1. A compound of the following formula or a salt or solvate thereof:

[0122]

Chemical formula

[0123] (wherein:

[0124] [Chem.]

[0125] represents a single bond, double bond, or triple bond; Y is a substituted aryl (e.g., substituted phenyl) or a substituted heteroaryl (e.g., a substituted quinolinyl such as substituted quinolin-3-yl), and Y is, at one or more ring positions, a substituent having the formula -X-R a and is substituted by a substituent having; X is selected from C1-C6-alkyl, C1-C6-alkenyl, and C1-C6-alkynyl; R a is selected from amino, alkylamino (e.g., methylamino), dialkylamino (e.g., dimethylamino), or a 4- to 6-membered heterocycle containing at least one nitrogen atom and optionally substituted at one or more positions by alkyl (e.g., methyl), alkoxy (e.g., ethoxy), or halo (e.g., fluoro); and here, Y is optionally substituted at one or more ring positions by halo (e.g., 2,3-difluoro-phenyl)).

[0126] Embodiment 2. A compound of Embodiment 1, wherein R a is pyrrolidinyl (e.g., pyrrolidin-2-yl) optionally substituted at one or more positions by alkyl (e.g., N-methyl-pyrrolidin-2-yl or 4-methyl-pyrrolidin-2-yl) or alkoxy (e.g., 4-ethoxy-pyrrolidin-2-yl) or halo (e.g., 4-fluoro-pyrrolidin-2-yl), azetidinyl (e.g., azetidin-2-yl) optionally substituted at one or more positions by alkyl (e.g., N-methyl-azetidin-2-yl), and morpholinyl (e.g., morpholin-3-yl) optionally substituted at one or more positions by alkyl (e.g., N-methyl-morpholin-3-yl).

[0127] Embodiment 3. The compound of Embodiment 1 having formula (I):

[0128]

Chemical formula

[0129] (wherein R 2 , R 3 , R 4 , and R 6 are each independently H or halogen (for example, fluoro)).

[0130] Embodiment 4. The compound of Embodiment 3, wherein R 3 is halogen (for example, fluoro).

[0131] Embodiment 5. The compound of Embodiment 3 or 4, wherein R 2 is halogen (for example, fluoro).

[0132] Embodiment 6. The compound of Embodiment 3, wherein R 2 is halogen (for example, fluoro), and R 3 is halogen (for example, fluoro).

[0133] Embodiment 7. The compound of any of the foregoing embodiments, wherein X is methyl, ethyl, or propyl.

[0134] Embodiment 8. The compound of any of the foregoing embodiments, wherein R a is dimethylamino.

[0135] Embodiment 9. The compound of any of the foregoing embodiments having the following formula:

[0136]

Chemical formula

[0137] (wherein R 2 , R 3 , R4 and R 6 each independently is H or halogen (e.g., fluoro).

[0138] Embodiment 10. A compound of any of the foregoing embodiments of the following formula:

[0139]

Chemical formula

[0140]

Chemical formula

[0141]

Chemical formula

[0142] Embodiment 11. A compound of any of the foregoing embodiments, wherein the compound has an effective permeability P e of at least about 5×10 -6 cm / s, 6×10 -6 cm / s, 7×10 -6 cm / s, 8×10 -6 cm / s, 9×10 -6 cm / s, 10×10 -6 cm / s, 11×10 -6 cm / s, 12×10 -6 cm / s, 13×10 -6 cm / s, 14×10 -6 cm / s, 15×10 -6 cm / s, 16×10 -6 cm / s, 17×10 -6 cm / s, 18×10 -6 cm / s, 19×10 -6 cm / s, or 20×10 -6 cm / s.

[0143] Embodiment 12. A compound of any of the foregoing embodiments, wherein the compound has a selectivity of nNOS over iNOS of at least about 30.

[0144] Embodiment 13. A compound according to any of the preceding embodiments, wherein the compound has a selectivity of nNOS over eNOS of at least about 1000.

[0145] Embodiment 14. A pharmaceutical composition comprising a compound according to any of the preceding embodiments and a pharmaceutically acceptable carrier.

[0146] Embodiment 15. A method of treating or preventing a disease or disorder associated with nitric oxide synthase in a subject in need thereof, comprising administering to the subject a compound according to any of Embodiments 1-13 or the pharmaceutical composition of Embodiment 14.

[0147] Embodiment 16. The method of Embodiment 15, wherein the disease or disorder is a neurodegenerative disease or disorder.

[0148] Embodiment 17. The method of Embodiment 15, wherein the disease or disorder is Alzheimer's disease.

[0149] Embodiment 18. The method of Embodiment 15, wherein the disease or disorder is Huntington's disease.

[0150] Embodiment 19. The method of Embodiment 15, wherein the disease or disorder is Parkinson's disease.

[0151] Embodiment 20. The method of Embodiment 15, wherein the disease or disorder is amyotrophic lateral sclerosis (ALS).

[0152] Embodiment 21. The method of Embodiment 15, wherein the disease or disorder is cerebral palsy.

[0153] Embodiment 22. The method of Embodiment 15, wherein the disease or disorder is migraine.

[0154] Embodiment 23. A method of inhibiting nitric oxide synthase (NOS) in a cell, comprising contacting the cell with any of the compounds of Embodiments 1-13.

Examples

[0155] Examples The following examples are illustrative and are not intended to limit the scope of the claimed subject matter.

[0156] Example 1

[0157] Title - Optimization of Blood-Brain Barrier Permeability by Reduced P-Glycoprotein Substrate Liability for Potent and Selective Human Neuronal Nitric Oxide Synthase Inhibitors Bearing a 2-Aminopyridine Scaffold for CNS Drug Discovery

[0158] See the manuscript by Do et al., "Optimization of Blood-Brain Barrier Permeability by Potent and Selective Human Neuronal Nitric Oxide Synthase Inhibitors with 2-Aminopyridine Scaffolds" (J. Med. Chem. 2019, 62, 5, 2690-2707), published on February 25, 2019, the content of which is hereby incorporated by reference in its entirety.

[0159] Neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and Huntington's disease, which are generally known, are characterized by the gradual degeneration and death of neurons in the central nervous system (CNS), which causes problems in the patient's muscle movement and mental function. Despite an urgent medical need, the comprehensive treatment of these diseases remains very limited. 1,2 One of the most difficult challenges in CNS drug discovery is the effective delivery of therapeutic agents to the human brain, mainly due to the presence of the blood-brain barrier (BBB) located at the boundary between blood vessels and brain tissue. 3 The BBB is composed of a layer of endothelial cells with tight junctions that prevent the access of foreign toxins, thereby protecting the brain and maintaining its optimal physiological environment. However, this cell layer also limits the access of beneficial therapeutic agents to the brain. 4 The main route for CNS drugs to cross the BBB is passive diffusion through its lipid membrane. In addition to the tight junctions of endothelial cells, the high expression levels of efflux transporters on the BBB, particularly P-glycoprotein (P-gp), significantly contribute to the limited brain exposure of CNS drugs. 5 As a result, it is necessary for CNS drug discovery to establish a strategy consisting of a combination between increased passive permeability and decreased P-gp-mediated excretion.3,6,7 .

[0160] Neuronal nitric oxide synthase (nNOS) has been identified as a promising therapeutic target in the development of novel treatments for neurodegenerative diseases 8-10 . In the brain, nitric oxide (NO) produced by nNOS participates in neuronal signaling 11 . However, the overproduction of intracellular NO is harmful. In particular, excessive NO formed by overactivated nNOS in the CNS can cause excessive nitration and nitrosylation of proteins, which leads to their misfolding and aggregation 12 . In addition, the reaction of NO with superoxide anions generates peroxynitrite, a powerful oxidant that damages DNA and causes lipid peroxidation. These processes lead to neuronal cell death and dysfunction in neuronal signaling 13,14 . Therefore, limiting NO production by inhibiting nNOS has emerged as an essential approach that may protect neurons and cure certain neurodegenerative diseases 15,16 .

[0161] nNOS is a homodimeric enzyme, and each monomer contains one C-terminal reductase domain and one N-terminal oxygenase domain. The C-terminal reductase domain consists of nicotinamide adenine dinucleotide phosphate (NADPH), flavin adenine dinucleotide (FAD), and flavin mononucleotide (FMN), whereas the N-terminal oxygenase domain contains non-catalytic zinc, tetrahydrobiopterin (H4B), and heme. These two domains are bound to each other by a calmodulin domain. When dimerization occurs, the electron flow from the reductase domain to the oxygenase domain is promoted, at which point L-Arg is oxidized to L-Cit and NO is released 15,17 . Promoting molecules that compete with L-Arg binding at the enzyme's active site is one of the basic approaches to inhibiting nNOS 16The problem with this task is related not only to the efficacy of the inhibitor but also to their binding selectivity for nNOS over both eNOS and iNOS, two isoforms that share very similar structural features with that of nNOS. 18,19 Since eNOS inhibition can cause circulatory failure while iNOS inhibition can trigger immune system destruction, it is necessary to avoid over-inhibiting these two NOS isoforms. 20 。

[0162] In recent years, the inventors' efforts to develop nNOS inhibitors with excellent efficacy and high isoform activity have led to a promising class of molecules bearing a 2-aminopyridine scaffold. Using this molecular scaffold, the inventors obtained nNOS inhibitors that exhibit excellent activity at concentrations below 30 nM. 15,21,22 However, the first-generation nNOS inhibitors bearing the inventors' 2-aminopyridine scaffold showed predicted poor permeation through the BBB, as revealed by their very low Caco-2 permeability. 23 Recently, the inventors were able to improve the cell membrane permeability of their 2-aminopyridine nNOS inhibitors while maintaining their high inhibitory activity. In the inventors' previous report, they obtained a novel lead compound (1, Figure 1) that showed excellent efficacy and selectivity for human nNOS (K i hnNOS = 30 nM; hnNOS / heNOS = 2799), and exhibited an efflux ratio (ER) of 5.9 in the Caco-2 assay. 24 To progress in CNS drug discovery, the cell membrane permeability of these 2-aminopyridine nNOS inhibitors must be further improved to accompany an ER < 2.5, which is required for a promising CNS(+) agent. 7,25 。

[0163] In this contribution, the inventors report on their optimization to use 1 as a lead compound for numerous chemical modifications to improve the cell membrane permeability of 2-aminopyridine nNOS inhibitors and to reduce P-gp substrate liability. Insights into the structure-activity for the analogs' activity and permeability were obtained through various medicinal chemistry approaches, including modulating the pKa of the basic amino group within the molecule and enhancing the lipophilicity and rigidity of the novel analogs (Figure 1). These structural modifications are focused on enhancing the disposition of the nNOS inhibitor to the brain while retaining their efficacy and selectivity comparable to those of 1. Further in this study, the inventors, for the first time, aimed to examine the inhibition tests of the potential compounds using all human NOS isoforms, which helps not only in the direct comparison of isoform selectivity but also in providing stronger data for clinical trials when proceeding with any tested nNOS inhibitor to the subsequent stages of drug discovery.

[0164] The structural modification of 1 (Figure 1) first went to the tail chain where an unsaturated C-C triple bond was incorporated into the novel analogs (Compounds 2 and 3) to enhance the rigidity of these molecules. To enhance the lipophilicity of 1 and as a result increase its permeability, analogs 4 - 9 with a pyrrolidine ring in the tail chain were designed by introducing more hydrocarbon groups by the lead molecule and reducing the rotatable bonds within the molecule. Different enantiomers of the pyrrolidine ring were also tested to examine the effect of chirality on the activity and selectivity of these molecules. Further, one fluorine atom was incorporated at the C4 position of the pyrrolidine ring (8 and 9), reducing the basicity of this cyclic amine and protecting it from metabolism. In another direction, the lipophilicity of the novel analogs was enhanced by incorporating additional fluorine atoms into the central fluorobenzene linker of 1. Various compounds (10 - 14) bearing two or three fluorine atoms at various relative positions of the central linker were designed and synthesized. The combination of the enhanced rigidity brought about by the unsaturated C-C triple bond and the increased lipophilicity associated with the multi-fluorobenzene linker as observed in Compounds 15 and 16 was also investigated to gain insights into the combined effect on the cell membrane permeability of the novel analogs. Subsequently, the inventors further performed structural modification by combining the optimal multi-fluorobenzene linker with a pyrrolidine tail chain that retained most of the nNOS inhibitory activity from 1, where Compounds 17 and 18 achieved a potential maximization of lipophilicity. Finally, the pKa of the cyclic amino group within the tail chain, i.e., the pyrrolidine ring, was adjusted using different heterocycles including morpholine (19) and azetidine (21), and by introducing electron-withdrawing groups to the pyrrolidine ring (20). All novel analogs were tested for their nNOS inhibition and selectivity over eNOS and iNOS using the NO-hemoglobin capture assay, while their cell membrane permeability was examined using the parallel artificial membrane permeability (PAMPA-BBB) assay regarding the blood-brain barrier. Compounds with high efficacy, selectivity, and permeability were further tested in the Caco-2 bidirectional assay to evaluate their P-gp substrate liability.

[0165] Results and Discussion

[0166] Chemistry

[0167] The syntheses of compounds 2 and 3 with enhanced rigidity in the tail chain are shown in Scheme 1. Deprotonation of pyrrole-protected 2,4-dimethylpyridine 22 with n-BuLi, followed by reaction of the resulting anion with the electrophile 23, provided intermediate 24 with a coupled 2-aminopyridine head and central linker. Sonogashira coupling of 24 with either N-Boc-N-methyl-propargylamine (25a) or 3-dimethylamino-1-propyne (25b) afforded an alkyne bearing a Boc-protected secondary amine (26a) or tertiary amine (26b). Boc-deprotection of 26a, followed by pyrrole deprotection, gave the target compound 2, whereas pyrrole deprotection of 26b produced compound 3.

[0168]

Chemical formula

[0169] Reagents and conditions: (a) (i) 1.6 M n-BuLi in THF, THF, -78 °C → -20 °C, 15 min, (ii) 23, THF, -78 °C → -20 °C, 20 min; (b) 25a or 25b, Pd(PPh3)4, CuI, TEA:DMF (9:1), microwave, 120 °C, 30 min; (c) 20% TFA, CH2Cl2, RT, 1 h; (d) NH2OH·HCl, EtOH / H2O (2:1), 100 °C, 20 h.

[0170] The synthesis of pyrrolidine analogs 4 - 9 was carried out through the preparation of pyrrolidinoalkynes 32a - c (Scheme 2). The pure enantiomers of these pyrrolidinoalkynes were synthesized directly from their corresponding aldehydes (30a - c) using the Seyferth - Gilbert homologation reaction. Aldehydes 30a - c were obtained either from commercially available sources (29a and 29b) or prepared from the oxidation of their corresponding alcohols, which were synthesized from carboxylic acid precursors (28) (29c). Scheme 3 shows the synthetic routes for 4 - 9 from 32a - c. The sequence of Sonogashira coupling of intermediate 24 with various pyrrolidinoalkynes (32a - c), followed by Boc - deprotection and hydrogenation, yields intermediates 34a - c. The pyrrole - deprotection of these intermediates provided secondary amine analogs 4, 6, and 8. In another route, methylation of the secondary amino group using formaldehyde / NaBH4, followed by removal of the pyrrole protecting group, produced tertiary amine analogs 5, 7, and 9.

[0171]

Chem.

[0172] Reagents and conditions: (a) BH3 1M / THF, THF, 0 °C → RT; (b) Dess - Martin periodinane, CH2Cl2, RT, 3 h; (c) 31, MeOH, RT, 15 h.

[0173]

Chem.

[0174] Reagents and conditions: (a) 32a - c, Pd(PPh3)4, CuI, TEA:DMF (9:1), microwave, 120 °C, 30 min; (b) 20% TFA, CH2Cl2, RT, 1 h; (c) Pd / C, H2, MeOH, RT, 20 h; (d) (i) 37% HCHO in H2O, (ii) NaBH4, MeOH; (e) NH2OH·HCl, EtOH / H2O (2:1), 100 °C, 20 h.

[0175] The synthesis of analogs (10 - 14) containing di- and tri-fluorobenzene linkers was initiated with the preparation of linker components (36a - e) from commercially available sources (see supporting information). Following the general synthetic route of nNOS inhibitors as shown in Scheme 1, the synthesized linkers (36a - e) were coupled with the pyrrole-protected 2-aminopyridine head 22 through carbon-carbon bond formation using n-BuLi. The resulting intermediates (37a - e) were then subjected to Sonogashira coupling with alkyne 25b, retaining the same tail chain as lead compound 1. The alkyne reduction of intermediates 38a - e, followed by pyrrole deprotection, yielded the desired products 10 - 14 (Scheme 4).

[0176]

Chem.

[0177] Reagents and conditions: (a) (i) n-BuLi 1.6M / THF, THF, -78 °C → -20 °C, 15 min, (ii) 36a - e, THF, -78 °C → -20 °C, 20 min; (b) 25b, Pd(PPh3)4, CuI, TEA:DMF (9:1), microwave, 120 °C, 30 min; (c) Pd / C, H2, MeOH, RT, 20 h; (d) NH2OH·HCl, EtOH / H2O (2:1), 100 °C, 20 h.

[0178] To obtain the highest possible improvement in cell membrane permeability, a new set of compounds with both enhanced lipophilicity and increased rigidity was synthesized. The new analogs were each designed to have a tail chain containing either a pyrrolidine ring or an alkynylamino group within the difluorobenzene central linker and the tail chain in order to increase their lipophilicity or decrease the number of rotatable bonds. The synthesis of these analogs (15 - 18) is shown in Scheme 5. The Sonogashira coupling reactions of the previously synthesized 37a with various alkynes (25a - b, 32a - b) yielded intermediates 39 and 41 - 43. The Boc - deprotection of 39 generated intermediate 40, which underwent pyrrole - deprotection together with 41 to yield the two desired compounds (15 and 16) bearing an alkynylamino group within the tail chain. On the other hand, 42 and 43 underwent a series of reactions including Boc - deprotection, hydrogenation with Pd / C, methylation with formaldehyde, and pyrrole deprotection to yield the target products (17 and 18) containing a pyrrolidine ring within their tail chains.

[0179] [Chemical formula]

[0180] Reagents and conditions: (a) 25a - b or 32a - b, Pd(PPh3)4, CuI, TEA:DMF (9:1), microwave, 120 °C, 30 min; (b) 20% TFA, CH2Cl2, RT, 1 h; (c) Pd / C, H2, MeOH, RT, 20 h; (d) (i) 37% HCHO in H2O, (ii) NaBH4, MeOH; (e) NH2OH·HCl, EtOH / H2O (2:1), 100 °C, 20 h.

[0181] The synthesis of compounds 19 - 21 to adjust the pKa of the amino group within the tail chain first involved the corresponding alkyne-functionalized tail chains (44a - c), the synthesis of which can be found in the "Supporting Information". The Sonogashira coupling was used again to attach these tail chains to intermediate 37a. The target compounds (19 - 21) were obtained by subsequent reactions including (i) Boc / Cbz deprotection, (ii) hydrogenation of the alkyne, (iii) methylation of the secondary amine to a tertiary amine with HCHO / NaBH4, and (iv) pyrrole deprotection. These reactions worked effectively for the synthesis of 19, but it is worth mentioning that the removal of the Cbz group in the syntheses of 20 and 21 required the use of Pd(OH)2 / C under H2 gas. The use of Pd / C, H2 to remove the Cbz group did not yield the desired product even at a high pressure of up to 110 psi of hydrogen gas. In addition, for the Sonogashira coupling of azetidine alkyne 44c to obtain reasonable yields and fewer inseparable by-products, the use of different Pd catalysts and bases (i.e., Pd(PPh3)2Cl2 and diethylamine, respectively) was required (Scheme 6).

[0182]

Chem.

[0183] Reagents and conditions: (a) 44a or 44b, Pd(PPh3)4, CuI, TEA:DMF (9:1), microwave, 120 °C, 30 min; (b) 44c, Pd(PPh3)2Cl2, CuI, PPh3, DEA:DMF (1:1), microwave, 120 °C, 20 min; (c) 45a → 46a: (i) 20% TFA, CH2Cl2, RT, 1 h, (ii) Pd / C, H2, MeOH, RT, 20 h, (iii) 37% HCHO in H2O, NaBH4, MeOH; (d) 45b - c → 46b - c: (i) Pd(OH)2 / C, H2 (1 atm), MeOH, RT, 20 h, (ii) 37% HCHO in H2O, NaBH4, MeOH; (e) NH2OH·HCl, EtOH / H2O (2:1), 100 °C, 20 h.

[0184] Biological Activity

[0185] The nNOS inhibitory activity and selectivity of the novel analog 2-21 were determined by a NO hemoglobin capture assay, and the results were summarized in Table 1 together with the results of Compound 1 for comparison. Based on the screening, these compounds were first tested against rat and human nNOS and mouse iNOS, and their efficacy and selectivity were evaluated due to the ease of expression and purification of these enzymes. In addition to the selectivity of nNOS over iNOS (n / i), the inhibitory activity of these compounds against rat and human nNOS (hn / rn ratio) was also compared as it is useful information when moving from preclinical to clinical trials. A parallel artificial membrane permeability (PAMPA-BBB) assay regarding the blood-brain barrier was used to evaluate the effect of the 2-21 structural modifications of the present inventors on cell membrane permeability, which helped to understand the structure-permeability relationship of these compounds.

[0186]

Table 1

[0187] The structural modification of 1 was first carried out on the tail chain, where the bismethylene group was replaced by an acetylene group (one), enhancing the rigidity of the molecule. The NOS inhibition test revealed that Compounds 2 and 3 showed a decrease in the efficacy of both rat and human nNOS, where the hnNOS / rnNOS ratio of K i was about 2, indicating a more significant effect on the human species. Compound 3 with a tertiary amino group in the tail chain exhibited lower efficacy than the secondary amine analog (2), where the efficacy of 3 decreased by more than 2-fold and 4-fold for rat and human nNOS, respectively, compared to the efficacy of 1, while the efficacy of 2 was only slightly attenuated. Despite the decrease in efficacy, 3 was Pe Value 18.8×10 -6 cm·s -1 and has excellent permeability, indicating that the decrease in flexibility of 1 may assist in enhancing its cell membrane permeability.

[0188] To increase the lipophilicity of 1 and potentially increase its permeability, compounds 4 - 9 with a pyrrolidine ring in the tail chain were designed to introduce more hydrocarbon groups into the lead molecule. Tests on the rat and human nNOS inhibition of 4 - 9 showed that all compounds except analogs 8 (K i =65 nM) and 9 (K i =128 nM) exhibited excellent efficacy (K i =~30 nM) against rat nNOS. The decrease in the rnNOS inhibitory activity of 8 and 9 is probably the result of the binding of the tail amino group or the decrease in pKa, which was also observed in the previous reports of the inventors 24 . Compounds 4 - 7 maintain moderate hnNOS efficacy compared to 1, while 8 and 9 show a significant drop in their activities (1 / 4 and 1 / 7 of 1 respectively). There is only a slight difference in the activities of these two pairs of enantiomers, and 4 and 5 show similar K i values compared to those of 6 and 7 for both rnNOS and hnNOS. However, the selectivity of these compounds over mouse iNOS is slightly enhanced when switching from the (R)-isomers (4, 5) to the (S)-isomers (6, 7). In the PAMPA - BBB assay, compounds with a tertiary amino group in the tail show a tendency to permeate more lipid membranes than their corresponding secondary amine analogs (5, 7, and 9 vs. 4, 6, and 8 respectively). Furthermore, additional fluorine atoms on the pyrrolidine ring assist in improving the permeability of the nNOS inhibitors. For example, 8 and 9 exhibit higher P e values than 4 and 5 respectively. The presence of both a tertiary amino group and additional fluorine atoms results in excellent permeability of 18.9×10 -6 cm·s -1 for compound 9, which is that of 1 (Pe = 14.8×10 -6 cm·s -1 ) is significantly higher than. However, despite its high P e value, 9 represents a dramatic decrease in the efficacy and selectivity of nNOS.

[0189] Another direction for enhancing the cell membrane permeability of 1 was achieved by incorporating additional fluorine atoms onto the central fluorobenzene linker of our nNOS inhibitors. Compounds (10 - 14) possessing di - and tri - fluorobenzene linkers, in which the fluorine atoms are arranged in different relative positions to each other, were designed and synthesized. The results of the biological activity tests revealed that the relative positions between the fluorine atoms on the central benzene ring have a specific effect on the nNOS inhibitory activity and selectivity of the five resulting modified compounds. In particular, analogs containing ortho - (10) and meta - (11) difluorobenzene linkers exhibit higher efficacy against both rnNOS and hnNOS than the efficacy of the analog containing para - difluorobenzene linker (12). Compared to lead compound 1, analog 12 exhibits a loss exceeding 1 / 2 of the original efficacy, while 10 shows slightly better efficacy against both rnNOS and hnNOS. Compounds 13 and 14 containing a trifluorobenzene central linker exhibit a moderate decrease in nNOS efficacy in both rats and humans compared to 1 and 10. However, the addition of fluorine atoms to the central linker decreases the rat nNOS / mouse iNOS selectivity for both di - and tri - fluorobenzene analogs. On the other hand, the PAMPA - BBB assay indicates that the incorporation of one additional fluorine in 10 - 12, although slight, aids in the increase of the P e value compared to that of 1. These difluorobenzene - containing analogs have a P e value increase of these new analogs, although slight compared to the P -6 cm·s -1show similar effective permeability. The further incorporation of more fluorine into the central linker, as observed in trifluorobenzene-containing analogs 13 and 14, resulted in a significant improvement in cell membrane permeability. In particular, the P e values of 13 and 14 are approximately the same as those of 9, which is an analog with a fluoropyrrolidine group in the tail chain. Thus, these permeability tests by the PAMPA-BBB assay support the proper orientation of our modifications by the incorporation of additional fluorine atoms into the central linker to enhance permeability while maintaining sufficient nNOS inhibition and isoform selectivity of the modified compounds. e

[0190] The central linker associated with the ortho-difluorobenzene ring of 10 was then employed to construct new analogs due to its excellent nNOS inhibition and improved permeability shown in the PAMPA-BBB assay. These new analogs were each designed to have an alkynylamino group (15, 16) and a pyrrolidine ring (17, 18) incorporated into the tail chain to reduce the number of rotatable bonds or increase their lipophilicity. The directionality of these two modifications is expected to assist in enhancing the permeability of the resulting analogs. Our biological tests on the nNOS inhibition of these new compounds revealed that the incorporation of the alkynyl group causes a decline in the nNOS efficacy of 15 and 16, where compound 16 with a tertiary amino group in its tail chain inhibits a greater loss of efficacy than analog 15 with a secondary amine tail chain. Interestingly, 15 can restore the selectivity for rnNOS over mouse iNOS, which was previously attenuated by the introduction of the difluorobenzene linker. It is also worth noting that a similar trend is observed in compounds (2, 3) retaining the alkynyl tail chain and fluorobenzene central linker, where the secondary amine analog (2) exhibits better performance in both nNOS inhibition and selectivity than the tertiary amine analog (3). In the PAMPA-BBB assay, both 15 and 16 have a P e value of approximately 18×10 -6 cm·s -1 ​show an enhancement of their permeability. Interestingly, despite 15 having an excessive hydrogen bond donor, which often plays an important role in the cell membrane permeability of CNS agents, compared to 16, there is surprisingly negligible difference in cell membrane permeability between the two analogs 15 and 16. This finding suggests a significant effect of the structural rigidity on the permeability of the analogs. However, due to the attenuation of nNOS efficacy and isoform activity, the modification of nNOS inhibitors using this rigid alkyne-containing tail chain is not favorable for further research.

[0191] Analogs 17 and 18 were obtained through combined modifications of both the difluorobenzene central linker and the pyrrolidine ring of the tail chain. The hemoglobin NO capture assay revealed that both compounds exhibited excellent efficacy in nNOS inhibition, where the (S)-isomer 18 showed higher efficacy and selectivity than its counterpart, the (R)-isomer 17. Promisingly, analog 18 showed enhanced inhibition of rat and human nNOS compared to lead compound 1, where the inhibitory activities of 18 against rNOS and hNOS were approximately 2-fold and 1.5-fold that of 1 respectively. The cell membrane permeability tests by PAMPA-BBB assay for 17 and 18 indicated that both compounds exhibited enhanced permeability with P -6 cm·s -1 values close to 18.0×10 e These results support the inventors' proposed direction of improving the cell membrane permeability of nNOS inhibitors by increasing the lipophilicity and / or rigidity of the lead molecule.

[0192] Next, the inventors performed further modifications based on the structural scaffold of 18, considering its excellence in nNOS activity, selectivity, and cell membrane permeability. Various heterocycles containing morpholine, (4-ethoxy)pyrrolidine, and azetidine, as observed in compounds 19, 20, and 21 respectively, were utilized to modulate the basicity of the tertiary amino group of the pyrrolidine ring, which may enhance its permeability and protect it from metabolism. 26. Biological tests revealed that replacement of the 18 pyrrolidine rings with 19 morpholine rings resulted in a loss of more than half of the inhibitory activity of this compound against nNOS in both rats and humans. Similarly, introduction of an ethoxy group at C4 of the pyrrolidine ring caused a decrease in the efficacy of 20 nNOS. In contrast, use of an azetidine ring instead of the pyrrolidine ring led to high nNOS inhibition of the resulting compound 21, which had an rnNOS and hnNOS K i value equivalent to that of 18 and isoform selectivity. Regarding cell membrane permeability, despite the loss of nNOS inhibitory activity, 19 exhibited a significant improvement in permeability with a P e value of 21.1×10 -6 cm·s -1 , which is the highest permeability value the inventors obtained for their nNOS inhibitors based on the 2-aminopyridine scaffold and is comparable to that of the positive control compound verapamil (P e = 20.2×10 -6 cm·s -1 ). On the other hand, PAMPA-BBB tests for two analogs 20 and 21 indicated that the structural modifications by the inventors through replacement of the 18 pyrrolidine ring with (4-ethoxy)pyrrolidine (20) and azetidine (21) of the inventors maintained the permeability of these analogs, and the effective permeability changed little compared to that of 18, being 20 (P e = 17.7×10 -6 cm·s -1 ) and 21 (P e = 16.3×10 -6 cm·s -1 ).

[0193] In addition to the inventors' efforts to enhance the cell membrane permeability of nNOS inhibitors, the inventors also aimed to obtain the isoform selectivity of their nNOS inhibitors using all human NOS isoforms to provide an accurate comparison of selectivity. Bovine eNOS and murine iNOS were used previously for isoform selectivity, while the recent success in the preparation of the inventors' hiNOS enzyme has enabled the inventors to perform this selectivity test for the first time on all human NOS. Based on the previous bioactivity tests, six compounds (10, 14, 15, 17, 18, and 21) with excellent human nNOS inhibitory activity and their structural diversity were selected for a selectivity test superior to human eNOS (heNOS) and human iNOS (hiNOS), and the results of the isoform selectivity for the inhibition of human NOS by these compounds are summarized in Table 2. This data reveals that most of the selected compounds show excellent selectivity for hnNOS over heNOS, and with the exception of analog 15 with an hnNOS / heNOS selectivity ratio of 394, the hnNOS / heNOS ratio ranges from 900 to 1200. Regarding human iNOS, the use of a difluorobenzene linker tends to result in a decrease in the selectivity of the selected analogs compared to lead compound 1, which is consistent with the findings regarding rnNOS selectivity over murine iNOS (Table 1). Unlike the other analogs, 18 and 21 show some retention potential regarding hnNOS / hiNOS selectivity, with selectivity ratios of 106 and 77, respectively. As a result, these two compounds were selected for the inventors' tests, including P-gp substrate liability, to evaluate their potential as CNS agents.

[0194] [Table 2]

[0195] P-gp Substrate Liability

[0196] P-glycoprotein (P-gp) is an efflux transporter highly expressed in the BBB to rapidly efflux any harmful molecules and potential drugs out of the brain. 5,27 . Therefore, the evaluation of the potential of a compound as a P-gp substrate is one of the important steps in CNS drug discovery. The P-gp substrate liability of a compound can be measured by the Caco-2 bidirectional assay, which measures the ability of the compound to cross a monolayer of colon cells expressing P-gp in either of two directions: from the apical well to the basolateral well (A→B) or from the basolateral well to the apical well (B→A). 28 It can be evaluated through the efflux ratio (ER) obtained from it. Subsequently, the ER ratio is determined by the ratio of the apparent permeability (P app ) from B→A over A→B. If a compound has an ER greater than 3, it is often considered as a substrate of P-gp with limited penetration into the brain. 3 .

[0197] In our previous studies, the main drawback of lead compound 1 for further testing in CNS drug discovery was the undesirably high efflux ratio (ER = 5.9) of this compound, as revealed in the Caco-2 bidirectional assay. Therefore, the reduction of the ER of 1 is an important step for our 2-aminopyridine nNOS inhibitors before proceeding these compounds to the next stage of drug discovery, i.e., animal testing. Due to their excellent efficacy, maintained isoform selectivity, and high cell membrane permeability as determined by the PAMPA-BBB assay, analogs 18 and 21 were selected for the evaluation of their ER in the Caco-2 bidirectional assay. The results summarized in Table 3 clearly show that these two compounds exhibit very promising efflux ratios. Analogue 18 shows an ER of 2.1, which is much lower than that of lead compound 1 (ER = 5.9) and, more importantly, within the efflux ratio regime (less than 2.5) required for molecules that are likely CNS(+) agents. More interestingly, compound 21 shows a very low ER of 0.8, which is an indicator of very low liability to be a P-gp substrate. These results suggest a high potential for these two analogs to cross the BBB and penetrate into the brain. For reasons that are not clear, 18 exhibits low permeability, only 1.1×10 -6 cm·s -1 of P app (A→B) in the Caco-2 assay, which is worthy of note. Nevertheless, analog 21, which is the most promising compound for us, not only exhibits excellent nNOS inhibition and selectivity but also shows a high potential in its ability to cross the BBB and penetrate into the brain, as shown by both the PAMPA-BBB and Caco-2 assays. The P app (A→B) value of 17.0×10 -6 cm·s -1 in the Caco-2 assay of 21 also shows excellent consistency with the effective permeability (P e = 16.3×10 -6 cm·s -1 ) obtained from the PAMPA-BBB results.

[0198]

Table 3

[0199] Conclusion

[0200] In summary, the inventors focused on improving the cell membrane permeability of these inhibitors so that they can cross the blood-brain barrier while maintaining their excellent inhibitory activity and high isoform selectivity, and reported the optimization of nNOS inhibitors bearing a 2-aminopyridine scaffold. A new series of potent and selective human nNOS inhibitors were designed and synthesized by utilizing various medicinal chemistry approaches involved in enhancing the lipophilicity of the basic amino tail group of lead molecule 1, increasing molecular rigidity, and adjusting pKa. NO hemoglobin capture assay and PAMPA-BBB assay were used to understand the effect of structural modification on nNOS inhibitory efficacy and isoform selectivity, as well as the cell membrane permeability of the new analogs. The inventors recognized that the introduction of additional fluorine atoms into the fluorobenzene central linker and the use of an azetidine ring in the tail chain led to the discovery of compound 21, which not only showed excellent inhibition (K i = 23 nM) against human nNOS and high selectivity over human eNOS (hn / he = 956) and human iNOS (hn / hi = 77), but also indicated a high potential for brain penetration. The Caco-2 bidirectional assay revealed that 21 had an efflux of only 0.8, which was significantly lower than the required ER < 2.5 for lead compound 1 (ER = 5.9) and CNS(+) drugs. The Caco-2 bidirectional assay also showed that 21 had a high cell membrane permeability with a P app value of 17.0×10 -6 cm·s -1 , which was the effective permeability (P e = 16.3×10 -6 cm·s -1) was also clearly shown to be in good agreement. The results of the present inventors provide a basis for further exploration of 2-aminopyridine nNOS inhibitors in CNS drug discovery and insights into strategies to overcome the BBB using medicinal chemistry approaches.

[0201] References for Example 1

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[0253] Example 2

[0254] The following examples provide support information regarding the information disclosed in Example 1 as follows.

[0255] Refer to the manuscript of Do et al., "Optimization of Blood-Brain Barrier Permeability by Potent and Selective Human Neuronal Nitric Oxide Synthase Inhibitors with 2-Aminopyridine Scaffolds" (J. Med. Chem. 2019, 62, 5, 2690-2707), published on February 25, 2019, the content of which is incorporated herein by reference in its entirety.

[0256] General Procedures Unless otherwise specified, all reagents were obtained from Sigma-Aldrich, Combi-blocks, and Oakwood Chemical. Anhydrous solvents (THF, CH2Cl2, MeCN, and DMF) were purified by passing through a column composed of activated alumina and supported copper redox catalyst before use. The Sonogashira coupling was carried out on a Biotage microwave vial (0.5-2 mL, 2-5 mL, and 10-20 mL) using a Biotage Initiator microwave. Thin-layer chromatography (TLC) was performed on pre-coated plates (0.25 mm) of silica gel 60 F254 from Silicycle, and the components were visualized by ultraviolet light (254 nm) and / or KMnO4 or ninhydrin staining. Flash column chromatography was performed on an Agilent 971-FP automated flash purification system equipped with a Varian column station and various Silicycle cartridges (4-80 g, 40-63 μm, 60 Å). 1 H and 1313C NMR spectra were recorded on a Bruker Avance-III NMR instrument at 500 MHz and 126 MHz, using CDCl3 or CD3OD as the solvent, respectively. Chemical shifts were reported in ppm, and multiplicities were indicated as s = singlet, d = doublet, t = triplet, q = quartet, sep = septet, dd = doublet of doublets, dt = doublet of triplets, m = multiplet, br = broad resonance. Coupling constants ‘J’ were reported in Hz. High-resolution mass spectrometry data were obtained on an Agilent 6210 LC-TOF instrument in positive ion mode using electrospray ionization, equipped with an Agilent G1312A HPLC pump and an Agilent G1367B autoinjector, at the Integrated Molecular Structure Education and Research Center (IMSERC) of Northwestern University. The purity of the compounds was tested using a reverse-phase analytical Agilent Infinity 1260 HPLC equipped with an Agilent Poroshell 120 EC-C18 column, detecting at UV absorbance of 254 nm. All compounds tested biologically had a purity > 95%.

[0257] General Procedure A: Pyrrole Deprotection : To a microwave vial was added starting material 26b or 41 (1 equiv) and NH2OH·HCl (3 - 4 equiv). These were diluted with EtOH / H2O (2:1) to form a 0.16 M solution. The microwave vial was capped and the reaction mixture was allowed to proceed at 100 °C for 20 h. The cap was removed and the reaction mixture was concentrated under reduced pressure. The mixture of crude products was purified by reverse-phase flash chromatography to yield the final product 3 or 16.

[0258] General Procedure B: Boc and Pyrrole Deprotection: The starting material 26a or 39 (1 equiv) was dissolved in CH2Cl2 (0.1 M), and subsequently TFA (1.1 equiv) was added at 0 °C. The reaction was allowed to proceed at RT. After stirring at RT for 1 h, the crude product was concentrated under reduced pressure, redissolved and diluted with CH2Cl2, and washed with saturated NaHCO3. The organic layer was dried over Na2SO4, concentrated, giving the crude product, which was subjected to pyrrole deprotection according to the protocol of general procedure A, giving 2 or 15.

[0259] General Procedure C: Alkyne Reduction and Pyrrole Deprotection : The starting materials 38a-e (1 equiv) were dissolved in MeOH (0.1 M). The solution was degassed for 5 min and 10% wt. Pd / C was added. The reaction was allowed to proceed at RT for 20 h under a hydrogen balloon (1 atm). The crude mixture was then filtered through a Celite pad and the filtrate was concentrated under reduced pressure. Without purification, the crude product was subjected to pyrrole deprotection according to the protocol of general procedure A, giving 10-14.

[0260] General Procedure D: Boc Deprotection, Alkyne Reduction, Reductive Amination, Pyrrole Deprotection: The starting material (33a-c, 42, 43, or 45a) was dissolved in CH2Cl2 (0.1 M), and TFA (1.1 eq) was added at 0 °C. The reaction was allowed to proceed at RT for 1 h. Then, the solvent and TFA were removed under reduced pressure. The crude mixture was taken up in CH2Cl2 and diluted, and washed with saturated Na2CO3. Next, the organic layer was concentrated, and the crude product was subjected to alkyne reduction without purification. The crude product (1 eq) was dissolved in MeOH (0.1 M), the solution was degassed for 5 min, and 10% wt. Pd / C was subsequently added. The reaction was allowed to proceed at RT for 20 h under a hydrogen balloon (1 atm). Then, the crude mixture was filtered through a Celite pad, and the filtrate was concentrated under reduced pressure. The crude reduction product was diluted in MeOH (0.24 M), and 37% HCHO in H2O (3 eq) was subsequently added. The reaction was allowed to proceed at RT for 5 min. Then, the reaction was cooled to 0 °C, and NaBH4 (3 eq) was added slowly. The reaction was allowed to proceed for an additional 2 h at RT. Upon completion, the reaction was quenched with water, and the methanol was removed under reduced pressure. This aqueous mixture was extracted three times with ethyl acetate, the organic layers were combined, dried over Na2SO4, concentrated, and the crude product was obtained, which was subjected to pyrrole deprotection without purification. Then, the crude product was subjected to pyrrole deprotection according to the protocol of General Procedure A, and 5, 7, 9, 17, 18, and 19 were obtained. For compounds 4, 6, and 8, which carry only secondary amines in the tail, their synthesis followed the same protocol except that reductive amination with HCHO was omitted.

[0261] General Procedure E: Cbz Deprotection, Reductive Amination, Pyrrole Deprotection : The starting material 45b-c (1 eq) was dissolved in MeOH (0.1 M). The solution was degassed for 5 min, and 10% Pd(OH)2 / C was added. The reaction was allowed to proceed at RT for 24 h under a hydrogen balloon (1 atm). After completion, the reaction mixture was filtered through a Celite pad, and the filtrate was concentrated under reduced pressure to give the crude product, which was subjected to reductive amination with formaldehyde and pyrrole deprotection according to the same protocol described in General Procedure D, and 20 - 21 were obtained.

[0262]

Chemical Structure

[0263] 6-(3-Fluoro-5-(3-(methylamino)prop-1-yn-1-yl)phenyl)ethyl)-4-methylpyridin-2-amine (2). Compound 2 (74 mg, 50% for 2 steps) was prepared from 26b (237 mg, 0.5 mmol) according to the general procedure B. 1 H NMR (500 MHz, CD3OD) δ 7.08 (s, 1H), 6.98 - 6.91 (m, 2H), 6.29 (s, 1H), 6.27 (s, 1H), 3.58 (s, 2H), 2.92 (dd, J = 6.5, 9.4 Hz, 2H), 2.79 (dd, J = 6.3, 9.2 Hz, 2H), 2.48 (s, 3H), 2.17 (s, 3H). 13 C NMR (126 MHz, CD3OD) δ 162.4 (d, J C-F = 246.2 Hz), 157.6, 154.4, 147.9, 143.2 (d, J C-F = 8.1 Hz), 127.8 (d, J C-F = 2.9 Hz), 123.1 (d, J C-F = 10.1 Hz), 116.5 (d, J C-F = 21.5 Hz), 116.3 (d, J C-F = 23.5 Hz), 113.6, 109.6, 86.5 (d, J C-F = 3.6 Hz), 79.3, 38.0, 33.7, 33.6, 31.5, 20.6. HRMS-ESI:C 18 H 20 FN3[M + H] + Calculated value for 298.1714, found 298.1716.

[0264]

Chemical formula

[0265] 6-(3-(3-(Dimethylamino)prop-1-yn-1-yl)-5-fluorophenethyl)-4-methylpyridin-2-amine (3). Compound 3 (132 mg, 90%) was prepared from 26b (184 mg, 0.47 mmol) according to the general procedure A. 1 H NMR (500 MHz, CD3OD) δ 7.34 (s, 1H), 7.23 - 7.14 (m, 2H), 6.69 (s, 1H), 6.62 (s, 1H), 4.36 (s, 2H), 3.38 - 3.28 (m, 2H), 3.13 - 2.97 (m, 8H), 2.36 (s, 3H). 13 C NMR (126 MHz, CD3OD) δ 166.4 (d, J C-F = 246.4 Hz), 161.6, 158.4, 151.9, 147.2 (d, J C-F = 8.1 Hz), 131.8 (d, J C-F = 2.8 Hz), 126.7 (d, J C-F = 10.2 Hz), 120.8 (d, J C-F = 21.8 Hz), 120.4 (d, J C-F = 23.7 Hz), 117.5, 113.5, 92.1 (d, J C-F = 3.5 Hz), 81.8, 69.4, 45.5 (2C), 37.6, 37.5, 24.5. HRMS-ESI:C 19 H 22 FN3[M + H] + The calculated value of 312.1871, the measured value of 312.1874.

[0266]

Chemical Structure

[0267] (R)-6-(3-Fluoro-5-(2-(pyrrolidin-2-yl)ethyl)phenethyl)-4-methylpyridin-2-amine (4). Compound 4 (78 mg, 33% for 3 steps) was prepared from 33a (362 mg, 0.7 mmol) according to the general procedure D. 11H NMR (500 MHz, CD3OD) δ 7.06 (s, 1H), 6.94 - 6.86 (m, 2H), 6.68 (s, 1H), 6.63 (s, 1H), 3.56 - 3.50 (m, 1H), 3.36 - 3.34 (m, 1H), 3.23 (q, J = 7.3 Hz, 1H), 3.11 - 2.98 (m, 4H), 2.76 (t, J = 8.0 Hz, 2H), 2.36 (s, 3H), 2.31 - 2.22 (m, 1H), 2.17 - 1.94 (m, 4H), 1.79 - 1.66 (m, 1H). 13 13C NMR (126 MHz, CD3OD) δ 161.5 (d, J C-F = 244.8 Hz), 156.1, 152.9, 146.8, 141.8 (d, J C-F = 7.8 Hz), 141.0 (d, J C-F = 7.9 Hz), 122.7, 112.1, 111.4 (d, J C-F = 21.6 Hz), 111.3 (d, J C-F = 21.4 Hz), 107.9, 58.5, 43.3, 32.5, 32.4, 31.9, 30.5, 28.2, 21.6, 19.0. HRMS-ESI:C 20 H 26 FN3[M + H] + calculated value 328.2184, measured value 328.2185.

[0268]

Chemical Structure

[0269] (R)-6-(3-Fluoro-5-(2-(1-methylpyrrolidin-2-yl)ethyl)phenethyl)-4-methylpyridin-2-amine (5). Compound 5 (41 mg, 15% for 4 steps) was prepared from 33a (401 mg, 0.8 mmol) according to the general procedure D. 11H NMR (500 MHz, CD3OD) δ 7.06 (s, 1H), 6.91 (dd, J = 9.6, 20.1 Hz, 2H), 6.68 (s, 1H), 6.63 (s, 1H), 3.72 - 3.68 (m, 1H), 3.37 - 3.36 (m, 1H), 3.23 - 3.12 (m, 1H), 3.09 - 2.99 (m, 4H), 2.94 (s, 3H), 2.84 - 2.67 (m, 2H), 2.47 - 2.38 (m, 1H), 2.36 (s, 3H), 2.34 - 2.27 (m, 1H), 2.22 - 2.03 (m, 2H), 1.97 - 1.78 (m, 2H). 13 13C NMR (126 MHz, CD3OD) δ 163.1 (d, J C-F = 244.7 Hz), 157.7, 154.4, 148.4, 143.3 (d, J C-F = 7.5 Hz), 142.6 (d, J C-F = 7.8 Hz), 124.2, 113.6, 113.0 (d, J C-F = 21.4 Hz), 112.8 (d, J C-F = 21.6 Hz), 109.5, 68.7, 55.9, 38.5, 34.0, 33.9, 31.9, 31.7, 29.2, 21.1, 20.5. HRMS-ESI:C 21 H 28 FN3[M + H] + calculated value 342.2340, measured value 342.2342.

[0270]

Chemical Structure

[0271] (S)-6-(3-Fluoro-5-(2-(pyrrolidin-2-yl)ethyl)phenethyl)-4-methylpyridin-2-amine (6). Compound 6 (47 mg, 28% for 3 steps) was prepared from 33b (257 mg, 0.5 mmol) according to the general procedure D. 11H NMR (500 MHz, CD3OD) δ 7.05 (s, 1H), 6.89 (t, J = 10.9 Hz, 2H), 6.68 (s, 1H), 6.63 (s, 1H), 3.59 - 3.45 (m, 2H), 3.40 - 3.34 (m, 1H), 3.10 - 2.98 (m, 4H), 2.76 (t, J = 8.0 Hz, 2H), 2.36 (s, 3H), 2.32 - 2.23 (m, 1H), 2.18 - 1.91 (m, 4H), 1.79 - 1.62 (m, 1H). 13 13C NMR (126 MHz, CD3OD) δ 163.1 (d, J C-F = 244.9 Hz), 157.7, 154.4, 148.4, 143.4 (d, J C-F = 7.8 Hz), 142.6 (d, J C-F = 7.8 Hz), 124.2, 113.6, 112.9 (d, J C-F = 21.8 Hz), 112.8 (d, J C-F = 21.6 Hz), 109.4, 60.1, 44.9, 34.0, 33.9, 33.4, 32.0, 29.7, 23.1, 20.5. HRMS-ESI:C 20 H 26 FN3[M + H] + Calculated value for 328.2184, measured value 328.2180.

[0272]

Chemical Structure

[0273] (S)-6-(3-Fluoro-5-(2-(1-methylpyrrolidin-2-yl)ethyl)phenyl)ethyl)-4-methylpyridin-2-amine (7). Compound 7 (42 mg, 24% for 4 steps) was prepared from 33b (257 mg, 0.5 mmol) according to the general procedure D. 11H NMR (500 MHz, CD3OD) δ 7.07 (s, 1H), 6.95 - 6.88 (m, 2H), 6.70 (s, 1H), 6.63 (s, 1H), 3.71 (ddd, J = 5.0, 8.0, 11.5 Hz, 1H), 3.34 - 3.30 (m, 1H), 3.17 (dt, J = 8.4, 11.4 Hz, 1H), 3.05 (s, 4H), 2.94 (s, 3H), 2.84 - 2.67 (m, 2H), 2.45 - 2.38 (m, 1H), 2.36 (s, 3H), 2.33 - 2.26 (m, 1H), 2.21 - 2.04 (m, 2H), 1.98 - 1.80 (m, 2H). 13 13C NMR (126 MHz, CD3OD) δ 163.0 (d, J C-F = 244.7 Hz), 157.6, 154.4, 148.3, 143.4 (d, J C-F = 7.8 Hz), 142.6 (d, J C-F = 7.8 Hz), 124.2, 113.6, 113.0 (d, J C-F = 21.4 Hz), 112.8 (d, J C-F = 21.6 Hz), 109.5, 68.7, 55.9, 38.5, 34.1, 33.9, 31.9, 31.7, 29.3, 21.1, 20.6. HRMS-ESI:C 21 H 28 FN3[M + H] + calculated value 342.2340, measured value 342.2341.

[0274]

Chemical Structure

[0275] 6-(3-Fluoro-5-(2-((2R,4S)-4-fluoropyrrolidin-2-yl)ethyl)phenyl)ethyl)-4-methylpyridin-2-amine (8). Compound 8 (62 mg, 30% for 3 steps) was prepared from 33c (310 mg, 0.6 mmol) according to the general procedure D.1 1H NMR (500 MHz, CD3OD) δ 7.08 (s, 1H), 6.88 (dd, J = 17.5, 9.6 Hz, 2H), 6.69 (s, 1H), 6.61 (s, 1H), 5.44 (dt, J = 3.4, 52.3 Hz, 1H), 3.89 - 3.80 (m, 1H), 3.71 (ddd, J = 3.9, 13.9, 34.7 Hz, 1H), 3.64 - 3.49 (m, 1H), 3.09 - 2.99 (m, 4H), 2.85 - 2.71 (m, 2H), 2.60 - 2.48 (m, 1H), 2.34 (s, 3H), 2.20 (ddt, J = 7.0, 9.3, 14.0 Hz, 1H), 2.14 - 1.91 (m, 2H). 13 13C NMR (126 MHz, CD3OD) δ 162.9 (d, J C-F = 244.5 Hz), 157.5, 154.2, 148.2, 143.0 (d, J C-F = 7.4 Hz), 142.4 (d, J C-F = 7.6 Hz), 124.1, 113.5, 112.9 (d, J C-F = 21.6 Hz), 112.8 (d, J C-F = 21.8 Hz), 109.4, 91.9 (d, J C-F = 175.9 Hz), 58.5, 51.0 (d, J C-F = 24.7 Hz), 37.5 (d, J C-F = 20.9 Hz), 33.9, 33.8, 32.9, 31.9, 20.5. HRMS-ESI: C 20 H 25 F2N3 [M + H] + Calculated value for [M + H] is 346.2089, measured value is 346.2088.

[0276]

Chemical Structure

[0277] 6-(3-Fluoro-5-(2-((2R,4S)-4-fluoro-1-methylpyrrolidin-2-yl)ethyl)phenyl)ethyl)-4-methylpyridin-2-amine (9). Compound 9 (30 mg, 24% for 4 steps) was prepared from 33c (180 mg, 0.35 mmol) according to the general procedure D. 1 H NMR (500 MHz, CD3OD) δ 7.08 (s, 1H), 6.94 (d, J = 9.9 Hz, 1H), 6.90 (d, J = 9.7 Hz, 1H), 6.68 (s, 1H), 6.64 (s, 1H), 5.45 (dd, J = 5.1, 52.5 Hz, 1H), 3.94 (t, J = 15.0 Hz, 1H), 3.58 - 3.46 (m, 2H), 3.07 - 3.03 (m, 4H), 3.02 (s, 3H), 2.97 - 2.69 (m, 3H), 2.36 (s, 3H), 2.24 - 2.09 (m, 2H), 2.01 - 1.94 (m, 1H). 13 C NMR (126 MHz, CD3OD) δ 163.1 (d, J C-F = 244.7 Hz), 157.6, 154.4, 148.3, 143.0 (d, J C-F = 7.7 Hz), 142.7 (d, J C-F = 7.7 Hz), 124.2, 113.6, 113.0 (d, J C-F = 21.4 Hz), 112.9 (d, J C-F = 21.6 Hz), 109.5, 90.6 (d, J C-F = 176.0 Hz), 67.5, 61.6 (d, J C-F = 23.5 Hz), 38.4, 36.8 (d, J C-F = 22.7 Hz), 34.1, 33.9, 32.6, 31.5, 20.5. HRMS-ESI:C 21 H 27 F2N3[M + H] + Calculated value for 360.2246, measured value 360.2246.

[0278] [Chemical formula]

[0279] 6-(5-(3-(Dimethylamino)propyl)-2,3-difluorophenethyl)-4-methylpyridin-2-amine (10). Compound 10 (25 mg, 39% for 2 steps) was prepared from 38a (80 mg, 0.2 mmol) according to the general procedure C. 1 H NMR (500 MHz, CD3OD) δ 7.13 - 7.06 (m, 2H), 6.71 (s, 1H), 6.60 (s, 1H), 3.21 - 3.15 (m, 2H), 3.15 - 3.03 (m, 4H), 2.92 (s, 6H), 2.70 (t, J = 7.7 Hz, 2H), 2.36 (s, 3H), 2.11 - 2.01 (m, 2H). 13 C NMR (126 MHz, CD3OD) δ 157.6, 154.4, 150.1 (dd, J C-F = 246.6, 13.3 Hz), 147.9, 147.3 (dd, J C-F = 244.4, 12.6 Hz), 137.4 - 137.2 (m), 128.7 (d, J C-F = 12.5 Hz), 125.3 (t, J C-F = 3.0 Hz), 115.3 (d, J C-F = 17.5 Hz), 113.6, 109.6, 56.9, 42.1, 32.7, 31.1, 27.5, 25.7, 20.5. HRMS-ESI: C 19 H 25 F2N3[M + H] + Calculated value for 334.2089, measured value 334.2089.

[0280] [Chemical formula]

[0281] 6-(3-(3-(Dimethylamino)propyl)-2,6-difluorophenethyl)-4-methylpyridin-2-amine (11). Compound 11 (35 mg, 35% for 2 steps) was prepared from 38b (122 mg, 0.3 mmol) according to the general procedure C. 1 H NMR (500 MHz, CD3OD) δ 7.27 (q, J = 7.9 Hz, 1H), 6.91 (t, J = 8.8 Hz, 1H), 6.73 (s, 1H), 6.49 (s, 1H), 3.24 - 3.19 (m, 2H), 3.14 (t, J = 7.3 Hz, 2H), 3.02 (t, J = 7.3 Hz, 2H), 2.92 (s, 6H), 2.73 (t, J = 7.7 Hz, 2H), 2.32 (s, 3H), 2.04 (t, J = 8.2 Hz, 2H). 13 C NMR (126 MHz, CD3OD) δ 160.0 (dd, J C-F = 244.8, 8.2 Hz), 159.2 (dd, J C-F = 245.6, 8.2 Hz), 157.5, 154.4, 147.8, 129.3 (dd, J = 6.6, 10.0 Hz), 123.0 (dd, J = 3.7, 17.0 Hz), 114.4 (t, J = 20.6 Hz), 113.7, 110.7 (dd, J = 3.6, 22.3 Hz), 109.7, 56.9, 42.1, 31.9, 25.1 (d, J = 2.4 Hz), 24.8, 21.4, 20.5. HRMS-ESI:C 19 H 25 F2N3[M + H] + The calculated value of 334.2089, the measured value of 334.2090.

[0282]

Chem.

[0283] 6-(3-(3-(Dimethylamino)propyl)-2,5-difluorophenethyl)-4-methylpyridin-2-amine (12). Compound 12 (32 mg, 42% for two steps) was prepared from 38c (93 mg, 0.23 mmol) according to the general procedure C. 1 H NMR (500 MHz, CD3OD) δ 7.11 - 6.92 (m, 2H), 6.72 (s, 1H), 6.59 (s, 1H), 3.25 - 3.18 (m, 2H), 3.14 - 3.01 (m, 4H), 2.92 (s, 6H), 2.75 (t, J = 7.9 Hz, 2H), 2.36 (s, 3H), 2.13 - 2.01 (m, 2H). 13 C NMR (126 MHz, CD3OD) δ 158.4 (d, J C-F = 242 Hz), 157.6, 155.2 (d, J C-F = 241 Hz), 154.4, 147.9, 128.9 (dd, J = 8.1, 19.2 Hz), 128.2 (dd, J = 8.2, 19.3 Hz), 115.0 (ddd, J = 4.7, 20.3, 24.5 Hz, 2C), 113.6, 109.6, 56.9, 42.1, 32.5, 27.7, 25.4, 24.5, 20.6. HRMS-ESI:C 19 H 25 F2N3[M + H] + The calculated value of 334.2089, the measured value of 334.2092.

[0284]

Chemical formula

[0285] 6-(3-(3-(Dimethylamino)propyl)-2,5,6-trifluorophenethyl)-4-methylpyridin-2-amine (13). Compound 13 (10 mg, 24% for two steps) was prepared from 38d (50 mg, 0.12 mmol) according to the general procedure C. 11H NMR (500 MHz, CD3OD) δ 7.24 (ddd, J = 6.9, 8.8, 10.6 Hz, 1H), 6.71 (s, 1H), 6.54 (s, 1H), 3.23 - 3.13 (m, 4H), 3.03 (t, J = 7.4 Hz, 2H), 2.91 (s, 6H), 2.73 (t, J = 7.8 Hz, 2H), 2.34 (s, 3H), 2.08 - 1.96 (m, 2H). 13 13C NMR (126 MHz, CD3OD) δ 156.1, 153.0, 152.9 (ddd, J C-F = 1.3, 5.0, 242.0 Hz), 146.0, 145.8 (ddd, J C-F = 8.8, 13.9, 245.7 Hz), 145.1 (ddd, J C-F = 2.5, 12.6, 244.4 Hz), 121.9 (dt, J C-F = 5.1, 19.2 Hz), 115.2 (dd, J C-F = 16.6, 23.1 Hz), 114.5 (dd, J C-F = 5.9, 19.5 Hz), 112.2, 108.3, 55.3, 40.6, 30.2, 23.3, 23.0, 20.2, 19.0. HRMS-ESI:C 19 H 24 F3N3[M + H] + calculated value 352.1995, measured value 352.1996.

[0286]

Chemical Structure

[0287] 6-(5-(3-(Dimethylamino)propyl)-2,3,4-trifluorophenethyl)-4-methylpyridin-2-amine (14). Compound 14 (48 mg, 36% for two steps) was prepared from 38e (160 mg, 0.37 mmol) according to the general procedure C. 11H NMR (500 MHz, CD3OD) δ 7.15 (td, J = 7.6, 2.4 Hz, 1H), 6.64 (s, 1H), 6.58 (s, 1H), 3.24 - 3.16 (m, 2H), 3.12 - 2.98 (m, 4H), 2.91 (s, 6H), 2.77 (t, J = 7.7 Hz, 2H), 2.34 (s, 3H), 2.11 - 2.01 (m, 2H). 13 13C NMR (126 MHz, CD3OD) δ 156.5, 155.2, 149.3, 148.1 (dd, J C-F = 245.7, 10.1 Hz, 2C), 139.6 (td, J C-F = 250.7, 16.4 Hz), 124.8 (dd, J C-F = 7.6, 3.8 Hz), 124.3 (dd, J C-F = 13.9, 5.0 Hz), 124.0 (dd, J C-F = 13.9, 3.8 Hz), 113.6, 109.2, 56.8, 42.1, 33.4, 27.4, 24.7, 24.6, 20.4. HRMS-ESI:C 19 H 24 F3N3[M + H] + Calculated value for 352.1995, measured value 352.1997.

[0288]

Chemical Structure

[0289] 6-(2,3-Difluoro-5-(3-(methylamino)prop-1-yn-1-yl)phenyl)ethyl)-4-methylpyridin-2-amine (15). Compound 15 (80 mg, 63% for two steps) was prepared from 39 (198 mg, 0.4 mmol) according to the general procedure B. 11H NMR (500 MHz, CD3OD) δ 7.42 - 7.33 (m, 2H), 6.72 (s, 1H), 6.59 (s, 1H), 4.18 (s, 2H), 3.16 (dd, J = 6.3, 9.0 Hz, 2H), 3.06 (dd, J = 6.2, 9.0 Hz, 2H), 2.85 (s, 3H), 2.35 (s, 3H). 13 13C NMR (126 MHz, CD3OD) δ 157.6, 154.5, 149.9 (dd, J C-F = 248.4, 13.6 Hz), 149.6 (dd, J C-F = 250.6, 13.0 Hz), 147.5, 129.8 (d, J C-F = 13.5 Hz), 129.6 - 129.4 (m), 118.9 (d, J C-F = 19.3 Hz), 117.9 - 117.8 (m), 113.6, 109.8, 85.7, 79.1, 38.0, 32.4, 31.5, 27.2, 20.5. HRMS-ESI:C 18 H 19 F2N3[M + H] + Calculated value for 316.1620, measured value 316.1618.

[0290]

Chemical Structure

[0291] 6-(5-(3-(Dimethylamino)prop-1-yn-1-yl)-2,3-difluorophenethyl)-4-methylpyridin-2-amine (16). Compound 16 (65 mg, 66%) was prepared from 41 (122 mg, 0.3 mmol) according to the general procedure A. 1 1H NMR (500 MHz, CD3OD) δ 7.45 - 7.38 (m, 2H), 6.70 (s, 1H), 6.60 (s, 1H), 4.35 (s, 2H), 3.15 (t, J = 7.7 Hz, 2H), 3.10 - 2.98 (m, 8H), 2.36 (s, 3H). 1313C NMR (126 MHz, CD3OD) δ 157.7, 154.5, 150.0 (dd, J C-F = 248.8, 13.7 Hz), 149.8 (dd, J C-F = 250.9, 12.9 Hz), 147.6, 129.8 (d, J C-F = 13.5 Hz), 129.7 (t, J C-F = 3.5 Hz), 119.1 (d, J C-F = 19.5 Hz), 117.5 (dd, J C-F = 8.8, 4.7 Hz), 113.6, 109.8, 87.4, 77.7, 46.9, 41.5 (2C), 32.4, 27.2, 20.5. HRMS-ESI: C 19 H 21 F2N3[M + H] + Calculated value for 330.1776, measured value 330.1777.

[0292]

Chemical formula

[0293] (R)-6-(2,3-Difluoro-5-(2-(1-methylpyrrolidin-2-yl)ethyl)phenyl)ethyl)-4-methylpyridin-2-amine (17). Compound 17 (55 mg, 46% for 4 steps) was prepared from 42 (173 mg, 0.33 mmol) according to the general procedure D. 1 1H NMR (500 MHz, MeOD) δ 7.18 - 7.06 (m, 2H), 6.71 (s, 1H), 6.60 (s, 1H), 3.77 - 3.67 (m, 1H), 3.35 - 3.30 (m, 1H), 3.21 - 3.15 (m, 1H), 3.15 - 3.02 (m, 4H), 2.95 (s, 3H), 2.79 - 2.65 (m, 2H), 2.47 - 2.38 (m, 1H), 2.36 (s, 3H), 2.33 - 2.24 (m, 1H), 2.22 - 2.03 (m, 2H), 1.95 - 1.80 (m, 2H).13 13C NMR (126 MHz, MeOD) δ 157.6, 154.4, 150.1 (dd, J C-F = 246.9, 13.0 Hz), 147.9, 147.3 (dd, J C-F = 244.4, 13.9 Hz), 137.8 - 137.1 (m), 128.7 (d, J C-F = 12.4 Hz), 125.3 (d, J C-F = 3.2 Hz), 115.2 (d, J C-F = 17.3 Hz), 113.6, 109.6, 68.6, 55.9, 38.5, 32.7, 31.9, 31.2, 29.2, 27.5, 21.1, 20.5. HRMS-ESI: C 21 H 27 F2N3 [M + H] + Calculated value for 360.2246, measured value 360.2247.

[0294]

Chemical formula

[0295] (S)-6-(2,3-Difluoro-5-(2-(1-methylpyrrolidin-2-yl)ethyl)phenyl)ethyl)-4-methylpyridin-2-amine (18). Compound 18 (64 mg, 38% for 4 steps) was prepared from 43 (244 mg, 0.47 mmol) according to the general procedure D. 1 1H NMR (500 MHz, CD3OD) δ 7.15 - 7.07 (m, 2H), 6.70 (s, 1H), 6.60 (s, 1H), 3.71 (ddd, J = 5.0, 8.0, 11.6 Hz, 1H), 3.21 - 3.09 (m, 4H), 3.05 (dd, J = 6.6, 9.3 Hz, 2H), 2.95 (s, 3H), 2.80 - 2.63 (m, 2H), 2.46 - 2.38 (m, 1H), 2.34 - 2.24 (m, 1H), 2.21 - 2.04 (m, 2H), 1.94 - 1.80 (m, 2H). 1313C NMR (126 MHz, CD3OD) δ 157.7, 154.4, 150.1 (dd, J C-F = 246.8, 13.4 Hz), 147.9, 147.3 (dd, J C-F = 244.4, 13.9 Hz), 137.5, 128.7 (d, J C-F = 12.5 Hz), 125.2, 115.2 (d, J C-F = 17.3 Hz), 113.6, 109.6, 68.7, 55.9, 38.5, 32.7, 31.9, 31.2, 29.2, 27.5, 21.1, 20.5. HRMS-ESI: C 21 H 27 F2N3[M + H] + Calculated value for 360.2246, measured value 360.2245.

[0296]

Chemical formula

[0297] (S)-6-(2,3-Difluoro-5-(2-(4-methylmorpholin-3-yl)ethyl)phenyl)ethyl)-4-methylpyridin-2-amine (19). Compound 19 (68 mg, 28% for 4 steps) was prepared from 45a (346 mg, 0.65 mmol) according to the general procedure D. 11H NMR (500 MHz, CD3OD) δ 7.19 - 7.03 (m, 2H), 6.70 (s, 1H), 6.59 (s, 1H), 4.15 (dd, J = 13.2, 3.5 Hz, 1H), 4.09 - 4.01 (m, 1H), 3.93 - 3.77 (m, 1H), 3.63 (dd, J = 13.2, 10.4 Hz, 1H), 3.50 (dd, J = 12.9, 2.2 Hz, 1H), 3.30 - 3.25 (m, 1H), 3.17 - 3.02 (m, 4H), 2.99 (s, 3H), 2.80 - 2.59 (m, 2H), 2.36 (s, 3H), 2.32 - 2.23 (m, 1H), 1.93 - 1.78 (m, 1H). 13 13C NMR (126 MHz, CD3OD) δ 157. 7, 154.4, 150 (dd, J C-F = 248.2, 13.9 Hz), 147.9, 147.4 (dd, J C-F = 244.4, 12.6 Hz), 137.2 (d, J C-F = 5.0 Hz), 128.8 (d, J C-F = 12.5 Hz), 125.3 (d, J C-F = 3.3 Hz), 115.3 (d, J C-F = 17.8 Hz), 113.6, 109.6, 67.6, 63.7, 63.3, 54.0, 39.9, 32.7, 30.1, 28.0, 27.5, 20.5. HRMS-ESI:C 21 H 27 F2N3O [M + H] + Calculated value for 376.2195, measured value 376.2197.

[0298]

Chem.

[0299] 6-(5-(2-((2S,4R)-4-Ethoxy-1-methylpyrrolidin-2-yl)ethyl)-2,3-difluorophenethyl)-4-methylpyridin-2-amine (20). Compound 20 (22 mg, 41% for 3 steps) was prepared from 45b (75 mg, 0.13 mmol) according to the general procedure E. 1 H NMR (500 MHz, CD3OD) δ 6.96 (ddd, J = 11.3, 7.3, 2.2 Hz, 1H), 6.82 (t, J = 5.1, 2.2 Hz, 1H), 6.28 (s, 1H), 6.27 (s, 1H), 3.98 (td, J = 6.6, 6.1, 3.5 Hz, 1H), 3.47 (q, J = 6.9 Hz, 2H), 3.18 (d, J = 11.0 Hz, 1H), 3.00 (t, J = 7.8 Hz, 2H), 2.82 (d, J = 7.8 Hz, 2H), 2.68 - 2.59 (m, 1H), 2.54 - 2.47 (m, 1H), 2.42 (dt, J = 13.5, 7.4 Hz, 1H), 2.35 (dd, J = 11.0, 5.8 Hz, 1H), 2.31 (s, 3H), 2.17 (s, 3H), 2.06 - 1.97 (m, 1H), 1.60 - 1.51 (m, 2H), 1.19 (t, J = 7.0 Hz, 3H). 13 C NMR (126 MHz, CD3OD) δ 159.3, 157.5, 150.1 (dd, J C-F = 245.6, 13.3 Hz), 149.7, 147.0 (dd, J C-F = 242.9, 12.8 Hz), 138.3 (d, J C-F = 5.2 Hz), 130.3 (d, J C-F = 12.7 Hz), 125.1 (t, J C-F = 3.3 Hz), 114.2 (d, J C-F= 17.3 Hz), 113.2, 106.7, 76.3, 65.4, 63.9, 62.2, 39.0, 38.4, 37.3, 34.4, 31.6, 28.6, 19.6, 14.3. HRMS-ESI: C 23 H 31 F2N3O [M + H] + Calculated value 404.2508, measured value 404.2510.

[0300]

Chem.

[0301] (S)-6-(2,3-Difluoro-5-(2-(1-methylazetidin-2-yl)ethyl)phenyl)ethyl)-4-methylpyridin-2-amine (21). Compound 21 (15 mg, 11% for 3 steps) was prepared from 45c (213 mg, 0.4 mmol) according to the general procedure E. 1 H NMR (500 MHz, CD3OD) δ 6.93 (ddd, J = 11.3, 7.3, 2.2 Hz, 1H), 6.79 (d, J = 6.1 Hz, 1H), 6.27 (s, 2H), 3.38 (td, J = 7.7, 2.3 Hz, 1H), 3.05 (qd, J = 7.9, 5.9 Hz, 1H), 2.99 (t, J = 7.8 Hz, 2H), 2.89 - 2.84 (m, 1H), 2.82 (d, J = 7.8 Hz, 2H), 2.58 - 2.45 (m, 2H), 2.32 (s, 3H), 2.17 (s, 3H), 2.08 - 2.01 (m, 1H), 1.93 - 1.84 (m, 1H), 1.83 - 1.67 (m, 2H). 13 C NMR (126 MHz, CD3OD) δ 159.4, 157.6, 150.1 (dd, J C-F = 245.6, 13.2 Hz), 149.6, 147.0 (dd, J C-F = 242.9, 12.8 Hz), 138.2 - 138.1 (m), 130.3 (d, J C-F= 12.7 Hz), 125.1 (t, J C-F = 3.2 Hz), 114.2 (d, J C-F = 17.1 Hz), 113.2, 106.7, 68.0, 52.4, 43.4, 37.3, 37.2, 30.5, 28.6, 23.6, 19.6. HRMS-ESI: C 20 H 25 F2N3[M + H] + Calculated value of 346.2089, measured value of 346.2091.

[0302] General Procedure SA: Suzuki Coupling . In a microwave vial, aryl bromide 24 or 37a - e (1 equiv) and alkyne 25a - b, 32a - c, or 44a - c (1.5 - 2 equiv) were added. This mixture was diluted with Et3N / DMF (9:1) to form a 0.16 M solution. After degassing this mixture for 5 minutes, Pd(PPh3)4 (5 - 10 mol%) and CuI (5 - 10 mol%) were added all at once. The microwave vial was capped and the reaction mixture was allowed to proceed in a Biotage microwave reactor at 120 °C for 30 - 40 minutes. The cap was removed and the reaction mixture was filtered through a pad of celite. The filtrate was diluted with ethyl acetate, washed with water, ammonium chloride, and brine, dried over Na2SO4, and concentrated under reduced pressure. The mixture of crude products was purified by flash column chromatography to give 26a - b, 33a - c, 38a - e, 39, 41, 42, 43, or 45a - c.

[0303]

Chemical Structure

[0304] tert-Butyl (3-(3-(2-(6-(2,5-dimethyl-1H-pyrrol-1-yl)-4-methylpyridin-2-yl)ethyl)-5-fluorophenyl)prop-2-yn-1-yl)(methyl)carbamate (26a). Compound 26a was synthesized according to the general procedure SA using aryl bromide 24 (200 mg, 0.65 mmol), alkyne 25a (164 mg, 0.97 mmol), Pd(PPh3)4 (38 mg, 0.0325 mmol), and CuI (6.2 mg, 0.0325 mmol). 26a was isolated as a yellow oil (250 mg, 81%) after flash column chromatography with 10% ethyl acetate / hexane. 1 1H NMR (500 MHz, CDCl3): δ 7.02 (s, 1H), 6.91 (d, J = 9.0 Hz, 1H), 6.88 (s, 1H), 6.84 (s, 1H), 6.81 (d, J = 9.5 Hz, 1H), 5.86 (s, 2H), 4.24 (brs, 2H), 3.04 - 3.02 (m, 4H), 2.94 (s, 3H), 2.35 (s, 3H), 2.10 (s, 6H), 1.47 (s, 9H). 13 13C NMR (125 MHz, CDCl3): δ 162.4 (d, J C-F = 244.5 Hz), 160.1, 155.3, 151.7, 149.6, 144.1 (d, J C-F = 7.5 Hz), 128.5, 127.8 (d, J C-F = 2.4 Hz), 124.4 (d, J C-F = 10.0 Hz), 122.7, 120.3, 116.1 (d, J C-F = 22.9 Hz), 115.8 (d, J C-F = 21.0 Hz), 106.7, 85.4, 82.5 (d, J C-F = 3.4 Hz), 80.2, 39.0, 35.1, 35.0, 33.6, 28.4, 21.0, 13.2.

[0305]

Chemical Structure

[0306] 3-(3-(2-(6-(2,5-Dimethyl-1H-pyrrol-1-yl)-4-methylpyridin-2-yl)ethyl)-5-fluorophenyl)-N,N-dimethylprop-2-yn-1-amine (26b). Compound 26b was synthesized according to the general procedure SA using aryl bromide 24 (194 mg, 0.5 mmol), alkyne 25b (63 mg, 0.75 mmol), Pd(PPh3)4 (29 mg, 0.025 mmol), and CuI (4.7 mg, 0.025 mmol). 26b was isolated as a yellow oil (183 mg, 94%) after flash column chromatography with 5% methanol / dichloromethane. 1 H NMR (500 MHz, CDCl3): δ 7.03 (s, 1H), 6.93 (d, J = 9.0 Hz, 1H), 6.87 (s, 1H), 6.84 (s, 1H), 6.80 (d, J = 9.5 Hz, 1H), 5.87 (s, 2H), 3.43 (s, 2H), 3.04 - 3.02 (m, 4H), 2.35 (s, 3H), 2.34 (s, 6H), 2.10 (s, 6H). 13 C NMR (125 MHz, CDCl3): δ 162.4 (d, J C-F = 244.4 Hz), 160.2, 151.8, 149.6, 144.1 (d, J C-F = 8.0 Hz), 128.5, 127.8 (d, J C-F = 2.4 Hz), 124.7 (d, J C-F = 10.0 Hz), 122.7, 120.3, 116.1 (d, J C-F = 22.8 Hz), 115.5 (d, J C-F = 21.0 Hz), 106.8, 85.4, 84.3 (d, J C-F = 3.4 Hz), 48.5, 44.3, 39.1, 35.1, 21.0, 13.2. MS ESI [M+H] + = 390.04.

[0307] [Chemistry]

[0308] tert-Butyl (S)-2-((3-(2-(6-(2,5-dimethyl-1H-pyrrol-1-yl)-4-methylpyridin-2-yl)ethyl)-5-fluorophenyl)ethynyl)pyrrolidine-1-carboxylate (33a). Compound 33a was synthesized according to the general procedure SA using aryl bromide 24 (387 mg, 1 mmol), alkyne 32a (292 mg, 1.5 mmol), Pd(PPh3)4 (58 mg, 0.05 mmol), and CuI (9.5 mg, 0.05 mmol). 33a was isolated as a yellow oil (374 mg, 75%) after flash column chromatography with 10% ethyl acetate / hexane. 1 H NMR (500 MHz, CDCl3) δ 6.99 (s, 1H), 6.93 - 6.82 (m, 3H), 6.78 (d, J = 9.5 Hz, 1H), 5.87 (s, 2H), 4.80 - 4.49 (m, 1H), 3.56 - 3.21 (m, 2H), 3.08 - 2.95 (m, 4H), 2.35 (s, 3H), 2.20 - 2.00 (m, 9H), 1.91 (s, 1H), 1.47 (s, 9H). 13 C NMR (126 MHz, CDCl3) δ 162.3 (d, J C-F = 245.5 Hz), 160.2, 154.1, 151.7, 149.6, 144.0, 128.5, 127.6, 124.8 (d, J C-F = 10.0 Hz), 122.6, 120.3, 115.9 (d, J C-F = 21.9 Hz), 115.5 (d, J C-F = 20.8 Hz), 106.7, 90.7, 80.6, 79.7, 48.7, 45.6, 39.1, 35.1, 33.8, 28.6, 23.8, 21.0, 13.2.

[0309] [Chemistry]

[0310] tert-Butyl (R)-2-((3-(2-(6-(2,5-dimethyl-1H-pyrrol-1-yl)-4-methylpyridin-2-yl)ethyl)-5-fluorophenyl)ethynyl)pyrrolidine-1-carboxylate (33b). Compound 33b was synthesized according to the general procedure SA using aryl bromide 24 (387 mg, 1 mmol), alkyne 32b (292 mg, 1.5 mmol), Pd(PPh3)4 (58 mg, 0.05 mmol), and CuI (9.5 mg, 0.05 mmol). 33b was isolated as a yellow oil (258 mg, 51%) after flash column chromatography with 10% ethyl acetate / hexane. 1 H NMR (500 MHz, CDCl3) δ 6.99 (s, 1H), 6.93 - 6.82 (m, 3H), 6.78 (d, J = 9.4 Hz, 1H), 5.87 (s, 2H), 4.80 - 4.50 (m, 1H), 3.58 - 3.23 (m, 2H), 3.11 - 2.95 (m, 4H), 2.34 (s, 3H), 2.14 - 2.00 (m, 9H), 1.91 (s, 1H), 1.47 (s, 9H). 13 C NMR (126 MHz, CDCl3) δ 162.3 (d, J C-F = 246.3 Hz), 160.2, 154.1, 151.7, 149.6, 144.1, 128.5, 127.6, 124.8 (d, J C-F = 9.6 Hz), 122.6, 120.3, 115.9 (d, J C-F = 21.5 Hz), 115.5 (d, J C-F = 21.0 Hz), 106.7, 90.6, 80.5, 79.7, 48.7, 45.6, 39.1, 35.1, 33.8, 28.5, 23.8, 21.0, 13.2.

[0311] [Chemistry]

[0312] tert-Butyl (2S,4S)-2-((3-(2-(6-(2,5-dimethyl-1H-pyrrol-1-yl)-4-methylpyridin-2-yl)ethyl)-5-fluorophenyl)ethynyl)-4-fluoropyrrolidine-1-carboxylate (33c). Compound 33c was synthesized according to the general procedure SA using aryl bromide 24 (235 mg, 0.6 mmol), alkyne 32c (194 mg, 0.9 mmol), Pd(PPh3)4 (35 mg, 0.03 mmol), and CuI (5.7 mg, 0.03 mmol). 33c was isolated as a yellow oil (180 mg, 58%) after flash column chromatography with 20% ethyl acetate / hexane. 1 H NMR (500 MHz, CDCl3) δ 7.00 (s, 1H), 6.90 (d, J = 9.5 Hz, 1H), 6.86 (s, 1H), 6.84 (s, 1H), 6.78 (d, J = 9.5 Hz, 1H), 5.86 (s, 2H), 5.26 (dt, J = 4.2, 52.6 Hz, 1H), 4.83 (d, J = 58.0 Hz, 1H), 3.88 - 3.72 (m, 1H), 3.62 (ddd, J = 4.4, 13.2, 35.2 Hz, 1H), 3.05 - 2.98 (m, 4H), 2.47 (t, J = 16.2 Hz, 1H), 2.41 - 2.23 (m, 4H), 2.10 (s, 6H), 1.48 (s, 9H). 13 C NMR (126 MHz, CDCl3) δ 162.9 (d, J C-F = 245.5 Hz), 160.2, 154.1, 151.7, 149.6, 144.0, 128.5, 127.6, 124.8 (d, J C-F = 11.0 Hz), 122.7, 120.3, 115.9 (d, J C-F = 21.6 Hz), 115.5 (d, J C-F= 20.1 Hz), 106.7, 90.5 (d, J C-F = 174.3 Hz), 90.7, 80.4, 79.7, 58.5, 51.0, 39.1, 35.1, 28.5, 23.8, 21.0, 13.2. MS ESI [M+H] + = 520.07。

[0313]

Chem.

[0314] 3-(3-(2-(6-(2,5-Dimethyl-1H-pyrrol-1-yl)-4-methylpyridin-2-yl)ethyl)-4,5-difluorophenyl)-N,N-dimethylprop-2-yn-1-amine (38a). Compound 38a was synthesized according to the general procedure SA using aryl bromide 37a (103 mg, 0.5 mmol), alkyne 25b (83 mg, 1 mmol), Pd(PPh3)4 (58 mg, 0.05 mmol), and CuI (9.5 mg, 0.05 mmol). 38a was isolated as a yellow oil (156 mg, 77%) after flash column chromatography with 10% methanol / dichloromethane. 1 H NMR (500 MHz, CDCl3) δ 7.04 (ddd, J = 2.1, 7.1, 10.5 Hz, 1H), 6.97 (dd, J = 3.0, 4.9 Hz, 1H), 6.88 (s, 1H), 6.84 (s, 1H), 5.87 (s, 2H), 3.40 (s, 2H), 3.12 - 2.99 (m, 4H), 2.35 (s, 3H), 2.32 (s, 6H), 2.10 (s, 6H). 13 C NMR (126 MHz, CDCl3) δ 159.9, 151.7, 150.0 (dd, J C-F = 13.9, 248.2 Hz), 149.6, 149.1 (dd, J C-F = 12.6, 249.5 Hz), 132.1 (d, J C-F = 9.9 Hz), 130.9 (d, J C-F= 13.4 Hz), 129.0, 128.5, 122.6, 120.3, 118.2 (d, J C-F = 18.5 Hz), 106.7, 85.0, 83.4, 48.5, 44.3, 37.8, 28.6, 21.0, 13.2. MS ESI [M+H] + = 408.88。

[0315]

Chem.

[0316] 3-(3-(2-(6-(2,5-Dimethyl-1H-pyrrol-1-yl)-4-methylpyridin-2-yl)ethyl)-2,4-difluorophenyl)-N,N-dimethylprop-2-yn-1-amine (38b). Compound 38b was synthesized according to the general procedure SA using aryl bromide 37b (405 mg, 1 mmol), alkyne 25b (166 mg, 2 mmol), Pd(PPh3)4 (115 mg, 0.1 mmol), and CuI (19 mg, 0.1 mmol). 38b was isolated as a yellow oil (245 mg, 60%) after flash column chromatography with 10% methanol / dichloromethane. 1 H NMR (500 MHz, CDCl3) δ 7.24 - 7.19 (m, 1H), 6.84 (s, 2H), 6.75 (td, J = 1.4, 8.7 Hz, 1H), 5.86 (s, 2H), 3.48 (s, 2H), 3.11 - 2.98 (m, 4H), 2.35 (s, 6H), 2.33 (s, 3H), 2.09 (s, 6H). 13 C NMR (126 MHz, CDCl3) δ 161.7 (dd, J C-F = 8.8, 252.0 Hz), 161.1 (dd, J C-F = 7.6, 249.5 Hz), 160.1, 151.7, 149.4, 131.3 (dd, J C-F = 2.7, 10.0 Hz), 128.5, 122.4, 120.2, 117.2 (t, JC-F = 20.5 Hz), 111.0 (dd, J C-F = 3.9, 23.4 Hz), 107.7 (dd, J C-F = 3.8, 17.6 Hz), 106.6, 89.2, 78.1, 48.6, 44.1, 37.3, 22.7, 20.9, 13.2。

[0317]

Chem.

[0318] 3-(3-(2-(6-(2,5-Dimethyl-1H-pyrrol-1-yl)-4-methylpyridin-2-yl)ethyl)-2,5-difluorophenyl)-N,N-dimethylprop-2-yn-1-amine (38c). Compound 38c was synthesized according to the general procedure SA using aryl bromide 37c (203 mg, 0.5 mmol), alkyne 25b (83 mg, 1 mmol), Pd(PPh3)4 (58 mg, 0.05 mmol), and CuI (9.5 mg, 0.05 mmol). 38c was isolated as a yellow oil (191 mg, 94%) after flash column chromatography with 10% methanol / dichloromethane. 1 1H NMR (500 MHz, CDCl3) δ 6.92 (ddd, J = 3.2, 5.1, 8.3 Hz, 1H), 6.88 (s, 1H), 6.85 (s, 1H), 6.76 (ddd, J = 3.2, 5.5, 8.6 Hz, 1H), 5.87 (s, 2H), 3.50 (s, 2H), 3.09 - 3.00 (m, 4H), 2.35 (s, 6H), 2.35 (s, 3H), 2.09 (s, 6H). 13 13C NMR (126 MHz, CDCl3) δ 159.9, 157.6 (ddd, J C-F = 3.8, 18.9, 243.2 Hz), 151.7, 149.6, 131.2 (ddd, J C-F = 8.2, 18.5, 234.4 Hz), 128.5 (d, J C-F= 12.6 Hz), 128.5, 122.6, 120.3, 117.3, 117.1, 112.5 (dd, J C-F = 10.4, 19.6 Hz), 106.7, 90.8 (d, J C-F = 3.8 Hz), 78.1 (d, J C-F = 2.7 Hz), 48.6, 44.2, 37.6, 29.0, 21.0, 13.2。

[0319]

Chem.

[0320] 3-(3-(2-(6-(2,5-Dimethyl-1H-pyrrol-1-yl)-4-methylpyridin-2-yl)ethyl)-2,4,5-trifluorophenyl)-N,N-dimethylprop-2-yn-1-amine (38d). Compound 38d was synthesized according to the general procedure SA using aryl bromide 37d (211 mg, 0.5 mmol), alkyne 25b (83 mg, 1 mmol), Pd(PPh3)4 (58 mg, 0.05 mmol), and CuI (9.5 mg, 0.05 mmol). 38d was isolated as a yellow oil (106 mg, 50%) after flash column chromatography with 50% ethyl acetate / dichloromethane. 1 H NMR (500 MHz, CDCl3) δ 7.06 (ddd, J = 6.5, 8.6, 10.0 Hz, 1H), 6.89 - 6.80 (m, 2H), 5.86 (s, 2H), 3.47 (s, 2H), 3.14 - 2.98 (m, 4H), 2.34 (s, 3H), 2.34 (s, 6H), 2.09 (s, 6H). 13 C NMR (126 MHz, CDCl3) δ 162.5, 159.7, 159.7 - 158.2 (m), 151.8, 150.1 - 147.7 (m), 149.5, 146.4 (ddd, J C-F = 244.2, 13.1, 2.5 Hz), 128.5, 122.4, 120.4, 119.2 (dd, JC-F = 21.8, 17.4 Hz), 117.9 (d, J C-F = 20.6 Hz), 106.7, 90.5 (2xC), 48.6, 44.2, 37.1, 23.0, 20.9, 13.2。

[0321]

Chem.

[0322] 3-(5-(2-(6-(2,5-Dimethyl-1H-pyrrol-1-yl)-4-methylpyridin-2-yl)ethyl)-2,3,4-trifluorophenyl)-N,N-dimethylprop-2-yn-1-amine (38e). Compound 38e was synthesized according to the general procedure SA using aryl bromide 37e (411 mg, 0.97 mmol), alkyne 25b (161 mg, 1.94 mmol), Pd(PPh3)4 (112 mg, 0.097 mmol), and CuI (18 mg, 0.097 mmol). 38e was isolated as a yellow oil (380 mg, 92%) after flash column chromatography with 40% ethyl acetate / dichloromethane. 1 H NMR (500 MHz, CDCl3) δ 6.94 (td, J = 7.3, 2.4 Hz, 1H), 6.88 (s, 1H), 6.85 (s, 1H), 5.87 (s, 2H), 3.47 (s, 2H), 3.08 - 2.98 (m, 4H), 2.35 (s, 3H), 2.33 (s, 6H), 2.09 (s, 6H). 13 C NMR (126 MHz, CDCl3) δ 159.7, 151.8, 151.4 - 149.2 (m), 150.7 - 148.5 (m), 149.7, 140.0 (dt, J C-F = 251.0, 15.8 Hz), 128.5, 127.6 (t, J C-F = 4.3 Hz), 125.4 (dd, J C-F = 13.9, 4.1 Hz), 122.6, 120.4, 108.6 (dd, J C-F= 12.7, 4.0 Hz), 106.8, 90.7 (2xC), 48.5, 44.1, 37.7, 28.3, 21.0, 13.2. MS ESI [M+H] + = 426.46。

[0323]

Chem.

[0324] tert-Butyl (3-(3-(2-(6-(2,5-dimethyl-1H-pyrrol-1-yl)-4-methylpyridin-2-yl)ethyl)-4,5-difluorophenyl)prop-2-yn-1-yl)(methyl)carbamate (39). Compound 39 was synthesized according to the general procedure SA using aryl bromide 37a (203 mg, 0.5 mmol), alkyne 25a (169 mg, 1 mmol), Pd(PPh3)4 (58 mg, 0.05 mmol), and CuI (9.5 mg, 0.05 mmol). 39 was isolated as a yellow oil (197 mg, 80%) after flash column chromatography with 20% ethyl acetate / hexane. 1 H NMR (500 MHz, CDCl3) δ 7.03 (ddd, J = 2.0, 7.2, 9.9 Hz, 1H), 6.97 (dt, J = 1.8, 6.0 Hz, 1H), 6.88 (s, 1H), 6.85 (s, 1H), 5.87 (s, 2H), 4.22 (s, 2H), 3.11 - 3.00 (m, 4H), 2.92 (s, 3H), 2.35 (s, 3H), 2.10 (s, 6H), 1.46 (s, 9H). 13 C NMR (126 MHz, CDCl3) δ 159.9, 155.3, 151.7, 149.9 (dd, J C-F = 14.0, 248.6 Hz), 149.6, 149.1 (dd, J C-F = 12.4, 249.7 Hz), 132.1 (d, J C-F = 9.8 Hz), 131.0 (d, J C-F= 13.9 Hz), 129.1, 128.5, 122.6, 120.4, 118.2 (d, J C-F = 18.8 Hz), 106.7, 85.0, 81.7, 80.2, 39.7, 37.7, 33.6, 28.6, 28.4, 21.0, 13.2。

[0325]

Chem.

[0326] tert-Butyl (S)-2-((3-(2-(6-(2,5-dimethyl-1H-pyrrol-1-yl)-4-methylpyridin-2-yl)ethyl)-4,5-difluorophenyl)ethynyl)pyrrolidine-1-carboxylate (42). Compound 42 was synthesized according to the general procedure SA using aryl bromide 37a (203 mg, 0.5 mmol), alkyne 32a (195 mg, 1 mmol), Pd(PPh3)4 (58 mg, 0.05 mmol), and CuI (9.5 mg, 0.05 mmol). 42 was isolated as a yellow oil (170 mg, 66%) after flash column chromatography with 15% ethyl acetate / hexane. 1 1H NMR (500 MHz, chloroform-d) δ 7.00 (s, 1H), 6.95 (s, 1H), 6.88 (s, 1H), 6.84 (s, 1H), 5.87 (s, 2H), 4.63 (d, J = 65.1 Hz, 1H), 3.40 (d, J = 69.7 Hz, 2H), 3.13 - 2.96 (m, 4H), 2.35 (s, 3H), 2.10 (s, 6H), 2.08 - 1.84 (m, 4H), 1.46 (s, 9H). 13 13C NMR (126 MHz, CDCl3) δ 160.0, 154.1, 151.8, 149.8 (dd, J C-F = 13.4, 248.6 Hz), 149.6, 149.0 (dd, J C-F = 12.4, 249.7 Hz), 132.0 (d, J C-F= 9.5 Hz), 131.0 (d, J C-F = 13.9 Hz), 129.0, 128.5, 122.5, 120.3, 118.2 (d, J C-F = 18.5 Hz), 106.7, 90.6, 80.5, 79.7, 48.5, 45.6, 37.8, 33.6, 33.1, 28.6, 28.5, 21.0, 13.2。

[0327]

Chem.

[0328] tert-Butyl (R)-2-((3-(2-(6-(2,5-dimethyl-1H-pyrrol-1-yl)-4-methylpyridin-2-yl)ethyl)-4,5-difluorophenyl)ethynyl)pyrrolidine-1-carboxylate (43). Compound 43 was synthesized according to the general procedure SA using aryl bromide 37a (297 mg, 0.7 mmol), alkyne 32b (205 mg, 1 mmol), Pd(PPh3)4 (81 mg, 0.07 mmol), and CuI (13 mg, 0.07 mmol). 43 was isolated as a yellow oil (242 mg, 67%) after flash column chromatography with 15% ethyl acetate / hexane. 1 H NMR (500 MHz, CDCl3) δ 7.00 (s, 1H), 6.95 (s, 1H), 6.88 (s, 1H), 6.84 (s, 1H), 5.87 (s, 2H), 4.63 (d, J = 64.7 Hz, 1H), 3.40 (d, J = 70.6 Hz, 2H), 3.10 - 2.95 (m, 4H), 2.35 (s, 3H), 2.10 (s, 6H), 2.07 - 1.88 (m, 4H), 1.46 (s, 9H). 13 C NMR (126 MHz, CDCl3) δ 160.0, 154.1, 151.8, 149.8 (dd, J C-F = 12.4, 249.5 Hz), 149.6, 149.0 (dd, J C-F= 13.0, 248.5 Hz), 132.0 (d, J C-F = 9.4 Hz), 131.0 (d, J C-F = 13.1 Hz), 129.0, 128.5, 122.5, 120.4, 118.2 (d, J C-F = 18.4 Hz), 106.7, 90.2, 80.5, 79.7, 48.6, 45.6, 37.8, 33.8, 33.2, 28.6, 28.5, 21.0, 13.2。

[0329]

Chem.

[0330] tert-Butyl (S)-3-((3-(2-(6-(2,5-Dimethyl-1H-pyrrol-1-yl)-4-methylpyridin-2-yl)ethyl)-4,5-difluorophenyl)ethynyl)morpholine-4-carboxylate (45a). Compound 45a was synthesized according to the general procedure SA using aryl bromide 37a (460 mg, 1.1 mmol), alkyne 44a (286 mg, 1.3 mmol), Pd(PPh3)4 (130 mg, 0.011 mmol), and CuI (21 mg, 0.011 mmol). 45a was isolated as a yellow oil (435 mg, 72%) after flash column chromatography with 20% ethyl acetate / hexane. 11H NMR (500 MHz, CDCl3) δ 7.06 (ddd, J = 10.4, 7.1, 2.0 Hz, 1H), 7.01 (dt, J = 6.0, 1.8 Hz, 1H), 6.88 (s, 1H), 6.85 (s, 1H), 5.86 (s, 2H), 4.89 (s, 1H), 3.94 (d, J = 11.3 Hz, 1H), 3.90 (dd, J = 11.8, 3.1 Hz, 1H), 3.72 (d, J = 12.9 Hz, 1H), 3.63 (dd, J = 11.3, 3.2 Hz, 1H), 3.47 (td, J = 11.8, 2.7 Hz, 1H), 3.32 (t, J = 11.7 Hz, 1H), 3.11 - 2.99 (m, 4H), 2.35 (s, 3H), 2.10 (s, 6H), 1.47 (s, 9H). 13 13C NMR (126 MHz, CDCl3) δ 159.9, 154.4, 151.8, 149.8 (dd, J C-F = 13.0, 248.5 Hz), 149.6, 149.0 (dd, J C-F = 13.0, 245.5 Hz), 130.9 (d, J C-F = 13.2 Hz), 129.3 (t, J C-F = 3.4 Hz), 128.5, 122.6, 120.4, 118.7 - 118.6 (m), 118.5, 118.4, 106.7 (2xC), 86.3, 81.9, 80.9, 70.0, 66.9, 60.4, 37.8, 28.7, 28.4, 21.0, 13.2. MS ESI [M+H] + = 536.11。

[0331]

Chem.

[0332] Benzyl (2R,4R)-2-((3-(2-(6-(2,5-dimethyl-1H-pyrrol-1-yl)-4-methylpyridin-2-yl)ethyl)-4,5-difluorophenyl)ethynyl)-4-ethoxypyrrolidine-1-carboxylate (45b). Compound 45b was synthesized according to the general procedure SA using aryl bromide 37a (300 mg, 0.74 mmol), alkyne 44b (223 mg, 0.81 mmol), Pd(PPh3)4 (85 mg, 0.074 mmol), and CuI (14 mg, 0.074 mmol). 45b was isolated as a yellow oil (221 mg, 50%) after flash column chromatography with 25% ethyl acetate / hexane. 1 H NMR (500 MHz, CDCl3) δ 7.43 - 7.24 (m, 5H), 7.02 (d, J = 17.7 Hz, 1H), 6.95 - 6.90 (m, 1H), 6.87 (s, 1H), 6.84 (s, 1H), 5.87 (s, 2H), 5.24 (d, J = 12.4 Hz, 1H), 5.08 (d, J = 12.9 Hz, 1H), 4.81 - 4.71 (m, 1H), 4.10 (q, J = 7.2 Hz, 2H), 4.08 (m, 1H), 3.66 - 3.46 (m, 2H), 3.11 - 2.95 (m, 4H), 2.35 (s, 3H), 2.29 (m, 2H), 2.10 (s, 6H), 1.24 (t, J = 7.1 Hz, 3H). 13 C NMR (126 MHz, CDCl3) δ 160.0, 154.4, 151.8, 149.8 (dd, J C-F = 13.0, 248.5 Hz), 149.6, 149.0 (dd, J C-F = 12.9, 248.0 Hz), 136.7 (d, J C-F = 11.9 Hz), 130.8, 129.0 (d, J C-F = 24.5 Hz), 128.5, 128.4, 128.2 - 127.9 (m, 1C), 127.7, 122.5, 120.3, 119.2, 118.3 (d, J C-F= 18.6 Hz), 106.7, 89.8, 80.5, 76.3, 67.1, 64.3, 60.4, 52.1, 47.1, 37.8, 28.6, 21.1, 15.4, 13.2。

[0333]

Chem.

[0334] Benzyl (R)-2-((3-(2-(6-(2,5-dimethyl-1H-pyrrol-1-yl)-4-methylpyridin-2-yl)ethyl)-4,5-difluorophenyl)ethynyl)azetidine-1-carboxylate (45c). Compound 45c was synthesized following the general procedure SA with some modifications. Aryl bromide 37a (122 mg, 0.3 mmol) and alkyne 44c (71 mg, 0.33 mmol) were dissolved in DEA:DMF (1:1), and the mixture was degassed for 5 minutes. Pd(PPh3)2Cl2 (10.5 mg, 0.015 mmol), CuI (3 mg, 0.015 mmol), and PPh3 (16 mg, 0.06 mmol) were added sequentially. The reaction was carried out in a microwave reactor at 120 °C for 20 minutes. 45c was isolated as a yellow oil (123 mg, 76%) after flash column chromatography with 25% ethyl acetate / hexane. 1 H NMR (500 MHz, CDCl3) δ 7.40 - 7.24 (m, 5H), 7.06 - 6.96 (m, 2H), 6.88 (s, 1H), 6.85 (s, 1H), 5.87 (s, 2H), 5.19 (d, J = 12.6 Hz, 1H), 5.07 (d, J = 12.4 Hz, 1H), 5.00 (dd, J = 8.8, 6.0 Hz, 1H), 4.05 (td, J = 8.7, 5.6 Hz, 1H), 3.95 (td, J = 8.7, 6.5 Hz, 1H), 3.12 - 2.98 (m, 4H), 2.66 - 2.56 (m, 1H), 2.42 - 2.31 (m, 1H), 2.35 (s, 3H), 2.10 (s, 6H). 1313C NMR (126 MHz, CDCl3) δ 159.9, 156.0, 151.8, 150.0 (dd, J C-F = 13.8, 248.6 Hz), 149.6, 149.0 (dd, J C-F = 12.8, 249.0 Hz) 136.6, 134.7 (d, J C-F = 6.6 Hz), 131.0 (d, J C-F = 13.7 Hz), 130.1 (d, J C-F = 11.3 Hz), 129.2, 128.5, 128.4, 128.0, 122.5, 120.4, 118.4 (d, J C-F = 18.9 Hz), 106.7, 88.0, 83.7, 66.7, 51.0, 47.1, 37.8, 28.6, 24.4, 21.0, 13.2. MS ESI [M+H] + = 540.07。

[0335] General Procedure SB: Coupling of Pyrrolyl-Lutidine and Aryl Bromide . In a flame-dried round-bottom flask, n-BuLi 1.6 M / THF (1.1 - 1.2 equivalents) was added, diluted with THF to form a 0.8 M solution. At -78 °C, 22 (1 M in THF, 1 equivalent) was added dropwise to the n-BuLi solution. Next, the reaction was allowed to proceed at -20 °C for 15 minutes. Then, the reaction mixture was cooled back to -78 °C, and aryl bromide 23 or 36a - e (1 M in THF, 1.1 - 1.2 equivalents) was added dropwise to this reaction mixture. The reaction was allowed to proceed at -20 °C for 20 minutes and then quenched with saturated NH4Cl solution. The crude reaction mixture was partitioned between ethyl acetate and water, and the organic layer was washed with H2O and brine, dried over Na2SO4, and concentrated under reduced pressure. The crude product was purified by flash column chromatography to give 24 or 37a - e.

[0336]

Chemical Structure

[0337] 2-(3-Bromo-5-fluorophenethyl)-6-(2,5-dimethyl-1H-pyrrol-1-yl)-4-methylpyridine (24). Compound 24 was synthesized according to the general procedure SB using 22 (2 g, 10 mmol) and 23 (3.08 g, 11 mmol). 24 was isolated as a pale-yellow oil (2.55 g, 66%) after flash column chromatography with 5% ethyl acetate / hexane. 1 H NMR (500 MHz, CDCl3) δ 7.10 (t, J = 1.6 Hz, 1H), 7.06 (dt, J = 8.2, 2.1 Hz, 1H), 6.90 (s, 1H), 6.87 (s, 1H), 6.82 (dt, J = 9.4, 2.2 Hz, 1H), 5.89 (s, 2H), 3.05 - 2.98 (m, 4H), 2.37 (s, 3H), 2.12 (s, 6H). 13 C NMR (126 MHz, CDCl3) δ 162.6 (d, J C-F =.9, 151. 250.1 Hz), 1598, 149.7, 145.6 (d, J C-F = 7.8 Hz), 128.5, 127.6 (d, J C-F = 2.9 Hz), 122.7, 122.3 (d, J C-F = 10.1 Hz), 120.4, 116.7 (d, J C-F = 24.5 Hz), 114.4 (d, J C-F = 20.9 Hz), 106.8, 39.0, 35.0, 21.0, 13.2.

[0338]

Chem.

[0339] 2-(5-Bromo-2,3-difluorophenethyl)-6-(2,5-dimethyl-1H-pyrrol-1-yl)-4-methylpyridine (37a). Compound 37a was synthesized according to the general procedure SB using 22 (1 g, 5 mmol) and 36a (1.57 g, 5.5 mmol). After flash column chromatography with 5% ethyl acetate / hexane, 37a was isolated as a pale-yellow oil (1.14 g, 56%). 1 H NMR (500 MHz, CDCl3) δ 7.14 (ddd, J = 2.5, 6.7, 9.3 Hz, 1H), 7.01 (dd, J = 2.6, 5.0 Hz, 1H), 6.88 (s, 1H), 6.86 (s, 1H), 5.88 (s, 2H), 3.17 - 2.87 (m, 4H), 2.36 (s, 3H), 2.10 (s, 6H). 13 C NMR (126 MHz, CDCl3) δ 159.6, 151.8, 150.5 (dd, J C-F = 13.9, 252.0 Hz), 149.7, 132.5 (d, J C-F = 13.8 Hz), 148.4 (dd, J C-F = 11.3, 245.7 Hz), 128.5, 128.4, 122.6, 120.4, 118.5 (d, J C-F = 20.2 Hz), 106.8, 37.6, 28.6, 21.0, 13.2.

[0340]

Chemical formula

[0341] 2-(3-Bromo-2,6-difluorophenethyl)-6-(2,5-dimethyl-1H-pyrrol-1-yl)-4-methylpyridine (37b). Compound 37b was synthesized according to the general procedure SB using 22 (500 mg, 2.5 mmol) and 36b (786 mg, 2.75 mmol). After flash column chromatography with 5% ethyl acetate / hexane, 37b was isolated as a pale-yellow oil (725 mg, 71%).1 1H NMR (500 MHz, CDCl3) δ 7.37 - 7.30 (m, 1H), 6.87 - 6.83 (m, 2H), 6.74 (td, J = 1.7, 8.7 Hz, 1H), 5.86 (s, 2H), 3.15 - 3.08 (m, 2H), 3.07 - 2.97 (m, 2H), 2.34 (s, 3H), 2.10 (s, 6H). 13 13C NMR (126 MHz, CDCl3) δ 160.6 (dd, J C-F = 7.6, 248.2 Hz), 159.9, 157.7 (dd, J C-F = 9.1, 247.3 Hz), 151.7, 149.5, 130.8 (d, J C-F = 9.6 Hz), 128.5, 122.4, 120.3, 118.6 (t, J C-F = 21.3 Hz), 112.2 (dd, J C-F = 3.8, 23.9 Hz), 106.7, 103.7 (dd, J C-F = 4.0, 22.3 Hz), 37.2, 23.2, 20.9, 13.2.

[0342]

Chem.

[0343] 2-(3-Bromo-2,5-difluorophenethyl)-6-(2,5-dimethyl-1H-pyrrol-1-yl)-4-methylpyridine (37c). Compound 37c was synthesized according to the general procedure SB using 22 (1 g, 5 mmol) and 36c (1.57 g, 5.5 mmol). 37c was isolated as a pale-yellow oil (1.27 g, 63%) after flash column chromatography with 5% ethyl acetate / hexane. 11H NMR (500 MHz, CDCl3) δ 7.10 (ddd, J = 3.1, 5.1, 7.8 Hz, 1H), 6.89 (s, 1H), 6.86 (s, 1H), 6.79 (ddd, J = 3.1, 5.3, 8.5 Hz, 1H), 5.88 (s, 2H), 3.15 - 3.01 (m, 4H), 2.36 (s, 3H), 2.10 (s, 6H). 13 13C NMR (126 MHz, CDCl3) δ 159.7, 157.9 (dd, J C-F = 2.8, 246.1 Hz), 154.0 (dd, J C-F = 3.1, 241.7 Hz), 151.8, 149.7, 131.1 (dd, J C-F = 8.0, 19.4 Hz), 128.5, 122.6, 120.4, 118.0 (d, J C-F = 26.4 Hz), 116.3 (dd, J C-F = 4.3, 23.4 Hz), 109.0 (dd, J C-F = 10.7, 24.6 Hz), 106.8, 37.5, 29.3, 21.0, 13.2.

[0344] [Chemical formula]

[0345] 2-(3-Bromo-2,5,6-trifluorophenethyl)-6-(2,5-dimethyl-1H-pyrrol-1-yl)-4-methylpyridine (37d). Compound 37d was synthesized according to the general procedure SB using 22 (1.06 g, 5.3 mmol) and 36d (1.77 g, 5.82 mmol). 37d was isolated as a pale-yellow oil (1.45 g, 65%) after flash column chromatography with 5% ethyl acetate / hexane. 11H NMR (500 MHz, CDCl3) δ 7.30 - 7.20 (m, 1H), 6.92 - 6.85 (m, 2H), 5.88 (s, 2H), 3.21 - 3.00 (m, 4H), 2.37 (s, 3H), 2.11 (s, 6H). 13 13C NMR (126 MHz, CDCl3) δ 159.5, 159.3, 154.7 - 152.4 (m), 151.8, 149.7, 149.6, 148.2 (ddd, J C-F = 249.2, 13.5, 7.4 Hz), 146.8 (ddd, J C-F = 249.2, 14.3, 3.9 Hz), 128.5, 122.4, 120.4, 118.3 (d, J C-F = 21.8 Hz), 102.6 (ddd, J C-F = 24.2, 7.7, 4.4 Hz), 37.0, 23.5, 21.0, 13.2.

[0346]

Chem.

[0347] 2-(5-Bromo-2,3,4-trifluorophenethyl)-6-(2,5-dimethyl-1H-pyrrol-1-yl)-4-methylpyridine (37e). Compound 37e was synthesized according to the general procedure SB using 22 (1 g, 5 mmol) and 36e (1.67 g, 5.5 mmol). 37e was isolated as a pale-yellow oil (1.19 g, 57%) after flash column chromatography with 5% ethyl acetate / hexane. 1 1H NMR (500 MHz, CDCl3) δ7.15 (ddd, J = 9.3, 6.7, 2.5 Hz, 1H), 7.02 (dt, J = 5.4, 2.1 Hz, 1H), 6.89 (s, 1H), 6.87 (s, 1H), 5.88 (s, 2H), 3.12 - 3.00 (m, 4H), 2.36 (s, 3H), 2.11 (s, 6H). 1313C NMR (126 MHz, CDCl3) δ 159.7, 151.8, 150.5 (dd, J C-F = 13.9, 243.2 Hz), 149.7, 148.4 (dd, J C-F = 10.1, 245.7 Hz), 128.5, 128.4 (t, J C-F = 3.5 Hz ), 122.6, 120.5, 118.6, 118.5, 115.3 (dd, J C-F = 8.2, 4.4 Hz), 106.8, 37.6, 28.6, 21.0, 13.2. MS-ESI [M+H] + = 425.33。

[0348] General Procedure SC: Synthesis of Alkynes 32a-c and 44a-c 。Alkynes 32a-c and 44a-c were synthesized from their corresponding aldehydes according to the previously reported protocol using the Seyferth-Gilbert homologation reaction 1 。Generally, a dried round-bottom flask was charged with p-toluenesulfonyl azide (1.3 equiv) and K2CO3 (4.5 equiv). The mixture was dissolved in MeCN (0.1 M), and subsequently dimethyl-2-oxopropylphosphonate (1.2 mmol) was added. The mixture was stirred at RT for 2 h, and then an aldehyde (1.0 equiv) dissolved in MeOH (0.1 M) was added dropwise. The reaction was then stirred at RT for 15 h. Upon completion, the reaction mixture was filtered through Celite, and the solvent was removed under reduced pressure. The residue was partitioned between Et2O and water, and the organic layer was washed with water, brine, and dried over anhydrous Na2SO4. The solvent was removed, and the crude mixture was purified by flash chromatography to afford alkynes 32a-c

[0349]

Chemical Structure

[0350] tert-Butyl (S)-2-ethynylpyrrolidine-1-carboxylate (32a). Compound 32a was synthesized from 30a (647 mg, 3.25 mmol) according to the general procedure SC. 32a (428 mg, 67%) was isolated with 10% ethyl acetate / hexane. 1 H NMR (500 MHz, CDCl3, mixture of rotamers) δ (4.49 (s) + 4.33 (s), 1H), 3.47 - 3.28 (m, 2H), 2.16 - 2.01 (m, 1H), 1.88 - 1.64 (m, 4H), (1.42 (s) + 1.38 (s), 9H). 13 C NMR (126 MHz, CDCl3, mixture of rotamers) δ 154.0, 84.4, 79.8, (69.8 + 69.4, 1C), (48.0 + 47.8, 1C), (45.9 + 45.5, 1C), (33.7 + 33.0, 1C), 28.5, (24.4 + 23.6, 1C).

[0351]

Chemical Structure

[0352] tert-Butyl (R)-2-ethynylpyrrolidine-1-carboxylate (32b). Compound 32b was synthesized from 30b (1 g, 5 mmol) according to the general procedure SC. 32b (821 mg, 84%) was isolated with 10% ethyl acetate / hexane. 1 H NMR (500 MHz, CDCl3, mixture of rotamers) δ (4.50 (s) + 4.39 (s), 1H), 3.46 - 3.43 (m, 1H), 3.34 - 3.30 (m, 1H), 2.18 (brs, 1H), 2.04 - 1.99 (m, 3H), 1.87 brs, 1H) 1.45 (s, 9H). 1313C NMR (126 MHz, CDCl3, mixture of rotamers) δ 154.0, (84.4 + 84.1, 1C), 79.8, (69.8 + 69.4, 1C), (48.0 + 47.8, 1C), (45.9 + 45.5, 1C), (33.7 + 33.0, 1C), 28.5, (24.4 + 23.6, 1C).

[0353]

Chem.

[0354] tert-Butyl (2S,4S)-2-ethynyl-4-fluoropyrrolidine-1-carboxylate (32c). Compound 32c was synthesized from 30c (1.57 g, 7.23 mmol) according to the general procedure SC. 32c (1 g, 67%) was isolated by 20% ethyl acetate / hexane. 1 1H NMR (500 MHz, CDCl3) δ 5.27 (brs, 1H), 4.62 (brd, J = 53.6 Hz, 1H), 3.79 - 3.73 (m, 1H), 3.58 (dd, J = 35.5, 10.7 Hz, 1H), 2.42 (t, J = 16.2 Hz, 1H) 2.29 - 2.22 (m, 2H), 1.46 (s, 9H). 13 13C NMR (126 MHz, CDCl3, mixture of rotamers) δ 153.6, (93.0 (d, J C-F = 119.7 Hz) + 91.5 (d, J C-F = 119.6 Hz), 1C), 83.2 (d, J C-F = 33.5 Hz), 80.5, 70.4 (d, J C-F = 45.7 Hz), 52.7, 46.5 (d, J C-F = 22.8 Hz), 39.4 (d, J C-F = 77.0 Hz), 28.4.

[0355]

Chem.

[0356] tert-Butyl (S)-3-ethynylmorpholine-4-carboxylate (44a). Compound 44a was synthesized from S1 (336 mg, 1.56 mmol) according to the general procedure SC. 44a (200 mg, 61%) was isolated with 20% ethyl acetate / hexane. 1 H NMR (500 MHz, CDCl3) δ 4.71 (s, 1H), 4.01 - 3.83 (m, 2H), 3.68 (d, J = 13.0 Hz, 1H), 3.57 (dd, J = 11.3, 3.2 Hz, 1H), 3.44 (td, J = 11.8, 2.8 Hz, 1H), 3.29 (t, J = 13.5 Hz, 1H), 2.28 (d, J = 2.3 Hz, 1H), 1.46 (s, 9H). 13 C NMR (126 MHz, CDCl3) δ 154.4, 80.8, 80.4, 71.9, 69.9, 66.8, 44.4, 40.3, 28.3.

[0357]

Chemical formula

[0358] Benzyl (2R,4R)-4-ethoxy-2-ethynylpyrrolidine-1-carboxylate (44b). Compound 44b was synthesized from S2 (480 mg, 1.73 mmol) according to the general procedure SC. 44b (220 mg, 45%) was isolated with 30% ethyl acetate / hexane. 1 H NMR (500 MHz, CDCl3) δ 7.32 - 7.13 (m, 5H), 5.15 - 4.94 (m, 2H), 4.56 - 4.44 (m, 1H), 3.96 (brs, 1H), 3.61 - 3.49 (m, 1H), 3.48 - 3.42 (m, 2H), 3.40 - 3.31 (m, 1H), 2.26 - 2.10 (m, 3H), 1.11 (t, J = 7.0 Hz, 3H). 1313C NMR (126 MHz, CDCl3, mixture of rotamers) δ 154.4, (136.7 + 136.6, 1C), 128.4, 128.1, 127.9, 83.8, 83.4, 76.4, 70.5, 67.1, 64.4, (52.1 + 52.0, 1C), (46.9 + 46.5, 1C), (38.2 + 37.1, 1C), 15.2. MS-ESI [M+H] + = 274.28

[0359]

Chem.

[0360] Benzyl (R)-2-ethynylazetidine-1-carboxylate (44c). Compound 44c was synthesized from S3 (926 mg, 4.23 mmol) according to the general procedure SC. 44c (500 mg, 55%) was isolated by 30% ethyl acetate / hexane. 1 1H NMR (500 MHz, CDCl3) δ 7.28 - 7.16 (m, 5H), 5.01 (d, J = 6.4 Hz, 2H), 4.70 (ddd, J = 8.6, 5.9, 2.0 Hz, 1H), 3.92 (td, J = 8.8, 5.8 Hz, 1H), 3.81 (td, J = 8.7, 6.4 Hz, 1H), 2.49 - 2.41 (m, 1H), 2.43 (s, 1H), 2.25 - 2.17 (m, 1H). 13 13C NMR (126 MHz, CDCl3) δ 156.0, 136.5, 128.4, 128.0, 127.9, 82.5, 73.8, 66.8, 50.3, 47.3, 24.1. MS-ESI [M+H] + = 216.04.

[0361]

Chem.

[0362] tert-Butyl-methyl(prop-2-ynyl)carbamate (25a). 25a was synthesized in the same manner as previously reported. 2 To a solution of N-methylpropargylamine (1 equiv) in methanol, di-tert-butyl dicarbonate (1.05 equiv) was slowly added at RT. The reaction mixture was stirred overnight at RT. Upon completion, the reaction mixture was concentrated under reduced pressure, yielding 25a as a pale-yellow oil (87%). 1 H NMR (500 MHz, CDCl3) δ 4.00 (brs, 2H), 2.86 (s, 3H), 2.17 (s, 1H), 1.42 (s, 9H). 13 C NMR (125 MHz, CDCl3) δ 155.2, 80.1, 79.2, 77.3, 71.6, 33.4, 28.3; MS ESI [M+H] + = 170.03.

[0363]

Chem.

[0364] tert-Butyl (S)-2-formylpyrrolidine-1-carboxylate (30a). Aldehyde 30a was synthesized from commercially available alcohol 29a using Dess-Martin periodinane (DMP) oxidation. A dried round-bottom flask was charged with alcohol 29a (1 g, 4.97 mmol, 1 equiv) and diluted with CH2Cl2 (0.3 M). At 0 °C, DMP (2.3 g, 5.46 mmol, 1.1 equiv) was added and the reaction was allowed to proceed for 3 h at RT. Upon completion, the reaction was diluted with CH2Cl2, washed with saturated NaHCO3 solution and brine, and dried over Na2SO4. The organic layer was concentrated under reduced pressure and the crude mixture was purified by flash chromatography column with 30% ethyl acetate / hexane, yielding 30a (0.84 g, 85%). 11H NMR (500 MHz, CDCl3, mixture of rotamers) δ (9.52 (d, J = 2.2 Hz) + 9.43 (d, J = 3.0 Hz), 1H), (4.17 (td, J = 3.2, 6.1, 7.0 Hz) + 4.01 (ddd, J = 3.0, 6.3, 9.0 Hz), 1H), 3.47 (m, 2H), 2.16 - 1.76 (m, 4H), (1.44 (s) + 1.39 (s), 9H).

[0365]

Chem.

[0366] tert-Butyl (2S,4S)-4-fluoro-2-(hydroxymethyl)pyrrolidine-1-carboxylate (29c). To a dry round-bottom flask was added 28 (2 g, 8.57 mmol, 1 equiv), and diluted with THF (0.5 M). At 0 °C, BH3 1 M / THF (11 mL, 11 mmol, 1.3 equiv) was added dropwise. The reaction was allowed to proceed at RT for 24 h and then quenched with H2O. The reaction mixture was extracted three times with ethyl acetate, the combined organic layers were washed with brine, dried over Na2SO4, and concentrated under reduced pressure. The crude product 29c (1.8 g, 97%) was used in the next step without purification.

[0367] tert-Butyl (2S,4S)-4-fluoropyrrolidine-1-carboxylate-2-formyl (30c). A dry round-bottom flask was charged with alcohol 29c (1.8 g, 8.35 mmol, 1 equiv) and diluted with CH2Cl2 (0.3 M). At 0 °C, DMP (3.9 g, 9.2 mmol, 1.1 equiv) was added and the reaction was allowed to proceed at RT for 3 h. Upon completion, the reaction was diluted with CH2Cl2, washed with saturated NaHCO3 solution and brine, and dried over Na2SO4. The organic layer was concentrated under reduced pressure and the crude mixture was purified by flash chromatography column with 30% ethyl acetate / hexane to give 30c (1.57 g, 87%). 11H NMR (500 MHz, DMSO-d6, rotamer mixture) δ 9.50 (d, J = 10.1 Hz, 1H), 5.31 (dt, J = 3.2, 52.3 Hz, 1H), 4.26 (t, J = 10.3 Hz, 1H), 3.71 - 3.47 (m, 2H), 2.47 - 2.18 (m, 2H), (1.44 (s) + 1.38 (s), 9H). 13 13C NMR (126 MHz, DMSO-d6, rotamer mixture) δ (202.7 + 202.3, 1C), (154.4 + 153.6, 1C), (93.5 (d, J C-F = 121.9 Hz) + 92.2 (d, J C-F = 121.7 Hz), 1C), 80.1, (63.9 + 63.7, 1C), (53.7 (d, J C-F = 22.1 Hz) + 53.4 (d, J C-F = 22.0 Hz), 1C), (35.4 (d, J C-F = 20.8 Hz) + 34.6 (d, J C-F = 20.4 Hz), 1C), (28.5 + 28.3, 1C).

[0368]

Chem.

[0369] tert-Butyl (S)-3-(hydroxymethyl)morpholine-4-carboxylate (S1b). S1a (500 mg, 2.2 mmol, 1 equiv) was added into a dried round-bottom flask and diluted with THF (0.5 M). At 0 °C, BH3 1 M / THF (3 mL, 3 mmol, 1.3 equiv) was added dropwise. The reaction was allowed to proceed at RT for 24 h and then quenched with H2O. The reaction mixture was extracted three times with ethyl acetate, the combined organic layers were washed with brine, dried over Na2SO4, and concentrated under reduced pressure. The crude product S1b (449 mg, 94%) was used in the next step without purification. 11H NMR (500 MHz, CDCl3) δ 3.98 (s, 1H), 3.90 (d, J = 11.9 Hz, 1H), 3.87 - 3.77 (m, 3H), 3.74 - 3.69 (m, 1H), 3.54 (dd, J = 11.8, 3.5 Hz, 1H), 3.44 (td, J = 11.8, 3.1 Hz, 1H), 3.16 (t, J = 13.4 Hz, 1H), 1.45 (s, 9H). 13 13C NMR (126 MHz, CDCl3) δ 155.4, 80.5, 66.7, 66.4, 60.6, 52.1, 40.1, 28.4. MS-ESI [M+H] + = 218.14.

[0370] tert-Butyl (R)-3-formylmorpholine-4-carboxylate (S1). A dried round-bottom flask was charged with alcohol S1b (449 mg, 2 mmol, 1 equiv) and diluted with CH2Cl2 (0.3 M). At 0 °C, DMP (965 mg, 2.3 mmol, 1.1 equiv) was added and the reaction was allowed to proceed at RT for 3 h. Upon completion, the reaction was diluted with CH2Cl2, washed with saturated NaHCO3 solution, brine, and dried over Na2SO4. The organic layer was concentrated under reduced pressure and the crude mixture was purified by flash chromatography column with 30% ethyl acetate / hexane to yield S1 (336 mg, 75%). 1 1H NMR (500 MHz, CDCl3) δ 9.6 (s, 1H), 4.5 - 4.2 (m, 2H), 3.9 - 3.7 (m, 2H), 3.6 (dd, J = 12.0, 4.2 Hz, 1H), 3.5 (dd, J = 8.0, 4.2 Hz, 1H), 3.3 - 3.0 (m, 1H), 1.5 (s, 9H). 1313C NMR (126 MHz, CDCl3, mixture of rotamers) δ (199.3 + 190.0, 1CH), (155.6 + 155.1, 1C), 81.03 (1C), (66.6 + 66.3, 1CH2), (64.5 + 64.4, 1CH2), (61.7 + 60.5, 1CH), (42.42 + 41.08, 1CH2), 28.2。

[0371]

Chem.

[0372] (2R,4R)-1-((Benzyloxy)carbonyl)-4-ethoxypyrrolidine-2-carboxylic acid (S2c). A round-bottom flask was charged with S2b (2.2 g, 7.18 mmol, 1 equiv) and diluted with THF (0.25 M). At 0 °C, 1 M LiOH (11 mL, 11 mmol, 1.5 equiv) was added dropwise. The reaction was allowed to proceed at 0 °C for 3 h. Upon completion, THF was removed under vacuum and the reaction mixture was acidified to pH = 1 with 2 M HCl. The reaction mixture was extracted with ethyl acetate, the combined organic layers were washed with brine, dried over Na2SO4, and concentrated under reduced pressure. The crude product S2c (1.7 g, 80%) was used in the next step without purification. MS-ESI [M+H] + = 294.34.

[0373] Benzyl (2R,4R)-4-ethoxy-2-(hydroxymethyl)pyrrolidine-1-carboxylate (S2d). S2c (1.7 g, 5.8 mmol, 1 equiv) was added to a dry round-bottom flask and diluted with THF (0.5 M). At 0 °C, 1 M BH3 / THF (8 mL, 8 mmol, 1.4 equiv) was added dropwise. The reaction was allowed to proceed at RT for 24 h and then quenched with H2O. The reaction mixture was extracted with ethyl acetate, the combined organic layers were washed with brine, dried over Na2SO4, and concentrated under reduced pressure. The crude product S2d (1.26 g, 79%) was used in the next step without purification. 1 1H NMR (500 MHz, CDCl3) δ 7.29 - 7.18 (m, 5H), 5.02 (d, J = 6.0 Hz, 2H), 4.12 - 4.06 (m, 1H), 3.96 - 3.84 (m, 1H), 3.70 - 3.63 (m, 1H), 3.48 (dd, J = 11.9, 5.1 Hz, 2H), 3.41 (d, J = 10.6 Hz, 1H), 3.38 - 3.33 (m, 1H), 2.17 - 2.05 (m, 1H), 1.84 - 1.69 (m, 1H), 1.07 (t, J = 7.2 Hz, 3H). 1313C NMR (126 MHz, CDCl3) δ 156.5, 136.4, 128.5, 128.1, 128.0, 76.8, 67.3, 66.4, 64.6, 59.6, 52.6, 34.4, 15.2. MS-ESI [M+H] + = 280.30.

[0374] Benzyl (2R,4R)-4-ethoxy-2-formylpyrrolidine-1-carboxylate (S2). A dried round-bottom flask was charged with S2d (1.2 g, 4.5 mmol, 1 equiv) and diluted with CH2Cl2 (0.3 M). At 0 °C, DMP (2.1 g, 5 mmol, 1.1 equiv) was added and the reaction was allowed to proceed at RT for 3 h. Upon completion, the reaction was diluted with CH2Cl2, washed with saturated NaHCO3 solution and brine, and dried over Na2SO4. The organic layer was concentrated under reduced pressure and the crude product S2 (0.96 g, 77%) was used in the next step without purification. MS-ESI [M+H] + = 278.26.

[0375]

Chemical formula

[0376] Benzyl (2R,4R)-4-ethoxy-2-(hydroxymethyl)pyrrolidine-1-carboxylate (S3b). In a dried round-bottom flask, S3a (2 g, 8.5 mmol, 1 equiv) was added and diluted with THF (0.5 M). At 0 °C, BH3 1 M / THF (12 mL, 12 mmol, 1.4 equiv) was added dropwise. The reaction was allowed to proceed at RT for 24 h and then quenched with H2O. The reaction mixture was extracted with ethyl acetate, the combined organic layers were washed with brine, dried over Na2SO4 and concentrated under reduced pressure. The crude product S3b (1.2 g, 64%) was used in the next step without purification. MS-ESI [M+H] + = 222.15.

[0377] Benzyl (2R,4R)-4-ethoxy-2-formylpyrrolidine-1-carboxylate (S3). A dry round-bottom flask was charged with S3b (1.3 g, 5.8 mmol, 1 equiv) and diluted with CH2Cl2 (0.3 M). At 0 °C, DMP (2.7 g, 6.4 mmol, 1.1 equiv) was added and the reaction was allowed to proceed at RT for 3 h. Upon completion, the reaction was diluted with CH2Cl2, washed with saturated NaHCO3 solution and brine, and dried over Na2SO4. The organic layer was concentrated under reduced pressure and the crude mixture was purified by flash chromatography column with 40% ethyl acetate / hexane to give S3 (0.92 g, 72%). 1 H NMR (500 MHz, CDCl3) δ 9.71 (s, 1H), 7.20 - 7.13 (m, 5H), 5.01 (d, J = 6.5 Hz, 2H), 4.67 - 4.56 (m, 1H), 3.86 - 3.61 (m, 2H), 2.45 - 2.34 (m, 1H), 2.24 - 2.15 (m, 1H). MS-ESI [M+H] + = 220.04.

[0378]

Chemical Structure

[0379] (5-Bromo-2,3-difluorophenyl)methanol (S4c). In a dry round-bottom flask, S4b (1.1 g, 5 mmol, 1 equiv) was added and diluted with MeOH (0.2 M). At 0 °C, NaBH4 (265 mg, 7 mmol, 1.4 equiv) was added. The reaction was allowed to proceed at RT for 3 h and then quenched with H2O. The reaction mixture was extracted with ethyl acetate, the combined organic layers were washed with brine, dried over Na2SO4, and concentrated under reduced pressure. The crude mixture was purified by flash chromatography column with 20% ethyl acetate / hexane to give S4c (1 g, 81%). 1 1H NMR (500 MHz, CDCl3) δ 7.35 (dt, J = 2.2, 5.1 Hz, 1H), 7.24 (ddd, J = 2.4, 7.1, 9.3 Hz, 1H), 4.73 (s, 2H), 2.15 (s, 1H). 13 13C NMR (126 MHz, CDCl3) δ 150.1 (dd, J C-F = 13.6, 253.1 Hz), 147.4 (dd, J C-F = 12.9, 248.6 Hz), 131.7 (d, J C-F = 12.5 Hz), 126.5 (t, J C-F = 3.3 Hz), 119.8 (d, J C-F = 20.1 Hz), 115.9 (dd, J C-F= 4.4, 8.0 Hz), 58.3 (t, J C-F = 3.6 Hz).

[0380] 5-Bromo-1-(bromomethyl)-2,3-difluorobenzene (36a). In a dried round-bottom flask, S4c (1 g, 4.5 mmol, 1 equiv) was added and diluted with CH2Cl2 (0.2 M). At 0 °C, PBr3 1 M / CH2Cl2 (5.4 mL, 5.4 mmol, 1.2 equiv) was added dropwise. The reaction was allowed to proceed at 0 °C for 1 h and then quenched with saturated NaHCO3 solution. The reaction mixture was extracted with ethyl acetate, and the combined organic layers were washed with brine, dried over Na2SO4, and concentrated under reduced pressure. The crude mixture was purified by flash chromatography column with 5% ethyl acetate / hexane to give 36a (0.7 g, 60%). 1 1H NMR (500 MHz, CDCl3) δ 7.29 - 7.19 (m, 2H), 4.37 (s, 2H). 13 13C NMR (126 MHz, CDCl3) δ 150.4 (dd, J C-F = 13.5, 254.2 Hz), 148.0 (dd, J C-F = 13.1, 252.6 Hz), 129.0 (d, J C-F = 12.4 Hz), 128.8 (d, J C-F = 3.5 Hz), 121.0 (d, J C-F = 20.3 Hz), 115.8 (dd, J C-F = 4.8, 8.1 Hz), 23.4 (t, J C-F = 3.7 Hz).

[0381]

Chemical Structure

[0382] 1-Bromo-3-(bromomethyl)-2,4-difluorobenzene (36b). In a dry round-bottom flask, S6 (2 g, 9.7 mmol, 1 equiv), NBS (2 g, 12 mmol, 1.2 equiv) and (PhCO)2O2 (75 mg, 0.3 mmol, 0.03 equiv) were added. The mixture was diluted with CCl4 (0.125 M) and irradiated with a mercury lamp at RT for 1 h. Upon completion, the reaction mixture was filtered through a Celite pad, the filtrate was diluted with CH2Cl2, washed with water and brine, and concentrated under reduced pressure. The crude mixture was purified by flash chromatography column with 100% hexane to give 36b (2.34 g, 85%). 1 1H NMR (500 MHz, CDCl3) δ 7.48 (td, J = 5.9, 8.3 Hz, 1H), 6.83 (td, J = 1.8, 8.8 Hz, 1H), 4.50 (s, 2H). 13 13C NMR (126 MHz, CDCl3) δ 160.0 (dd, J C-F = 5.8, 252.8 Hz), 157.3 (dd, J C-F = 7.2, 252.2 Hz), 133.5 (dd, J C-F = 1.8, 9.9 Hz), 116.1 (t, J C-F = 19.5 Hz), 112.7 (dd, J C-F = 4.1, 22.7 Hz), 104.2 (dd, J C-F = 4.1, 21.4 Hz), 17.5 (t, J C-F = 4.4 Hz).

[0383]

Chemical Structure

[0384] 1-Bromo-3-(bromomethyl)-2,5-difluorobenzene (36c). In a dried round-bottom flask, S7 (3.4 g, 16.4 mmol, 1 equiv), NBS (3.2 g, 18 mmol, 1.1 equiv) and (PhCO)2O2 (120 mg, 0.5 mmol, 0.03 equiv) were added. The mixture was diluted with CCl4 (0.125 M) and irradiated with a mercury lamp at RT for 1 h. Upon completion, the reaction mixture was filtered through a Celite pad, the filtrate was diluted with CH2Cl2, washed with water and brine, and concentrated under reduced pressure. The crude mixture was purified by flash chromatography column with 100% hexane to give 36c (1.64 g, 35%). 1 1H NMR (500 MHz, CDCl3) δ 7.24 (ddd, J = 3.1, 5.4, 7.6 Hz, 1H), 7.07 (ddd, J = 3.1, 5.3, 8.2 Hz, 1H), 4.43 (s, 2H). 13 13C NMR (126 MHz, CDCl3) δ 159.0 - 156.4 (m), 153.5 (dd, J C-F = 2.7, 245.8 Hz), 127.5 (dd, J C-F = 8.4, 17.8 Hz), 120.7 (d, J C-F = 26.4 Hz), 116.8 (dd, J C-F = 2.5, 24.1 Hz), 109.8 (dd, J C-F = 10.4, 23.8 Hz), 24.4 (d, J C-F = 3.8 Hz).

[0385]

Chemical formula

[0386] In another dry round-bottom flask, S8a (422 mg, 2 mmol, 1 equiv) was added and diluted with 4 mL of THF (0.5 M). At -78 °C, freshly generated LDA was added dropwise to S8a via cannula, and the reaction mixture was stirred at the same temperature for 10 minutes. Then dry ice in Et2O was added, and the reaction was warmed to RT and allowed to proceed for 30 minutes, then quenched with 1 M HCl. The reaction mixture was extracted with ethyl acetate, the combined organic layers were washed with brine, dried over Na2SO4, and concentrated under reduced pressure. The crude product (453 mg, 88%) was used in the next step without purification. 1 1H NMR (500 MHz, CDCl3) δ 8.45 (s, broad, 1H), 7.59 (td, J = 6.2, 8.4 Hz, 1H).

[0387] (3-Bromo-2,5,6-trifluorophenyl)methanol (S8c). In a dry round-bottom flask, S8b (450 mg, 1.76 mmol, 1 equiv) was added and diluted with THF (0.5 M). At 0 °C, BH3 1 M / THF (2.5 mL, 2.5 mmol, 1.4 equiv) was added dropwise. The reaction was allowed to proceed at RT for 24 h, then quenched with H2O. The reaction mixture was extracted three times with ethyl acetate, the combined organic layers were washed with brine, dried over Na2SO4, and concentrated under reduced pressure. The crude product S8c (327 mg, 77%) was used in the next step without purification. 1 1H NMR (500 MHz, CDCl3) δ 7.46 - 7.29 (m, 1H), 4.79 (s, 2H), 2.29 (s, 1H).

[0388] 1-Bromo-3-(bromomethyl)-2,4,5-trifluorobenzene (36d). S8c (241 mg, 1 mmol, 1 equiv) was added into a dried round-bottom flask and diluted with CH2Cl2 (0.2 M). At 0 °C, PBr3 1 M / CH2Cl2 (1.2 mL, 1.2 mmol, 1.2 equiv) was added dropwise. The reaction was allowed to proceed at 0 °C for 1 h and then quenched with saturated NaHCO3 solution. The reaction mixture was extracted with ethyl acetate, and the combined organic layers were washed with brine, dried over Na2SO4, and concentrated under reduced pressure. The crude mixture was purified by a flash chromatography column with 100% hexane to give 36d (273 mg, 90%). 1 1H NMR (500 MHz, CDCl3) δ 7.25 (td, J = 6.4, 8.6 Hz, 1H), 4.36 (t, J = 1.5 Hz, 2H). 13 13C NMR (126 MHz, CDCl3) δ 153.1 (dt, J C-F = 4.1, 248.8 Hz), 147.9 (ddd, J C-F = 6.3, 13.9, 255.8 Hz), 146.8 (ddd, J C-F = 5.0, 13.9, 250.7 Hz), 120.9 (d, J C-F = 21.5 Hz), 117.4 (dd, J C-F = 15.9, 21.3 Hz), 103.2 (ddd, J C-F = 4.8, 7.9, 23.8 Hz), 16.9 (t, J C-F = 3.3 Hz).

[0389]

Chemical formula

[0390] In another dry round-bottom flask, S8a (500 mg, 2.4 mmol, 1 equiv) was added and diluted with 4 mL of THF (0.5 M). At -78 °C, freshly generated LDA was added dropwise to S8a via cannula, and the reaction mixture was stirred at the same temperature for 10 minutes. Then dry ice in Et2O was added, the reaction was warmed to RT and allowed to proceed for 30 minutes, and then quenched with 1 M HCl. The reaction mixture was extracted with ethyl acetate, the combined organic layers were washed with brine, dried over Na2SO4, and concentrated under reduced pressure. The crude product (410 mg, 67%) was used in the next step without purification. 1 H NMR (500 MHz, CDCl3) δ 8.66 (broad, s, 2H), 8.05 (td, J = 6.9, 2.4 Hz, 1H). 13 C NMR (126 MHz, CDCl3) δ 166.8 (t, J C-F = 3.3 Hz), 152.3 (ddd, J C-F = 258.9, 11.4, 3.1 Hz), 151.4 (ddd, J C-F = 268.2, 11.3, 2.8 Hz), 141.1 (dt, J C-F = 257.5, 15.9 Hz), 129.8 (d, J C-F = 3.9 Hz), 115.6 (dd, J C-F = 7.4, 4.0 Hz), 104.9 (dd, J C-F = 18.6, 4.6 Hz).

[0391] (3-Bromo-2,5,6-trifluorophenyl)methanol (S9c). S9b (3.6 g, 14.12 mmol, 1 equiv) was added into a dried round-bottom flask and diluted with THF (0.5 M). At 0 °C, BH3 1 M / THF (19.8 mL, 19.8 mmol, 1.4 equiv) was added dropwise. The reaction was allowed to proceed at RT for 24 h and then quenched with H2O. The reaction mixture was extracted three times with ethyl acetate, the combined organic layers were washed with brine, dried over Na2SO4, and concentrated under reduced pressure. The crude mixture was purified by flash chromatography column with 20% ethyl acetate / hexane to yield S9c (3.16 g, 93%). 1 1H NMR (500 MHz, CDCl3) δ 7.24 (td, J = 6.9, 2.5 Hz, 1H), 4.56 (s, 2H), 3.70 (s, 1H). 13 13C NMR (126 MHz, CDCl3) δ 148.2 (dt, J C-F = 250.2, 3.4 Hz), 148.1 (ddd, J C-F = 250.1, 5.0, 3.4 Hz), 140.2 (dt, J C-F = 255.7, 16.1 Hz), 129.7 (d, J C-F = 3.8 Hz), 125.8 (dd, J C-F = 12.8, 4.1 Hz), 125.6 (t, J C-F = 4.0 Hz), 104.5 (dd, J C-F = 18.1, 4.3 Hz), 58.0 (t, J C-F = 3.0 Hz).

[0392] 1-Bromo-3-(bromomethyl)-2,4,5-trifluorobenzene (36e). S9c (3.16 g, 13 mmol, 1 equiv) was added into a dried round-bottom flask and diluted with CH2Cl2 (0.2 M). At 0 °C, PBr3 1 M / CH2Cl2 (6.6 mL, 6.6 mmol, 0.5 equiv) was added dropwise. The reaction was allowed to proceed at 0 °C for 1 h and then quenched with saturated NaHCO3 solution. The reaction mixture was extracted with ethyl acetate, the combined organic layers were washed with brine, dried over Na2SO4, and concentrated under reduced pressure. The crude mixture was purified by flash chromatography column with 100% hexane to yield 36e (1.68 g, 42%). 1 1H NMR (500 MHz, CDCl3) δ 7.38 (td, J = 6.9, 2.6 Hz, 1H), 4.42 (s, 2H). 13 13C NMR (126 MHz, CDCl3) δ 148.9 (dd, J C-F = 2.5, 252.0 Hz), 148.8 (dd, J C-F = 2.5, 252.0 Hz), 140.5 (dt, J C-F = 256.7, 16.0 Hz), 127.8 (t, J C-F = 3.2 Hz), 123.6 (dd, J C-F = 12.7, 4.2 Hz), 104.7 (dd, J C-F = 18.4, 4.6 Hz), 23.2 (t, J C-F = 3.1 Hz).

[0393] NOS Enzyme Inhibition Assay . The NOS inhibitory activities of 2 - 21 were measured by hemoglobin (Hb) NO capture assay according to the protocol described previously 24,29. Briefly, this assay was performed at 37 °C in 100 mM HEPES buffer (pH 7.4) containing 10% glycerol, in the presence of 10 μM L-Arg, 10 μM H4B, 100 μM NADPH, 0.83 mM CaCl2, 320 units / mL calmodulin, and 3 μM human oxyhemoglobin. The 10 μM concentration of L-Arg was sufficient so as not to cause NOS uncoupling and was used because it is close to the K m value of all three NOS isoforms where competitive inhibitors can be effectively detected. This assay was performed in a 96-well plate using a Biotek Gen5 (trademark) microplate reader. NO production was monitored kinetically at 410 nm for 6 minutes. Rat nNOS 30 , human nNOS 31 , mouse macrophage iNOS 32 , human iNOS 33 and human eNOS 34 were expressed and purified in E. coli as previously reported. The inhibition constant (K i ) for all NOS was calculated from the IC 50 value of the dose–response curve using the Cheng–Prusoff equation: K i = IC 50 / (1 + [S] / K m ) 35 , and K m (human nNOS: 1.6 μM; rat nNOS: 1.3 μM; mouse iNOS: 8.2 μM; bovine eNOS: 1.7 μM; human eNOS: 3.9 μM; human iNOS: 8.0 μM) 36,37 . The dose–response curves were composed of 7–9 test concentrations (200 μM to 50 nM), and the IC 50 values were calculated by non-linear regression using GraphPad Prism software. The standard deviation calculated from the dose–response curves of this assay was less than 10% for all NOS.

[0394] PAMPA-BBB Assay . The PAMPA-BBB assay was performed according to the protocol described previously 24. Briefly stated, this assay was performed in 10 mM PBS buffer (pH = 7.5), and the compounds were tested at a concentration of 200 μM. The donor plate was first coated with 4 μL of porcine brain lipid (20 mg / mL in dodecane), and then 250 μL of the test compound was added. The acceptor plate was filled with 250 μL of PBS, and the donor plate was carefully placed on top of the acceptor plate to create a "sandwich". The plate was incubated at 25 °C for 17 h with swirling agitation at 100 rpm in an atmosphere of saturated humidity. Verapamil and theophylline were used as the positive control and negative control, respectively. After incubation, 150 μL of the test solution was collected from each well on both sides (donor and acceptor) and transferred to a UV plate for measurement. The effective permeability (P e ) was calculated using the following formula 38 :

Equation

Equation

[0395] Caco-2 Assay. The bidirectional Caco-2 assay was performed by Sai Life Sciences (Pune, India) or Chempartner (Shanghai, China). Briefly, this assay was conducted in a buffer of Hank's balanced salt solution (HBSS) (pH = 7.4) at 37 °C for 90 minutes. The compounds were tested at a concentration of 5 μM (0.1% DMSO). The tested compounds were applied either to the apical side (A→B direction) or the basolateral side (B→A direction). The apparent permeability (P app ) was calculated using the following formula: P app = (dQ / dt) / C0·A, where dQ / dt is the change in the concentration of the test compound in the receiver chamber over time, C0 is the initial concentration of the compound in the donor well, and A is the filter well area (0.7 cm 2 ). The excretion ratio is defined as the ratio of the apparent permeability of B→A to that of A→B. An ER value greater than 3 suggests that the compound may be a substrate of P-gp or other active excretion transporters.

[0396] Preparation of Inhibitor Complex Crystals . Using the sitting-drop vapor diffusion method, crystallization was carried out at 4 °C for the heme domains of rat nNOS (8 mg / mL containing 20 mM histidine), human nNOS K301R / R354A / G357D mutant (10 mg / mL), and human eNOS (7 mg / mL). The crystallization conditions have been described previously 39 . Fresh crystals were first passed through a cryoprotectant solution stepwise, then immersed in 5 - 10 mM inhibitor at 4 °C for 3 - 4 h, then flash-cooled with liquid nitrogen, and stored until data collection. The presence of acetate ions near the heme active site of bovine eNOS caused interference in the binding mode for some inhibitors 40 . The high concentration of magnesium acetate in the heNOS crystallization conditions may also introduce acetate near the active site, which can affect the binding mode of the inhibitor. To avoid having this acetate in the structure, magnesium acetate in the cryoprotectant solution was replaced with MgCl2.

[0397] X-Ray Diffraction Data Collection, Data Processing, and Structure Refinement At extremely low temperature (100 K), X-ray diffraction data were remotely collected at the Stanford Synchrotron Radiation Lightsource (SSRL) or the Advanced Light Source (ALS) using the data collection control software Blu-Ice 41 and a crystal-mounting robot. When using a CCD detector, 100 - 125° of the data were typically collected at 0.5° per frame. When using a Pilatus pixel array detector, 140 - 160° of the finely sliced data were collected at 0.2° per frame. The raw CCD data frames were indexed, integrated, and scaled using iMOSFLM 42 while the pixel array data were processed using XDS 43 and scaled using Aimless 44 . The binding of the inhibitor was detected by an anomalous difference Fourier map calculated using REFMAC 45 . Next, the inhibitor molecule was modeled using Coot 46 and refined using REFMAC or PHENIX 47 . The crystal packing of the MgCl2-soaked heNOS crystal was slightly changed, which resulted in a symmetry change from the previously reported orthorhombic P212121 48 to the monoclinic P21, and the β angle was slightly smaller by 0.6 - 0.7° compared to the initial 90°. Therefore, molecular replacement calculations using PHASER-MR 49 were necessary to elucidate the structure. In the P21 space group, there were two heNOS dimers in the asymmetric unit. Partial disorder of the inhibitor bound at the NOS active site was often observed, which sometimes led to poor density quality. However, the partial structural features were usually still clear when the equivalent position level of the sigma A-weighted 2m|Fo|-D|Fc| map was dropped to 0.5σ, which enabled the construction of a reasonable model for the disordered region. Water molecules were added using PHENIX and checked using Coot. The TLS protocol50 This was carried out at the final stage of refinement by each subunit as one TLS group. The Fo-Fc density omit map was calculated by removing the inhibitor coordinates from the input PDB file before attempting another round of TLS refinement in PHENIX (simulating an annealing protocol at an initial temperature of 2000K). The resulting map coefficients DELFWT / PHDELWT were used to create the map. For some of the latest structures, the Polder map facility in PHENIX was used to calculate the density omit map for the combined inhibitor. 51 The refined structure was verified in Coot before deposition in the Protein Data Bank.

[0398] References

[0399] 1. Muller, S.; Liepold, B.; Roth, G. J.; Bestmann, H. J., An Improved One-pot Procedure for the Synthesis of Alkynes from Aldehydes. Synlett, 1996, 1996, 521-522.

[0400] 2. Wang, H.-Y.; Qin, Y.; Li, H.; Roman, L. J.; Martasek, P.; Poulos, T. L.; Silverman, R. B. Potent and Selective Human Neuronal Nitric Oxide Syntherhase Inhibition by Optimization of the 2-Aminopyridine-Based Scaffold with a Pyridine Linker. Journal of Medicinal Chemistry, 2016, 59, 4913-4925

[0401] Example 3

[0402] In the brain, nitric oxide (NO) produced by neuronal nitric oxide synthase (nNOS) contributes to neurotransmission. However, excessive production of NO by over-activated nNOS is harmful and is involved in many neurodegenerative diseases, including Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis, cerebral palsy, and ischemic stroke. Therefore, inhibition of nNOS holds promise as a therapeutic approach for developing novel drugs for these diseases. In CNS drug discovery, effective delivery of therapeutic agents to the human brain is one of the most challenging tasks due to the presence of the blood-brain barrier (BBB), which excludes most potential molecules from entering the brain. In this study, the inventors focused on enhancing their BBB permeability and report the design and synthesis by the inventors of novel human neuronal nitric oxide synthase (nNOS) inhibitors. The inventors have not only demonstrated excellent efficacy (K i <30 nM) and high isoform selectivity in nNOS inhibition, but also shown significant improvement in brain penetration, as indicated by high passive permeability in the PAMPA-BBB assay and low efflux ratio in the Caco-2 bidirectional assay. Several novel analogs have been developed.

[0403] The treatment of neurodegenerative diseases commonly known as Alzheimer's disease (AD), Parkinson's disease (PD) and amyotrophic lateral sclerosis (ALS) remains very limited. Current treatments merely assist in delaying symptoms. In addition, the increasing number of patients (over 5 million living Americans with AD in 2017) and the huge costs required for treatment emphasize the urgent medical need for these diseases. Inhibition of human nNOS holds promise as a valuable therapeutic tool for treating these disorders. The human nNOS inhibitors represented by the present invention not only exhibit high efficacy and selectivity, but also enhanced brain penetration, indicating high potential for further development of novel therapeutic agents for neurodegeneration.

[0404]

Table 4-1

Table 4-2

[0405] The compounds in Table 4 were prepared using a procedure similar to the procedure outlined in Example 2. Similarly, these compounds were tested according to the procedure outlined in Example 2.

[0406] In the foregoing description, it will be readily apparent to those skilled in the art that various substitutions and modifications can be made to the invention disclosed herein without departing from the scope and spirit of the invention. The invention illustratively described herein can preferably be practiced without the presence of one or more elements or one or more limitations specifically not disclosed herein. The terms and expressions used are used as terms of description and not of limitation, and there is no intention to exclude any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention. Accordingly, it should be understood that the invention is illustrated by specific embodiments and any features thereof, but that modifications and / or variations of the concepts disclosed herein may be made by those skilled in the art, and such modifications and variations are considered to be within the scope of the invention.

[0407] Numerous patent and non-patent documents are cited herein. The cited references are hereby incorporated by reference in their entirety. In the event of a conflict between the definition of a term herein and the definition of the term in the cited reference, the term shall be construed based on the definition herein.

Claims

**Claim 1** A compound of the following formula, or a salt or solvate thereof: 【Chemical 1】 (In the formula: 【Chemical 2】 represents a single bond, double bond, or triple bond; Y is a substituted aryl or a substituted heteroaryl, and Y is substituted at one or more ring positions with a substituent having the formula -X-R ａ ; and X is C １ -C ６ -alkyl, C １ -C ６ -alkenyl, and C １ -C ６ -alkynyl; is selected from R ａ is selected from 4- to 6-membered heterocycles containing amino, alkylamino, dialkylamino, or at least one nitrogen atom, and whose heterocycle is optionally substituted at one or more positions by alkyl, alkoxy, or halo; and where Y is optionally substituted by halo at one or more ring positions). **Claim 2** Said R ａ is selected from pyrrolidinyl optionally substituted by alkyl, alkoxy or halo at one or more positions, azetidinyl optionally substituted by alkyl at one or more positions, and morpholinyl optionally substituted by alkyl at one or more positions, the compound according to claim 1. **Claim 3** The compound according to claim 1, having formula (I): 【Chemical Formula 3】 (wherein, R ２ , R ３ , R ４ , and R ６ are each independently H or halogen). **Claim 4** Said R ３ The compound according to claim 3, wherein R is a halogen. **Claim 5** Said R ２ The compound according to claim 3 or 4, wherein R is a halogen. **Claim 6** Said R ２ is a halogen, and R ３ is a halogen (for example, fluoro), the compound according to claim 3. **Claim 7** The compound according to any one of claims 1 to 6, wherein X is methyl, ethyl, or propyl. **Claim 8** Said R ａ is dimethylamino, the compound according to any one of claims 1 to 7. **Claim 9** The compound according to any one of claims 1 to 8, having the following formula: 【Chemical Formula 4】 (wherein R ２ , R ３ , R ４ , and R ６ are each independently H or halogen). **Claim 10** The compound according to any one of claims 1 to 9, having the following formula: 【Chemical Formula 5】 【Chemical Formula 6】 [Chemical Formula 7] **Claim 11** The compound has an effective permeability P －６ across the blood-brain barrier of at least about 8 × 10 ｅ cm / s, the compound according to any one of claims 1 to 10. **Claim 12** The method according to any one of claims 1 to 11, wherein the compound has a selectivity of nNOS over iNOS of at least about 30. **Claim 13** The compound according to any one of claims 1 to 12, wherein the compound has a selectivity of nNOS over eNOS of at least about 1000. **Claim 14** A pharmaceutical composition comprising a compound according to any one of claims 1 to 13 and a pharmaceutically acceptable carrier. **Claim 15** A method of treating or preventing a disease or disorder associated with nitric oxide synthase in a subject in need thereof, comprising administering a compound according to any one of claims 1 to 13 or the pharmaceutical composition according to claim 14. **Claim 16** The method according to claim 15, wherein the disease or disorder is a neurodegenerative disease or disorder. **Claim 17** The method according to claim 15, wherein the disease or disorder is Alzheimer's disease. **Claim 18** The method according to claim 15, wherein the disease or disorder is Huntington's disease. **Claim 19** The method according to claim 15, wherein the disease or disorder is Parkinson's disease. **Claim 20** The method according to claim 15, wherein the disease or disorder is amyotrophic lateral sclerosis (ALS). **Claim 21** The method according to claim 15, wherein the disease or disorder is cerebral palsy. **Claim 22** The method according to claim 15, wherein the disease or disorder is migraine. **Claim 23** A method of inhibiting intracellular nitric oxide synthase (NOS), comprising contacting a cell with any one of the compounds according to claims 1 to 13.

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