Charged ion channel blocker and method of use

Abstract

The present invention provides a compound of the following formula (I) or a pharmaceutically acceptable salt thereof: The compounds, compositions, methods, and kits of the present invention are useful for the treatment of pain, cough, itching, and neurological inflammation.

Description

Technology Field

[0001] Related applications

[0002] This application claims the benefit of U.S. Provisional Application Serial No. 62 / 816,434 filed March 11, 2019 and U.S. Provisional Application Serial No. 62 / 931,590 filed November 6, 2019. The entire contents of the said applications are incorporated herein by reference.

[0003] Technology field

[0004] The present invention relates to a quaternary ammonium compound, a pharmaceutical composition, and a method that are generally useful as a selective inhibitor of pain, cough, and itching-sensing neurons (nociceptors, cough receptors, and pruritus receptors) and in the treatment of neuroinflammatory diseases. Background Technology

[0005] The present invention features compounds, compositions, and methods for treating neuroinflammatory disease by selectively inhibiting sensory neurons (nociceptors, cough receptors, and itch receptors) and targeting nociceptors with small molecule drugs while minimizing effects on non-nociceptor neurons or other types of cells. According to the method of the present invention, small cationic drug molecules access the intracellular compartments of sensory neurons through entry via large pore receptor / ion channels that are present in pain-sensing, cough-sensing, and itch-sensing neurons but are present to lesser extent or not at all in other types of neurons or other types of tissues.

[0006] Local anesthetics, such as lidocaine and articaine, act by inhibiting voltage-dependent sodium channels in neurons. By blocking sodium channels, these anesthetics block the excitability of all neurons, not just pain-sensing neurons (nociceptors). Therefore, while the goal of local or regional anesthesia is to prevent pain by blocking nociceptor signaling, the administration of local anesthetics also causes unwanted or harmful effects, such as generalized numbness due to low threshold pressure and blockade of contact receptors, paralysis due to motor deficits and / or motor axon blockade, and other complications due to autonomic fiber blockade. Local anesthetics are relatively hydrophobic molecules that diffuse through the cell membrane to access blockade sites on sodium channels. Charged derivatives of these compounds, which are not membrane-permeable, do not affect neuronal sodium channels when applied to the outer surface of the nerve membrane; however, they can block sodium channels if they are somehow introduced into the cell, for example, by diffusion from a micropipette used for whole-cell electrophysiological recording from isolated neurons. Pain-sensing, cough-sensing, and itch-sensing neurons differ from other types of neurons (in most cases) in that they express TRPV1 receptors / channels that are activated by painful heat or capsaicin, the spicy component of chili peppers. Other types of channels selectively expressed in various types of pain-sensing, cough-sensing, and itch-sensing (itch receptor) neurons include, but are not limited to, TRPV2-4, TRPA1, TRPM8, ASIC, and P2X(2 / 3) channels. It is well established that some cationic small molecules, such as QX-314, can enter cells by passing through activated large porous channels such as TRPV1.

[0007] Neuropathic, inflammatory, and nociceptive pain differ in their etiology, pathophysiology, diagnosis, and treatment. Nociceptive pain occurs in response to the activation of nociceptors, a specific subset of high-threshold peripheral sensory neurons, by intense or noxious stimuli. It is generally acute, self-limiting, and serves a protective biological function by acting as a warning against potential or ongoing tissue damage. It is typically well-localized. Examples of nociceptive pain include, but are not limited to, traumatic or surgical pain, analgesia, sprains, fractures, burns, bumps, contusions, injections, dental procedures, skin biopsies, and closures.

[0008] Inflammatory pain is pain associated with damage to joints, muscles, and tendons, such as pain occurring in the presence of tissue damage or inflammation, including post-operative pain (i.e., acute pre- and post-operative pain resulting from inflammation caused by tissue trauma (e.g., surgical incision, resection, burn) or direct nerve damage (e.g., nerve transection, stretching, or compression)), post-traumatic pain, arthritis pain (rheumatic; or osteoarthritis (i.e., joint pain and stiffness due to the progressive deterioration of articular cartilage; risk factors including aging, injury, and obesity; generally affected joints are the hands, wrists, neck, knees, hips, and spine)), pain, and axial low back pain (i.e., a pervasive and painful condition affecting the lower part of the back; common causes include muscle strain, vertebral fractures, bulging or ruptured discs, and arthritis), and severe nociceptive pain can transition into inflammatory pain if there is associated tissue damage.

[0009] Neuropathic pain is a common type of chronic, non-malignant pain resulting from damage or dysfunction of the peripheral or central nervous system that does not provide a protective biological function. It is estimated that more than 1.6 million people in the U.S. population are affected. Neuropathic pain has many different etiologies and can occur, for example, due to trauma, surgery, herniated discs, spinal cord injury, diabetes, herpes zoster infection, HIV / AIDS, terminal cancer, amputation (including mastectomy), carpal tunnel syndrome, chronic alcohol use, radiation exposure, and as an unintended side effect of neurotoxic agents such as certain anti-HIV and chemotherapy drugs. Peripheral neuropathy is caused by peripheral nerve damage from injury, trauma, or prolonged pressure, or by inflammation and pain in the affected part of the body that causes numbness.

[0010] Neuropathic pain is often naturally described as "burning," "electric," "tingling," or "stinging." It is often characterized by chronic dynamic allodynia (defined as pain caused by moving stimuli that do not typically trigger a painful response, such as light contact) and hyperalgesia (generally defined as increased sensitivity to painful stimuli), and can persist for months or years even after the apparent healing of any damaged tissue.

[0011] Pain can occur in patients with cancer and can be caused by various factors such as inflammation, compression, invasion, or the spread of metastasis to bone or other tissues.

[0012] There are some conditions in which pain occurs in the absence of harmful stimuli, tissue damage, or lesions to the nervous system referred to as dysfunctional pain, and these include, but are not limited to, fibromyalgia, tension headache, and irritable bowel disorder.

[0013] Migraine is a headache associated with the activation of sensory fibers that innervate the meninges of the brain.

[0014] Pruritus is a dermatological condition that can be localized or systemic and may be associated with skin lesions (rash, atopic eczema, urticaria). Pruritus is associated with many conditions, including but not limited to stress, anxiety, UV radiation from the sun, metabolic and endocrine disorders (e.g., liver or kidney disease, hyperthyroidism), cancer (e.g., lymphoma), reactions to drugs or food, parasitic and fungal infections, allergic reactions, blood disorders (e.g., polycythemia vera), and dermatological conditions. Pruritus is mediated by pruritus receptors, which are a subset of small-diameter primary sensory neurons that share many features of nociceptive neurons, including but not limited to the expression of TRPV1 channels and other large porous channels (e.g., TRPV2-4, TRPA1, TRPM8, ASIC, and P2X(2 / 3)). Certain pruritic mediators, such as eicosanoids, histamine, bradykinin, ATP, and various neurotrophins, possess endovaniloid functions. Topical capsaicin inhibits histamine-induced itching. Therefore, like nociceptors, pruritus receptors are suitable targets for the aforementioned method of delivering ion channel blockers.

[0015] Coughing is a defensive reflex designed to protect the airways from foreign substances and aid in clearing lumen debris. However, in many diseases, this reflex becomes abnormal, which can lead to an unproductive dry cough in which a hyper-tussive or allo-tussive state is present. Hyper-tussive and allo-tussive states are often chronic in nature, lasting for more than three months, and can occur in many airway disease conditions, including asthma, COPD, asthma-COPD overlap syndrome (ACOS), interstitial pulmonary fibrosis (IPF), and lung cancer. Additionally, an inappropriate cough reflex can appear acutely or chronically following a viral infection. Furthermore, chronic cough can be idiopathic, meaning the etiology is unknown.

[0016] Neuroinflammation is a mode of inflammation mediated by the efferent (motor) function of sensory neurons, where pro-inflammatory mediator molecules released from the periphery by pain sensory neurons (nociceptors) activate various inflammatory pathways in immune cells and also act on the vascular system to alter blood flow and capillary permeability.

[0017] Neuroinflammation is attributed to peripheral inflammation in various tissues triggered by tissue damage, autoimmune diseases, infections, allergies, and exposure to irritants, and is thought to play a significant role in the pathogenesis of numerous disorders (e.g., migraines, arthritis, rhinitis, gastritis, colitis, cystitis, and sunburn). One method to reduce neuroinflammation is to block the excitability of nociceptors, thereby preventing the activation of nociceptive terminals and the release of pro-inflammatory chemicals.

[0018] Despite the development of various therapies for pain, itching, and neuroinflammation, additional agents are needed.

[0019] The present invention relates to the following chemical formula that can be used to treat or prevent pain, itching, and neuroinflammatory disease ( I Provides a compound represented by ):

[0020]

[0021] In the above formula,

[0022] Y - is a pharmaceutically acceptable anion;

[0023] R A , R B , and R C is H, D, halogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, OR I , CN, NRJ R K , NR L C(O)R M , S(O)R N , S(O)2R N , SO2R O R P , SO2NR Q R R , SO3R S , CO2R T ; C(O)R U , and C(O)NR V R W Each is independently selected from (preferably H, F, Cl, or CN and more preferably H);

[0024] R I , R J , R K , R L , R M , R N , R O , R P , R Q , R R , R S , R U , R V , and R W Each is independently selected from H, D, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, and substituted or unsubstituted heterocycloalkyl; R J and R K or R V and R W or R Q and R R Silver can also be taken together with the nitrogen to which they are attached to form substituted or unsubstituted 5, 6, 7, or 8-membered rings;

[0025] R A , R B , and / or R CThese can be taken together with the phenyl ring to which they are attached to form a fused bicyclic or tricyclic ring system, such as naphthyl, dihydrodenyl, tetrahydronaphthyl, quinolinyl, indolyl, etc.;

[0026] X 1 ―CR X R Y ―, ―NR Z C(O)―, ―OC(O)―, ―SC(O)―, ―C(O)NR 1A ―, ―C(O)O―, -C(O)-, ―(O)CS―, ―NR 1A S(O)―, ―S(O)NR 1A ―, ―NR 1A C(O)NR 1A Selected from ―, ―S(O)― and ―S(O)2―; X 1 Eun also ―NR Z C(O)CR X R Y ―It could be;

[0027] R X , R Y , R Z , and R 1A Each is independently selected from H, D, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, and substituted or unsubstituted heteroalkyl;

[0028] R D and R E Each is independently selected from H, D, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted heteroalkyl, and substituted or unsubstituted cycloalkyl, or R D and R E ...forms a substituted or unsubstituted 3-6-membered cycloalkyl (C3-C6 cycloalkyl), a substituted or unsubstituted heterocyclic, or a substituted or unsubstituted heteroalkyl ring together with the carbon to which they are attached;

[0029] R Dand R Z They form a 5-8-membered lactam that is selectively substituted with the carbon to which they are attached and -NC(O)-;

[0030] R F and R G is N + Forming an optionally substituted heterocyclic ring having one or more nitrogen atoms together with; R F and R G Each is a substituted or unsubstituted alkyl, a substituted or unsubstituted alkenyl, a substituted or unsubstituted alkynyl, a substituted or unsubstituted heteroalkyl, and a substituted or unsubstituted C 3-6 Independently selected from cycloalkyls;

[0031] R H is a substituted or unsubstituted aryl ring or a substituted or unsubstituted heteroaryl ring.

[0032] In another implementation example, R H may be a substituted alkyl. The substituent is preferably an ester group, e.g., -OC(O)R 1B and, here R 1B is a substituted or unsubstituted alkyl, a substituted or unsubstituted alkenyl, a substituted or unsubstituted alkynyl, and a substituted or unsubstituted heteroalkyl, a substituted or unsubstituted aryl, or a substituted or unsubstituted heteroaryl. 1B is preferably a substituted or unsubstituted phenyl. R H It is preferably ―CH2OC(O)―phenyl. Specific details for implementing the invention

[0033] The present invention is based on the chemical formula as described above ( I The present invention provides a compound represented by ) or a pharmaceutically acceptable salt, stereoisomer, solvate, hydrate, or combination thereof. The present invention also provides the formula ( IA composition comprising a compound having ) or a pharmaceutically acceptable salt thereof, for example, an effective amount of formula ( I The present invention provides a composition comprising a compound of ) or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient. The composition of the present invention may further comprise the compound of the present invention and a biological active agent. The composition may be formulated for oral, intravenous, intramuscular, rectal, skin, subcutaneous, topical, transdermal, sublingual, nasal, inhalation, vaginal, intradural, epidural, or ocular administration.

[0034] The present invention also has a chemical formula ( I A method for treating a patient's pain, cough, itching, or neuroinflammatory disorder comprises administering to the patient a composition comprising a compound having a) said compound, wherein the compound inhibits one or more voltage-gated ion channels present in nociceptors and / or cough receptors and / or itch receptors when exposed to or applied to the inner surface of a channel, but does not substantially inhibit the channel when applied to the outer surface of a channel, said compound may enter the nociceptors, cough receptors, or itch receptors through a large porous channel when the channel is activated and may inhibit one or more voltage-gated ion channels present in the nociceptors, cough receptors, or itch receptors.

[0035] In a specific embodiment, the large porous channel is a transient receptor potential ion channel (TRP channel). In another embodiment, the TRP channel is activated by an exogenous or endogenous agonist. In yet another embodiment, the large porous channel is TRPA1, TRPV1-4, TRPM8, ASIC, or P2X. In a specific embodiment, the compound can enter a nociceptor, cough receptor, or itch receptor through the TRPA1, TRPV1-4, TRPM8, ASIC, or P2X receptor / channel when the receptor / channel is activated. In yet another embodiment, the compound inhibits a voltage-gated sodium channel. In another embodiment, the type of pain treated by the method, composition and kit of the present invention is selected from the group consisting of neuropathic pain, inflammatory pain, nociceptive pain, pain due to infection, and procedural pain, or the neuroinflammatory disorder is selected from the group consisting of allergic inflammation, asthma, chronic cough, conjunctivitis, rhinitis, psoriasis, inflammatory bowel disease, interstitial cystitis, and atopic dermatitis.

[0036] The inventors have the following chemical formula, which is expressed in nociceptors and / or cough receptors and / or itchiness receptors but is not expressed in motor neurons and can pass through an open, large porous channel ( I We identified compounds having ):

[0037]

[0038] Since the ion channel blocking compounds of the present invention are positively charged, they are not membrane-permeable and therefore cannot enter cells that do not express large porous channels. Since large porous channels are often more active in tissue conditions associated with pain (e.g., inflammation) caused by the release of endogenous ligands or activation by thermal stimulation, the ion channel blockers of the present invention may be used alone to selectively target activated nociceptors to effectively treat (e.g., eliminate or alleviate) pain, cough, itching, or neuroinflammatory conditions. The ion channel blockers of the present invention may also be used in combination with one or more exogenous large porous channel agonists to selectively target nociceptors to effectively treat (e.g., eliminate or alleviate) pain, itching, or neuroinflammatory conditions.

[0039] Voltage-dependent ion channels in pain-sensing neurons are currently attracting significant attention in the development of drugs to treat pain. Blocking voltage-dependent sodium channels in pain-sensing neurons can block pain signals by interfering with the initiation and transmission of action potentials. Additionally, blocking voltage-dependent sodium channels in nociceptors can reduce or eliminate neuroinflammation by preventing the activation of nociceptive terminals and the release of pro-inflammatory chemicals.

[0040] To date, the limitation in treating with molecules that block sodium or calcium channels is that the majority of the aforementioned external application molecules are hydrophobic and can pass through membranes. Because of this, they enter all cells and lack selectivity to affect only nociceptors.

[0041] The inhibitor of the present invention is membrane-impermeable and effective only when present inside nociceptor cells; therefore, to produce an effect, it must pass through the cell membrane via channels or receptors such as large porous channels (e.g., TRPAV1-4, TRPA1, TRPM8, ASIC, and P2X(2 / 3)). Under normal circumstances, most large porous channels of nociceptors are inactive and require harmful thermal, mechanical, or chemical stimuli to activate them. For example, TRP channels of nociceptors can be activated by exogenous TRP ligands (i.e., TRP agonists), such as capsaicin, which open TRPV1 channels. Therefore, one approach to selectively targeting nociceptors is to co-administer a membrane-impermeable ion channel inhibitor with an exogenous TRP ligand that allows the inhibitor to pass into the cell through TRP channels. In addition to capsaicin, the exogenous TRP ligand may also be another capsaicinoid, mustard oil, or lidocaine. In another example, TRP channels can be activated in response to exogenous stimulant activators, such as acrolein inhaled from smoke or chemical warfare agents like tear gas.

[0042] Under certain circumstances, large porous channels can be activated by endogenous inflammatory activators generated by tissue damage, infection, autoimmunity, atopy, ischemia, hypoxia, cellular stress, immune cell activation, immune mediator production, and oxidative stress in the absence of exogenous large porous channel agonists / ligands. Under these conditions, endogenous molecules (e.g., protons, lipids, and reactive oxygen species) can activate large porous channels expressed in nociceptors, allowing membrane-impermeable, voltage-gated ion channel blockers to access the interior of nociceptors through the endogenously activated large porous channels. Endogenous inflammatory activators of large porous channels include, for example, prostaglandins, nitric oxide (NO), peroxide (H2O2), cysteine-reactive inflammatory mediators, for example, 4-hydroxynonenal, endogenous alkenylaldehyde, endocannabinoids, and immune mediators (e.g., interleukin 1 (IL-1), nerve growth factor (NGF), and bradykinin, whose receptor is coupled to the large porous channel).

[0043] definition

[0044] As used herein, singular terms mean including one or more unless otherwise specified.

[0045] "Biological activity" refers to the exertion of biological, physical, or chemical effects or activity by a molecule, including biological molecules such as nucleic acids, peptides, polypeptides, and proteins, on proteins, enzymes, receptors, ligands, antigens themselves, or other molecules. For example, a "biologically active" molecule may possess, for instance, enzymatic activity, protein-binding activity, or pharmacological activity.

[0046] Biological active agents that may be used in the methods and kits described herein include, but are not limited to, TRP1A receptor agonists, TRPV1-4 receptor agonists, ASIC agonists, TRPM8 agonists, P2X receptor agonists, NSAIDs, glucocorticoids, narcotics, anti-inflammatory agents and inflammation modulators, antibodies or antibody fragments, antibiotics, polynucleotides, polypeptides, proteins, anticancer agents, growth factors, and vaccines.

[0047] "Inflammation" refers to any type of inflammation, such as inflammation caused by the immune system (immune-mediated inflammation) and the nervous system (neuro-inflammation), and any symptoms of inflammation including redness, heat, swelling, pain, and / or loss of function.

[0048] "Neuro-inflammatory" refers to any type of inflammation mediated by or caused by neurons (e.g., nociceptors) or any other component of the central or peripheral nervous system.

[0049] The term "pain" is used in its broadest sense herein and refers to all types of pain, including nociceptive pain, e.g., somatic pain and visceral pain; inflammatory pain, dysfunctional pain, idiopathic pain, neuropathic pain, e.g., centrally originating pain and peripherally originating pain; and acute and chronic pain such as migraines and cancer pain.

[0050] The term "nociceptive pain" is used to encompass all pain caused by noxious stimuli that threaten or actually damage body tissues, including, but not limited to, those resulting from amputations, contusions, fractures, crush injuries, burns, etc. Pain receptors (nociceptors) for tissue damage are primarily located in the skin, musculoskeletal system, or internal organs.

[0051] The term "somatic pain" is used to refer to pain occurring in bones, joints, muscles, skin, or connective tissue. This type of pain is typically well localized.

[0052] The term "visceral pain" is used herein to refer to pain occurring in internal organs such as the respiratory system, gastrointestinal tract and pancreas, urinary tract, and reproductive organs. Visceral pain includes pain resulting from tumor invasion of the tracheal capsule. Another type of visceral pain, typically caused by obstruction of the hollow intestine, is characterized by intermittent cramping pain and non-localized pain. Visceral pain may be associated with inflammation, such as in cystitis or reflux esophagitis.

[0053] The term "inflammatory pain" includes pain associated with active inflammation that can be caused by trauma, surgery, infection, and autoimmune diseases.

[0054] The term "neuropathic pain" is used herein to refer to pain resulting from the abnormal processing of sensory input by the peripheral or central nervous systems caused by lesions in these systems.

[0055] The term "procedure pain" refers to pain that occurs during medical, dental, or surgical procedures that are typically planned or associated with acute trauma.

[0056] The term "pruritus" is used in its broadest sense at this institution and refers to all types of itching and stinging that are localized, generalized, acute, intermittent, or persistent. Pruritus may be idiopathic, allergic, metabolic, infectious, or drug-induced, and may be caused by liver or kidney disease or cancer. "Pruritus" refers to severe itching.

[0057] "Patient" means any animal. In one embodiment, the patient is a human. Other animals that can be treated using the method, composition and kit of the present invention include, but are not limited to, non-human primates (e.g., monkeys, gorillas, chimpanzees), livestock (e.g., horses, pigs, goats, rabbits, sheep, cattle, llamas), and companion animals (e.g., guinea pigs, rats, mice, lizards, snakes, dogs, cats, fish, hamsters, and birds).

[0058] Compounds useful for the present invention include, but are not limited to, any of the compounds described herein in any pharmaceutically acceptable forms, such as their isomers, such as diastereomers and enantiomers, salts, esters, amides, thioesters, solvates, and polymorphs, as well as racemic mixtures and pure isomers of the compounds described herein. As used herein, the term “pharmaceutically acceptable anion” refers to the conjugate base of a pharmaceutically acceptable acid. Such acid is described in the literature [Stahl, PH and Wermuth, CG (eds.), Handbook of Pharmaceutical Salts: Properties, Selection and Use, Wiley VCH (2008)]. Pharmaceutically acceptable acids are acetic acid, dichloroacetic acid, adipic acid, alginic acid, L-ascorbic acid, L-aspartic acid, benzenesulfonic acid, 4-acetamidobenzoic acid, benzoic acid, p-bromophenylsulfonic acid, (+)-camponic acid, (+)-camphor-10-sulfonic acid, capric acid, caproic acid, caprylic acid, carbonic acid, cinnamic acid, cyclamic acid, dodecyl sulfuric acid, ethane-1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, sulfuric acid, boric acid, citric acid, formic acid, fumaric acid, galactar acid, gentisic acid, D-glucoheptonic acid, D-gluconic acid, D-glucuronic acid, glutamic acid, glutaric acid, 2-oxoglutaric acid, glycerophosphate, glycolic acid, hippuric acid, hydrochloric acid, Hydrobromide, hydroiodide, isobutyric acid, DL-lactic acid, lactobionic acid, lauric acid, maleic acid, (-)-L-malic acid, malonic acid, DL-mandelic acid, methanesulfonic acid, naphthalene-1,5-disulfonic acid, naphthalene-2-sulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid, nitric acid, oleic acid, orotic acid, oxalic acid, palmitic acid, parmoic acid, phosphoric acid, propionic acid, (-)-L-pyroglutamic acid, salicylic acid, 4-aminosalicylic acid, sebactic acid, stearic acid, succinic acid, (+)-L-tartaric acid, thiocyanate, p-toluenesulfonic acid, and undecylenic acid, but are not limited thereto.Pharmaceutically acceptable anions include the conjugate base of any acid presented above.

[0059] The term "pharmaceuticalally acceptable salt" refers to a salt that is suitable for use in contact with human and lower animal tissues without excessive toxicity, irritation, or allergic reactions within the scope of sound medical judgment, and corresponds to a reasonable benefit / risk ratio. The salt may be prepared in situ during the final separation and purification of the compound of the present invention, or separately by reacting a free base functional group with a suitable organic acid. Representative acid salts include acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, isethionate, lactobionate, lactate, laurate, lauryl sulfate, maleate, maleate, malonate, mesylate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, and oxalate. Includes, but is not limited to, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, toluenesulfonate, undecanoate, valerate salts, etc.

[0060] In the general description of the compounds of the present invention, the number of atoms of a specific type in the substituent group is generally in a range, for example, an alkyl group containing 1 to 4 carbon atoms or C 1-4 C of alkyl 1-It is provided as a C4 alkyl. References to the above range are intended to include specific references to groups having each of the integer atoms within the specified range. For example, alkyl groups of 1 to 4 carbon atoms include C1, C2, C3, and C4 alkyls, respectively. Different numbers of atoms and different types of atoms may be indicated in a similar manner.

[0061] "D" is deuterium.

[0062] As used herein, the term "alkyl" and the prefix "alk-" include both straight-chain and branched-chain groups and cyclic groups, namely cycloalkyl. Cyclic groups may be monocyclic or polycyclic and, preferably, may have 3 to 6 cyclic carbon atoms or 3 to 7 carbon atoms. Exemplary cyclic groups include cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl groups.

[0063] "C 1-4 "alkyl" or "C1-C4alkyl" refers to a branched or unbranched hydrocarbon group having 1 to 4 carbon atoms. Similarly, "C 1-6 "Alkyl" or "C1-C6" is a branched or unbranched hydrocarbon group having 1 to 6 carbon atoms. For example, C 1-4 alkyl or C 1-6Alkyl groups containing alkyl groups may be substituted or unsubstituted. Exemplary substituents include, but are not limited to, alkoxy, aryloxy, sulfhydryl, alkylthio, arylthio, halide, hydroxyl, fluoroalkyl, perfluoroalkyl, amino, alkylamino, disubstituted amino, quaternary amino, alkylcarboxyl, and carboxyl groups. Exemplary substituents are also alkoxy, aryloxy, sulfhydryl, alkylthio, arylthio, halide (F, Cl, Br or I), hydroxyl, fluoroalkyl, perfluoroalkyl, oxo, amino, alkylamino, disubstituted amino, quaternary amino, amido, ester, alkylcarboxyl, alkoxycarbonyl, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxyl, alkylcarbonyl, arylcarbonyl, alkylthiocarbonyl, phosphate, phosphonato, phosphinato, acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amido, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfate, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, It includes cyano, azido, aryl, heterocyclil, alkylaryl, or aromatic or heteroaromatic moiety. C 1-4 Alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, cyclopropyl, cyclopropylmethyl, n-butyl, iso-butyl, sec-butyl, tert-butyl, and cyclobutyl. C 1-6 Alkyl includes methyl, ethyl, n-propyl, isopropyl, cyclopropyl, cyclopropylmethyl, n-butyl, iso-butyl, sec-butyl, tert-butyl, n-pentyl, n-hexyl, cyclobutyl, cyclopentyl, and cyclohexyl.

[0064] An example of a substituted alkyl is a heteroalkyl. "Heteroalkyl" means a branched or unbranched alkyl, cycloalkyl, alkenyl, or alkynyl group having 1 to 7 or more carbon atoms in addition to 1, 2, 3, or 4 heteroatoms independently selected from the group consisting of N, O, S, and P. "C 1-7 "Heteroalkyl" means a branched or unbranched alkyl, alkenyl, or alkynyl group having 1 to 7 carbon atoms in addition to 1, 2, 3, or 4 heteroatoms independently selected from the group consisting of N, O, and S. Heteroalkyl may include, but is not limited to, tertiary amines, secondary amines, ethers, thioethers, amides, thioamides, carbamates, thiocarbamates, hydrazones, imines, phosphodiesters, phosphoramidates, sulfonamides, and disulfides. Heteroalkyl may optionally include monocyclic, bicyclic, or tricyclic rings, each ring preferably having 3 to 6 members. The heteroalkyl group may be substituted or unsubstituted. Examples of substituents include alkyl, alkoxy, aryloxy, sulfhydryl, alkylthio, arylthio, and halide (F, Cl, Br, or I), hydroxyl, fluoroalkyl, perfluoroalkyl, oxo, amino, alkylamino, disubstituted amino, quaternary amino, amido, ester, alkylcarboxy, alkoxycarbonyl, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxyl, alkylcarbonyl, arylcarbonyl, alkylthiocarbonyl, phosphate, phosphonato, phosphinato, acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amido, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfate, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido, aryl, heterocyclil, alkylaryl, or includes aromatic or heteroaromatic moiety. C 1-7Examples of heteroalkyls include, but are not limited to, methoxymethyl and ethoxyethyl.

[0065] Alkenyls are branched or unbranched hydrocarbon groups containing one or more double bonds. For example, "C 2-6 "Alkenyl" or "C2-C6 alkenyl" refers to a branched or unbranched hydrocarbon group containing one or more double bonds and having 2 to 6 carbon atoms. The alkenyl may optionally comprise a monocyclic or polycyclic ring, wherein each ring preferably has 3 to 6 members. The alkenyl group may be substituted or unsubstituted. Exemplary substituents include those described above for the alkyl group, specifically including alkoxy, aryloxy, sulfhydryl, alkylthio, arylthio, halide, hydroxyl, fluoroalkyl, perfluoroalkyl, amino, alkylamino, disubstituted amino, quaternary amino, alkylcarboxyl, and carboxyl groups. C 2-6 Alkenyls include, but are not limited to, vinyl, allyl, 2-cyclopropyl-1-ethenyl, 1-propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 2-methyl-1-propenyl and 2-methyl-2-propenyl.

[0066] Alkynyl is a branched or unbranched hydrocarbon group containing one or more triple bonds. For example, "C 2-6"Alkynyl" or "C2-C6-alkynyl" refers to a branched or unbranched hydrocarbon group containing one or more triple bonds and having 2 to 6 carbon atoms. The alkynyl may optionally comprise a monocyclic, bicyclic, or tricyclic ring, wherein each ring preferably has 5 or 6 members. The alkynyl group may be substituted or unsubstituted. Exemplary substituents include those described above for the alkyl group, specifically including alkoxy, aryloxy, sulfhydryl, alkylthio, arylthio, halide, hydroxy, fluoroalkyl, perfluoroalkyl, amino, alkylamino, disubstituted amino, quaternary amino, alkylcarboxyl, and carboxyl groups. C 2-6 Alkynyls include, but are not limited to, ethinyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, and 3-butynyl.

[0067] "Heterocyclil," "heterocyclic," or "heterocycloalkyl" means a stable monocyclic or polycyclic (including bicyclic or tricyclic) heterocyclic ring that is saturated, partially unsaturated, or unsaturated (including heteroaryl or aromatic), and comprises two or more carbon atoms and one, two, three, or four or more heteroatoms independently selected from N, O, and S, and any of the heterocyclic rings defined above comprises any bicyclic or polycyclic group fused to a benzene ring, heteroaryl, cycloalkyl, or heterocycloalkyl ring. In certain embodiments, the heterocyclil is a 3 to 15-membered ring system, a 3 to 12-membered ring system, or a 3 to 9-membered ring system. "C 2-6"Heterocyclile" means a stable 5 to 7-membered monocyclic or 7 to 14-membered bicyclic heterocyclic ring that is saturated, partially unsaturated, or unsaturated (including heteroaryl or aromatic) and consists of 2 to 6 carbon atoms and 1, 2, 3, or 4 heteroatoms independently selected from N, O, and S, and any of the heterocyclic rings defined above comprises any bicyclic group fused to a benzene ring, heteroaryl, cycloalkyl, or heterocycloalkyl ring. The heterocyclile or heteroaryl group may be substituted or unsubstituted. Exemplary substituents are substituted or unsubstituted alkyl, aryl, cycloalkyl, heterocycloalkyl, heteroaryl, alkoxy, aryloxy, sulfhydryl, alkylthio, arylthio, halide, hydroxy, fluoroalkyl, perfluoroalkyl, amino, alkylamino, It includes disubstituted amino, quaternary amino, alkylcarboxyl, oxo, and carboxyl groups. Nitrogen and sulfur heteroatoms may be optionally oxidized. The heterocyclic ring may be covalently attached through any heteroatom or carbon atom to form a stable structure; for example, the imidazolinyl ring may be connected at any one of the ring-carbon atom positions or at the nitrogen atom. The nitrogen atom of the heterocycle may be quaternized. Preferably, when the total number of S and O atoms in the heterocycle exceeds 1, these heteroatoms are not adjacent to each other. The heterocycle includes 1H-indazole, 2-pyrrolidonyl, 2H,6H-1,5,2-dithiazinyl, 2H-pyrrolyl, 3H-indolyl, 4-piperidonyl, 4aH-carbazole, 4H-quinolidinyl, 6H-1,2,5-Thiadiazinyl, Acridinyl, Azocinyl, Benzimidazolyl, Benzofuranyl, Benzothiofuranyl, Benzothiophenyl, Benzoxazolyl, Benzthiazolyl, Benztriazolyl, Benztetrazollyl, Benzisoxazolyl, Benzisothiazolyl, Benzimidazalonil, Carbazollyl, 4aH-Carbazollyl, b-Carvolinyl, Cromanyl, Cromenyl, Cinnolinyl, Decahydroquinolinyl, 2H,6H-1,5,2-Dithiazinyl, Dihydrofuro[2,3-b]tetrahydrofuran, Furanyl, Furazanyl, Imidazolidinyl, Imidazolininyl, Imidazolill, 1H-Indazolyl, Indolenyl, Indolinyl, Indoliginyl, Indolyl, Isobenzofuranyl, Isochromanyl, Isoindazolill, Isoindolinyl, Isoindolyl, Isoquinolinyl, Isothiazolill, Isoxazolillil, Morfollinyl, Naphthiridinyl, Octahydroisoquinolinyl, Oxadiazolyl, 1,2,3-Oxadiazolyl, 1,2,4-Oxadiazolyl, 1,2,5-Oxadiazolyl, 1,3,4-Oxadiazolyl, Oxazolidinyl, Oxazolill, Oxazolidinylperimidinyl, Fenanthridinil, Fenanthrolinil, Fenarsazinil, Fenazinil, Fenothiazinil, Fenoxatiinil, Fenoxazinil, Phthalazinil, Piperazinil, Piperidinil, Pteridinil, Piperidonil, 4-Piperidonil, Pteridinil, Purinil, Pyranil, Pyrazinil, Pyrazolidinil, Pyrazolinil, Pyrazolil, Pyridazinil, Pyridoxazole, Pyridomidazole, Pyridothiaazole, Pyridinil, Pyridyl, Pyrimidinil, Pyrrolidinil, Pyrrolinil, Pyrrolilil, Quinazolinil, Quinolinil, 4H-Quinolidinil, Quinoxalinil, Quinuclidinil, Carbolinil, Tetrahydrofuranil, Tetrahydroisoquinolinil, Includes, but is not limited to, tetrahydroquinolinyl, 6H-1,2,5-thiadiazinyl, 1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl, 1,3,4-thiadiazolyl, thiantrenyl, thiazolyl, thienyl, thienothiazolyl, thienooxazolyl, thienoimidazolyl, thiophenyl, triazinyl, 1,2,3-triazolyl, 1,2,4-triazolyl, 1,2,5-triazolyl, 1,3,4-triazolyl, xanthenyl, β-lactam, γ-lactam and δ-lactam. Preferred 5 to 10-membered heterocycles include pyridinyl, pyrimidinyl, triazinyl, furanyl, thienyl, thiazolyl, pyrrolyl, pyrazolyl, imidazolyl, oxazolyl, isoxazolyl, tetrazolyl, benzofuranyl, benzothiofuranyl, indolyl, benzimidazolyl, 1H-indazolyl, oxazolidinyl, isoxazolidinyl, benzotriazolyl, benzisoxazolyl, oxindolyl, benzoxazolidinyl,It includes, but is not limited to, quinolinyl and isoquinolinyl. Preferred 5 to 6-membered heterocycles include, but are not limited to, pyridinyl, quinolinyl, pyrimidinyl, triazinyl, furanyl, thienyl, thiazolyl, pyrrolyl, piperazinyl, piperidinyl, pyrazolyl, imidazolyl, oxazolyl, isoxazolyl, and tetrazolyl. Preferred substituents include phenyl, methyl, ethyl, propyl, butyl, chloro, bromo, iodo, and iodine.

[0068] "Aryl" refers to an aromatic group (e.g., phenyl) having a ring system containing a carbon atom with conjugated π electrons. "C 6- C 12 "Aryl" or "C6-C 10 "Aryl" is an aryl group having 6 to 12 carbon atoms or 6 to 10 carbon atoms, respectively. The aryl group may optionally comprise a monocyclic, bicyclic, or tricyclic ring, wherein each ring preferably has 5 or 6 members. The bicyclic or tricyclic ring system may be fused (e.g., naphnyl) or unfused (e.g., biphenyl). The aryl group may be substituted or unsubstituted. Exemplary substituents include substituted or unsubstituted alkyl, hydroxyl, alkoxy, aryloxy, sulfhydryl, alkylthio, arylthio, halide, fluoroalkyl, carboxyl, alkylcarboxyl, amino, alkylamino, monosubstituted amino, disubstituted amino, and quaternary amino groups. A preferred aryl group is phenyl.

[0069] "Aralkyl" means a substituted or unsubstituted alkyl group substituted with a substituted or unsubstituted aryl group (e.g., including benzyl, phenethyl, or 3,4-dichlorophenethyl).

[0070] "C 7-14"Aralkyl" means an alkyl group substituted with an aryl group having 7 to 14 carbon atoms (e.g., benzyl, phenethyl, or 3,4-dichlorophenethyl).

[0071] "C 3-10 "Heterocycloalkyl" means an alkyl-substituted heterocyclic group having 3 to 10 carbon atoms in addition to one or more heteroatoms (e.g., 3-furanylmethyl, 2-furanylmethyl, 3-tetrahydrofuranylmethyl, or 2-tetrahydrofuranylmethyl).

[0072] "Halide" or "halogen" refers to bromine, chlorine, iodine, or fluorine.

[0073] "Fluoroalkyl" refers to an alkyl group substituted with a fluorine atom.

[0074] "Alkyl carboxy" refers to a chemical moiety having the chemical formula -(R)-COOH, where R is C 1-7 Alkyl, C 2-7 Alkenyl, C 2-7 Alkinyl, C 2-6 Heterocyclil, C 6-12 Aril, C 7-14 Aralkyl, C 3-10 Heterocycloalkyl or C 1-7 It is selected from heteroalkyls.

[0075] "Alkoxy" refers to a chemical substituent having the chemical formula -OR, where R is a substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, or substituted or unsubstituted alkynyl, or R is C 1-7 Alkyl, C 2-7 Alkenyl, C 2-7 Alkinyl, C 2-6 Heterocyclil, C 6-12 Aril, C 7-14 Aralkyl, C 3-10 Heterocycloalkyl or C 1-7 It can be selected from heteroalkyls.

[0076] "Aryloxy" refers to a chemical substituent of the chemical formula -OR, where R is C 6-12It is Aril Gi.

[0077] "Alkylthio" refers to a chemical substituent of the chemical formula -SR, where R is C 1-7 Alkyl, C 2-7 Alkenyl, C 2-7 Alkinyl, C 2-6 Heterocyclil, C 6-12 Aril, C 7-14 Aralkyl, C 3-10 Heterocycloalkyl or C 1-7 It is selected from heteroalkyls.

[0078] "Arylthio" refers to a chemical substituent of the chemical formula -SR, where R is C 6-12 It is Aril Gi.

[0079] "Charged moiety" means a moiety that gains a proton at physiological pH and becomes positively charged (e.g., ammonium, guanidinium, or amidinium) or a moiety containing a net positive charge without protonation (e.g., quaternary ammonium). A charged moiety may be permanently charged or temporarily charged.

[0080] "Therapeutic effective dose" or "effective dose" means an amount sufficient to produce the desired result, e.g., reduction or elimination of pain, cough, itching, or neuroinflammatory inflammation, in a patient (e.g., human) suffering from a condition, disease, or disorder caused wholly or partially by neuroinflammatory inflammation (e.g., asthma, arthritis, colitis, contact dermatitis, diabetes, eczema, cystitis, chronic intractable cough, postviral cough, gastritis, migraine, psoriasis, rhinitis, rosacea, or sunburn).

[0081] "Solvent" refers to a form of solvent addition containing stoichiometric or non-stoichiometric amounts of solvent.

[0082] The compound of the present invention, comprising a salt of the compound, may exist in a solvated form including a hydrated form and a non-hydrated form, as well as in a non-solvent form. Generally, the solvated form is equivalent to the non-solvent form and is included within the scope of the present invention. Non-limiting examples of hydrates include monohydrates, dihydrates, hemihydrates, etc. In certain embodiments, the compound is a hemihydrate. Non-limiting examples of solvates include ethanol solvates, acetone solvates, etc.

[0083] The compounds of the present invention may exist in polycrystalline or amorphous forms. Generally, all physical forms are intended to be equivalent for the uses considered by the present invention and to be within the scope of the present invention.

[0084] The compounds that can be used in the compositions, kits, and methods of the present invention are those with the following chemical formula ( I It includes a compound having ) or a pharmaceutically acceptable salt thereof:

[0085]

[0086] In the above formula,

[0087] Y - is a pharmaceutically acceptable anion;

[0088] R A , R B , and R C is H, D, halogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, OR I , NR J R K , NR L C(O)R M , S(O)R N , S(O)2R N , SO2R O R P , SO2NR Q R R , SO3R S , CO2R T , C(O)R U, and C(O)NR V R W Each is selected independently from;

[0089] R I , R J , R K , R L , R M , R N , R O , R P , R Q , R R , R S , R T , R U , R V , and R W Each is independently selected from H, D, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, and substituted or unsubstituted alkynyl;

[0090] X 1 ―CR X R Y ―, ―NR Z C(O)―, ―NR Z C(O)CR X R Y ―,―OC(O)―, ―SC(O)―, ―C(O)NR 1A ―, ―C(O)O―, -C(O)-, ―(O)CS―, ―NR 1A S(O)―, ―S(O)NR 1A ―, ―NR 1A C(O)NR 1A Selected from ―, ―S(O)― and ―S(O)2―;

[0091] R X , R Y , R Z , and R 1A Each is independently selected from H, D, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, and substituted or unsubstituted alkynyl;

[0092] R D and R EEach is independently selected from H, D, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, and substituted or unsubstituted cycloalkyl; R D and R E They form a substituted or unsubstituted C3-C6 cycloalkyl or a substituted or unsubstituted heterocyclic (e.g., a 5 to 7-membered heterocyclic ring) together with the carbon to which they are attached;

[0093] R F and R G is N to which they are attached + N together with + In addition to 0, forming an optionally substituted heterocyclic ring having one or more nitrogen atoms, or R F and R G Each is a substituted or unsubstituted alkyl, a substituted or unsubstituted alkenyl, a substituted or unsubstituted alkynyl, a substituted or unsubstituted heterocyclyl, and a substituted or unsubstituted C 3-6 Independently selected from cycloalkyls;

[0094] R H is a substituted or unsubstituted aryl, or a substituted or unsubstituted heteroaryl.

[0095] In a preferred embodiment, R H is substituted or unsubstituted C 5-10 Aryl or substituted or unsubstituted C 5-10 It is selected independently from heteroaryls.

[0096] In a further preferred embodiment, R H is substituted or unsubstituted C 6-10 It is independently selected from aryls or substituted or unsubstituted 5 to 10 heteroaryls.

[0097] In some implementations, R H is C 1-6 Alkan, C 1-6Heteroalkanes, carbocycles, substituted carbocycles, heterocarbocycles, substituted heterocarbocycles, phenyl, substituted phenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, carboxamide, hydroxyl, ether, amide, ester, sulfonamide, sulfone, amino, aminoalkyl, urea, nitrile, or substituted C optionally substituted with a halogen 5-10 Aryl or substituted C 5-10 It is a heteroaryl. In a preferred embodiment, C 1-6 The alkanes are selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, and n-hexyl. In a preferred embodiment, C 1-6 The heteroalkanes are selected from -O-methyl, -O-ethyl, -O-propyl, -O-isopropyl, -O-butyl, -O-isobutyl, -O-cyclohexyl, -O-cyclopentyl, and -ethyl-O-methyl. In a preferred embodiment, the carbocycle is selected from cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl. In a preferred embodiment, the heterocarbocycle is selected from aziridine, azetidine, furan, pyrrolidine, pyran, piperidine, piperazine, azepine, and diazapine.

[0098] In another additional preferred embodiment, R H is substituted or unsubstituted C 6-10 It is an aryl or a substituted or unsubstituted 5 to 10-membered heteroaryl. In certain embodiments, R H C that is not substituted 6-10 It is an aryl or an unsubstituted 5 to 10-membered heteroaryl. In additional embodiments, R H is C 6-10 It is an aryl or a 5 to 10-membered heteroaryl, each optionally substituted with a substituted or unsubstituted C1-C6 alkyl, halo, nitrile, hydroxyl, and alkoxy group. In a further embodiment, R H is C 6-10aryl or 5 to 10-membered heteroaryls, each being a substituted or unsubstituted C1-C6 alkyl, halo, nitrile, and OR 2B It is optionally substituted as, where R 2B is hydrogen or a substituted or unsubstituted C1-C6 alkyl. In a further preferred embodiment, R H is an unsubstituted phenyl. In a further embodiment, R H is a phenyl substituted with a substituent selected from the group consisting of substituted or unsubstituted C1-C6 alkyl, halo, nitrile, hydroxyl, and alkoxy. In additional embodiments, R H is substituted or unsubstituted C1-C6 alkyl, halo, nitrile and OR 2B It is a phenyl substituted with a substituent selected from the group consisting of, wherein R 2B is hydrogen or a substituted or unsubstituted C1-C6 alkyl. In further embodiments, R H is a phenyl substituted with an unsubstituted C1-C6 alkyl, halo, nitrile, hydroxyl, or alkoxy group. In further embodiments, R H is unsubstituted C1-C6 alkyl, halo, nitrile and OR 2B It is a phenyl substituted with a substituent selected from the group consisting of, where R 2B is hydrogen or a substituted or unsubstituted C1-C6 alkyl.

[0099] In an additional mode, R H is selected from the Z group presented in Tables 1 to 3.

[0100] In a preferred embodiment, X 1 is ―NHC(O)― or -C(O)NH-. In another preferred embodiment, X 1 It is ―NHC(O)―.

[0101] In a preferred embodiment, R A , R B and R C is H, D, halogen, substituted or unsubstituted C 1-4 Alkyl and NRJ R K Selected independently from each; R J and R K Each is H and substituted or unsubstituted C 1-4 It is independently selected from alkyls.

[0102] In a preferred embodiment, R A and R B Each is CH3, and R C is H, CH 3, It is a halogen, nitrile, methoxy, or ethoxy.

[0103] Also, in an additional desirable embodiment, R A , R B and R C is independently H, D, halogen, OR I , substituted or unsubstituted C1-C4 alkyl and NR J R K Selected from; R I , R J and R K Each is independently selected from H and substituted or unsubstituted C1-C4 alkyls. In a preferred embodiment, R A and R B Each is CH3, and R C is selected from the group consisting of H, CH3, halogen, nitrile (cyano), methoxy, and ethoxy, in a further preferred embodiment, R A and R B Each is CH3, and R C is selected from the group consisting of H, CH3, fluoro, chloro, nitrile, methoxy, and ethoxy. In a further preferred embodiment, R A and R B Each is CH3, and R C is hydrogen.

[0104] In a specific other implementation example, R D is halogen, oxygen, C 3-8 C optionally substituted with a substituent selected from the group consisting of cycloalkyl, aryl, and heteroaryl. 1-4Alkyl and / or R E is H, or halogen, oxygen, C 3-8 C optionally substituted with a substituent selected from the group consisting of cycloalkyl, aryl, or heteroaryl 1-4 It is an alkyl.

[0105] In a preferred embodiment, R D and R E Each is H, D, CH3, CH2CH3, (CH2)2CH 3, and It is independently selected from (CH2)3CH3. In a more preferred embodiment, R E is hydrogen, and R D is CH3, CH2CH3, (CH2)2CH 3, or (CH2)3CH3. In other specific preferred embodiments, R D and R E It forms C3-C6 cycloalkyl groups that are substituted or unsubstituted together.

[0106] In a specific preferred embodiment, R D and R E Both are hydrogen. Also, in an additional preferred embodiment, R D is hydrogen, and R E is an alkyl, for example, a C1-C6 alkyl or C1-C4 alkyl including, butyl, methyl, ethyl, propyl, and butyl. In certain additional preferred embodiments, R D and R E These are taken together with the carbon atoms to which they are attached to form C3-C6 cycloalkyls, including cyclopropyl or cyclobutyl, to a non-limiting extent.

[0107] In a preferred embodiment, R F and R G is N to which they are attached + Together with, form a 5, 6, 7, or 8-membered heterocyclic ring that is optionally substituted to produce a compound of the following formula Ia:

[0108]

[0109] In the above formula, each variable is as defined above and includes preferred or alternative embodiments, where n is 1, 2, 3, 4 or 5; R 1B is H or a substituent, e.g., H, D, halogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, OR 1I , CN, NR 1J R 1K , NR 1L C(O)R 1M , S(O)R 1N , S(O)2R 1N , SO2R 1O R 1P , SO2NR 1Q R 1R , SO3R 1S , CO2R 1T , C(O)R 1U and C(O)NR 1V R 1W is; R 1I , R 1J , R 1K , R 1L , R 1M , R 1N , R 1O , R 1P , R 1Q , R 1R , R 1S , R 1T , R 1U , R 1V and R 1W Each is independently selected from H, D, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, and substituted or unsubstituted heterocycloalkyl. R 1J and R 1K or R 1Vand R 1W or R 1Q and R 1R Silver can also be taken together with the nitrogen to which they are attached to form a substituted or unsubstituted 5, 6, 7, or 8-membered ring.

[0110] In a preferred embodiment, R F and R G is N to which they are attached + Forming a 5, 6, 7, or 8-membered nitrogen-containing heterocyclic ring together with, and including, but not limited to, the following,

[0111]

[0112] Each of these is optionally substituted. Preferred substituents are phenyl and CO2R 1T and C(O)NR 1V R 1W Includes

[0113] In an additional mode, R F and R G is independently a C1-C4 alkyl. In another embodiment, R F and R G is independently selected from CH3 and CH2CH3. In other specific embodiments, R F and R G is the same and is a substituted or unsubstituted C1-C4 alkyl. In additional embodiments, R F and R G is identical and is methyl, ethyl, propyl, or butyl. In another additional embodiment, R F and R G It is the same, and is CH3 or CH2CH3.

[0114] In some implementations, Y - is a halide anion, carboxylate, or sulfonate. Y -It may be, for example, a halide ion, a substituted or unsubstituted alkyl sulfonate, a substituted or unsubstituted aryl sulfonate, a substituted or unsubstituted alkyl or aliphatic carboxylate, a substituted or unsubstituted aryl carboxylate, or a substituted or unsubstituted heterocyclyl carboxylate.

[0115] In a specific implementation example, Y - trifluoroacetate, sulfate, phosphate, acetate, fumarate, formate, carbonate, maleate, citrate, pyruvate, succinate, oxalate, sulfonate (e.g., methanesulfonate, trifluoromethanesulfonate, toluenesulfonate, e.g., p-toluenesulfonate, benzenesulfonate, ethanesulfonate, camphorsulfonate, 2-methylenesulfonate, or naphthalenesulfonate, e.g., 2-naphthalenesulfonate), bisulfate, malonate, cinnapoate, ascorbate, oleate, nicotinate, saccharinate, adipate, formate, glycolate, L-lactate, D-lactate, aspartate, maleate, L-tartrate, D-tartrate, stearate, 2-furoate, It is selected from the group consisting of 3-furoate, napadisilate (naphthalene-1,5-disulfonate or naphthalene-1-(sulfonic acid)-5-sulfonate), edicilate (ethane-1,2-disulfonate or ethane-1-(sulfonic acid)-2-sulfonate), isethionate (2-hydroxyethylsulfonate), D-mandelate, L-mandelate, propionate, tartrate, phthalate, hydrochlorate, hydrobromate, and nitrate. In one embodiment, Y - is a halide anion.

[0116] In a preferred embodiment, the anion is selected from halide ions, bromide, chloride, or iodide.

[0117] The aforementioned desirable group may be taken in combination with one, some, or all other desirable groups.

[0118] In a preferred embodiment, the present invention relates to a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein R H is an optionally substituted aryl or an optionally substituted heteroaryl selected from one of the following:

[0119]

[0120] .

[0121] In a specific preferred embodiment, R H is a substituted or unsubstituted phenyl.

[0122] In certain additional embodiments, the compound has the following chemical formula ( II has:

[0123]

[0124] In the above formula,

[0125] Y - is a pharmaceutically acceptable anion;

[0126] q is 0, 1, 2, or 3;

[0127] R D is hydrogen, methyl, or ethyl;

[0128] R 1B is as defined above.

[0129] In some embodiments, the compound is Y - It has the chemical formula (II) in which is a halide anion, a carboxylate, or a sulfonate. Y - It may be, for example, a halide ion, a substituted or unsubstituted alkyl sulfonate, a substituted or unsubstituted aryl sulfonate, a substituted or unsubstituted alkyl or aliphatic carboxylate, a substituted or unsubstituted aryl carboxylate, or a substituted or unsubstituted heterocyclyl carboxylate.

[0130] In a specific embodiment, the compound has the chemical formula ( IIWith ), here Y - trifluoroacetate, sulfate, phosphate, acetate, fumarate, formate, carbonate, maleate, citrate, pyruvate, succinate, oxalate, sulfonate (e.g., methanesulfonate, trifluoromethanesulfonate, toluenesulfonate, e.g., p-toluenesulfonate, benzenesulfonate, ethanesulfonate, camphorsulfonate, 2-methylenesulfonate or naphthalenesulfonate, e.g., 2-naphthalenesulfonate), bisulfate, malonate, cinnapoate, ascorbate, oleate, nicotinate, saccharinate, adipate, formate, glycolate, L-lactate, D-lactate, aspartate, maleate, L-tartrate, D-tartrate, stearate, 2-furoate, It is selected from the group consisting of 3-furoate, napadisilate (naphthalene-1,5-disulfonate or 2-naphthalene-1-(sulfonic acid)-5-sulfonate), edicilate (ethane-1,2-disulfonate or ethane-1-(sulfonic acid)-2-sulfonate), isethionate (2-hydroxyethylsulfonate), D-mandelate, L-mandelate, propionate, tartarate, phthalate, hydrochlorate, hydrobromate, and nitrate.

[0131] In one embodiment, the compound is Y - It has the chemical formula (II) in which is a halide anion. In a preferred embodiment, Y - The halide ion is selected from bromide, chloride, or iodide.

[0132] As is understood, when q is 0, 1, 2, or 3, N of formula (II) + - The containing rings are as follows:

[0133] .

[0134] In a specific preferred embodiment, q is 0. In another preferred embodiment, q is 1. In another preferred embodiment, q is 2. In an additional preferred embodiment, q is 3.

[0135] In a further preferred embodiment, the compound has the chemical formula ( II Having ), R D is hydrogen.

[0136] In an additional embodiment, the compound has the chemical formula ( II Having ), R D is methyl.

[0137] In additional other embodiments, the compound has the chemical formula ( II Having ), R D is ethyl.

[0138] In an additional embodiment, the compound has the chemical formula ( II having ), q is 0, and R D is hydrogen.

[0139] In a further preferred embodiment, the compound has the chemical formula ( II Having ), q is 1, and R D is hydrogen.

[0140] In a further preferred embodiment, the compound has the chemical formula ( II It has ), q is 2, and R D is hydrogen.

[0141] In an additional embodiment, the compound has the chemical formula ( II It has ), q is 3, and R D is hydrogen.

[0142] In a further preferred embodiment, in a further aspect, the compound is of the formula ( II having ), q is 0, and R D is methyl.

[0143] In a further preferred embodiment, the compound has the chemical formula ( II Having ), q is 1, and R D is methyl.

[0144] In a further preferred embodiment, the compound has the chemical formula ( II It has ), q is 2, and R D is methyl.

[0145] In an additional embodiment, the compound has the chemical formula ( II It has ), q is 3, and R D is methyl.

[0146] In a further preferred embodiment, in a further aspect, the compound is of the formula ( II having ), q is 0, and R D is ethyl.

[0147] In a further preferred embodiment, the compound has the chemical formula ( II Having ), q is 1, and R D is ethyl.

[0148] In a further preferred embodiment, the compound has the chemical formula ( II It has ), q is 2, and R D is ethyl.

[0149] In an additional embodiment, the compound has the chemical formula ( II It has ), q is 3, and R D is ethyl.

[0150] In a further embodiment, the compound is selected from Table A below or pharmaceutically acceptable salts thereof, wherein Y - is a pharmaceutically acceptable anion.

[0151] Table A

[0152]

[0153]

[0154]

[0155]

[0156]

[0157]

[0158] In a further preferred embodiment, the compound is selected from the compounds of the table below or pharmaceutically acceptable salts thereof, wherein Y - is an anion as indicated or pharmaceutically acceptable:

[0159] Table B

[0160]

[0161]

[0162]

[0163]

[0164]

[0165]

[0166]

[0167]

[0168]

[0169]

[0170]

[0171]

[0172]

[0173]

[0174]

[0175]

[0176] Representative compounds according to the present invention, their enantiomers, and pharmaceutically acceptable salts thereof are selected from Table C below, wherein Y - is a pharmaceutically acceptable anion as defined above, and Z is an aryl or heteroaryl selected from one of the structures in Table 1, or a substituted aryl or substituted heteroaryl selected from one of the structures in Tables 2-3.

[0177] Table C - Representative compounds of the present invention

[0178]

[0179]

[0180]

[0181]

[0182]

[0183]

[0184]

[0185]

[0186]

[0187]

[0188]

[0189]

[0190]

[0191]

[0192]

[0193] Table 1 - Representative Z structures

[0194]

[0195]

[0196] Table 2 - Representative Z structures

[0197]

[0198]

[0199]

[0200]

[0201]

[0202]

[0203]

[0204]

[0205] Table 3 - Representative Z structures

[0206]

[0207]

[0208]

[0209] The preferred compounds according to the present invention, their enantiomers, and pharmaceutically acceptable salts thereof are represented by the following chemical formula (III):

[0210]

[0211] In the above formula, the preferred substituent combination R C , R D , N + / R F / R G , and Z are as defined in Table 4, and Y - is a pharmaceutically acceptable anion as defined above. The compound can be prepared according to the method generally described below.

[0212] Table 4 - R according to chemical formula (III) C , R D , N + / R F / R G , and a preferred combination of Z substituents

[0213]

[0214]

[0215]

[0216]

[0217]

[0218]

[0219]

[0220]

[0221]

[0222]

[0223]

[0224]

[0225]

[0226]

[0227]

[0228]

[0229]

[0230]

[0231]

[0232]

[0233]

[0234]

[0235]

[0236]

[0237]

[0238]

[0239]

[0240]

[0241]

[0242]

[0243]

[0244]

[0245]

[0246]

[0247]

[0248]

[0249] Each preferred embodiment described herein may be taken in combination with one, any, or all other preferred embodiments as presented herein in all permutations.

[0250] The composition of the present invention may comprise a racemic mixture, a pure enantiomer, or an excess of one enantiomer relative to the others. For example, the composition may comprise an excess of at least 5, 10, 20, 30, 40, 50, 60, 70, 80, or 90% of the enantiomer. In one embodiment, the excess of the enantiomer is at least 95%.

[0251] The compounds of the present invention include all enantiomers that can be defined as (R)- or (S)- in terms of absolute stereochemistry, as well as their racemic and optically pure forms, and are not limited to any of the pharmaceutically acceptable forms described herein, including enantiomers, salts, solvates, polymorphs, solvatomorphs, hydrates, anhydrous and other crystalline forms, and combinations thereof. Likewise, all tautomer forms are intended to be included.

[0252] Preferably, the pharmaceutical composition comprises the compound of the present invention as a substantially pure R enantiomer; the pharmaceutical composition comprises the compound of the present invention as a substantially pure S enantiomer; or the pharmaceutical composition comprises the compound of the present invention as an enantiomer mixture containing an excess of the R enantiomer or an excess of the S enantiomer. It is particularly preferable that the pharmaceutical composition contains the compound of the present invention as a substantially pure optical isomer. To avoid any doubt, the compound of the present invention may be used in the form of a solvate if desired.

[0253] synthesis

[0254] Chemical formula ( I Compounds having ) can be prepared using a method similar to the following general synthesis reaction scheme:

[0255]

[0256] Reaction Equation A

[0257] and

[0258]

[0259] Reaction Equation B.

[0260] for example,

[0261]

[0262] and

[0263] .

[0264] Compounds having chemical formula (II) can be prepared using a method similar to that described in the examples and the synthesis formula below:

[0265]

[0266] and

[0267] .

[0268] Additional biological active agents and exogenous large porous channel agonists

[0269] As described above, the compounds or compositions of the present invention may be administered together with biological activators. For example, one or more additional biological activators, including those typically used to treat neuroinflammatory diseases, may be used in combination with the compounds or compositions of the present invention described herein. Biological activators include, but are not limited to, TRP1A receptor agonists, TRPV1-4 receptor agonists, TRPM8 agonists, ASIC agonists, P2X receptor agonists, acetaminophen, NSAIDs, glucocorticoids, narcotics, tricyclic antidepressants, amine transporter inhibitors, anticonvulsants, antiproliferative agents and immunomodulators, antibodies or antibody fragments, antibiotics, polynucleotides, polypeptides, proteins, anticancer agents, growth factors, and vaccines.

[0270] TRPV1 agonists that may be used in the methods, kits, and compositions of the present invention include, but are not limited to, any that activate TRPV1 receptors on nociceptors and allow entry of at least one inhibitor (e.g., compounds of the present invention) into voltage-gated ion channels. Suitable TRPV1 agonists are capsaicin or other capsaicinoids that are members of the vanilloid family of molecules. Naturally occurring capsaicinoids are capsaicin itself, dihydrocapsaicin, nordihydrocapsaicin, homodihydrocapsaicin, homocapsaicin, and nonivamid. Other suitable capsaicinoids and capsaicinoid analogs and derivatives for use in the compositions and methods of the present invention are, for example, vanilloids (e.g., N-vanillyl-alkanidienamide, N-vanillyl-alkanidienyl, and N-vanillyl-cis-monounsaturated alkenamide), capsiates, dihydrocapsiates, nordihydrocapsiates and other capsinoids, capsiconiates, dihydrocapsiconiates and other coniferyl esters, capsiconinoids, resiniferatoxin, tinyatoxin, civamide, N-phenylmethylalkenamide capsaicin derivatives, olvanil, N-[(4-(2-aminoethoxy)-3-methoxyphenyl)methyl]-9Z-octa-decaneamide, N-oleyl-homovanilamid, triprenyl It includes naturally occurring and synthetic capsaicin derivatives and analogs, including phenol (e.g., scutigeral), gingerol, piperine, shogaol, guaiacol, eugenol, zingerone, nuvanil, NE-19550, NE-21610, and NE-28345. Additional capsaicinoids, their structures, and methods for their preparation are described in U.S. Patent Nos. 7,446,226 and 7,429,673, which are incorporated herein by reference.

[0271] Additional suitable TRPV1 agonists include eugenol, arvanil (N-arachidonoylvanilamin), anandamide, 2-aminoethoxydiphenyl borate (2APB), AM404, resininiferatoxin, phorbol 12-phenylacetate 13-acetate 20-homovanilate (PPAHV), olvanil (NE 19550), OLDA (N-oleoyldopamine), N-arachidonyldopamine (NADA), 6'-iodoresiniferatoxin (6'-IRTX), C18 N-acylethanolamine, lipoxygenase derivatives, e.g., 12-hydroperoxyeicosatetraenoic acid, inhibitor cysteine ​​knot (ICK) peptide (vanillotoxin), piperine. MSK195 (N-[2-(3,4-dimethylbenzyl)-3-(pivaloyloxy)propyl]-2-[4-(2-aminoethoxy)-3-methoxyphenyl]acetamide), JYL79 (N-[2-(3,4-dimethylbenzyl)-3-(pivaloyloxy)propyl]-N'-(4-hydroxy-3-methoxybenzyl)thiourea), hydroxy-alpha-sanshool, 2-aminoethoxydiphenyl borate, 10-shogaol, oleylgingerol, oleylshogaol and SU200 (N-(4-tert-butylbenzyl)-N'-(4-hydroxy-3-methoxybenzyl)thiourea), but is not limited thereto.Other TRPV1 agonists include amylocaine, articaine, benzocaine, bupivacaine, carbocaine, carticaine, chloroprocaine, cyclomethicaine, dibucaine (cinchocaine), dimethocaine (larocaine), etidocaine, hexylcaine, levobupivacaine, lidocaine, mepivacaine, meprylcaine (oracaine), metabutoxycaine, piperocaine, prilocaine, procaine (novacaine), proparacaine, propoxycaine, and risocaine. Includes ropivacaine, tetracaine (amethocaine) and trimecaine.

[0272] Suitable TRPV2-4 agonists are 2-APB, cannabinol, diphenylboronic anhydride, insulin-like growth factor 1, lysophosphatidylcholine, lysophosphatidylinositol, probenecid, Δ9-tetrahydrocannabinol, vanillin, eugenol, cinnamaldehyde, camphor, carvacrol, thymol, citral, farnesyl diphosphate, tetrahydrocannabivarin, Incenseol acetate, diphenylboronic anhydride, 6-tert-butyl-m-cresol, dihydrocarbeocarbeol, borneol, (-)-menthol, GSK1016790A, 4α-PDH, 5,6-epoxyeicosatrienoic acid, 4α-PDD, bisandrographolide, citric acid, phorbol 12-myristate 13-acetate and Includes, but is not limited to, RN1747.

[0273] Suitable TRPM8 agonists include, but are not limited to, menthol, icilin, eucalyptus, linalool, geraniol, hydroxy-citronellal, WS-3, WS-23, Frescolat MGA, Frescolat ML, PMD 38, CPS125, Coolact P, M8-Ag, AITC, cryosim-3, and Cooling Agent 10.

[0274] Suitable ASIC agonists include, but are not limited to, chlorophenylguanidine hydrochloride, GMQ hydrochloride, tetrahydropapaverolin (THP), reticulin, polyamine agmatine, lysophosphatidylcholine, arachidonic acid, and neuropeptide SF.

[0275] Other biological activators that may be used in the methods, compositions, and kits of the present invention include any that activate TRP1A receptors on nociceptors or itchiness receptors and allow the entry of at least one inhibitor of voltage-gated ion channels. Suitable TRP1A agonists include, but are not limited to, cinnamaldehyde, allyl-isothiocyanate (mustard oil), diallyl disulfide, icyl, cinnamon oil, wintergreen oil, clove oil, acrolein, hydroxy-alpha-sanshool, 2-aminoethoxydiphenyl borate, 4-hydroxynonenal, methyl p-hydroxybenzoate, and 3'-carbamoylbiphenyl-3-yl cyclohexylcarbamate (URB597).

[0276] P2X agonists that may be used in the methods, compositions, and kits of the present invention include any that activate P2X receptors on nociceptors or itchiness receptors and allow entry of at least one inhibitor of voltage-gated ion channels. Suitable P2X agonists include, but are not limited to, ATP, α,β-methylene ATP, 2-methylthio-ATP, 2' and 3'-O-(4-benzoylbenzoyl)-ATP, and ATP5'-O-(3-thiotriphosphate).

[0277] Other biological active agents that can be used in combination with the compounds of the present invention include NSAIDs, glucocorticoids, narcotics, tricyclic antidepressants, amine transporter inhibitors, anticonvulsants, antiproliferative agents and immunomodulators, antibodies or antibody fragments, antibiotics, polynucleotides, polypeptides, proteins, anticancer agents, growth factors, and vaccines.

[0278] Non-steroidal anti-inflammatory drugs (NSAIDs) that may be administered to a patient (e.g., human) suffering from neuroinflammatory disease in combination with the composition of the present invention are acetylsalicylic acid, amoxiprin, benorylate, benorylate, choline magnesium salicylate, diflunisal, ethenzamide, faislamine, methyl salicylate, magnesium salicylate, salicyl salicylate, salicylamide, diclofenac, aceclofenac, acemethacin, aclofenac, bromfenac, etodolac, indomethacin, nabumetone, oxametacin, Proglumetacin, Sulindac, Tolmetin, Ibuprofen, Alminoprofen, Benoxaprofen, Carprofen, Dexibuprofen, Dexketoprofen, Fenbufen, Fenoprofen, Flunoxaprofen, Flurbiprofen, Ibuproxam, Indoprofen, Ketoprofen, Ketorolac, Loxoprofen, Naproxen, Oxaprozin, Pirprofen, Suprofen, Tiaprofenic acid, Mefenamic acid, flufenamic acid, meclofenamic acid, tolfenamic acid,phenylbutazone, ampyrone, azapropazone, clofezone, kebuzone, metamizole, mofebutazone, oxyphenbutazone, phenazone, sulfinpyrazone, piroxicam, droxicam, lornoxicam, meloxicam, tenoxicam, and COX-2 inhibitors celecoxib, etoricoxib, lumiracoxib, parecoxib, rofecoxib, valdecoxib, and pharmaceutically acceptable salts thereof, wherein It is not limited.

[0279] Glucocorticoids that may be administered to a patient (e.g., human) suffering from neuroinflammatory disease in combination with the composition of the present invention include, but are not limited to, hydrocortisone, cortisone acetate, prednisone, prednisolone, methylprednisolone, dexamethasone, betamethasone, triamcinolone, beclomethasone, fludrocortisone acetate, deoxycorticosterone acetate, aldosterone, and pharmaceutically acceptable salts thereof.

[0280] Narcotic agents that may be administered to a patient (e.g., human) suffering from neuroinflammatory disease in combination with the composition of the present invention include, but are not limited to, tramadol, hydrocodone, oxycodone, morphine, and pharmaceutically acceptable salts thereof.

[0281] Antiproliferative agents and immunomodulators that may be administered to a patient (e.g., human) suffering from neuroinflammatory disease in combination with the composition of the present invention include, but are not limited to, alkylating agents, platinum agents, antimetabolites, focal isomerase inhibitors, dihydrofolate reductase inhibitors, antitumor antibiotics, antimitotic agents, aromatase inhibitors, thymidylate synthase inhibitors, DNA antagonists, farnesyltransferase inhibitors, pump inhibitors, histone acetyltransferase inhibitors, metalloproteinase inhibitors, ribonucleoside reductase inhibitors, TNF-alpha agonists, TNF-alpha antagonists or scavengers, interleukin 1 (IL-1) antagonists or scavengers, endothelin A receptor antagonists, retinoic acid receptor agonists, hormones, antihormones, photodynamic agents, and tyrosine kinase inhibitors.

[0282] The biological active agent may be administered before, simultaneously with, or after the administration of the composition of the present invention using any formulation, dosage, or administration known in the art that is therapeutically effective.

[0283] Formulation of the composition

[0284] The administration of the compound of the present invention may be carried out by any suitable means that causes a reduction in perceived pain sensation in the target area. The compound of the present invention may be contained in any suitable amount in any suitable carrier material, which is generally present in an amount of 1-99% by weight of the total weight of the composition. The composition may be provided in a form of administration suitable for oral, parenteral (e.g., intravenous, intramuscular), rectal, skin, subcutaneous, topical, transdermal, sublingual, nasal, vaginal, intrathecal, epidural, or ocular administration, or may be provided by injection, inhalation, or direct contact with the nasal or oral mucosa.

[0285] Accordingly, the composition may be in the form of, for example, tablets, capsules, pills, powders, granules, suspensions, emulsions, solutions, gels including hydrogels, pastes, ointments, creams, plasters, drenches, osmotic delivery devices, suppositories, enemas, injections, implants, sprays, or aerosols. The composition may be formulated according to ordinary pharmaceutical practice (e.g., the literature [Remington: The Science and Practice of Pharmacy, 22nd edition, 2013, ed. LV Allen, Pharmaceutical Press, Philadelphia, and Encyclopedia of Pharmaceutical Technology, 4 th Edition, ed. J. Swarbrick, 2013, CRC Press, New York].

[0286] Each compound may be formulated in various ways known in the art. For example, the compounds of the present invention and biological active agents as defined herein may be formulated together or separately. Preferably, the compounds and biological active agents of the present invention are formulated together for simultaneous or near-simultaneous administration thereof. In another embodiment, two or more biological active agents may be formulated together with or separately from the compounds of the present invention. Other examples include, but are not limited to, two or more compounds of the present invention formulated together, said compounds being formulated with or without one or more biological active agents.

[0287] Preparations formulated individually or separately may be packaged together as a kit. Non-limiting examples include, but are not limited to, a kit containing, for example, two pills, a pill and a powder, a suppository and a liquid in a vial, two topical creams, etc. The kit may include optional components that aid in administering a unit dose to a patient, such as a vial for reconstitution of the powder form, an injectable syringe, a customized IV delivery system, an inhaler, etc. Additionally, the unit dose kit may include instructions for the preparation and administration of the composition.

[0288] The kit may be manufactured as a single-use unit dose for one patient or as a multiple-use for a specific patient (at a constant dose or where the efficacy of individual compounds may vary as the regimen progresses); or the kit may contain multiple doses suitable for administration to a number of patients (“bulk packaging”). Kit components may be assembled in a carton, blister pack, bottle, tube, etc.

[0289] Controlled-release formulation

[0290] Each compound of the present invention, either alone or in combination with one or more of the biological active agents described herein, may be formulated for controlled-release (e.g., sustained or measured) administration as described in U.S. Patent Application Publications No. 2003 / 0152637 and 2005 / 0025765, each incorporated herein by reference. For example, a compound of the present invention, either alone or in combination with one or more of the biological active agents described herein, may be incorporated into a capsule or tablet administered to a patient.

[0291] Any pharmaceutically acceptable vehicle or formulation suitable for topical application and / or injection to a site to be treated (e.g., painful surgical incision, wound, or joint) capable of providing sustained release of the compound of the present invention, either alone or in combination with one or more of the biological active agents described herein, may be used as needed to provide long-term clearance or relief of inflammation. Control-release formulations known in the art comprise specially coated pellets, polymer formulations, or matrices for surgical insertion, or sustained-release microparticles, e.g., microspheres or microcapsules, for implantation, insertion, infusion, or injection, wherein the slow release of the active agent occurs through sustained or controlled diffusion outside the matrix and / or selective degradation of the product coating or selective degradation of the polymer matrix. Other formulations or vehicles for controlled, sustained, or immediate delivery of the agent to a desired local site of a patient include, e.g., suspensions, emulsions, gels, liposomes, and any other suitable delivery vehicle or formulation known in the art that is acceptable for subcutaneous or intramuscular administration.

[0292] A wide variety of biocompatible materials may be used as controlled-release carriers to provide controlled release of the compounds of the present invention, either alone or in combination with one or more biological activators as described herein. Any pharmaceutically acceptable biocompatible polymer known to those skilled in the art may be used. It is desirable that the biocompatible controlled-release material degrades in vivo within about one year, preferably within about three months, and more preferably within about two months. More preferably, the controlled-release material will degrade significantly within one to three months, at least 50% of the material will degrade into non-toxic residues and be eliminated by the body, and 100% of the compounds of the present invention will be released within about two weeks, preferably within about two to seven days. The degradable controlled-release material should preferably degrade by hydrolysis due to surface erosion or bulk erosion to provide not only sustained release but also a desirable release rate. However, the pharmacokinetic release profiles of these formulations may be primary, zero, secondary, or multiple to provide the desired reversible local anti-nourishing effect over the desired period.

[0293] Suitable biocompatible polymers may be used as controlled-release materials. Polymeric materials may include biocompatible, biodegradable polymers, and in certain preferred embodiments, preferably copolymers of lactic acid and glycolic acid. Preferred controlled-release materials useful for the formulations of the present invention include polyanhydrides, polyesters, copolymers of lactic acid and glycolic acid (preferably, the weight ratio of lactic acid to glycolic acid is 4:1 or less, i.e., 80 weight% or less of lactic acid to 20 weight% or more of glycolic acid), and polyorthoesters containing a catalyst or degradation-enhancing compound, and, for example, at least 1 weight% of an anhydride catalyst such as maleic anhydride. Examples of polyesters include polylactic acid, polyglycolic acid, and polylactic acid-polyglycolic acid copolymers. Other useful polymers include protein polymers such as collagen, gelatin, fibrin, and fibrinogen, and polysaccharides such as hyaluronic acid.

[0294] Polymeric materials may be prepared by any method known to those skilled in the art. For example, if the polymeric material comprises a copolymer of lactic acid and glycolic acid, such copolymer may be prepared by the procedure described in U.S. Patent No. 4,293,539, incorporated herein by reference. Alternatively, copolymers of lactic acid and glycolic acid may be prepared by any other procedure known to those skilled in the art. Other useful polymers include polylactide, polyglycolide, polyanhydride, polyorthoester, polycaprolactone, polyphosphazene, polyphosphoester, polysaccharide, proteinaceous polymer, soluble derivative of polysaccharide, soluble derivative of proteinaceous polymer, polypeptide, polyester and polyorthoester, or mixtures or blends of any of these.

[0295] The pharmaceutically acceptable polyanhydrides useful in the present invention have anhydrous bonds that are unstable in water. The drug release rate can be controlled by the specific polyanhydride polymer used and its molecular weight. The polysaccharide may be a poly-1,4-glucan, for example, starch glycogen, amylose, amylopectin, and mixtures thereof. The biodegradable hydrophilic or hydrophobic polymer may be a water-soluble derivative of poly-1,4-glucan, including hydrolyzed amylopectin, derivatives of hydrolyzed amylopectin, for example, hydroxyethyl starch (HES), hydroxyethyl amylose, dialdehyde starch, etc. The polyanhydride polymer may be branched or linear.

[0296] Examples of polymers useful in the present invention include (in addition to homopolymers and copolymers of poly(lactic acid) and / or poly(glycolic acid)) poly[bis(p-carboxyphenoxy)propane anhydride] (PCPP), poly[bis(p-carboxy)methane anhydride] (PCPM), polyanhydrides of oligomerized unsaturated aliphatic acids, polyanhydride polymers prepared from amino acids modified to include additional carboxylic acids, aromatic polyanhydride compositions, and copolymers of polyanhydrides with other materials, e.g., fatty acid-terminated polyanhydrides, e.g., polyanhydrides polymerized from monomers of dimers and / or trimers of unsaturated fatty acids or unsaturated aliphatic acids. Polyanhydrides may be prepared according to the method described in U.S. Patent No. 4,757,128, which is incorporated herein by reference. Polyorthoester polymers may be manufactured, for example, as described in U.S. Patent No. 4,070,347, incorporated herein by reference. Polyphosphoesters may be manufactured and used as described in U.S. Patent Nos. 6,008,318, 6,153,212, 5,952,451, 6,051,576, 6,103,255, 5,176,907, and 5,194,581, each incorporated herein by reference.

[0297] Protein polymers may also be used. Protein polymers and their soluble derivatives include gelling biodegradable synthetic polypeptides, elastin, alkylated collagen, alkylated elastin, etc. Biodegradable synthetic polypeptides include copolymers of poly-(N-hydroxyalkyl)-L-asparagine, poly-(N-hydroxyalkyl)-L-glutamine, N-hydroxyalkyl-L-asparagine, and N-hydroxyalkyl-L-glutamine with other amino acids. The proposed amino acids include L-alanine, L-lysine, L-phenylalanine, L-valine, L-tyrosine, etc.

[0298] In additional embodiments, a release control material that acts as a carrier for a compound of the present invention, either alone or in combination with one or more biological active agents as described herein, may further comprise a bioadhesive polymer, e.g., pectin (polygalacturonic acid), mucopolysaccharide (hyaluronic acid, mucin), or non-toxic lectin, or the polymer itself may be a bioadhesive, e.g., a polysaccharide such as polyanhydride or chitosan.

[0299] In an embodiment in which a biodegradable polymer comprises a gel, one of the useful polymers is a heat-gelling polymer, e.g., a polyethylene oxide, polypropylene oxide (PEO-PPO) block copolymer, e.g., BASF Wyandotte’s Pluronic™ F127. In the above case, the local anesthetic formulation may be injected via a syringe as a free-flowing liquid that gels rapidly at 30°C or higher (e.g., when injected into a patient). Subsequently, the gel system releases a steady dose of the compound of the present invention at the site of administration, either alone or in combination with one or more biological active agents as described herein.

[0300] Dosage form for oral use

[0301] Formulations for oral use comprise tablets containing the active ingredient(s) mixed with non-toxic, pharmaceutically acceptable excipients. These excipients are, for example, inactive diluents or fillers (e.g., sucrose, sorbitol, sugars, mannitol, microcrystalline cellulose, starch including potato starch, calcium carbonate, sodium chloride, lactose, calcium phosphate, calcium sulfate, or sodium phosphate); granulizing agents and disintegrants (e.g., cellulose derivatives including microcrystalline cellulose, starch including potato starch, sodium croscarmellose, alginate, or alginic acid); Binders (e.g., sucrose, glucose, sorbitol, acacia, alginic acid, sodium alginate, gelatin, starch, pregelatinized starch, microcrystalline cellulose, magnesium aluminum silicate, sodium carboxymethylcellulose, methylcellulose, hydroxypropyl methylcellulose, ethylcellulose, polyvinylpyrrolidone or polyethylene glycol); and lubricants, lubricants, and anti-adhesives (e.g., magnesium stearate, zinc stearate, stearic acid, silica, hydrogenated vegetable oil, or talc). Other pharmaceutically acceptable excipients may be colorants, flavorings, plasticizers, wetting agents, buffers, taste masking agents (e.g., hydroxypropyl methylcellulose, hydroxypropyl cellulose), etc.

[0302] One or more compounds of the present invention and one or more biological active agents as defined herein may be mixed with or divided into tablets, capsules, or other vehicles. In one example, the compound of the present invention is contained inside a tablet and the biological active agent is contained outside the tablet, so that a substantial portion of the biological active agent is released before the release of the compound of the present invention.

[0303] Formulations for oral use may also be provided as chewable tablets, or as hard gelatin capsules in which the active ingredient is mixed with an inactive solid diluent (e.g., potato starch, lactose, microcrystalline cellulose, calcium carbonate, calcium phosphate, or kaolin), or as soft gelatin capsules in which the active ingredient is mixed with a water or oil medium, e.g., peanut oil, liquid paraffin, or olive oil. Powders, granules, and pellets may be prepared using the aforementioned ingredients in tablets and capsules in a conventional manner, for example, using a mixer, a fluid bed device, or spray drying equipment.

[0304] Formulations for oral administration into the mouth may also be provided as mouthwash, oral spray, oral rinse solution, oral ointment, or oral gel.

[0305] Dissolution or diffusion-controlled release may be achieved by a suitable coating of a tablet, capsule, pellet, or granule formulation of the compound, or by incorporating the compound into a suitable matrix. The controlled release coating may comprise one or more of the coating materials mentioned above and / or, for example, shellac, beeswax, glycowax, castor wax, carnauba wax, stearyl alcohol, glyceryl monostearate, glyceryl distearate, glycerol palmitostearate, ethylcellulose, acrylic resin, dl-polylactic acid, cellulose acetate butyrate, polyvinyl chloride, polyvinyl acetate, vinylpyrrolidone, polyethylene, polymethacrylate, methyl methacrylate, 2-hydroxymethacrylate, methacrylate hydrogel, 1,3-butylene glycol, ethylene glycol methacrylate, and / or polyethylene glycol. In a controlled-release matrix formulation, the matrix material may also include, for example, hydrated methylcellulose, carnauba wax and stearyl alcohol, carbopol 934, silicone, glyceryl tristearate, methyl acrylate-methyl methacrylate, polyvinyl chloride, polyethylene, and / or halogenated fluorocarbons.

[0306] Liquid forms in which compounds and compositions of the present invention may be incorporated for oral administration include aqueous solutions, suitably flavored syrups, aqueous or oil suspensions, and flavored emulsions having edible oils such as cottonseed oil, sesame oil, coconut oil or peanut oil, as well as elixirs and similar pharmaceutical vehicles.

[0307] Generally, when administered to a human, the oral dosage of any compound of the combination of the present invention will depend on the properties of the compound and can be easily determined by a person skilled in the art. Typically, the dosage is generally about 0.001 mg to 2000 mg per day, preferably about 1 mg to 1000 mg per day, more preferably about 5 mg to 500 mg per day. A maximum dosage of 200 mg per day may be required.

[0308] As described herein, in combination therapy, the administration of each drug may be independent, from one to four times daily for one day to one year, and even for the patient's lifetime. In many cases, chronic long-term administration will be indicated.

[0309] parenteral formulation

[0310] Formulations suitable for parenteral administration (e.g., by injection) comprise aqueous or non-aqueous, isotonic, pyrogen-free, sterile liquids (e.g., solutions, suspensions), wherein the compound is dissolved, suspended, or otherwise provided (e.g., in liposomes or other microparticles). The liquid may further contain other pharmaceutically acceptable components, e.g., antioxidants, buffers, preservatives, stabilizers, bacteriostatic agents, suspending agents, thickeners, and solutes that make the formulation isotonic with the blood (or other related body fluids) of the intended recipient. Examples of excipients include, e.g., water, alcohol, polyol, glycerol, vegetable oil, etc. Examples of suitable isotonic carriers for use in the formulation include sodium chloride injection, Ringer's solution, or lactated Ringer's injection. Typically, the concentration of the compound in the liquid is about 1 ng / ml to about 10 μg / ml, for example, about 10 ng / ml to about 1 μg / ml. The formulation may be provided in unit-dose or multi-dose sealed containers, for example, ampoules and vials, and may be stored under freeze-drying conditions requiring only the addition of a sterile liquid carrier, for example, water for injection, immediately before use. Immediate injection solutions and suspensions may be prepared from sterile powders, granules, and tablets.

[0311] Topical formulations

[0312] Compositions of the present invention, alone or in combination with one or more of the biological active agents described herein, may also be adapted for topical use with a topical vehicle containing 0.0001% to 25% (w / w) or more of active ingredient(s).

[0313] In a preferred combination, the active ingredient is preferably 0.0001% to 10% (w / w) each, more preferably 0.0005% to 4% (w / w) of active ingredient. Topical formulations, including but not limited to creams, gels, or ointments, may be applied 1 to 4 times a day or as needed. By carrying out the method described herein, a topical vehicle containing a composition of the present invention or a combination therapy containing a composition of the present invention is preferably applied to the inflamed area of ​​a patient. For example, a cream may be applied to the hand of a patient suffering from an arthritic finger.

[0314] The composition may be formulated using any dermatologically acceptable carrier. Exemplary carriers include solid carriers, e.g., alumina, clay, microcrystalline cellulose, silica, or talc; and / or liquid carriers, e.g., alcohol, glycol, or aqueous alcohol / glycol blends. The therapeutic agent may also be administered in a liposomal formulation that allows the therapeutic agent to enter the skin. The liposomal formulations are U.S. Patent Nos. 5,169,637; 5,000,958; 5,049,388; 4,975,282; 5,194,266; 5,023,087; 5,688,525; 5,874,104; 5,409,704; 5,552,155; 5,356,633; 5,032,582; It is described in Patent No. 4,994,213; Patent No. 8,822,537, and PCT Publication No. WO 96 / 40061. Examples of other suitable vehicles are described in U.S. Patent No. 4,877,805, Patent No. 8,822,537, and EP Publication No. 0586106A1. Suitable vehicles of the present invention may also include inorganic oil, petroleum jelly, polydecene, stearic acid, isopropyl myristate, polyoxyl 40 stearate, stearyl alcohol, or vegetable oil.

[0315] The composition may further comprise a skin penetration enhancer such as that described in the literature ["Percutaneous Penetration enhancers", (eds. Smith EW and Maibach H I. CRC Press 1995)]. Exemplary skin penetration enhancers are alkyl (N,N-disubstituted aminoalkanoate) esters described in U.S. Patent Nos. 6,083,996 and 6,118,020, both of which are incorporated herein by reference, e.g., dodecyl 2-(N,N dimethylamino)propionate (DDAIP); Water-dispersible acid polymers, e.g., polyacrylic acid polymers, carbomers (e.g., Carbopol™ or Carbopol 940P™, available from BF Goodrich Company (Akron, Ohio)), copolymers of polyacrylic acid (e.g., Pemulen™ from BF Goodrich Company or Polycarbophil™ from AH Robbins, Richmond, Va.); polysaccharide gums, e.g., agar gum, alginate, carrageenan gum, ghatti gum, karaya gum, kadaya gum, ramicane gum, xanthan gum, and galactomannan gum (e.g., guar gum, carob gum, and locust bean gum), as well as other gums known in the art (e.g., Industrial Gums: Polysaccharides & Their Derivatives, Whistler RL, BeMiller JN (eds.), 3rd Ed. Academic Press (1992) and Davidson, RL, Handbook of Water-Soluble [See Gums & Resins, McGraw-Hill, Inc., NY (1980)]); or a combination thereof.

[0316] Other suitable polymeric skin penetration enhancers are cellulose derivatives, e.g., ethyl cellulose, methyl cellulose, and hydroxypropyl cellulose. Additionally, known transdermal penetration enhancers may also be added if desired. Examples include dimethyl sulfoxide (DMSO) and dimethyl acetamide (DMA), 2-pyrrolidone, N,N-diethyl-m-toluamide (DEET), 1-dodecyl azacycloheptan-2-one (Azone™, a registered trademark of Nelson Research), N,N-dimethylformamide, N-methyl-2-pyrrolidone, calcium thioglycolate, and other enhancers, e.g., dioxolane, cyclic ketones, and derivatives thereof.

[0317] Also examples are a group of biodegradable absorption enhancers that are alkyl N,N-2-(disubstituted amino)alkanoates, such as those described in U.S. Patent No. 4,980,378 and U.S. Patent No. 5,082,866, both of which are incorporated herein by reference, including tetradecyl (N,N-dimethylamino) acetate, dodecyl (N,N-dimethylamino) acetate, decyl (N,N-dimethylamino) acetate, octyl (N,N-dimethylamino) acetate, and dodecyl (N,N-diethylamino) acetate.

[0318] Particularly preferred skin penetration enhancers are isopropyl myristate; isopropyl palmitate; dimethyl sulfoxide; decyl methyl sulfoxide; dimethylalanine amide of a medium-chain fatty acid; dodecyl 2-(N,N-dimethylamino)propionate as described in U.S. Patent No. 6,118,020 or a salt thereof, e.g., its organic salts (e.g., hydrochloric acid, hydrobromide, sulfuric acid, phosphoric acid, and nitric acid addition salts) and inorganic salts (e.g., acetic acid, benzoic acid, salicylic acid, glycolic acid, succinic acid, nicotinic acid, tartaric acid, maleic acid, malic acid, parmolic acid, methanesulfonic acid, cyclohexanesulfamic acid, picric acid, and lactic acid addition salts); and includes alkyl 2-(N,N-disubstituted amino)-alkanoates as described in U.S. Patent No. 4,980,378 and U.S. Patent No. 5,082,866.

[0319] The skin penetration enhancer of this composition will be in the range of 0.5% to 10% (w / w) by weight. The most preferred range will be 1.0% to 5% (w / w). In another embodiment, the skin penetration enhancer constitutes 0.5%-1%, 1%-2%, 2%-3%, 3%-4%, or 4%-5% (w / w) of the composition.

[0320] The composition may be provided in any useful form. For example, the composition of the present invention may be formulated as a solution, emulsion (including microemulsion), suspension, cream, ointment, foam, lotion, gel, powder, or other typical solid, semi-solid, or liquid composition (e.g., topical spray) used for application to skin or other tissues where the composition may be used. The above composition comprises other ingredients typically used in the product, e.g., colorants, fragrances, thickeners (e.g., xanthan gum, fatty acids, fatty acid salts or esters, fatty alcohols, modified cellulose, modified inorganic materials, Krisgel 100™, or synthetic polymers), antimicrobial agents, solvents, surfactants, detergents, gelling agents, antioxidants, fillers, dyes, viscosity modifiers, preservatives, wetting agents, emollients (e.g., natural or synthetic oils, hydrocarbon oils, waxes or silicones), wettable agents, chelating agents, emollients, solubilizing excipients, adjuvants, dispersants, skin penetration enhancers, plasticizers, preservatives, stabilizers, anti-emulsifiers, humidifiers, UV blockers, emulsifiers, moisturizers, astringents, deodorizers, and optionally anesthetics, anti-itch agents, plant extracts, conditioning agents, darkening or lightening agents, glitter, wetting agents, mica, inorganic materials, polyphenols, It may include silicone or derivatives thereof, sunscreens, vitamins, and phytomedicines.

[0321] The composition may also include other similar ingredients to provide additional benefits and to improve the feel and / or appearance of the topical formulation. Specific classes of additives commonly used in these formulations include isopropyl myristate, sorbic acid NF powder, polyethylene glycol, phosphatidylcholine (a mixture of phosphatidylcholine, e.g., Phospholipone G), Krisgel 100™ distilled water, sodium hydroxide, decyl methyl sulfoxide (as a skin penetration enhancer), menthol crystals, lavender oil, butylated hydroxytoluene, ethyl diglycol reagent, and 95% (190 proof) ethanol.

[0322] Ophthalmic formulation

[0323] The compounds of the present invention may also be formulated with an ophthalmologically acceptable carrier at a concentration sufficient to deliver an effective amount of the active compound or compounds to the optic nerve area of ​​the eye. Preferably, the ophthalmic therapeutic solution contains one or more active compounds in a concentration range of approximately 0.0001% to approximately 5% (weight / volume), more preferably approximately 0.0005% to approximately 0.1% (weight / volume).

[0324] Ophthalmologically acceptable carriers do not cause significant irritation to the eye and do not negate the pharmacological activity and properties of charged sodium channel blockers.

[0325] Ophthalmologically acceptable carriers are generally sterile and essentially free of foreign particles, and generally have a pH in the range of 5 to 8. Preferably, the pH is as close as possible to the pH of tears (7.4). Ophthalmologically acceptable carriers are, for example, sterile isotonic solutions, for example, isotonic sodium chloride or boric acid solutions. The carrier is typically an aqueous solution containing sodium chloride or boric acid. Additionally, phosphate-buffered saline (PBS) solutions are useful.

[0326] Various preservatives may be used in ophthalmic formulations. Preferred preservatives include, but are not limited to, potassium benzalkonium, chlorobutanol, thimerosal, phenylmercury acetate, and phenylmercury nitrate. Likewise, various preferred vehicles may be used in the ophthalmic formulations. These vehicles include, but are not limited to, polyvinyl alcohol, povidone, hydroxypropyl methylcellulose, poloxamer, carboxymethylcellulose, and hydroxyethylcellulose.

[0327] Tonic modifiers may be added as needed or for convenience. These include, but are not limited to, salts, particularly sodium chloride, potassium chloride, etc., mannitol and glycerin, or any other suitable ophthalmologically acceptable tonic modifiers.

[0328] Various buffers and means for adjusting pH may be used as long as the resulting product is sufficiently acceptable. Thus, buffers include, but are not limited to, acetate buffers, citrate buffers, phosphate buffers, and borate buffers. Acids or bases may be used to adjust the pH of these formulations as needed. Ophthalmologically acceptable antioxidants may also be included. Antioxidants include, but are not limited to, sodium metabisulfite, sodium thiosulfate, acetylcysteine, butylated hydroxyanisole, and butylated hydroxytoluene.

[0329] Formulations for nasal and inhalation administration

[0330] The pharmaceutical composition of the present invention may be formulated for nasal or intranasal administration. When the carrier is a solid, a formulation suitable for nasal administration comprises, for example, a coarse powder having a particle size in the range of approximately 20 to 500 microns, which is administered by rapid inhalation through the nasal cavity. When the carrier is a liquid, for example, a nasal spray or nasal drop, one or more formulations may be mixed in an aqueous or oily solution and may be inhaled or sprayed into the nasal cavity.

[0331] In the case of administration by inhalation, the active ingredient may be conveniently delivered in the form of an aerosol spray from a pressurized pack or nebulizer with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, or other suitable gas. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve for delivering a metered amount. Capsules and cartridges, for example, of gelatin, containing a powder mixture of the compound and a suitable powder base, e.g., lactose or starch, may be formulated for use in an inhaler or blower.

[0332] A dry powder composition for local delivery to the lungs by inhalation may be provided in capsules and cartridges, for example, of laminated aluminum foil, for example, gelatin, or blister, for use in an inhaler or blower. Powder blend formulations generally contain a powder mixture for inhalation of the compounds of the present invention and a suitable powder base (carrier / diluent / excipient material), for example, a monosaccharide, disaccharide, or polysaccharide (e.g., lactose or starch). The use of lactose is preferred. In one embodiment, each capsule or cartridge contains about 2 µg to about 100 mg of the formula (optionally combined with another therapeutic active ingredient) IIt may contain a compound of ). In a preferred embodiment, each capsule or cartridge contains about 10 µg to about 50 mg of the formula (optionally combined with another therapeutic active ingredient) I It may contain a compound of ). In another embodiment, each capsule or cartridge contains about 20 µg to about 10 mg of the formula (optionally combined with another therapeutic active ingredient) I It may contain a compound of ). Alternatively, the compound of the present invention may be delivered without excipients.

[0333] Suitablely, the packaging / drug dispenser is a type selected from the group consisting of reservoir dry powder inhalers (RDPI), unit dose dry powder inhalers (e.g., capsule or blister pack inhalers), multi-dose dry powder inhalers (MDPI), and metered-dose inhalers (MDI).

[0334] A solution or suspension for use in a pressurized vessel, pump, spray, atomizer, or nebulizer may be formulated to contain an aqueous medium, ethanol, aqueous ethanol, or a suitable alternative agent for dispersing, solubilizing, or extending the release of the active ingredient(s); a propellant as a solvent; and / or a surfactant, e.g., sorbitan trioleate, oleic acid, or oligolactic acid.

[0335] Compositions formulated for nasal or inhalation administration may comprise one or more taste masking agents, e.g., flavoring agents, sweeteners, and other agents, e.g., sucrose, dextrose, and lactose, carboxylic acids, menthol, amino acids or amino acid derivatives, e.g., arginine, lysine, and monosodium glutamate, and / or synthetic flavoring oils and flavoring agents and / or natural oils, extracts from plants, leaves, flowers, fruits, etc., and combinations thereof. These may comprise cinnamon oil, wintergreen oil, peppermint oil, clover oil, bay oil, anise oil, eucalyptus, vanilla, citrus oils, e.g., lemon oil, orange oil, grape and grapefruit oil, and fruit essences including apple, peach, pear, strawberry, raspberry, cherry, plum, pineapple, apricot, etc. Additional sweeteners include sucrose, dextrose, aspartame, acesulfame-K, sucralose and saccharin, and organic acids (non-limiting examples include citric acid and aspartic acid). The flavoring agent may be present in an amount of about 0.05 to about 4 weight percent and may be present in a smaller or larger amount due to one or more factors of efficacy of the effect on the flavoring agent, the solubility of the flavoring agent, the solubility of other formulation components or the effect of the flavoring agent on other physicochemical or pharmacokinetic properties, or other factors.

[0336] Indications

[0337] The compounds, compositions, methods, and kits of the present invention are for trigeminal neurotrophic syndrome, erythematous limb pain, back and neck pain, low back pain, cancer pain, gynecological pain and analgesia, abdominal wall pain, chronic abdominal wall pain, fibromyalgia, allergic rhinitis, arthritis, rheumatoid arthritis, osteoarthritis, rheumatic pain, orthopedic pain, acute and post-herpetic neuralgia and other neuropathic pain (including peripheral neuropathy), sickle cell crisis, myalgia, vulvodynia, rectal pain, levator ani syndrome, transient rectal pain, perianal pain, hemorrhoidal pain, stomach pain, ulcer, inflammatory bowel disease, irritable bowel disease, irritable bowel syndrome, oral mucositis, esophagitis, interstitial cystitis, urethritis and other urological pain, toothache, burn pain, headache, ophthalmic irritation, conjunctivitis (e.g., allergic conjunctivitis), eye hyperemia, dry eye, dry eye syndrome (chronic eye pain), complex It can be used to treat pain, cough, or itching associated with any of the following conditions, including regional pain syndrome, acute postoperative pain, postoperative pain, postoperative ocular pain, and procedure pain (i.e., injection, abscess drainage, surgery, dental procedure, ophthalmic procedure, ophthalmic irritation, conjunctivitis (e.g., allergic conjunctivitis), eye redness, dry eye, pain associated with the use of arthroscopy and other medical devices, cosmetic surgery procedures, dermatological procedures, bone setting, biopsy, etc.).

[0338] Since a subclass of nociceptors mediates the sensation of itching, the compounds, compositions, methods, and kits of the present invention may also be used to treat itching in patients with conditions such as pruritus (including but not limited to brachioradialis muscle, chronic idiopathic, genital / anal, paresthetic back pain, and scalp), allergic dermatitis, atopic dermatitis, contact dermatitis, poison ivy, infection, parasite, insect bite, pregnancy, metabolic disorder, hepatic or renal failure, drug reaction, allergic reaction, eczema, hand eczema, genital and anal itching, hemorrhoidal itching, and cancer.

[0339] Since subclasses of nociceptors can initiate an abnormal cough reflex, the compounds, compositions, methods, and kits of the present invention can also be used to treat cough in patients with diseases such as asthma, COPD, asthma-COPD overlap syndrome (ACOS), interstitial pulmonary fibrosis (IPF), idiopathic pulmonary fibrosis, postviral cough, post-infectious cough, chronic idiopathic cough, and lung cancer.

[0340] The compounds, compositions, methods, and kits of the present invention may also be used to treat neuroinflammatory and neuroinflammatory disorders. Inflammation is a complex set of responses to noxious stimuli that cause localized redness, swelling, and pain. Inflammation can be innate or adaptive; the latter is induced by antigens and mediated by immune cells (immune-mediated inflammation). Neuroinflammatory inflammation arises from the efferent function of pain-sensing neurons (nociceptors), and neuropeptides and other chemicals that are pro-inflammatory mediators are released from them when the peripheral terminals of nociceptors are activated. This release process is mediated by calcium influx and the exocytosis of peptide-containing vesicles, and pro-inflammatory neuropeptides include substance P, neurokinins A and B (collectively known as tachykinins), calcitonin gene-associated peptide (CGRP), and vasoactive intestinal polypeptide (VIP).

[0341] The release of peripheral endogenous chemicals stimulates various inflammatory responses. First, the release of substance P increases capillary permeability, causing plasma proteins to leak from intravascular compartments into the extracellular space (plasma leakage), which can induce edema. This can be detected as wheals (firm, raised swelling of the skin), which are one of the three components of the inflammatory response known as the Lewis triple reaction: wheals, red spots, and redness. Second, the release of CGRP causes vasodilation, resulting in increased blood flow. This can be detected as redness, another component of the Lewis triple reaction.

[0342] Substance P also exerts pro-inflammatory effects on immune cells (e.g., macrophages, T-cells, mast cells, and dendritic cells) through neurokinin-1 (NK1) receptors. These effects have been recorded in allergic rhinitis, gastritis, and colitis, representing the interface between the neurogenic and immune-mediated components of inflammation. Substance P released from one nociceptor can also act on NK1 receptors on neighboring nociceptors to sensitize or activate them, thereby inducing activation and the diffusion of afferent / efferent functions. These efferent functions of nociceptors include 1) direct activation of the nociceptor terminal by appropriate peripheral stimulation (e.g., stinging pain) applied to the terminal; 2) indirect reverse activation of an unstimulated nociceptor terminal by an axonal reflex, wherein an action potential input from one nociceptor terminal reaches an axonal branch converging at the periphery, thereby generating an action potential moving from the branch to the unstimulated terminal's periphery; and 3) activation as a result of activation at the nociceptor central terminal of the CNS moving to the periphery (e.g., primary afferent depolarization of the central terminal generated by GABA may be sufficient to initiate an action potential moving in the "wrong direction").

[0343] Genomic analysis of lung-resident ILC2 cells revealed the expression of receptors for various neuropeptides released by sensory neurons, including SP, CGRP, and VIP, providing an opportunity for nociceptors to communicate directly with these cells. In particular, VIP was found to be expressed in NaV1.8+ nodular ganglion neurons, including lung afferents, in OVA-exposed mice. Cultured nodular ganglion neurons stimulated with capsaicin or IL5 also released VIP, whereas BALF from OVA-exposed mice contained elevated VIP compared to vehicle-attacked mice (Talbot et al., Neuron. 2015 July 15; 87(2): 341-354). These data show that VIP is released in inflamed lungs and can be blocked by silencing neurons using the charged sodium channel blocker of the present invention. Also, T H 2. When CD4+ T cells cultured under biased conditions were exposed to recombinant mouse VIP, transcript levels of IL-13 and IL-5 increased, indicating that VIP is responsible for the transcription of these type II regulatory cytokines in T cells. H 2. It suggests that it contributes to the ability of cells.

[0344] The release of immune mediators from immune cells can also activate nociceptors. Mast cells are found near primary nociceptor neurons and contribute to nociceptor sensitization in many situations. Injection of the secretagogue compound 48 / 80 promotes the degranulation of mast cells in the dura mater and excites meningeal nociceptors. Mast cell degranulation also contributes to the rapid onset of nerve growth factor-induced heat hyperalgesia. Macrophages contribute to nociceptor sensitization by releasing various soluble mediators. The expression of chemokine macrophage inflammatory protein-1α (MIP-1α) and its receptors CCR1 and CCR5 increases in macrophages and Schwann cells following partial ligation of the sciatic nerve, which contributes to the development of neuropathic pain. Lymphocytes contribute to the sensitization of peripheral nociceptors. T cells infiltrate the sciatic nerve and dorsal root ganglion (DRG) after nerve injury. Neuroinjury-induced hyperalgesia and allodynia are significantly attenuated or abolished in T-cell-deficient rodents, and the immunosuppressant rapamycin attenuates neuropathic pain in rats, partly due to its effect on T cells. Among the subsets of T cells, type 1 and type 2 helper T cells (T H 1 and T H 2 cells) were shown to have different roles in neuropathic pain. HWhile 1 cells promote neuropathic pain behavior by releasing pro-inflammatory cytokines (IL-2 and interferon-γ (IFNγ)), T H 2. Cells inhibit this by releasing anti-inflammatory cytokines (IL-4, IL-10, and IL-13). The complement system also plays a role in inflammatory hyperalgesia and neuropathic pain. The anaphylactic toxin C5a is an important effector of the complement cascade, and upon binding to the C5aR1 receptor on neutrophils, it becomes a potent neutrophil attractor (Ren & Dubner, Nat. Med. 16:1267-1276 (2010)).

[0345] Bacterial infection directly activates nociceptors, and immune responses mediated through TLR2, MyD88, T cells, B cells, neutrophils, and monocytes in mice Staphylococcus aureus ( Staphylococcus aureus It was found that it is not necessary for induced pain (Chiu et al., Nature Mechanical and thermal hyperalgesia in mice correlates with live bacterial load rather than tissue swelling or immune activation. Bacteria induce calcium flux and action potentials in nociceptor neurons through bacterial N-formylated peptides and porosiogenic toxins α-hemolysins, partly via distinct mechanisms. Specific depletion of Nav1.8-lineage neurons containing nociceptors abolishes pain during bacterial infection but simultaneously increased local immune infiltration and lymphadenopathy of draining lymph nodes. Thus, bacterial pathogens generate pain by directly activating sensory neurons that regulate inflammation, which represents an unexpected role of the nervous system in host-pathogen interactions. Literature [Talbot et al., ( Neuron Data from . 2015 July 15; 87(2): 341-354.)] also suggested that nociceptors are activated during exposure to allergens in sensitized animals.

[0346] In certain disorders, neuroinflammatory disorders are attributed to peripheral inflammation caused by tissue damage, autoimmune diseases, infections, and exposure to irritants in soft tissues, skin, respiratory system, joints, genitourinary and gastrointestinal tracts, liver, and brain. Neuroinflammatory disorders include, but are not limited to, allergic inflammation as described herein, inflammatory bowel disease, interstitial cystitis, atopic dermatitis, asthma, conjunctivitis, arthritis, colitis, contact dermatitis, diabetes mellitus, eczema, cystitis, gastritis, migraine, psoriasis, rhinitis, rosacea, sunburn, pancreatitis, chronic cough, chronic rhinosinusitis, traumatic brain injury, polybacterial sepsis, tendinopathy, chronic urticaria, rheumatic disease, acute lung injury, exposure to irritants, and inhalation of irritants, contaminants, or chemical warfare agents.

[0347] Evaluation of pain, cough, itching, and neuroinflammation

[0348] Measurement indices may be used to assess the efficacy of any compound, composition, method, and kit of the present invention in the treatment of pain associated with musculoskeletal, immuno-inflammatory, and neuropathic disorders. Useful indices include visual analog scales (VAS), Likert scales, categorical pain scales, descriptors, Lequesne indices, WOMAC indices, and AUSCAN indices, each of which is widely known in the art. These indices may be used to measure pain, itching, function, stiffness, or other variables.

[0349] Visual Analog Scales (VAS) provide one-dimensional quantitative measurements. VAS typically utilizes distance representations, such as line drawings with hash marks drawn at regular distance intervals, for example, at 10 intervals of 1-cm. For example, a patient may be asked to rank the sensation of pain or itching by selecting the point on the line that best corresponds to the sensation of pain or itching, where one end of the line corresponds to "no pain" (score of 0 cm) or "no itching," and the other end corresponds to "unbearable pain" or "unbearable itching" (score of 10 cm). This procedure provides a simple and rapid approach to obtaining quantitative information about how a patient experiences pain or itching. VAS scales and their uses are described, for example, in U.S. Patents No. 6,709,406 and 6,432,937.

[0350] The Likert scale similarly provides one-dimensional positive measurements. Generally, the Likert scale has discrete integer values ​​ranging from a low value (e.g., 0, meaning no pain) to a high value (e.g., 7, meaning severe pain). Patients experiencing pain are asked to select a number between the low and high values ​​to indicate the degree of pain experienced. The Likert scale and its uses are described, for example, in U.S. Patent Nos. 6,623,040 and 6,766,319.

[0351] The Requisnais Index and the Western Ontario and McMaster Universities (WOMAC) Osteoarthritis Index use self-management questionnaires to assess pain, function, and stiffness in the knees and hips of OA patients. While both the knee and hip are included by WOMAC, there is a single Requisnais questionnaire for the knee and a separate questionnaire for the hip. These questionnaires are useful because they contain more informative content compared to the VAS or Likert scales. Both the WOMAC Index and the Requisnais Index questionnaires have been extensively validated in OA, including in surgical settings (e.g., knee and hip arthroplasty). Their metric characteristics do not differ significantly.

[0352] The AUSCAN (Australia-Canada Hand Arthritis) index uses validated, reliable, and responsive patient self-report questionnaires. In one example, these questionnaires include 15 questions in three dimensions (pain, 5 questions; stiffness, 1 question; and physical function, 9 questions). The AUSCAN index may use, for example, Likert or VAS scales.

[0353] Indices useful in the method, composition, and kit of the present invention for measuring pain include the Pain Descriptor Scale (PDS), Visual Analog Scale (VAS), Verbal Descriptor Scale (VDS), Numeric Pain Intensity Scale (NPIS), Neuropathic Pain Scale (NPS), Neuropathic Pain Symptom Inventory (NPSI), Present Pain Inventory (PPI), Geriatric Pain Measure (GPM), McGill Pain Questionnaire (MPQ), Mean Pain Intensity (Descriptor Differential Scale), Numeric Pain Scale (NPS), Global Evaluation Score (GES), Short-Form McGill Pain Questionnaire, Minnesota Multiphasic Personality Inventory, Pain Profile, and Multidimensional Pain Inventory. It includes the Multidimensional Pain Inventory, the Child Health Questionnaire, and the Child Assessment Questionnaire.

[0354] Itching can be measured using subjective scales (VAS, Likert, Technician). Another approach is to measure scratching, which is an objective correlation of itching, using vibration transducers or motion detection sensors.

[0355] Coughing can be measured using standard questionnaires such as the Leicester Cough Questionnaire, as well as validated objective tools that measure cough frequency (e.g., VitaloJAK).

[0356] Examples

[0357] The following examples are intended to illustrate the invention and are not intended to limit it.

[0358] Definition of common abbreviations

[0359] ACN Acetonitrile

[0360] AcOH Acetic acid

[0361] aq. Mercury

[0362] Bn benzyl

[0363] Boc tertiary-butyloxycarbonyl

[0364] brine Saturated sodium chloride solution in water

[0365] ℃ degrees Celsius

[0366] δ Chemical shift (ppm)

[0367] d heavy hydrogen

[0368] DCM dichloromethane

[0369] DIPEA diisopropylethylamine

[0370] DMAP 4-Dimethylaminopyridine

[0371] DMSO dimethyl sulfoxide

[0372] ESI Electrospray ionization

[0373] Et2 O diethyl ether

[0374] EtOAc ethyl acetate

[0375] g gram

[0376] h hour

[0377] HATU 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyri Didium-3-oxide

[0378] MeOH methanol

[0379] mHz megahertz

[0380] min minute

[0381] ml milliliter

[0382] mmol millimoles

[0383] MS Molar spectrometer

[0384] m / z Mass to charge ratio

[0385] NMR nuclear magnetic resonance

[0386] Pet Ether petroleum ether

[0387] RT room temperature

[0388] TEA triethylamine

[0389] TLC Thin-layer chromatography

[0390] UV ultraviolet light

[0391] 1. Synthesis of N-Benzyl-2-((2,6-Dimethylphenyl)amino)-N,N-Diethyl-2-Oxoethane-1-Amium Bromide:

[0392]

[0393] Compound 1A

[0394] Benzyl bromide (0.164 g, 0.960 mmol, 1.5 equivalents) was added to a stirred solution of 2-(diethylamino)-N-(2,6-dimethylphenyl)acetamide (0.15 g, 0.640 mmol, 1 equivalent; Comb-Blocks, Inc., San Diego, CA Catalog No.: QA-3221) in ACN (3 mL), and the reaction mixture was stirred at 75°C for 12 h while monitoring the progress of the reaction by TLC (mobile phase DCM in 10% methanol; UV visualization). The reaction mixture was evaporated under reduced pressure to obtain an unpurified product, which was ground with EtOAc (2 x 5 mL) to obtain the product N-benzyl-2-((2,6-dimethylphenyl)amino)-N,N-diethyl-2-oxoethane-1-aminium bromide (0.08 g) as a white solid. MS (ESI): m / z 325.2 [M] + . 1 H NMR (400 MHz, DMSO-d6) δ 10.03 (br s, 1H), 7.66 - 7.39 (m, 5H), 7.13 (s, 3H), 4.82 (s, 2H), 4.17 (s, 2H), 3.52 (q, J = 6.7 Hz, 4H), 2.20 (s, 6H), 1.42 (br t, J = 6.9 Hz, 6H).

[0395] 2. Synthesis of N-benzyl-2-((2,6-dimethylphenyl)amino)-N,N-dimethyl-2-oxoethane-1-amium bromide:

[0396]

[0397] Compound 2A

[0398] · Synthesis of the intermediate 2-bromo-N-(2,6-dimethylphenyl)acetamide.

[0399] Bromoacetyl bromide (23.8 mL, 272.31 mmol, 1.1 equivalents) was added to a suspension of 2,6-dimethylaniline (30.5 mL, 247.56 mmol, 1.0 equivalent) in water (300 mL) at 10°C. The reaction mixture was maintained at pH 9–10 for 1 hour in a 15% Na2CO3 (aqueous) solution while monitoring the reaction progress by TLC (mobile phase: 30% EtOAc in hexane, UV visualization). The reaction mixture EtOAc Extracted with (2 x 600 mL), the combined organic extracts were washed with brine (200 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure. The resulting crude product Et 2 O 2-bromo-N-(2,6-dimethylphenyl)acetamide (21 g) was obtained as a white solid by grinding with (2 x 200 mL). MS (ESI): m / z 244.02 [M + 2] + . 1 H NMR (400 MHz, CDCl3) δ 7.75 (br s, 1H), 7.20 - 7.02 (m, 3H), 4.07 (s, 2H), 2.24 (s, 6H).

[0400] · Synthesis of the intermediate 2-(dimethylamino)-N-(2,6-dimethylphenyl)acetamide.

[0401] A 2.0 M solution of dimethylamine (51.8 mL, 103.5 mmol, 5 equivalents) in THF was added to a stirred solution of 2-bromo-N-(2,6-dimethylphenyl)acetamide (5 g, 20.7 mmol, 1.0 equivalent) in EtOAc (75 mL), and the reaction mixture was stirred in a steel boom at 75°C for 18 hours. The reaction mixture was quenched with water (100 mL) and extracted with EtOAc (2 x 200 mL). The combined organic extract was dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain 2-(dimethylamino)-N-(2,6-dimethylphenyl)acetamide (4.1 g). 1¹H NMR (400 MHz, CDCl₃- d ) δ ppm 8.63 (1 H, br s), 7.02 - 7.15 (3 H, m), 3.15 (2 H, s), 2.45 (6 H, s),2.24 (6 H, s).

[0402] · Synthesis of N-benzyl-2-((2,6-dimethylphenyl)amino)-N,N-dimethyl-2-oxoethane-1-amium bromide.

[0403] Benzyl bromide (0.23 mL, 1.936 mmol, 2.0 equivalents) was added to a stirred solution of 2-(dimethylamino)-N-(2,6-dimethylphenyl)acetamide (0.2 g, 0.969 mmol, 1.0 equivalent) in ACN (3 mL), and the reaction mixture was stirred at 90°C for 16 hours while monitoring the reaction progress by TLC (mobile phase: 10% MeOH in DCM, UV visualization). The reaction mixture was cooled to room temperature under reduced pressure and concentrated to obtain an unpurified product, which was ground with EtOAc (5 mL) to yield the product N-benzyl-2-((2,6-dimethylphenyl)amino)-N,N-dimethyl-2-oxoethane-1-aminium bromide (0.14 g). MS (ESI): m / z 297.2 [M] + . 1 H NMR (400 MHz, DMSO-d6) δ ppm 9.95 (1 H, s), 7.49 - 7.66 (5 H, m), 7.06 - 7.19 (3 ​​H, m), 4.81 (2 H, s), 4.30 (2 H, s), 3.26 (6 H, s), 2.19 (6 H, s).

[0404] 3. Synthesis of 1-Benzyl-1-(2-((2,6-Dimethylphenyl)amino)-2-oxoethyl)azepan-1-um bromide:

[0405]

[0406] Compound 3A

[0407] · Synthesis of the intermediate 2-(azepan-1-yl)-N-(2,6-dimethylphenyl)acetamide.

[0408] The synthesis of the intermediate 2-bromo-N-(2,6-dimethylphenyl)acetamide was as described above for Compound 2. K2CO3 (0.639 g, 4.6 mmol, 2.5 equivalents) and azepan (0.4 mL, 4.0 mmol, 2.0 equivalents) were added to a stirred solution of 2-bromo-N-(2,6-dimethylphenyl)acetamide (0.5 g, 1.9 mmol, 1.0 equivalent) in ACN (5.0 mL), and the mixture was stirred at 90°C for 20 hours while monitoring the reaction progress by TLC (mobile phase: 5% MeOH in DCM, UV visualization). The unpurified reaction product was concentrated under reduced pressure, and the resulting residue was distributed between water and EtOAc (2 x 50 mL). The combined organic extracts were washed with a saline solution, dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain an unpurified product, which was ground with EtOAc (20 mL) to obtain 2-(azepan-1-yl)-N-(2,6-dimethylphenyl)acetamide (0.47 g). MS (ESI): m / z 261.57 [M + H] + . 1 H NMR (400 MHz, CHLOROFORM-d) δ 8.83 (br s, 1H), 7.09 (s, 3H), 3.32 (s, 2H), 2.93 - 2.73 (m, 4H), 2.24 (s, 6H), 1.87 - 1.52 (m, 9H).

[0409] Synthesis of 1-benzyl-1-(2-((2,6-dimethylphenyl)amino)-2-oxoethyl)azepan-1-um bromide.

[0410] Benzyl bromide (0.1 mL, 0.6 mmol, 2.0 equivalents) was added to a stirred solution of 2-(azepan-1-yl)-N-(2,6-dimethylphenyl)acetamide (0.2 g, 0.8 mmol, 1.0 equivalents) in ACN (1.0 mL), and the reaction mixture was heated and refluxed for 16 hours while monitoring the reaction progress by TLC (mobile phase: 10% MeOH in DCM, UV visualization). The crude reaction mixture was concentrated under reduced pressure, and the product was ground with EtOAc (15 mL) to obtain the product 1-benzyl-1-(2-((2,6-dimethylphenyl)amino)-2-oxoethyl)azepan-1-um bromide (0.12 g). MS (ESI): m / z 351.2 [M] + . 1 H NMR (400 MHz, DMSO-d6) δ 10.04 (s, 1H), 7.71 - 7.62 (m, 2H), 7.60 - 7.48 (m, 3H), 7.20 - 7.03 (m, 3H), 4.93 (s, 2H), 4.15 (s, 2H), 3.86 - 3.70 (m, 2H), 3.66 - 3.42 (m, 2H), 2.23 (s, 6H), 1.98 (br d, J = 4.8 Hz, 4H), 1.67 (br s, 4H).

[0411] 4. Synthesis of 1-Benzyl-1-(2-((2,6-Dimethylphenyl)amino)-2-oxoethyl)piperidine-1-um bromide:

[0412]

[0413] Compound 4A

[0414] · Synthesis of the intermediate N-(2,6-dimethylphenyl)-2-(piperidine-1-yl)acetamide.

[0415] The synthesis of the intermediate 2-bromo-N-(2,6-dimethylphenyl)acetamide was as described above for Compound 2. Piperidine (0.851 g, 10.0 mmol, 2.0 equivalents) was added to a stirred solution of 2-bromo-N-(2,6-dimethylphenyl)acetamide (1.2 g, 5.0 mmol, 1.0 equivalent) in EtOAc (40 mL), and the reaction mixture was stirred at 75°C for 16 hours while monitoring the progress of the reaction by TLC (mobile phase: 50% EtOAc in petroleum ether, visualization by UV). The reaction mixture was diluted with EtOAc (20 mL) and washed with water (2 x 50 ml). The organic extracts were combined, dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain an unpurified product, which was ground with n-pentane (25 mL) and dried under vacuum to obtain the product N-(2,6-dimethylphenyl)-2-(piperidin-1-yl)acetamide (1.05 g). MS (ESI): m / z 247.36 [M + H] + . 1 H NMR (400 MHz, CDCl3-d) δ ppm 8.81 (1 H, br s), 7.04 - 7.11 (3 H, m), 3.14 (2 H, s), 2.62 (4 H, br s), 2.24 (6 H, s), 1.65 (4 H, quin, J=5.61 Hz), 1.44 - 1.54 (2 H, m).

[0416] Synthesis of 1-benzyl-1-(2-((2,6-dimethylphenyl)amino)-2-oxoethyl)piperidine-1-um bromide.

[0417] Benzyl bromide (0.205 g, 1.2 mmol, 2.0 equivalents) was added to a stirred solution of N-(2,6-dimethylphenyl)-2-(piperidin-1-yl)acetamide (0.15 g, 0.6 mmol, 1.0 equivalents) in ACN (3 mL), and the reaction mixture was stirred in a sealed tube at 80°C for 16 hours while monitoring the reaction progress by TLC (mobile phase: 10% MeOH in DCM, visualization by UV). The reaction mixture was cooled to room temperature and concentrated under reduced pressure to yield an unpurified product, which was ground with EtOAc (15 mL) and dried under vacuum to obtain the product 1-benzyl-1-(2-((2,6-dimethylphenyl)amino)-2-oxoethyl)piperidin-1-um bromide (0.15 g). MS (ESI): m / z 337.2 [M] + . 1 H NMR (400 MHz, DMSO-d6) δ ppm 10.07 (1 H, s), 7.49 - 7.66 (5 H, m), 7.07 - 7.21 (3 H, m), 4.96 (2 H, s), 4.21 (2 H, s), 3.65 (2 H, br d, J=12.50 Hz), 3.50 (2 H, dt, J=12.93, 6.69 Hz), 2.21 (6 H, s), 1.91 - 2.05 (4 H, m), 1.64 - 1.76 (1 H, m), 1.55 (1 H, dt, J=13.76, 7.04 Hz).

[0418] 5. Synthesis of 1-Benzyl-1-(2-((2,6-Dimethylphenyl)amino)-2-oxoethyl)piperidine-1-um bromide:

[0419]

[0420] Compound 5A

[0421] · Synthesis of the intermediate N-(2,6-dimethylphenyl)-2-(pyrrolidin-1-yl)acetamide.

[0422] The synthesis of the intermediate 2-bromo-N-(2,6-dimethylphenyl)acetamide was as described above for Compound 2. Pyrrolidine (0.533 g, 7.5 mmol, 1.5 equivalents) was added to a stirred solution of 2-bromo-N-(2,6-dimethylphenyl)acetamide (1.2 g, 5.0 mmol, 1.0) in EtOAc (40 mL), and the reaction mixture was stirred at 75°C for 3 hours while monitoring the progress of the reaction by TLC (mobile phase: 50% EtOAc in petroleum ether, visualization by UV). The reaction mixture was diluted with EtOAc (25 mL), washed with water (50 mL), dried over NaSO4, filtered, and concentrated under reduced pressure to obtain N-(2,6-dimethylphenyl)-2-(pyrrolidin-1-yl)acetamide (1.0 g). MS (ESI): m / z 233.44 [M + H] + . 1 ¹H NMR (400 MHz, CDCl₃- d ) δ ppm 8.63 (1 H, br s), 7.00 - 7.15 (3 H, m), 3.35 (2 H, s), 2.69 - 2.84 (4 H, m), 2.24 (6 H, s), 1.80 - 1.93 (4 H, m).

[0423] · Synthesis of 1-Benzyl-1-(2-((2,6-Dimethylphenyl)amino)-2-oxoethyl)piperidine-1-um bromide:

[0424] Benzyl bromide (0.205 g, 1.2 mmol, 2.0 equivalents) was added to a stirred solution of N-(2,6-dimethylphenyl)-2-(pyrrolidin-1-yl)acetamide (0.15 g, 0.6 mmol, 1.0 equivalents) in ACN (5 mL), and the reaction mixture was stirred in a sealed tube at 80°C for 16 hours while monitoring the reaction progress by TLC (mobile phase: 10% MeOH in DCM, visualization by UV). The reaction mixture was cooled to room temperature and concentrated under reduced pressure to yield an unpurified product, which was ground with EtOAc (15 mL) and dried under vacuum to obtain 1-benzyl-1-(2-((2,6-dimethylphenyl)amino)-2-oxoethyl)pyrrolidin-1-um bromide (0.14 g). MS (ESI): m / z 323.2 [M] + . 1 H NMR (400 MHz, DMSO-d6) δ ppm 2.11 - 2.29 (10 H, m), 3.60 - 3.74 (2 H, m), 3.76 - 3.88 (2 H, m), 4.20 (2 H, s), 4.83 (2 H, s), 7.08 - 7.18 (3 H, m), 7.50 - 7.66 (5 H, m), 9.97 (1 H, s).

[0425] 6. Synthesis of 1-Benzyl-1-(1-((2,6-Dimethylphenyl)amino)-1-oxobutane-2-yl)piperidine-1-um bromide:

[0426]

[0427] Compound 6A

[0428] · Synthesis of the intermediate 2-bromobutanoyl chloride.

[0429] Thionyl chloride (150 mL) was added to 2-bromobutanoic acid (25 g, 149.7 mmol, 1.0 equivalent) at 0°C, and the resulting solution was subsequently stirred at 80°C for 2 hours. The reaction mixture was cooled to room temperature and concentrated under reduced pressure to obtain unpurified 2-bromobutanoyl chloride (27.7 g, 99.5%) as a brown residue, which was used immediately in the next step without further purification. 1H NMR (400 MHz, CDCl3) δ 4.52 - 4.44 (m, 1H), 2.29 - 2.16 (m, 1H), 2.15 - 2.02 (m, 1H), 1.10 (t, J = 7.3 Hz, 3H).

[0430] · Synthesis of the intermediate 2-bromo-N-(2,6-dimethylphenyl)butanamide.

[0431] A solution of 2,6-dimethylaniline (15 g, 121.18 mmol, 1.0 equivalent) and pyridine (15 mL, 189.6 mmol, 1.5 equivalent) in DCM (400 mL) was cooled to 0°C in an ice bath. A solution of 2-bromobutanoyl chloride (27.5 g, 148.6 mmol, 1.2 equivalent) in DCM (50 mL) was slowly added to this mixture, and the resulting mixture was heated to room temperature while stirring for 2 hours. The reaction solution was adjusted to a pH of ~5 to 6 using 2 N HCl and extracted with DCM (2 x 200 mL). The combined organic extract was washed with water (250 mL) and brine (200 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure. The resulting crude product was ground with n-pentane (150 mL) to obtain 2-bromo-N-(2,6-dimethylphenyl)butanamide (30 g). MS (ESI): m / z 272.13 [M + 2] + . 1 H NMR (400 MHz, DMSO-d6) δ 9.69 (s, 1H), 7.15 - 7.02 (m, 3H), 4.51 (t, J = 7.3 Hz, 1H), 2.22 - 2.04 (m, 7H), 2.03 - 1.91 (m, 1H), 0.98 (t, J = 7.3 Hz, 3H).

[0432] · Synthesis of the intermediate N-(2,6-dimethylphenyl)-2-(piperidine-1-yl)butanamide.

[0433] Piperidine (54 mL, 544.1 mmol, 2.1 equivalents) was added at room temperature to a stirred solution of 2-bromo-N-(2,6-dimethylphenyl)butanamide (50 g, 259.1 mmol, 1.0 equivalent) in toluene (1 L). The reaction mixture was stirred at 110°C for 16 hours while monitoring the reaction progress by TLC (mobile phase: 30% EtOAc in hexane, UV visualization). The reaction mixture was concentrated under reduced pressure to yield an unpurified product, which was ground with n-pentane (500 mL) to obtain N-(2,6-dimethylphenyl)-2-(piperidine-1-yl)butanamide (55 g). MS (ESI): m / z 275.27 [M + H]+. 1H NMR (400 MHz, DMSO-d6) δ 9.16 (s, 1H), 7.06 (s, 3H), 3.06 (dd, J = 5.7, 8.6 Hz, 1H), 2.60 (br t, J = 4.8 Hz, 4H), 2.16 (s, 6H), 1.79 - 1.59 (m, 2H), 1.51 (br s, 4H), 1.44 - 1.35 (m, 2H), 0.93 (t, J = 7.5 Hz, 3H).

[0434] · Synthesis of 1-benzyl-1-(2-((2,6-dimethylphenyl)amino)-1-oxobutane-2-yl)piperidine-1-um bromide.

[0435] Benzyl bromide (1.872 g, 10.9 mmol, 2.0 equivalents) was added to a stirred solution of N-(2,6-dimethylphenyl)-2-(piperidin-1-yl)butanamide (1.5 g, 5.5 mmol, 1.0 equivalent) in ACN (25.0 mL), and the reaction mixture was stirred at 80°C for 16 hours while monitoring the reaction progress by TLC (mobile phase: 10% MeOH in DCM, UV visualization). The reaction mixture was concentrated under reduced pressure, and the crude product was ground with EtOAc (3 x 10 mL) to yield 1-benzyl-1-(1-((2,6-dimethylphenyl)amino)-1-oxobutane-2-yl)piperidin-1-um bromide (1.2 g). MS(ESI): m / z 365.4 [M]. +. 1H NMR (400 MHz, DMSO-d6) δ 10.36 (s, 1 H), 7.57-7.54 (m, 5 H), 7.17-7.11 (m, 3 H), 5.41 (d, 1 H), 4.62 (d, 1 H), 4.39 (bs, 1 H), 3.72 (bs, 2 H), 3.22 (bs, 2 H), 2.43-2.39 (m, 1 H), 2.15-2.07 (m, 9 H), 1.96-1.95 (m, 2 H), 1.61-1.45 (m, 2 H), 1.18 (t, 3 H).

[0436] 7. Synthesis of Compounds 7A-27A: Table 5.

[0437] Table 5 provides additional representative examples of the present invention prepared from the intermediate N-(2,6-dimethylphenyl)-2-(piperidin-1-yl)butanamide and a suitable alkyl halide according to the method described for the synthesis of compound 6.

[0438] Table 5

[0439]

[0440]

[0441]

[0442]

[0443]

[0444] 28. Synthesis of 1-(1-((2,6-dimethylphenyl)amino)-1-oxobutane-2-yl)-1-(2-hydroxybenzyl)piperidine-1-um 2,2,2-trifluoroacetate:

[0445]

[0446] Compound 28

[0447] Boron tribromide (1 M in DCM) (4 mL, 4.0 mmol, 20 equivalents) was added to a stirred solution of 1-(1-((2,6-dimethylphenyl)amino)-1-oxobutane-2-yl)-1-(2-methoxybenzyl)piperidin-1-um bromide (compound 24, 0.1 g, 0.21 mmol, 1.0 equivalent) in DMC (2 mL) at 0°C, and the reaction mixture was stirred at room temperature for 16 hours while monitoring the reaction with TLC (mobile phase: 10% MeOH in DCM, UV visualization). The reaction mixture was concentrated under reduced pressure to obtain an unpurified product, which was purified by preparative HPLC (mobile phase A: 0.1% TFA (aqueous); mobile phase B: ACN; column: Synergy Polar 250 x 21 mm, 4.7 u (Phenomenex Inc.), flow rate: 18 mL / min, method: 0 / 15, 2 / 15, 10 / 50, 15 / 80, 15.2 / 98, 18 / 98, 18.2 / 15, 22 / 15, temperature: ambient). The pure fraction was freeze-dried to obtain the product 1-(1-((2,6-dimethylphenyl)amino)-1-oxobutane-2-yl)-1-(2-hydroxybenzyl)piperidine-1-um 2,2,2-trifluoroacetate (0.056 g). MS (ESI): m / z 381.4[M]+. 1H NMR (500 MHz, DMSO-d6) δ ppm 1.17 (3H, br t, J=7.02 Hz), 1.32 - 1.47 (1H, m), 1.55 - 1.67 (1H, m), 1.76 - 2.01 (3H, m), 2.05 - 2.19 (2H, m), 2.25-2.3 (6H, m), 2.30 - 2.41 (1H, m), 2.99 - 3.26 (1H, m), 3.64 - 3.85 (2H, m), 4.35 - 4.61 (2H, m), 5.41 - 5.50 (1H, m), 6.88 - 6.95 (1H, m), 6.99 (1H, d, J=8.24 Hz), 7.10 - 7.19 (3H, m), 7.31 - 7.45 (2H, m), 10.34 (2H, br d, J=10.07 Hz).

[0448] 29. Synthesis of 1-(1-((2,6-dimethylphenyl)amino)-1-oxobutane-2-yl)-1-(3-hydroxybenzyl)piperidine-1-ium carbonate:

[0449]

[0450] Compound 29

[0451] Boron tribromide (1 M in DCM) (2 mL) was added to a stirred solution of 1-(1-((2,6-dimethylphenyl)amino)-1-oxobutane-2-yl)-1-(3-methoxybenzyl)piperidin-1-um bromide (compound 23, 0.06 g, 0.1 mmol, 1 equivalent) in DMC (2 mL) at 0°C, and the reaction mixture was stirred at room temperature for 16 hours while monitoring the progress with TLC (mobile phase: 10% MeOH in DCM, visualization by UV). The reaction mixture was concentrated under reduced pressure to obtain an unpurified product, which was purified by reverse-phase preparative HPLC (Mobile phase (A): 10 mM ammonium bicarbonate (aqueous); Mobile phase (B): 100% CAN; Column: X-Select C18 (19*250) 5u; Method: 0 / 35, 2 / 40, 20 / 40; Flow rate: 18 ml / min). The combined pure fractions were freeze-dried to obtain the product 1-(1-((2,6-dimethylphenyl)amino)-1-oxobutane-2-yl)-1-(3-hydroxybenzyl)piperidine-1-ium carbonate (0.02 g). MS (ESI): m / z 381.38 [M] + . 1 H NMR (500 MHz, DMSO-d6) δ ppm 1.11 (3H, br s), 1.31 - 1.62 (2H, m), 1.76 - 2.25 (12H, m), 2.93 - 3.13 (1H, m), 3.43 - 3.54 (3H, m), 3.60 - 3.76 (1H, m), 3.92 - 4.17 (2H, m), 4.21 - 4.39 (1H, m), 5.29 - 5.63 (1H, m), 6.67 - 6.85 (4H, m), 6.91 (2H, br s), 7.19 (1H, br t, J=7.78 Hz).

[0452] 30. Synthesis of 1-Benzyl-1-(2-((2,6-Dimethylphenyl)amino)-2-oxobutane-2-yl)azepan-1-yum bromide:

[0453]

[0454] Compound 30A

[0455] · Synthesis of the intermediate 2-(azepan-1-yl)-N-(2,6-dimethylphenyl)butanamide.

[0456] The synthesis of the intermediate 2-bromo-N-(2,6-dimethylphenyl)butanamide was as described above for Compound 6. K2CO3 (0.393 g, 2.85 mmol, 1.5 equivalents) and azepan (0.376 g, 3.8 mmol, 2.0 equivalents) were added to a stirred solution of 2-bromo-N-(2,6-dimethylphenyl)butanamide (0.5 g, 1.9 mmol, 1.0 equivalent) in ACN (10 mL), and the reaction mixture was stirred at 75°C for 16 hours while monitoring the reaction progress by TLC (mobile phase: 10% MeOH in DCM, visualization by UV). The unpurified reaction mixture was quenched with water (10 mL) and extracted with EtOAc (2 x 15 mL). The combined organic extract was dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain the intermediate 2-(azepan-1-yl)-N-(2,6-dimethylphenyl)butanamide (0.20 g). MS (ESI): m / z 289.33 [M + H] + . 1 ¹H NMR (400 MHz, CDCl₃- d ) δ ppm 1.00 - 1.16 (3H, m), 1.57 - 1.86 (12H, m), 1.95 - 2.13 (2H, m), 2.18 - 2.29 (8H, m), 2.72 - 2.97 (4H, m), 3.24 (1H, br t, J=6.72 Hz), 3.46 - 3.55 (2H, m), 7.03 - 7.15 (4H, m), 8.75 (1H, br s).

[0457] · Synthesis of 1-Benzyl-1-(1-((2,6-Dimethylphenyl)amino)-1-Oxobutane-2-yl)azepan-1-yum bromide:

[0458] Benzyl bromide (0.459 g, 2.85 mmol, 1.5 equivalents) was added to a stirred solution of 2-(azepan-1-yl)-N-(2,6-dimethylphenyl)butanamide (0.2 g, 0.7 mmol, 1.0 equivalents) in ACN (5 mL), and the reaction mixture was stirred at 75°C for 16 hours while monitoring progress by TLC (mobile phase: 10% MeOH in DCM, visualization by UV). The reaction mixture was cooled to room temperature and concentrated under reduced pressure to obtain an unpurified product, which was ground with EtOAc (20 mL) and dried under vacuum to obtain 1-benzyl-1-(1-((2,6-dimethylphenyl)amino)-1-oxobutane-2-yl)azepan-1-um bromide (0.118 g). MS (ESI): m / z 379.52 [M]+. 1H NMR (500 MHz, DMSO-d6) δ ppm 1.16 (3H, t, J=7.32 Hz), 1.26 - 1.51 (4H, m), 1.60 (2H, br s), 1.77 - 1.94 (2H, m), 2.10 - 2.31 (7H, m), 2.34 - 2.48 (1H, m), 3.56 - 3.81 (4H, m), 4.26 (1H, br d, J=10.68 Hz), 4.66 (1H, d, J=13.12 Hz), 5.21 (1H, d, J=13.12 Hz), 7.06 - 7.22 (3H, m), 7.45 - 7.61 (3H, m), 7.61 - 7.69 (2H, m), 10.42 (1H, s).

[0459] 31. Synthesis of 1-Benzyl-1-(2-((2,6-Dimethylphenyl)amino)-2-oxoethyl)azocant-1-um bromide:

[0460]

[0461] Compound 31A

[0462] · Synthesis of the intermediate 2-(azokant-1-yl)-N-(2,6-dimethylphenyl)acetamide:

[0463] To a stirred solution of 2-bromo-N-(2,6-dimethylphenyl)acetamide (500 mg, 2.065 mmol) in ACN (5.0 mL), K2CO3 (713 mg, 5.159 mmol) was added, followed by azocan (467 mg, 4.125 mmol), and the resulting reaction mixture was stirred at 80°C for 16 hours while monitoring the reaction progress by TLC (5% MeOH in DCM, UV visualization). The unpurified reaction mixture was poured into ice-cold water (40 mL) and extracted with EtOAc (2 x 50 mL). The combined organic extract was washed with a brine solution (50 mL), dried over anhydrous Na2SO4, and then concentrated under reduced pressure to obtain 2-(azokan-1-yl)-N-(2,6-dimethylphenyl)acetamide (480 mg) as a grayish-white solid. Mass (ESI): m / z 275.1 [M + H]+. 1H NMR (400 MHz, CHLOROFORM-d) δppm 8.74 (br s, 1 H), 7.02 - 7.16 (m, 3H), 3.34 (s, 2H), 2.74 - 2.88 (m, 4H), 2.25 (s, 6H), 1.53 - 1.80 (m, 10H).

[0464] · Synthesis of 1-Benzyl-1-(2-((2,6-Dimethylphenyl)amino)-2-oxoethyl)azocant-1-um bromide:

[0465] Benzyl bromide (240 mg, 1.403 mmol) was added to a stirred solution of 2-(azokan-1-yl)-N-(2,6-dimethylphenyl)acetamide (200 mg, 0.728 mmol) in ACN (2.0 mL), and the resulting reaction mixture was stirred at 90°C for 16 hours while monitoring the reaction progress by TLC (5% MeOH in DCM, UV visualization). The reaction mixture was concentrated under reduced pressure to obtain an unpurified product, which was ground with a 1:1 mixture of EtOAc:Et2O (3 x 50 mL) to obtain 1-benzyl-1-(2-((2,6-dimethylphenyl)amino)-2-oxoethyl)azokan-1-um bromide as a light brown solid. Mass (ESI): m / z 365.54 [M] +. 1H NMR (400 MHz, DMSO-d6) δppm 10.02 (s, 1H), 7.46 - 7.70 (m, 5H), 7.05 - 7.22 (m, 3H), 4.89 (s, 2H), 4.07 (s, 2H), 3.64 - 3.76 (m, 2H), 3.48 - 3.62 (m, 2H), 2.23 (s, 6H), 1.94 - 2.14 (m, 4H), 1.50 - 1.83 (m, 6H).

[0466] Synthesis of 1-benzyl-1-(2-((2,6-dimethylphenyl)amino)-2-oxoethyl)-3,3-difluoropiperidine-1-um bromide:

[0467]

[0468] Compound 32A

[0469] · Synthesis of the intermediate 2-(3,3-difluoropiperidin-1-yl)-N-(2,6-dimethylphenyl)acetamide.

[0470] TEA (0.626 g, 6.195 mmol) was added at 0°C to a stirred solution of 3,3-difluoropiperidine hydrochloride salt (0.371 g, 0.309 mmol) in ACN (25 mL) at 0°C. After stirring for 10 minutes at 0°C, 2-bromo-N-(2,6-dimethylphenyl)acetamide (0.5 g, 2.065 mmol) was added at 0°C. The reaction mixture was stirred at room temperature for 16 hours while monitoring the progress of the reaction by TLC (30% EtOAc in petroleum ether, UV visualization). The reaction mixture was diluted with water (10 mL) and extracted with EtOAc (2 x 25 mL). The combined organic layer was concentrated under reduced pressure to obtain an unpurified product, which was purified by normal-phase flash chromatography (5% MeOH in DCM) to obtain 2-(3,3-difluoropiperidin-1-yl)-N-(2,6-dimethylphenyl)acetamide (0.4 g) as a white solid. MS (ESI): m / z 283.24 [M+H] + . 1 H NMR (400 MHz, CHLOROFORM-d) δ ppm 8.58 (s, 1H), 7.07-7.13 (m, 3H), 3.27 (s, 2H), 2.86 (t, 2H), 2.50 (d, 2H), 2.23 (s, 6H), 1.86-2.04 (m, 4H).

[0471] Synthesis of 1-benzyl-1-(2-((2,6-dimethylphenyl)amino)-2-oxoethyl)-3,3-difluoropiperidine-1-um bromide:

[0472] Benzyl bromide (0.145 g, 0.850 mmol) was added to a solution of 2-(3,3-difluoropiperidin-1-yl)-N-(2,6-dimethylphenyl)acetamide (0.2 g, 0.708 mmol) in ACN (10 mL) at room temperature in a sealed tube. The resulting reaction mixture was heated to 75°C for 16 hours while monitoring the progress of the reaction by TLC (5% MeOH in DCM, UV visualization). The reaction mixture was diluted with water (25 mL) and extracted with 10% methanol (2 x 100 mL) in DCM. The combined organic layer was concentrated under reduced pressure to yield an unpurified product, which was ground with ethyl acetate (10 mL) to obtain 1-benzyl-1-(2-((2,6-dimethylphenyl)amino)-2-oxoethyl)-3,3-difluoropiperidin-1-um bromide (0.060 g) as a grayish-white solid. MS (ESI): m / z 373.1 [M] + . 1 H NMR (400 MHz, DMSO-d6) δ ppm 10.10 (s, 1H), 7.56-7.62 (m, 5H), 7.07 - 7.18 (m, 3H), 5.16 (d, 1H), 5.01 (d, 1H), 4.31-4.40 (m, 2H), 3.98-4.18 (m, 2H), 3.61- 3.68 (m, 2 H), 2.11-2.48 (m, 10H).

[0473] Synthesis of 1-(2-((2,6-dimethylphenyl)amino)-2-oxoethyl)-1-((phenyl-d5)methyl)azepan-1-ium chloride:

[0474]

[0475] Compound 33A

[0476] Synthesis of ·1-(chloromethyl)benzene-2,3,4,5,6-d5:

[0477] Paraformaldehyde (5.3522 g, 178.23 mmol) was added to a stirred solution of benzene-d6 (5 g, 59.41 mmol) in concentrated HCl (15 mL) at room temperature, and the reaction mixture was stirred in a sealed tube at 80°C for 48 hours while monitoring the progress of the reaction by 1H NMR. The reaction mixture was extracted with Et2O (2 x 25 mL), the combined organic extract was washed with a saturated sodium bicarbonate solution (50 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure at 25°C to obtain 1-(chloromethyl)benzene-2,3,4,5,6-d5 (1.6 g) as a colorless liquid. 1 H NMR (400 MHz, CDCl3) δ ppm 4.6 (s, 2H).

[0478] Synthesis of 1-(2-((2,6-dimethylphenyl)amino)-2-oxoethyl)-1-((phenyl-d5)methyl)azepan-1-ium chloride:

[0479] 1-(chloromethyl)benzene-2,3,4,5,6-d5 (1.592 g, 12.1 mmol) was added to a stirred solution of 2-(azepan-1-yl)-N-(2,6-dimethylphenyl)acetamide (1.5 g, 5.76 mmol) in ACN (15 mL), and the resulting reaction mixture was stirred in a sealed tube at 80°C for 16 hours while monitoring the progress of the reaction by TLC (10% MeOH in DCM, UV visualization). The reaction mixture was cooled to room temperature, filtered, and the precipitated solid was washed with EtOAc to obtain 1-(2-((2,6-dimethylphenyl)amino)-2-oxoethyl)-1-((phenyl-d5)methyl)azepan-1-ium chloride (1.67 g) as a white solid. MS (ESI): m / z 356.36 [M] + . 1 ¹H NMR (400 MHz, DMSO- d6) δ ppm 10.55 (s, 1 H), 7.1 - 7.18 (m, 3 H), 4.95 (s, 2 H), 4.2 (s, 2 H), 3.72 - 3.81 (m, 1 H), 3.5 - 3.6 (m, 1 H), 2.25 (s, 6 H), 1.9 - 2.1 (m, 4 H), 1.1 - 1.2 (m, 4 H).

[0480] Synthesis of 1-(1-((2,6-dimethylphenyl)amino)-1-oxobutane-2-yl)-1-((1-methyl-1H-pyrazole-4-yl)methyl)piperidin-1-um bromide:

[0481]

[0482] Compound 34A

[0483] · Synthesis of 4-(bromomethyl)-1-methyl-1H-pyrazol hydrogen bromide:

[0484] 33% HBr in 12 mL of AcOH was added at room temperature to a stirred solution of (1-methyl-1H-pyrazole-4-yl)methanol (1.0 g, 8.918 mmol) in 5 mL of AcOH. The reaction mixture was heated at 100°C for 16 hours while monitoring the progress of the reaction by TLC (10% MeOH in DCM, UV visualization). The reaction mixture was concentrated under reduced pressure to obtain 4-(bromomethyl)-1-methyl-1H-pyrazole hydrogen bromide (1.5 g) as a light brown solid. 1 ¹H NMR (400 MHz, DMSO- d6 ) δ ppm 8.01 (s, 1 H), 7.50 (s, 1 H), 5.51 (s, 2 H), 3.81 (s, 3 H).

[0485] Synthesis of ·1-(1-((2,6-dimethylphenyl)amino)-1-oxobutane-2-yl)-1-((1-methyl-1H-pyrazole-4-yl)methyl)piperidin-1-um bromide:

[0486] N-(2,6-dimethylphenyl)-2-(piperidin-1)-yl)butanamide (0.783 g, 2.853 mmol) and potassium carbonate (0.473 g, 3.427 mmol) were added to a solution of 4-(bromomethyl)-1-methyl-1H-pyrazole hydrogen bromide (0.5 g, 1.953 mmol) in DMF (5 mL). The resulting reaction mixture was heated at 100°C in a sealed tube for 24 hours while monitoring the reaction progress by TLC (5% MeOH in DCM, UV visualization). The reaction mixture was diluted with water (25 mL) and extracted with 10% methanol (2 x 100 mL) in DCM. The combined organic layer was concentrated under reduced pressure to obtain an unpurified product, which was purified by normal-phase flash chromatography (5% MeOH in DCM) to obtain 1-(1-((2,6-dimethylphenyl)amino)-1-oxobutane-2-yl)-1-((1-methyl-1H-pyrazole-4-yl)methyl)piperidine-1-ium bromide (0.120 g) as a grayish-white solid. MS (ESI): m / z 369.48 [M] + . 1 H NMR (400 MHz, DMSO-d6) δ ppm 10.4 (s, 1 H), 8.01 (s, 1 H), 7.64 (s, 1 H), 7.10-7.14 (m, 3 H), 5.17 (d, 1 H), 4.48 (d, 1 H), 4.1-4.3 (m, 1 H) H), 3.90 (s, 3 H), 3.8-3.9 (m, 1 H), 3.20-3.45 (m, 2 H), 2.30-2.36 (m, 1 H), 2.16 (s, 6 H), 1.92-2.08 (m, 6 H), 1.54 (d, 2 H), 1.11-1.17 (m, 3 H).

[0487] Synthesis of 1-(2-((2,6-dimethylphenyl)amino)-2-oxoethyl)-1-(3-(methoxycarbonyl)benzyl)piperidine-1-um bromide:

[0488]

[0489] Compound 35A

[0490] Synthesis of 1-(2-((2,6-dimethylphenyl)amino)-2-oxoethyl)-1-(3-(methoxycarbonyl)benzyl)piperidine-1-um bromide:

[0491] Methyl 3-(bromomethyl)benzoate (241.6 mg, 1.055 mmol) was added at room temperature to a solution of N-(2,6-dimethylphenyl)-2-(piperidin-1-yl)acetamide (200 mg, 0.812 mmol) in ACN (3 mL). The resulting reaction mixture was stirred at 90°C for 16 hours while monitoring the progress of the reaction by TLC (5% MeOH in DCM, visualization: UV). The reaction mixture was cooled to room temperature and concentrated under reduced pressure to yield an unpurified product, which was ground with EtOAc (3 x 10 mL) to obtain pure 1-(2-((2,6-dimethylphenyl)amino)-2-oxoethyl)-1-(3-(methoxycarbonyl)benzyl)piperidin-1-um bromide (57.5 mg) as a grayish-white solid. MS (ESI): m / z 395.2 [M] + . 1 H NMR (400 MHz, DMSO-d6) δ ppm 10.09 (s, 1 H), 8.18 (s, 1 H), 8.13 (d, 1 H), 7.83-7.85 (m, 1 H), 7.69-7.72 (m, 1 H), 7.12-7.17 (m, 3 H), 5.06 (s, 2 H), 4.22 (s, 2 H), 3.88 (s, 3H), 3.52-3.65 (m, 2 H), 3.47-3.50 (m, 2 H), 2.21 (s, 6 H), 1.90-1.96 (m, 4 H), 1.71-1.74 (m, 1 H), 1.56-1.57 (m, 1 H).

[0492] Synthesis of 1-(2-((2,6-dimethylphenyl)amino)-2-oxoethyl)-1-(4-(methoxycarbonyl)benzyl)piperidine-1-um bromide:

[0493]

[0494] Compound 36A

[0495] Synthesis of 1-(2-((2,6-dimethylphenyl)amino)-2-oxoethyl)-1-(4-(methoxycarbonyl)benzyl)piperidine-1-um bromide:

[0496] Methyl 4-(bromomethyl)benzoate (0.743 g, 3.247 mmol) was added to a stirred solution of N-(2,6-dimethylphenyl)-2-(piperidin-1-yl)acetamide (0.4 g, 1.623 mmol) in ACN (5 mL), and the resulting reaction mixture was stirred at 80°C for 16 hours while monitoring the progress of the reaction by TLC (10% methanol in DCM, visualization: UV). The reaction mixture was cooled to room temperature and concentrated under reduced pressure to yield an unpurified product, which was purified by reflux with EtOAc (30 mL) at 70°C for 2 hours. The refluxed product was filtered and washed with hot ethyl acetate (30 mL) to obtain (1-(2-((2,6-dimethylphenyl)amino)-2-oxoethyl)-1-(4-(methoxycarbonyl)benzyl)piperidin-1-um bromide (43 mg) as a white solid. Mass (ESI): m / z 395.3 [M] + . 1 H NMR (400 MHz, DMSO-d6) δ ppm 10.06 (s, 1 H), 8.09 (d, 2 H), 7.72 (d, 2 H), 7.17-7.12 (m, 3 H), 5.03 (s, 2 H), 4.22 (s, 2 H), 3.89 (s, 3 H), 3.67-3.64 (m, 2 H), 3.54-3.49 (m, 2 H), 2.20 (s, 6 H), 2.0-1.97 (m, 4 H), 1.72-1.69 (m, 1 H), 1.58-1.53 ​​(m, 1 H).

[0497] Synthesis of 1-(2-((2,6-dimethylphenyl)amino)-2-oxoethyl)-1-(2-(methoxycarbonyl)benzyl)piperidine-1-um bromide:

[0498]

[0499] Compound 37A

[0500] Synthesis of 1-(2-((2,6-dimethylphenyl)amino)-2-oxoethyl)-1-(2-(methoxycarbonyl)benzyl)piperidine-1-um bromide:

[0501] Methyl 2-(bromomethyl)benzoate (0.929 g, 4.0592 mmol) was added to a solution of N-(2,6-dimethylphenyl)-2-(piperidin-1-yl)acetamide (0.500 g, 2.0296 mmol) in ACN (5.0 mL), and the resulting reaction mixture was heated at 80°C for 16 hours while monitoring the progress of the reaction by TLC (10% MeOH, visualization: UV). The reaction mixture was concentrated under reduced pressure to obtain an unpurified compound, which was then ground with 3 x 10 mL of ethyl acetate followed by 2 x 10 mL of diethyl ether to obtain pure 1-(2-((2,6-dimethylphenyl)amino)-2-oxoethyl)-1-(2-(methoxycarbonyl)benzyl)piperidine-1-um bromide (250 mg) as a grayish-white solid. MS (ESI): m / z 395.2 [M] + . 1 H NMR (400 MHz, DMSO-d6) δ ppm 10.11 (s, 1 H), 8.01 (d, 1 H), 7.69-7.76 (m, 3 H), 7.11-7.17 (m, 3 H), 5.37 (s, 2 H), 4.43-4.46 (m, 2 H), 3.85 (s, 3 H), 3.75 (d, 2 H), 3.17 (t, 2 H), 2.20 (s, 6 H), 1.96-2.07 (m, 2 H), 1.79-1.83 (m, 2 H), 1.66-1.70 (m, 1H), 1.37-1.40 (m, 1H).

[0502] Synthesis of 1-benzyl-1-(2-((2,6-dimethylphenyl)(methyl)amino)-2-oxoethyl)azepan-1-um formate.

[0503]

[0504] Compound 38A

[0505] Synthesis of ·2-iodo-N-(2,6-dimethylphenyl)-N-methylacetamide:

[0506] NaH (60%) (0.607 g, 25.295 mmol) was added at 0°C to a stirred solution of 2-chloro-N-(2,6-dimethylphenyl)acetamide (2.0 g, 10.118 mmol) in THF (16 mL), and the mixture was stirred at 0°C for 20 minutes. Methyl iodide (0.5 ml, 8.031 mmol) was added at 0°C, and the resulting reaction mixture was stirred at room temperature for 16 hours while monitoring the progress of the reaction by TLC (10% EtOAc in PET, UV visualization). The reaction mixture was poured into ice water (50 mL) and extracted with ethyl acetate (2 x 50 mL). The combined organic extract was washed with a brine solution (25 mL), dried over Na2SO4, and concentrated under reduced pressure to obtain an unpurified product, which was purified by column chromatography (eluting with 20-30% ethyl acetate in petroleum ether). The collected pure fraction was concentrated under reduced pressure to obtain N-(2,6-dimethylphenyl)-2-iodo-N-methylacetamide (0.55 g) as a yellow liquid. Mass (ESI): m / z 304.03 [M+H] + .

[0507] · Synthesis of 2-(azepan-1-yl)-N-(2,6-dimethylphenyl)-N-methylacetamide:

[0508] K2CO3 (0.569 g, 4.123 mmol) and azepan (0.327 ml, 3.298 mmol) were added at room temperature to a stirred solution of N-(2,6-dimethylphenyl)-2-iodo-N-methylacetamide (0.5 g, 1.649 mmol) in ACN (5.0 mL). The resulting reaction mixture was stirred at 90°C for 16 hours while monitoring the progress of the reaction by TLC (50% EtOAc in petroleum ether, UV visualization). The reaction mixture was poured into ice water (25 mL) and extracted with ethyl acetate (2 x 25 mL). The combined organic extract was washed with a saline solution (25 mL), dried over Na2SO4, and concentrated under reduced pressure to obtain an unpurified compound, which was purified by normal-phase flash chromatography (eluting with 10%–50% EtOAc in petroleum ether). The collected pure fraction was concentrated under reduced pressure to obtain 2-(azepan-1-yl)-N-(2,6-dimethylphenyl)-N-methylacetamide (0.23 g). MS (ESI): m / z 275.49 [M+H] + .

[0509] · Synthesis of 1-benzyl-1-(2-((2,6-dimethylphenyl)(methyl)amino)-2-oxoethyl)azepan-1-um formate

[0510] (Bromomethyl)benzene (0.124 g, 0.728 mmol) was added to a stirred solution of 2-(azepan-1-yl)-N-(2,6-dimethylphenyl)-N-methylacetamide (0.1 g, 0.364 mmol) in acetonitrile (1.0 mL). The resulting reaction mixture was stirred at 90°C for 16 hours while monitoring the progress of the reaction by TLC (10% MeOH in DCM, visualization: UV). The reaction mixture was concentrated under reduced pressure to obtain an unpurified product, which was purified by reverse-phase preparative HPLC. (Column: X-Select C18 (250*19) mm, 5 µ, Mobile phase A: 0.1% FA in water,

[0511] Mobile phase B: ACN: MeOH, flow rate: 13 ml / min, solubility: water + THF + ACN, method(T% of B): 0 / 10, 2 / 10, 10 / 50, 13 / 50, 13.2 / 98, 17 / 98, 17.2 / 10, 20 / 10, temperature: ambient).

[0512] The collected pure fraction was freeze-dried to obtain the product (1-benzyl-1-(2-((2,6-dimethylphenyl)(methyl)amino)-2-oxoethyl)azepan-1-ium formate (70 mg) as a white gum. MS (ESI): m / z 365.3 [M] + . 1 ¹H NMR (400 MHz, DMSO- d 6) δ ppm 8.43 (s, 1 H), 7.56-7.42 (m, 5 H), 7.26-7.17 (m, 3 H), 4.95 (s, 2 H), 4.41 (s, 1 H), 3.91-3.85 (m, 1 H), 3.62-3.50 (m, 3) H), 3.36 (s, 1 H), 3.14 (d, 3 H), 2.32-2.05 (s, 7 H), 1.95-1.65 (m, 2 H), 1.46-1.23 (m, 5 H).

[0513] Synthesis of 1-Benzyl-1-(1-(Mesitylamino)-1-oxobutane-2-yl)piperidine-1-um bromide:

[0514]

[0515] Compound 39A

[0516] Synthesis of ·2-Bromo-N-Mesitylbutanamide:

[0517] DIPEA (5.735 g, 44.373 mmol) was added to a stirred solution of 2,4,6-trimethylaniline (2.0 g, 14.791 mmol) in DCM (20 mL) at room temperature, and then 2-bromobutanoyl chloride (3.291 g, 17.749 mmol) was added dropwise at 0°C. The resulting reaction mixture was stirred for 3 hours while monitoring the progress of the reaction by TLC (20% ethyl acetate in petroleum ether, visualization: UV). The reaction mixture was diluted with water (100 mL) and extracted with EtOAc (2 x 100 mL). The combined organic extracts were washed with a saline solution, dried over Na2SO4, and concentrated under reduced pressure to obtain an unpurified product, which was purified by column chromatography (eluting with 10–50% ethyl acetate in petroleum ether). The pure fractions were combined and concentrated to obtain the desired product, 2-bromo-N-methylbutanamide (1.5 g). MS (ESI): m / z 286.11 [M+2] + . 1 H NMR (400 MHz, CDCl3) δ ppm 7.60 (s, 1 H), 6.95 (s, 2 H), 4.45 (t, 1 H), 2.25 (s, 3 H), 2.2 (s, 6 H), 1.60-1.40 (m, 2 H), 1.10 (t, 3 H).

[0518] Synthesis of N-meshtil-2-(piperidin-1-yl)butanamide:

[0519] Potassium carbonate (2.18 g, 15.834 mmol) and piperidine (0.943 g, 11.083 mmol) were added at room temperature to a stirred solution of 2-bromo-N-methityl butanamide (1.5 g, 5.278 mmol) in acetonitrile (30 ml). The resulting reaction was refluxed for 16 hours while monitoring the reaction progress with TLC (50% EtOAc / hexane, visualization: UV). The reaction mixture was diluted with water (50 mL) and extracted with EtOAc (3 x 20 mL). The combined organic extract was washed with a saline solution, dried over Na2SO4, and concentrated under reduced pressure to obtain an unpurified product, which was ground with ethyl acetate (40 mL) to obtain pure N-methityl-2-(piperidin-1-yl)butanamide (760 mg). MS (ESI): m / z 289.43 [M+H] + . 1 ¹H NMR (400 MHz, DMSO- d 6) δ ppm 9.07 (s, 1 H), 6.86 (s, 2 H), 3.06-3.01 (t, 1 H), 2.60-2.48 (m, 4 H), 2.21 (s, 3 H), 2.06 (s, 6 H), 1.80-1.25 (t, 8 H), 0.95 (t, 3 H).

[0520] Synthesis of 1-benzyl-1-(1-(methylamino)-1-oxobutane-2-yl)piperidine-1-um bromide:

[0521] Benzyl bromide (0.177 g, 1.04 mmol) was added to a stirred solution of N-methityl-2-(piperidin-1-yl)butanamide (0.15 g, 0.52 mmol) in acetonitrile (1 mL). The resulting reaction mixture was stirred at 80°C for 20 hours while monitoring the progress of the reaction by TLC (10% methanol in DCM, visualization: UV). The reaction mixture was concentrated under reduced pressure to yield a crude product, which was ground with EtOAc (20 mL) to deliver the target product, 1-benzyl-1-(1-(methitylamino)-1-oxobutane-2-yl)piperidin-1-um bromide (194 mg), as a white solid. Mass (ESI): m / z 379.3 [M] + . 1 H NMR (400 MHz, DMSO-d6) δ ppm 10.31 (s, 1 H), 7.62-7.50 (m, 5 H), 6.93 (s, 2 H), 5.42-5.39 (m, 1 H), 4.63-4.20 (m, 2 H), 4.05-3.55 (m, 2 H) H), 3.30-3.10 (m, 2 H), 2.41-2.31 (m, 1 H), 2.23 (s, 9 H), 2.15-2.06 (m, 2 H), 2.05-1.80 (m, 3H), 1.57-1.43 (m, 2 H), 1.23-1.07 (m, 3 H).

[0522] Synthesis of 1-benzyl-1-(2-((4-((tert-butoxycarbonyl)(butyl)amino)phenyl)amino)-2-oxoethyl)azepan-1-um bromide:

[0523]

[0524] Compound 40A

[0525] · Synthesis of 2-Bromo-N-(4-Nitrophenyl)acetamide:

[0526] Bromoacetyl bromide (29.226 g, 144.790 mmol) was added at 0°C to a stirred suspension of 4-nitroaniline (10 g, 72.395 mmol) in H2O (100 mL), and the reaction mixture was stirred at room temperature for 16 hours while monitoring the progress of the reaction by TLC (50% EtOAc in petroleum ether, visualization: UV). The reaction mixture was basicized with a saturated Na2CO3 solution (100 mL), and the precipitated solid was filtered and dried. The crude product was ground with diethyl ether (2 x 100 mL) to obtain 2-bromo-N-(4-nitrophenyl)acetamide (8 g) as a yellow solid. Mass (ESI): m / z 259.17 [M+1] + . 1 ¹H NMR (400 MHz, DMSO- d 6) δ ppm 10.97 (s, 1 H), 8.25 (d, 2 H), 7.83 (d, 2 H), 4.11 (s, 2 H).

[0527] Synthesis of ·2-(azepan-1-yl)-N-(4-nitrophenyl)acetamide:

[0528] To a stirred solution of 2-bromo-N-(4-nitrophenyl)acetamide (5 g, 19.300 mmol) in ACN (50 ml), K2CO3 (8 g, 57.900 mmol) was added, followed by azepan (3.8 g, 38.600 mmol, 2 equivalents). The reaction mixture was heated at 90°C in a sealed tube for 16 hours while monitoring the progress of the reaction by TLC (50% EtOAc in petroleum ether, visualization: UV). The reaction mixture was cooled to room temperature, then diluted with water (200 mL) and extracted with EtOAc (2 x 200 mL). The combined organic extract was washed with a brine solution (100 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to obtain an unpurified product. The crude product was ground with diethyl ether (2 x 50 mL) to obtain pure 2-(azepan-1-yl)-N-(4-nitrophenyl)acetamide (2.5 g). Mass (ESI): m / z 278.27 [M+1] + . 1 H NMR (400 MHz, DMSO-d6) δ ppm 10.26 (s, 1 H), 8.21 (d, 1 H), 7.91 (d, 2 H), 3.33-3.4 (m, 2 H), 2.72-2.81 (m, 4 H), 1.56-1.62 (m, 8 H).

[0529] Synthesis of ·N-(4-aminophenyl)-2-(azepan-1-yl)acetamide:

[0530] 10% Pd / C (2.5 g) was added to a stirred solution of 2-(azepan-1-yl)-N-(4-nitrophenyl)acetamide (2.5 g, 9.014 mmol) in 25 mL of MeOH at room temperature, and the reaction mixture was stirred at room temperature for 16 hours under H2 gas (balloon pressure) while monitoring the progress of the reaction by TLC (50% EtOAc / petroleum ether, visualization: UV). The reaction mixture was filtered, and the filtrate was concentrated under reduced pressure to obtain pure N-(4-aminophenyl)-2-(azepan-1-yl)acetamide (1.5 g). Mass (ESI): m / z 248.30 [M+H]. 1 H NMR (400 MHz, DMSO-d6) δ ppm 9.20 (s, 1 H), 7.22 (d, 2 H), 6.49 (d, 2 H), 4.85 (s, 1 H), 3.16-3.19 (m, 2 H), 2.68-2.72 (m, 4 H), 1.57 - 1.62 (m, 8 H).

[0531] Synthesis of ·2-(azepan-1-yl)-N-(4-(butylamino)phenyl)acetamide:

[0532] 10% Pd / C (1.8 g) was added to a stirred solution of N-(4-aminophenyl)-2-(azepan-1-yl)acetamide (1.8 g, 7.277 mmol) and butyronitrile (2.51 g, 36.385 mmol) in methanol (20 mL), and the reaction mixture was stirred at room temperature for 16 hours under an H2 gas atmosphere (balloon) while monitoring the progress of the reaction by TLC (50% EtOAc in petroleum ether, visualization: UV). The reaction mixture was filtered, and the filtrate was concentrated under reduced pressure to obtain the target product 2-(azepan-1-yl)-N-(4-(butylamino)phenyl)acetamide (1 g). Mass (ESI): m / z 304.43[M+1] + .

[0533] Synthesis of tert-butyl(4-(2-(azepan-1-yl)acetamido)phenyl)(butyl)carbamate

[0534] To a stirred solution of 2-(azepan-1-yl)-N-(4-(butylamino)phenyl)acetamide (1.4 g, 4.613 mmol) in ACN (20 mL), triethylamine (1.39 g, 13.839 mmol) was added, followed by di-tert-butyl dicarbonate (1.5 g, 6.919 mmol), and the reaction mixture was stirred at room temperature for 16 hours while monitoring the progress of the reaction by TLC (50% EtOAc in petroleum ether, visualization: UV). After the starter material was completed by TLC, the unpurified compound was diluted with water (100 mL), extracted with EtOAc (2 x 120 mL), the combined organic extract was washed with a brine solution (70 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The resulting crude product was purified by column chromatography (elution with 10%–20% EtOAc in petroleum ether) to obtain the product tertiary-butyl (4-(2-(azepan-1-yl)acetamido)phenyl)(butyl)carbamate (1 g). Mass (ESI): m / z 404.58 [M+H] . 1 H NMR (400 MHz, DMSO-d6) δ ppm 9.65 (s, 1 H), 7.59 (d, 2 H), 7.13 (d, 2 H), 3.53 (t, 2 H), 3.24 (s, 2 H), 2.70-2.75 (m, 4 H), 1.61-1.86 (m, 8 H), 1.35-1.44 (m, 9 H), 1.24 - 1.32 (m, 3 H), 1.17 - 1.22 (m, 3 H).

[0535] Synthesis of 1-benzyl-1-(2-((4-((ter-butoxycarbonyl)(butyl)amino)phenyl)amino)-2-oxoethyl)azepan-1-um bromide:

[0536] Benzyl bromide (127 mg, 0.742 mmol) was added at room temperature to a stirred solution of tertiary-butyl(4-(2-(azepan-1-yl)acetamido)phenyl)(butyl)carbamate (200 mg, 0.495 mmol) in ACN (2 mL), and the reaction mixture was stirred at 90°C for 16 hours while monitoring the progress of the reaction by TLC (10% MeOH in DCM, visualization: UV). The reaction mixture was concentrated under reduced pressure, and the unpurified compound was ground with diethyl ether (2 x 20 mL) and EtOAc (2 x 20 mL) to obtain 1-benzyl-1-(2-((4-((tertiary-butoxycarbonyl))(butyl)amino)phenyl)amino)-2-oxoethyl)azepan-1-um bromide (80 mg). Mass (ESI): m / z 494.56 [M] + . 1 1H NMR (500 MHz, DMSO- d 6) δ ppm 10.63 (s, 1 H), 7.66 (d, 2 H), 7.50-7.60 (m, 5 H), 7.24 (d, 2 H), 4.91 (s, 2 H), 4.00 (s, 2H), 3.52-3.76 (m, 6 H), 1.90-1.98 (m, 4 H), 1.63-1.76 (m, 4 H), 1.37-1.41 (m, 13 H), 0.85 (t, 3 H).

[0537] Synthesis of 1-benzyl-1-(2-((4-(butylamino)phenyl)amino)-2-oxoethyl)azepan-1-ium formate:

[0538]

[0539] Compound 41A

[0540] Synthesis of 1-benzyl-1-(2-((4-(butylamino)phenyl)amino)-2-oxoethyl)azepan-1-ium formate:

[0541] The reaction mixture of 1-benzyl-1-(2-((4-((tert-butoxycarbonyl)(butyl)amino)phenyl)amino)-2-oxoethyl)azepan-1-um bromide (50 mg, 0.087 mmol) in 47% aqueous HBr (1 mL) was stirred at 80°C for 16 hours while monitoring the progress of the reaction with LCMS. The reaction mixture was concentrated under reduced pressure to obtain an unpurified product, which was purified by reverse-phase preparative HPLC (Column: X-Select C18 (19X250) mm 5u, Mobile phase: 0.1% formic acid in H2O: CAN, Flow rate: 18 ml / min, Gradient method: 0 / 51, 7.1 / 50, 7.2 / 99, 9.2 / 99, 9.3 / 5, 12 / 5). The pure fraction was collected and freeze-dried to obtain 1-benzyl-1-(2-((4-(butylamino)phenyl)amino)-2-oxoethyl)azepan-1-ium formate (25 mg). Mass (ESI): m / z 394.53 [M] + . 1 ¹H NMR (400 MHz, DMSO- d 6) δ ppm 10.95 (s, 1 H), 8.56 (s, 1 H), 7.68 (d, 2 H), 7.49-7.54 (m, 3 H), 7.36 (d, 2 H), 6.54 (d, 2 H), 5.52 (t, 1 H), 4.89 (s, 2 H), 4.01 (s, 2 H), 3.67-3.69 (m, 2 H), 3.51-3.53 (m, 2 H), 2.97 (t, 2 H), 1.91-2.01 (m, 4 H), 1.49-1.62 (m, 2 H), 1.35-1.40 (m, 2 H), 1.36 (t, 3 H).

[0542] Synthesis of 1-Benzyl-1-(2-((4-(butylamino)phenyl)amino)-2-oxoethyl)piperidine-1-ium carbonate:

[0543]

[0544] Compound 42A

[0545] · Synthesis of N-(4-nitrophenyl)-2-(piperidine-1-yl)acetamide:

[0546] Potassium carbonate (4.801 g, 34.74 mmol) and piperidine (1.972 g, 23.16 mmol) were added to a stirred solution of 2-bromo-N-(4-nitrophenyl)acetamide (3.0 g, 11.580 mmol) in ACN (30.0 mL), and the resulting reaction mixture was stirred at 85°C for 1 hour while monitoring the progress of the reaction by TLC (50% ethyl acetate in petroleum ether, UV visualization). The reaction mixture was cooled to room temperature and concentrated under reduced pressure to obtain an unpurified residue. The crude residue was diluted with water (150 mL) and extracted with EtOAc (3 x 100 mL). The combined organic extract was washed with a brine solution (1 x 100 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a crude product. This product was purified by column chromatography (eluting with 5% EtOAc in petroleum ether) to obtain the product N-(4-nitrophenyl)-2-(piperidin-1-yl)acetamide (1.40 g) as a pale yellow solid. MS (ESI): m / z 264.26 [M + H + ] . 1 ¹H NMR (400 MHz, DMSO- d 6) δ ppm 10.28 (s, 1 H), 8.20-8.23 (m, 2 H), 7.89 - 7.93 (m, 2 H), 3.14 (s, 2 H), 2.44-2.51 (m, 4 H), 1.53-1.59 (m, 4 H), 1.40 (t, 2H).

[0547] · Synthesis of N-(4-aminophenyl)-2-(piperidine-1-yl)acetamide:

[0548] 10% Pd / C (1.4 g) was added to a solution of N-(4-nitrophenyl)-2-(piperidin-1-yl)acetamide (1.4 g, 5.3171 mmol) in MeOH (15.0 ml), and the resulting reaction mixture was stirred at room temperature under a hydrogen gas atmosphere (balloon pressure) for 16 hours while monitoring the progress of the reaction by TLC (50% EtOAc in petroleum ether, visualization: UV). The reaction mixture was filtered through Celite washed with excess methanol (2 x 20 ml), and the combined filtrate was concentrated under reduced pressure to obtain an unpurified product, which was ground with diethyl ether (3 x 20 mL) to obtain N-(4-aminophenyl)-2-(piperidin-1-yl)acetamide (1.20 g). MS (ESI): m / z 234.29 [M + ] . 1 1H NMR (500 MHz, DMSO- d 6) δ ppm 10.25 (s, 1 H), 8.22 (d, 2 H), 7.92 (t, 2 H), 3.32 (d, 2 H), 2.72-2.85 (m, 4 H), 1.52-1.62 (m, 8 H). Analysis data is disclosed.

[0549] · Synthesis of N-(4-(butylamino)phenyl)-2-(piperidin-1-yl)acetamide:

[0550] Butyronitrile (1.777 g, 25.7157 mmol) and 10% Pd / C (1.2 g) were added to a solution of N-(4-aminophenyl)-2-(piperidin-1-yl)acetamide (1.2 g, 5.1431 mmol) in methanol (15.0 mL) under a nitrogen atmosphere. The resulting reaction mixture was stirred at room temperature for 16 hours under a hydrogen gas atmosphere (balloon pressure) while monitoring the progress of the reaction by TLC (ethyl acetate in petroleum ether, UV visualization). The reaction mixture was filtered through Celite washed with excess methanol (2 x 20 mL) and concentrated to obtain an unpurified product, which was purified by column chromatography (eluting with 20% EtOAc in petroleum ether) to obtain pure N-(4-(butylamino)phenyl)-2-(piperidin-1-yl)acetamide (1.10 g). MS (ESI): m / z 290.42 [M+H] . 1 ¹H NMR (400 MHz, DMSO- d 6) δ ppm 9.21 (s, 1 H), 7.27 (d, 2 H), 6.48 (d, 2 H), 5.33 (t, 2 H), 2.88-2.95 (m, 4 H), 2.49-2.51 (m, 4 H), 1.47-1.58 (m, 6 H), 1.34-1.41 (m, 4 H), 0.90 (t, 3H).

[0551] · Synthesis of tertiary-butylbutyl(4-(2-(piperidin-1-yl)acetamido)phenyl)carbamate:

[0552] TEA (1.672 g, 16.527 mmol) and di-tert-butyl dicarbonate (1.803 g, 8.2635 mmol) were added to a stirred solution of N-(4-(butylamino)phenyl)-2-(piperidin-1-yl)acetamide (0.956 g, 3.30 mmol) in DCM (10.0 mL) at 0°C, and the resulting reaction mixture was stirred at room temperature for 16 hours while monitoring the progress of the reaction by TLC (100% EtOAc, visualization: UV). The reaction mixture was concentrated under reduced pressure, diluted with water (50 mL), and extracted with DCM (3 x 50 mL). The combined organic extracts were washed with a brine solution (1 x 50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain an unpurified product, which was purified by normal-phase flash chromatography (eluted in 30% petroleum ether). The collected pure fraction was concentrated under reduced pressure to obtain the product tertiary-butylbutyl(4-(2-(piperidin-1-yl)acetamido)phenyl)carbamate (1.15 g). MS (ESI): m / z 390.57 [M+H] + .

[0553] · Synthesis of 1-benzyl-1-(2-((4-((tert-butoxycarbonyl)(butyl)amino)phenyl)amino)-2-oxoethyl)piperidine-1-um bromide:

[0554] Benzyl bromide (0.197 g, 1.1552 mmol) was added in a microwave vial to a stirred solution of tert-butylbutyl(4-(2-(piperidin-1-yl)acetamido)phenyl)carbamate (0.225 g, 0.5776 mmol) in acetonitrile (3 mL). The reaction mixture was stirred in a microwave (CEM instrument) at 100°C for 1 hour while monitoring the progress of the reaction by LCMS and TLC (5% MeOH in DCM, detection: UV). The reaction mixture was allowed to cool to room temperature and concentrated under reduced pressure to obtain an unpurified product, which was then ground with diethyl ether (5 x 5 ml) to obtain the product 1-benzyl-1-(2-((4-((tertiary-butoxycarbonyl)(butyl)amino)phenyl)amino)-2-oxoethyl)piperidin-1-ium bromide (110 mg). MS (ESI): m / z 480.58 [M]+ . .

[0555] · Synthesis of 1-Benzyl-1-(2-((4-(butylamino)phenyl)amino)-2-oxoethyl)piperidine-1-ium carbonate:

[0556] The reaction mixture of 1-benzyl-1-(2-((4-((tert-butoxycarbonyl)(butyl)amino)phenyl)amino)-2-oxoethyl)piperidin-1-um bromide (105 mg, 0.2184 mmol) in aqueous hydrogen bromide (48%) (1 mL) was stirred at room temperature for 16 hours while monitoring the progress of the reaction with LCMS and TLC (80% ethyl acetate in petroleum ether, detection: UV). The reaction mixture was freeze-dried to obtain an unpurified product, which was purified by reverse-phase preparative HPLC (Column: X-Select C18 (19*250) 5 u, Mobile phase (A): 10 mM ammonium bicarbonate, Mobile phase (B): 100% ACN, Methods: 0 / 3, 2 / 40, 20 / 40, 20.50 / 100, 30 / 35, Flow rate: 18 ml / min, Solubility: CAN + THF + MeOH). The pure fractions were combined and freeze-dried to obtain 1-benzyl-1-(2-((4-(butylamino)phenyl)amino)-2-oxoethyl)piperidine-1-ium carbonate (15.1 mg). MS (ESI): m / z 380.2 [M]+. 1 ¹H NMR (400 MHz, DMSO- d 6) δ ppm 11.10 (br s, 1 H), 7.54-7.72 (m, 5 H), 7.34 (d, 2 H), 6.52 (d, 2 H), 5.44 (t, 1 H), 4.94 (s, 2 H), 4.09 (s, 2 H), 3.65 (d, 2) H), 3.33-3.5 (m, 2 H), 2.97-3.01 (m, 2 H), 1.92-1.97 (m, 4 H), 1.63 (q, 1 H), 1.48-1.55 (m, 3 H), 1.34-1.42 (m, 2 H), 0.91 (t, 3 H).

[0557] Synthesis of 1-benzyl-1-(1-((4-(butylamino)-2,6-dimethylphenyl)amino)-1-oxobutane-2-yl)piperidine-1-ium carbonate:

[0558]

[0559] Compound 43A

[0560] · Synthesis of N-(2,6-dimethylphenyl)-4-methylbenzenesulfonamide:

[0561] Tosyl chloride (94.396 g, 495.131 mmol) was added to a stirred solution of 2,6-dimethylaniline (50 g, 413 mmol) in pyridine (1.2 L), and the mixture was heated and refluxed at 115°C for 4 hours while monitoring the progress of the reaction mixture by TLC (30% ethyl acetate in petroleum ether, visualization: UV). The reaction mixture was concentrated under reduced pressure to obtain an unpurified residue, which was diluted with water (500 mL), the pH adjusted to 6 with 2N HCl (500 mL), and extracted with ethyl acetate (2 x 1 L). The combined organic extract was washed with a brine solution (1.0 L), dried over anhydrous Na2SO4, and concentrated under reduced pressure to obtain an unpurified product, which was ground with petroleum ether to obtain N-(2,6-dimethylphenyl)-4-methylbenzenesulfonamide (90 g) as a grayish-white solid. MS (ESI): m / z 276.23 [M+H] + . 1 ¹H NMR (400 MHz, DMSO- d 6) δ ppm 9.231 (s, 1 H), 7.530-7.551 (d, 2 H), 7.354-7.375 (d, 2 H), 6.985-7.069 (m, 3 H), 2.384 (s, 3 H), 1.940 (s, 6 H).

[0562] · Synthesis of N-(2,6-dimethyl-4-nitrophenyl)-4-methylbenzenesulfonamide:

[0563] NaNO2 (45.103 g, 653.666 mmol) was added to a solution of N-(2,6-dimethylphenyl)-4-methylbenzenesulfonamide (90 g, 326.8 mmol) in AcOH (675 mL) and water (450 mL), followed by the dropwise addition of concentrated HNO3 (41.181 g, 653.666 mmol) over a period of 15 minutes at room temperature. The resulting reaction mixture was heated at 110°C for 5 hours while monitoring the progress of the reaction mixture by TLC (30% ethyl acetate in petroleum ether, visualization: UV). The reaction mixture was diluted with ice-cold water (500 mL), basicized with 1N NaOH solution (800 mL), and extracted with ethyl acetate (2 x 1 L). The combined organic extract was washed with a brine solution (1 L), dried over anhydrous Na2SO4, and concentrated under reduced pressure to obtain an unpurified product, which was ground with petroleum ether (500 mL) to obtain the product N-(2,6-dimethyl-4-nitrophenyl)-4-methylbenzenesulfonamide (40 g) as a grayish-white solid. MS (ESI): m / z 321.18 [M+H] + . 1 ¹H NMR (400 MHz, DMSO- d 6) δ ppm 9.72 (s, 1 H), 7.925 (s, 2 H), 7.562-7.583 (d, 2 H), 7.390-7.409 (d, 2 H), 2.400 (s, 3 H), 2.073 (s, 6 H).

[0564] · Synthesis of 2,6-Dimethyl-4-Nitroaniline:

[0565] HClO4 (250 ml) was added at room temperature to a stirred solution of N-(2,6-dimethyl-4-nitrophenyl)-4-methylbenzenesulfonamide (25 g, 78.037 mmol) in AcOH (125 ml), and the mixture was heated to 100°C for 3 hours while monitoring the progress of the reaction mixture by TLC (30% ethyl acetate in petroleum ether, visualization: UV). The reaction mixture was poured into an icebreaker, basicized with an aqueous ammonia solution (pH ~11), and extracted with ethyl acetate (2 x 1 L). The combined organic extract was washed with a brine solution (300 mL), dried over anhydrous Na2SO4, and concentrated under reduced pressure to obtain an unpurified product, which was purified by column chromatography (eluting with 20% ethyl acetate in petroleum ether) to obtain 2,6-dimethyl-4-nitroaniline (10 g) as a yellow solid. MS (ESI): m / z 167.03 [M+H] + . 1 ¹H NMR (400 MHz, DMSO- d 6) δ ppm 7.787 (s, 2 H), 6.142 (s, 2 H), 2.155 (s, 6 H).

[0566] · Synthesis of 2-Bromo-N-(2,6-dimethyl-4-nitrophenyl)butanamide:

[0567] Pyridine (1.713 g, 21.66 mmol) was added to a cooled solution (0°C) of 2,6-dimethyl-4-nitroaniline (3 g, 18.05 mmol) in DCM (45 mL), followed by 2-bromobutanoyl chloride (4.017 g, 21.66 mmol). The resulting reaction mixture was stirred at room temperature for 16 hours while monitoring the progress of the reaction mixture by TLC (50% ethyl acetate in petroleum ether, visualization: UV). The reaction mixture was filtered, and the isolated solid was washed with DCM (20 mL) and dried under high vacuum to obtain 2-bromo-N-(2,6-dimethyl-4-nitrophenyl)butanamide (2 g) as a grayish-white solid. MS (ESI): m / z 315.26 [M]+ . 1 ¹H NMR (400 MHz, DMSO- d 6) δ ppm 10.05 (s, 1H), 8.0 (s, 2H), 4.55 (t, 1H), 2.279 (s, 6H), 1.940-2.143 (m, 2H), 0.99 (t, 3H).

[0568] · Synthesis of N-(2,6-dimethyl-4-nitrophenyl)-2-(piperidine-1-yl)butanamide:

[0569] K2CO3 (2.627 g, 19.03 mmol) and piperidine (1.080 g, 12.690 mmol) were added to a stirred solution of 2-bromo-N-(2,6-dimethyl-4-nitrophenyl)butanamide (2 g, 6.345 mmol) in ACN (30 mL). The resulting mixture was heated at 80°C for 16 hours while monitoring the progress of the reaction mixture by TLC (50% ethyl acetate in petroleum ether, visualization: UV). The reaction mixture was concentrated, diluted with ethyl acetate (150 mL), and washed with water (50 mL x 3) and a saline solution (60 mL). The combined organic extracts were dried over anhydrous Na2SO4 and concentrated under reduced pressure to obtain an unpurified product, which was purified by column chromatography (eluting with 50% ethyl acetate in petroleum ether) to obtain N-(2,6-dimethyl-4-nitrophenyl)-2-(piperidin-1-yl)butanamide (1.70 g). MS (ESI): m / z 320.42 [M+H] + . 1 1H NMR (500 MHz, DMSO- d 6) δ ppm 9.578 (s, 1 H), 7.992 (s, 2 H), 3.12 (t, 1H), 2.55-2.62 (m, 4H), 2.28 (s, 6H), 1.60-1.81 (m, 2H), 1.35-1.56 (m, 6H), 0.93 (t, 3H).

[0570] · Synthesis of N-(4-amino-2,6-dimethylphenyl)-2-(piperidine-1-yl)butanamide:

[0571] 10% Pd-C (0.900 g) was added to a stirred solution of N-(2,6-dimethyl-4-nitrophenyl)-2-(piperidin-1-yl)butanamide (1.7 g, 5.322 mmol) in EtOH (40 mL), and the mixture was stirred for 16 hours at room temperature under H2 gas (balloon pressure) while monitoring the progress of the reaction mixture by TLC (100% ethyl acetate, visualization: UV). The mixture was filtered through Celite, the filter bed was washed with MeOH (200 ml), and the filtrate was concentrated under reduced pressure to obtain an unpurified product, which was purified by normal-phase flash chromatography (eluting with EtOAc) to obtain N-(4-amino-2,6-dimethylphenyl)-2-(piperidin-1-yl)butanamide (1.0 g). MS (ESI): m / z 290.46 [M+H] + . 1 ¹H NMR (400 MHz, DMSO- d 6) δ ppm 8.78 (s, 1 H), 6.23 (s, 2 H), 4.82 (s, 2 H), 2.95 (t, 1 H), 2.45-2.6 (m, 4 H), 1.2-1.6 (m, 8 H), 0.910 (t, 3 H).

[0572] · Synthesis of N-(4-(butylamino)-2,6-dimethylphenyl)-2-(piperidine-1-yl)butanamide:

[0573] Butyronitrile (2.387 g, 34.551 mmol) and 10% Pd / C (1 g) were added to a solution of N-(4-amino-2,6-dimethylphenyl)-2-(piperidin-1-yl)butanamide (1.0 g, 3.455 mmol) in MeOH (20 mL), and the mixture was stirred at room temperature under H2 gas (balloon pressure) for 16 hours while monitoring the progress of the reaction mixture by TLC (ethyl acetate, visualization: UV). The reaction mixture was filtered through a Celite bed, the filter bed was washed with MeOH (150 ml), and the filtrate was concentrated under reduced pressure to obtain an unpurified product, which was purified by normal-phase flash chromatography (eluting with ethyl acetate) to obtain N-(4-(butylamino)-2,6-dimethylphenyl)-2-(piperidin-1-yl)butanamide (0.500 g). MS (ESI): m / z 346.51 [M+H] + . 1 1H NMR (500 MHz, DMSO- d 6) δ ppm 8.789 (s, 1 H), 6.242 (s, 2 H), 2.96 (t, 3 H), 2.50-2.57 (m, 3 H), 2.027 (s, 6 H), 1.498-1.518 (m, 2 H), 1.475-1.488 (m, 6) H), 1.22-1.387 (m, 6 H), 0.885-0.918 (m, 6 H).

[0574] · Synthesis of tertiary-butylbutyl(3,5-dimethyl-4-(2-(piperidin-1-yl)butanamido)phenyl)carbamate:

[0575] DIPEA (0.179 g, 1.389 mmol) and di-tert-butyl dicarbonate (0.758 g, 3.473 mmol) were added at 0°C to a stirred solution of N-(4-(butylamino)-2,6-dimethylphenyl)-2-(piperidin-1-yl)butanamide (0.400 g, 1.157 mmol) in EtOH (10 mL). The reaction mixture was stirred at room temperature for 16 hours while monitoring the progress of the reaction mixture by TLC (50% ethyl acetate in petroleum ether, visualization: UV). The mixture was concentrated under vacuum to obtain an unpurified residue, which was diluted with ethyl acetate (80 ml) and washed with water (40 ml) and a brine solution (40 ml). The organic layer was dried over anhydrous Na2SO4 and concentrated under reduced pressure to obtain an unpurified product, which was purified by normal-phase flash chromatography (eluting with 40% ethyl acetate in petroleum ether) to obtain tertiary-butylbutyl(3,5-dimethyl-4-(2-(piperidin-1-yl)butanamido)phenyl)carbamate (0.300 g). MS (ESI): m / z 446.66 [M+H] + . 1 H NMR data shows the product along with aliphatic impurities. Analytical data is disclosed.

[0576] · Synthesis of 1-benzyl-1-(1-((4-((tertiary-butoxycarbonyl)(butyl)amino)-2,6-dimethylphenyl)amino)-1-oxobutane-2-yl)piperidine-1-um bromide:

[0577] Benzyl bromide (0.191 g, 1.116 mmol) was added to a stirred solution of tert-butylbutyl(3,5-dimethyl-4-(2-(piperidin-1-yl)butanamido)phenyl)carbamate (0.250 g, 0.561 mmol) in ACN (5 mL), and the mixture was heated to 85°C for 24 hours while monitoring the progress of the reaction mixture by TLC (10% methanol in DCM, visualization: UV). The reaction mixture was concentrated under vacuum to obtain an unpurified product, which was ground with a mixture of diethyl ether (20 ml) and ethyl acetate (10 ml) to obtain 1-benzyl-1-(1-((4-((tertiary-butoxycarbonyl)(butyl)amino)-2,6-dimethylphenyl)amino)-1-oxobutane-2-yl)piperidin-1-ium bromide (0.200 g). MS (ESI): m / z 536.61 [M] + . 1 1H NMR (500 MHz, DMSO- d 6) δ ppm 10.317 (s, 1 H), 7.547 (s, 5 H), 7.002 (s, 2 H), 5.41 (d, 1 H), 4.62 (d, 1 H), 3.6-3.9 (m, 3 H), 3.1-3.4 (m, 2 H), 2.4-2.5 (m, 2 H), 1.953-2.184 (m, 12 H), 1.163-1.620 (m, 18 H), 0.857 (s, 3 H).

[0578] · Synthesis of 1-benzyl-1-(1-((4-(butylamino)-2,6-dimethylphenyl)amino)-1-oxobutane-2-yl)piperidine-1-ium carbonate:

[0579] 47% HBr aqueous solution (8 ml) was added to 1-benzyl-1-(1-((4-((tertiary-butoxycarbonyl)(butyl)amino)-2,6-dimethylphenyl)amino)-1-oxobutane-2-yl)piperidin-1-um bromide (0.150 g, 0.243 mmol) at 0°C, and the resulting mixture was stirred at room temperature for 16 hours while monitoring the progress of the reaction mixture by TLC. (10% methanol in DCM, visualization: UV). The reaction mixture was concentrated under reduced pressure, and the residue was washed with diethyl ether (5 ml) to obtain an unpurified product, which was purified by reverse-phase preparative HPLC (Column: Chromasil C18 (25X150) mm 10 u, Mobile phase: 10 mM ammonium bicarbonate in H2O: CAN, Flow rate: 25 ml / min, Gradient method: 0 / 40, 10 / 82, 10.1 / 99, 12 / 99, 12.1 / 40, 14 / 40). The pure fraction was collected and freeze-dried to obtain 1-benzyl-1-(1-((4-(butylamino)-2,6-dimethylphenyl)amino)-1-oxobutane-2-yl)piperidine-1-ium carbonate salt (0.050 g). MS (ESI): m / z 436.61 [M] + . 1 1H NMR (500 MHz, DMSO- d 6) δ ppm 10.826 (s, 1 H), 7.54 (t, 5 H), 6.271 (s, 2 H), 5.44 (t, 2 H), 4.60 (d, 2 H), 3.708 (s, 2 H), 2.96 (q, 2 H), 2.362 (s, 2 H). 2.027-2.066 (m, 8 H), 1.838-1.99 (m, 2 H), 1.492-1.549 (m, 6 H), 1.32-1.45 (m, 3 H), 1.12-1.25 (m, 3 H), 0.922 (t, 3 H).

[0580] Synthesis of 1-Benzyl-1-(2-((2,6-Dimethylphenyl)amino)-2-oxoethyl)-4-(Methoxycarbonyl)piperidine-1-um bromide:

[0581]

[0582] Compound 44A

[0583] · Synthesis of methyl 1-(2-((2,6-dimethylphenyl)amino)-2-oxoethyl)piperidine-4-carboxylate:

[0584] K2CO3 (0.712 g, 5.162 mmol) was added to a stirred solution of 2-bromo-N-(2,6-dimethylphenyl)acetamide (0.500 g, 2.065 mmol) and methylpiperidine-4-carboxylate (0.443 g, 3.097 mmol) in ACN (10 ml). The mixture was heated at 90°C for 16 hours while monitoring the progress of the reaction mixture by TLC (50% ethyl acetate in petroleum ether, visualization: UV). The reaction mixture was concentrated under reduced pressure, diluted with EtOAc (100 ml), and washed with water (50 ml x 3) and a brine solution (50 ml). The organic extract was dried and concentrated over anhydrous Na2SO4 to yield an unpurified product, which was purified by normal-phase flash chromatography (eluting in a 10%–30% ethyl acetate gradient in petroleum ether). The collected pure fraction was concentrated under reduced pressure to yield the target product, methyl 1-(2-((2,6-dimethylphenyl)amino)-2-oxoethyl)piperidine-4-carboxylate (0.400 g). MS (ESI): m / z 305.36 [M+H] + . 1 ¹H NMR (400 MHz, DMSO- d 6) δ ppm 9.171 (s, 1 H), 7.060-7.09 (m, 3 H), 3.607 (s, 3 H), 3.089 (s, 2 H), 2.879-2.908 (m, 2 H), 2.316-2.371 (m, 1 H), 2.198-2.261 (m, 2 H), 2.129 (s, 6 H), 1.816-1.856 (m, 2 H). 1.680-1.778 (m, 2 H).

[0585] Synthesis of 1-benzyl-1-(2-((2,6-dimethylphenyl)amino)-2-oxoethyl)4-(methoxycarbonyl)piperidin-1-um bromide:

[0586] Benzyl bromide (0.224 g, 1.314 mmol) was added to a stirred solution of methyl 1-(2-((2,6-dimethylphenyl)amino)-2-oxoethyl)piperidine-4-carboxylate (0.200 g, 0.657 mmol) in ACN (5 ml), and the mixture was heated to 90°C for 24 hours while monitoring the reaction progress by TLC (10% MeOH in DCM, visualization: UV). The reaction mixture was concentrated to obtain an unpurified product, which was ground with ethyl acetate (30 ml) and n-pentane (20 ml) to obtain pure 1-benzyl-1-(2-((2,6-dimethylphenyl)amino)-2-oxoethyl)-4-(methoxycarbonyl)piperidine-1-um bromide (0.080 g) as a white solid. MS (ESI): m / z 395.3 [M] + . 1 H NMR (400 MHz, DMSO-d6) δ ppm 10.048 (s, 1 H), 7.551-7.591 (m, 5 H), 7.144-7.159 (m, 3 H), 4.947 (s, 2 H), 4.237 (s, 2 H), 3.671-3.766 (m, 5 H), 3.541-3.549 (m, 2 H), 2.732-2.788 (m, 1 H), 2.128-2.212 (m, 10 H).

[0587] Synthesis of 1-Benzyl-1-(2-((2,6-Dimethylphenyl)amino)-2-oxoethyl)-3-(ethoxycarbonyl)piperidine-1-um bromide:

[0588]

[0589] Compound 45A

[0590] Synthesis of ethyl 1-(2-((2,6-dimethylphenyl)amino)-2-oxoethyl)piperidine-3-carboxylate:

[0591] K2CO3 (8.726 g, 63.237 mmol) and ethyl piperidine-3-carboxylate (5.964 g, 37.942 mmol) were added at room temperature to a stirred solution of 2-chloro-N-(2,6-dimethylphenyl)acetamide (5 g, 25.295 mmol) in ACN (100 ml). The resulting reaction mixture was stirred at room temperature for 16 hours while monitoring the progress of the reaction mixture by TLC (30% ethyl acetate in petroleum ether, visualization: UV). The reaction mixture was filtered to remove inorganic salts, and the filtrate was concentrated under reduced pressure to obtain an unpurified product, which was diluted with ethyl acetate (250 ml) and washed with water (80 ml x 3) and a brine solution (80 ml). The organic phase was dried on an anhydrous Na2SO4 phase and concentrated under reduced pressure to obtain ethyl 1-(2-((2,6-dimethylphenyl)amino)-2-oxoethyl)piperidine-3-carboxylate (6.5 g). MS (ESI): m / z 319.01 [M+H] + . 1 H NMR (400 MHz, CDCl3) δ ppm 8.751 (s, 1 H), 7.066-7.101 (m, 3 H), 4.075-4.168 (m, 2 H), 3.154-3.249 (m, 2 H), 2.927-2.949 (m, 1 H), 2.761-2.817 (m, 2 H), 2.613-2.664 (m, 1 H), 2.45-2.49 (m, 1 H), 2.23 (s, 6 H), 1.506-2.045 (m, 4 H), 1.209-1.277 (m, 3 H).

[0592] Synthesis of 1-benzyl-1-(2-((2,6-dimethylphenyl)amino)-2-oxoethyl)-3-(ethoxycarbonyl)piperidine-1-um bromide:

[0593] Benzyl bromide (0.322 g, 1.884 mmol) was added to a stirred solution of ethyl 1-(2-((2,6-dimethylphenyl)amino)-2-oxoethyl)piperidine-3-carboxylate (0.300 g, 0.942 mmol) in ACN (10 ml), and the mixture was heated at 90°C for 16 hours in a sealed tube while monitoring the reaction progress by TLC (10% MeOH in DCM, visualization: UV). The reaction mixture was concentrated under reduced pressure to obtain an unpurified product, which was purified by normal-phase flash chromatography (eluting with 2% methanol in DCM). The collected pure fraction was concentrated under reduced pressure to obtain the target product as a colorless gum (LCMS-85%). This compound was diluted with ethyl acetate (15 ml) and stirred for 1 hour, then filtered and washed with ethyl acetate (15 ml x 3) to obtain pure 1-benzyl-1-(2-((2,6-dimethylphenyl)amino)-2-oxoethyl)-3-(ethoxycarbonyl)piperidin-1-um bromide (70 mg) as a grayish-white solid. MS (ESI): m / z 409.2 [M] + . 1 ¹H NMR (400 MHz, DMSO- d 6) δ ppm 10.05 (s, 1H), 7.545-7.585 (m, 5 H), 7.125-7.178 (m, 3 H), 4.945-5.060 (m, 2 H), 4.109-4.342 (m, 4 H), 3.904-3.934 (m, 1 H), 3.644-3.677 (m, 1 H), 3.433-3.569 (m, 2 H), 3.230-3.262 (m, 1 H), 2.213 (s, 6 H), 1.948-2.195 (m, 3 H), 1.553-1.620 (m, 1) H), 1.24 (t, 3 H).

[0594] Synthesis of 1-Benzyl-1-(2-((2,6-Dimethylphenyl)amino)-2-oxoethyl)-3-(isopropoxycarbonyl)piperidine-1-um trifluoroacetate salt

[0595]

[0596] Compound 46A

[0597] Synthesis of 1-(2-((2,6-dimethylphenyl)amino)-2-oxoethyl)piperidine-3-carboxylic acid:

[0598] A mixture of ethyl 1-(2-((2,6-dimethylphenyl)amino)-2-oxoethyl)piperidine-3-carboxylate (5 g, 15.702 mmol) in concentrated HCl (100 ml) was heated at 100°C for 16 hours while monitoring the progress of the reaction by TLC (50% EtOAc in petroleum ether, visualization: UV). The reaction mixture was concentrated under reduced pressure to obtain 1-(2-((2,6-dimethylphenyl)amino)-2-oxoethyl)piperidine-3-carboxylic acid (4.2 g). MS(ESI): m / z 291.13 [M+H] + .

[0599] · Synthesis of isopropyl 1-(2-((2,6-dimethylphenyl)amino)-2-oxoethyl)piperidine-3-carboxylate:

[0600] Thionyl chloride (5 mL) was added dropwise at 0°C to a stirred solution of 1-(2-((2,6-dimethylphenyl)amino)-2-oxoethyl)piperidine-3-carboxylic acid (0.5 g, 1.722 mmol) in isopropanol (10 mL). The resulting reaction mixture was stirred at 90°C for 16 hours while monitoring the progress of the reaction by TLC (50% EtOAc in petroleum ether, visualization: UV). The reaction mixture was cooled to room temperature and concentrated under reduced pressure to obtain an unpurified residue, which was quenched with an ice-cold saturated sodium bicarbonate solution (20 mL) and extracted with EtOAc (2 x 50 mL). The combined organic extracts were dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain an unpurified product, which was purified by normal-phase flash chromatography (eluting with 20%–30% EtOAc / petroleum ether). The collected pure fraction was concentrated under reduced pressure to obtain 1-(2-((2,6-dimethylphenyl)amino)-2-oxoethyl)piperidine-3-carboxylate (0.54 g) as a pale yellow solid. MS (ESI): m / z 332.27 [M+H] + . 1H NMR (400 MHz, CDCl3) δ ppm 8.5 (br s, 1 H), 7.06-7.11 (m, 3 H), 4.97 - 5.03 (m, 1 H), 3.19 - 3.24 (m, 2 H), 2.93-2.95 (m, 1 H), 2.59-2.48 (m, 2 H), 2.48-2.40 (m, 2 H), 2.18-2.20 (m, 5 H), 1.41-1.65 (m, 3 H), 1.18-1.24 (m, 6 H).

[0601] Synthesis of 1-benzyl-1-(2-((2,6-dimethylphenyl)amino)-2-oxoethyl)-3-(isopropoxycarbonyl)piperidin-1-um trifluoroacetate salt:

[0602] Benzyl bromide (0.6173 g, 3.609 mmol) was added at room temperature to a stirred solution of isopropyl 1-(2-((2,6-dimethylphenyl)amino)-2-oxoethyl)piperidine-3-carboxylate (0.4 g, 1.203 mmol) in ACN (10 mL). The resulting reaction mixture was stirred in a sealed tube at 90°C for 16 hours while monitoring the progress of the reaction by TLC (50% EtOAc in petroleum ether, visualization: UV). The reaction mixture was cooled to room temperature and concentrated under reduced pressure to yield 430 mg (LCMS 51%) of unpurified compound, which was further purified by reverse-phase preparative HPLC (Column: X-Select csh C18 (250*19 ) mm, 5 µ, Mobile phase A: 0.1% TFA in water (aqueous), Mobile phase B: Acetonitrile, Flow rate: 22 ml / min, Method (T / %) of B: 0 / 10, 2 / 10, 10 / 50, 13 / 50, 13.1 / 100, 16 / 100, 16.1 / 10, 19 / 20, Solubility: ACN + H2O + THF, Temperature: Ambient). The combined pure fraction was freeze-dried to obtain pure 1-benzyl-1-(2-((2,6-dimethylphenyl)amino)-2-oxoethyl)-3-(isopropoxycarbonyl)piperidin-1-ium trifluoroacetate salt (168 mg). LCMS: 99.37% (77.95% + 21.42%, mixture of isomers), MS(ESI): m / z 423.2, [M]+ . 1 ¹H NMR (400 MHz, DMSO- d 6) δ ppm 10.01-10.05 (m, 1 H), 7.57-7.61 (m, 5 H), 7.12-7.17 (m, 3 H), 4.92-5.06 (m, 3 H), 4.14-4.22 (m, 2 H), 3.87-3.67 (dd, 1 H), 3.46-3.55 (m, 3 H), 3.15-3.21 (m, 1 H), 2.208 (d, 6 H), 1.85-2.07 (m, 3 H), 1.19-1.23 (m, 6 H).

[0603] Synthesis of 1-benzyl-1-(2-((2,6-dimethylphenyl)amino)-2-oxoethyl)-3-(propoxycarbonyl)piperidine-1-um bromide

[0604]

[0605] Compound 47A

[0606] · Synthesis of propylpiperidine-3-carboxylate:

[0607] SOCl2 (11.05 g, 92.908 mmol) was added to a stirred solution of piperidine-3-carboxylic acid (2.0 g, 15.484 mmol) in propan-1-ol (20.0 ml) at 0-5°C. The resulting reaction mixture was stirred at 80°C for 16 hours while monitoring the progress of the reaction using TLC (30% ethyl acetate / petroleum ether; visualization: ninhydrin). The reaction mixture was concentrated under reduced pressure to obtain an unpurified compound, which was dissolved in ethyl acetate (100 ml) and washed with a saturated bicarbonate solution (1 x 100 ml) and a brine solution (1 x 50 ml). The organic layer was dried over anhydrous Na2SO4 and concentrated under reduced pressure to obtain propylpiperidine-3-carboxylate (2.0 g) as a colorless liquid. This unpurified product is used in the next step without further purification or analysis.

[0608] · Synthesis of propyl 1-(2-((2,6-dimethylphenyl)amino)-2-oxoethyl)piperidine-3-carboxylate:

[0609] K2CO3 (4.14 g, 29.95 mmol) and propylpiperidine-3-carboxylate (3.47 g, 20.28 mmol) were added at room temperature to a stirred solution of 2-chloro-N-(2,6-dimethylphenyl)acetamide (2.0 g, 10.15 mmol) in ACN (20 ml). The resulting reaction mixture was stirred at 80°C for 16 hours while monitoring the progress of the reaction using TLC (50% ethyl acetate / petroleum ether. Visualization: UV active). After the reaction was complete on TLC, the reaction mixture was concentrated under reduced pressure to obtain an unpurified compound, which was diluted with water (30 ml) and extracted with ethyl acetate (2 x 100 ml). The combined organic extracts were washed with brine (50.0 mL), dried over anhydrous Na2SO4, and concentrated under reduced pressure to obtain an unpurified compound, which was purified by column chromatography (eluting with 20%–80% ethyl acetate / petroleum ether). The collected pure fraction was concentrated under reduced pressure to obtain propyl 1-(2-((2,6-dimethylphenyl)amino)-2-oxoethyl)piperidine-3-carboxylate (1.0 g) as a gum compound. MS (ESI): m / z 333.10 [M+H] + .

[0610] Synthesis of 1-benzyl-1-(2-((2,6-dimethylphenyl)amino)-2-oxoethyl)-3-(propoxycarbonyl)piperidine-1-um bromide:

[0611] Benzyl bromide (0.3113 g, 1.806 mmol) was added at room temperature to a solution of propyl 1-(2-((2,6-dimethylphenyl)amino)-2-oxoethyl)piperidine-3-carboxylate (0.3 g, 0.9030 mmol) in acetonitrile (10 mL). The resulting reaction mixture was stirred at 90°C for 16 hours while monitoring the reaction progress by TLC (10% MeOH / DCM, visualization: UV). After the starting material was consumed on the TLC, the reaction mixture was concentrated directly under reduced pressure to obtain an unpurified compound as a pale yellow semi-solid, which was purified by normal-phase flash chromatography (eluted with 0%-30% MeOH / DCM). The collected pure fraction was concentrated under reduced pressure to obtain 1-benzyl-1-(2-((2,6-dimethylphenyl)amino)-2-oxoethyl)-3-(propoxycarbonyl)piperidin-1-um bromide (70 mg) as a grayish-white solid. MS (ESI): m / z 423.3 [M] + . 1 ¹H NMR (400 MHz, DMSO- d 6) δ ppm 10.30 (s, 1 H), 7.55-7.60 (m, 5 H), 7.11-7.18 (m, 3 H), 4.97-5.15 (m, 2 H), 4.33 (dd, 2 H) 4.05 (t, 2 H), 3.88 (d, 1H), 3.66 (d, 1H), 3.31-3.54 (m, 3H), 2.09-2.21 (m, 8 H), 1.96 (d, 2 H), 1.55-1.64 (m, 3 H), 0.87-0.93 (m, 3 H).

[0612] The following example was prepared from piperidine-3-carboxylic acid, 2-chloro-N-(2,6-dimethylphenyl)acetamide and benzyl bromide according to the procedure described for the synthesis of compound 47A.

[0613]

[0614] Synthesis of 1-benzyl-1-(2-((2,6-dimethylphenyl)amino)-2-oxoethyl)-3-(propionyloxy)piperidine-1-um bromide

[0615]

[0616] Compound 52A

[0617] · Synthesis of tert-butyl 3-(propionyloxy)piperidine-1-carboxylate:

[0618] Propionyl chloride (1.103 g, 11.923 mmol) was added at 0°C to a stirred solution of tertiary-butyl 3-hydroxypiperidin-1-carboxylate (2 g, 9.936 mmol) and pyridine (2.357 g, 29.808 mmol) in DCM (30 ml), and the resulting reaction mixture was stirred at room temperature for 24 hours while monitoring the progress of the reaction by TLC (20% EtOAc in petroleum ether, visualization: UV). The reaction mixture was diluted with DCM (120 mL), washed twice with water (50 mL), dried over sodium sulfate, and concentrated under reduced pressure to obtain tertiary-butyl 3-(propionyloxy)piperidin-1-carboxylate (1.34 g) as a grayish-white solid. 1 H NMR (400 MHz, CDCl3) δ ppm 4.85-4.75 (m, 1 H), 3.51-3.43 (m, 3 H), 3.3-3.29 (m, 1 H), 2.41-2.39 (m, 2 H), 1.85-1.72 (m, 3 H), 1.45 (s, 9) H), 1.18-1.12 (m, 4 H).

[0619] · Synthesis of piperidine-3-yl propionate:

[0620] TFA (5 ml) was added at 0°C to a stirred solution of tert-butyl 3-(propionyloxy)piperidin-1-carboxylate (1.3 g, 5.05 mmol) in DCM (20 ml), and the resulting reaction mixture was stirred at room temperature for 16 hours while monitoring the progress of the reaction by TLC (50% EtOAc in petroleum ether, visualization: ninhydrin). The reaction mixture was concentrated under reduced pressure to yield an unpurified product, which was co-vanzated with toluene (2 x 15 mL) to obtain piperidin-3-yl propionate TFA salt (1.34 g) as a pale yellow oil. 1H NMR (400 MHz, CDCl3) δ ppm 5.16 (s, 1 H), 3.41 (d, 2 H), 3.24 (t, 1 H), 3.10 (d, 1 H), 2.42-2.36 (m, 2 H), 2.13-2.10 (m, 2 H), 2.02-2.01 (m, 2 H), 1.88-1.84 (m, 3 H).

[0621] Synthesis of 1-(2-((2,6-dimethylphenyl)amino)-2-oxoethyl)piperidine-3-ylpropionate:

[0622] DIPEA (0.5884 g, 4.551 mmol) and piperidine-3-yl propionate TFA salt (0.4937 g, 1.8204 mmol) were added at room temperature to a stirred solution of 2-chloro-N-(2,6-dimethylphenyl)acetamide (0.3 g, 1.517 mmol) in acetonitrile (15 mL). The resulting reaction mixture was stirred in a sealed tube at 80°C for 16 hours while monitoring the progress of the reaction by TLC (50% EtOAc in petroleum ether, visualization: UV). The reaction mixture was concentrated under reduced pressure to obtain the residue, which was diluted with water (50 ml) and extracted with ethyl acetate (2 x 25 ml). The combined organic extracts were dried over sodium sulfate and concentrated under reduced pressure to obtain an unpurified product, which was purified by normal-phase flash chromatography (eluting with 25%–30% EtOAc / petroleum ether). The collected pure fraction was concentrated under reduced pressure to obtain 2-(azepan-1-yl)-N-(3-methyl-[1,1'-biphenyl]-2-yl)acetamide (310 mg). Mass (ESI): 319.26 m / z, [M+H] + . 1H NMR (400 MHz, CDCl3) δ ppm 8.67 (s, 1 H), 7.12-7.06 (m, 3 H), 4.95-4.92 (m, 1 H), 3.20 (s, 2 H), 2.95-2.89 (m, 1 H), 2.73-2.56 (m, 1 H), 2.59-2.54 (m, 2 H), 2.31-2.23 (m, 8 H), 1.88-1.84 (m, 2 H), 1.68-1.56 (m, 3 H), 1.09 (t, 3 H).

[0623] Synthesis of 1-benzyl-1-(2-((2,6-dimethylphenyl)amino)-2-oxoethyl)-3-(propionyloxy)piperidin-1-um bromide

[0624] Benzyl bromide (0.4297 g, 2.5124 mmol) was added to a stirred solution of 1-(2-((2,6-dimethylphenyl)amino)-2-oxoethyl)piperidin-3-yl propionate (0.2 g, 0.6281 mmol) in acetonitrile (15 ml) in a sealed tube at room temperature, and the resulting reaction mixture was stirred at room temperature for 16 hours while monitoring the progress of the reaction by TLC (10% methanol in DCM. Visualization: UV). The reaction mixture was cooled to room temperature and concentrated under reduced pressure to obtain an unpurified product, which was ground with 1:1 EtOAc:Et2O (30:30 ml) to obtain 1-benzyl-1-(2-((2,6-dimethylphenyl)amino)-2-oxoethyl)-3-(propionyloxy)piperidin-1-um bromide (90 mg) (mixture of isomers, Peak-1: 35.75% + Peak-2: 63.34%). Mass (ESI): m / z 409.03 [M] + . 1 ¹H NMR (400 MHz, DMSO- d6) δ ppm 10.05 (d, 1 H), 7.60-7.56 (m, 5 H), 7.15-7.13 (m, 3 H), 5.45-5.25 (m, 1H), 5.05-4.97 (m, 1 H), 4.35-4.18 (m, 2H), 4.29 (d, 2 H), 3.87-3.52 (m, 4 H), 2.44-2.35 (m, 2 H), 2.21 (m, 6 H), 2.20-1.98 (m, 3 H), 1.80-1.6 (m, 1 H), 1.04 (t, 3 H).

[0625] Synthesis of 1-Benzyl-3-Carbamoyl-1-(2-((2,6-Dimethylphenyl)amino)-2-Oxoethyl)Piperidine-1-Yum Bromide:

[0626]

[0627] Compound 53A

[0628] Synthesis of 1-(2-((2,6-dimethylphenyl)amino)-2-oxoethyl)piperidine-3-carboxamide

[0629] Potassium carbonate (4.195 g, 30.3551 mmol) was added to a stirred solution of 2-chloro-N-(2,6-dimethylphenyl)acetamide (2.0 g, 10.1183 mmol) in ACN (20.0 ml), and the resulting mixture was stirred at room temperature for 10 minutes. The reaction mixture was treated with piperidine-3-carboxamide (2.6081 g, 20.2366 mmol) at room temperature and stirred for 16 hours while heating to 90°C, while monitoring the progress of the reaction by TLC (10% MeOH in DCM. Visualization: UV). The reaction mixture was allowed to cool to room temperature, diluted with 70 ml of ethyl acetate, and washed with 2 x 50 ml of water followed by 1 x 30 ml of saline solution. The organic phase was dried on an anhydrous Na2SO4 phase, filtered, and concentrated under reduced pressure to obtain 1-(2-((2,6-dimethylphenyl)amino)-2-oxoethyl)piperidine-3-carboxamide (2.5 g) as a white solid. MS (ESI): m / z 290.25 [M+H] + . 1H NMR (400 MHz, CHLOROFORM-d) δ ppm 8.57 (s, 1 H), 7.06-7.09 (m, 3 H), 5.95 (br s, 1 H), 5.45 (br s, 1 H), 3.15-3.30 (m, 2 H), 2.70-2.90 (m, 3 H), 2.45-2.60 (m, 2 H), 2.15-2.30 (m, 6), 1.81-1.86 (m, 2 H), 1.60-1.72 (m, 2 H).

[0630] Synthesis of 1-benzyl-3-carbamoyl-1-(2-((2,6-dimethylphenyl)amino)-2-oxoethyl)piperidine-1-um bromide:

[0631] Benzyl bromide (0.295 g, 1.730 mmol) was added to a stirred solution of 1-(2-((2,6-dimethylphenyl)amino)-2-oxoethyl)piperidine-3-carboxamide (0.250 g, 0.865 mmol) in ACN (3.0 ml), and the resulting reaction mixture was heated at 90°C for 16 hours while monitoring the progress of the reaction by TLC (10% MeOH in DCM, visualization: UV). The reaction mixture was directly concentrated under reduced pressure to obtain an unpurified compound, which was ground with 3 x 10 ml of ethyl acetate to obtain 1-benzyl-3-carbamoyl-1-(2-((2,6-dimethylphenyl)amino)-2-oxoethyl)piperidine-1-um bromide (130 mg) as a grayish-white solid (33.18% + 65.55%, mixture of isomers). MS (ESI): m / z 380.2 [M] + . 1 H NMR (400 MH-DMSO-d6) δ ppm 9.92-10.15 (m, 1 H), 7.45-7.60 (m, 6 H), 7.16-7.22 (m, 4 H), 4.94-5.06 (m, 2 H), 4.05-4.29 (m, 2 H), 3.41-3.83 (m, 4 H), 2.92 (t, 1 H), 2.19-2.21 (m, 6 H), 1.98-2.08 (m, 3 H), 1.50-1.54 (m, 1 H).

[0632] Synthesis of 1-benzyl-1-(2-((2,6-dimethylphenyl)amino)-2-oxoethyl)-3-(ethylcarbamoyl)piperidine-1-um bromide

[0633]

[0634] Compound 54A

[0635] Synthesis of 3-(ethylcarbamoyl)piperidine-1-carboxylate

[0636] Et3N (2.64 g, 26.166 mmol) and ethanolamine hydrochloride (1.42 g, 17.444 mmol) were added to a stirred solution of 1-(tertiary-butoxycarbonyl)piperidine-3-carboxylic acid (2 g, 8.722 mmol) in THF (20 mL), followed by HATU (4.97 g, 13.083 mmol). The reaction mixture was stirred at room temperature for 16 hours while monitoring the progress of the reaction by TLC (50% EtOAc-petroleum-ether, visualization: ninhydrin). Upon completion, the reaction mixture was concentrated under reduced pressure, quenched with 2N HCl, and extracted with EtOAc (2 x 200 mL). The combined organic extract was washed with brine (200 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to obtain an unpurified compound (2.2 g), which was purified by column chromatography (eluting with 40% EtOAc in petroleum ether) to obtain pure tert-butyl 3-(ethylcarbamoyl)piperidine-1-carboxylate (2.1 g) as a light brown liquid. 1 ¹H NMR (400 MHz, DMSO- d 6) δ ppm 6.12 (br s, 1 H), 3.83 (br s, 2 H), 2.80 (S, 5 H), 2.26-2.28 (m, 1 H), 2.04-1.05 (m, 2 H), 1.84-1.85 (m, 1 H), 1.46-1.48 (m, 11 H), 1.13 (t, 3H).

[0637] Synthesis of N-ethylpiperidin-3-carboxamide

[0638] TFA (10 ml) was added at 0°C to a stirred solution of tert-butyl 3-(ethylcarbamoyl)piperidine-1-carboxylate (2.5 g, 9.752 mmol) in DCM (20 ml), and the reaction mixture was stirred at room temperature for 3 hours while monitoring the progress of the reaction by TLC (10% MeOH-DCM, visualization: ninhydrin). The reaction mixture was concentrated under reduced pressure to obtain pure N-ethylpiperidine-3-carboxamide TFA salt (2.4 g) as a light brown liquid. LCMS purity: 99.89%, mass (ESI): m / z 157.09 [M+H] + .

[0639] Synthesis of 1-(2-((2,6-dimethylphenyl)amino)-2-oxoethyl)-N-ethylpiperidine-3-carboxamide

[0640] DIPEA (1.53 g, 11.89 mmol) was added to a solution of N-ethylpiperidin-3-carboxamide TFA salt (1 g, 3.96 mmol) in ACN (10 mL), followed by 2-bromo-N-(2,6-dimethylphenyl)acetamide (960 mg, 3.96 mmol). The reaction mixture was stirred in a sealed tube at 90°C for 16 hours while monitoring the progress of the reaction by TLC (10% MeOH-DCM, visualization: UV). The reaction mixture was concentrated under reduced pressure to obtain an unpurified compound (1.5 g), which was purified by column chromatography (eluting with 2% MeOH in DCM) to obtain the product 1-(2-((2,6-dimethylphenyl)amino)-2-oxoethyl)-N-ethylpiperidin-3-carboxamide (1 g) as a semi-solid. LCMS Purity: 39%, Mass (ESI): m / z 318.12 [M+H] + This product is used in the next step without further purification.

[0641] Synthesis of 1-benzyl-1-(2-((2,6-dimethylphenyl)amino)-2-oxoethyl)-3-(ethylcarbamoyl)piperidine-1-um bromide

[0642] Benzyl bromide (0.808 g, 4.724 mmol) was added to a stirred solution of 1-(2-((2,6-dimethylphenyl)amino)-2-oxoethyl)-N-ethylpiperidine-3-carboxamide (0.3 g, 0.945 mmol) in acetonitrile (2 ml), and the resulting reaction mixture was stirred at 90°C for 16 hours while monitoring the progress of the reaction by TLC (10% methanol in DCM, visualization: UV). The reaction mixture was cooled to room temperature and concentrated under reduced pressure to obtain an unpurified product, which was purified by normal-phase flash chromatography (eluting with 10%–15% MeOH in DCM) to obtain 1-benzyl-1-(2-((2,6-dimethylphenyl)amino)-2-oxoethyl)-3-(ethylcarbamoyl)piperidine-1-um bromide (100 mg) as a white solid. MS (ESI): m / z 408.19 [M] + . 1 H NMR (400 MHz, DMSO-d6) δ ppm 10.15 (s, 1 H), 7.98 (t, 1 H), 7.60-7.53 (m, 5 H), 7.18-7.12 (m, 3 H), 5.01-5.00 (m, 2 H), 4.22 (s, 2 H), 3.73-3.66 (m, 2 H), 3.51-3.42 (m, 2 H), 3.13-3.04 (m, 2 H), 2.9-2.78 (m, 1 H), 2.21 (s, 6 H), 2.02-1.98 (m, 3 H), 1.54-1.51 (m, 1) H), 0.99 (t, 3 H).

[0643] The following examples were prepared from 1-(tert-butoxycarbonyl)piperidine-3-carboxylic acid, 2-bromo-N-(2,6-dimethylphenyl)acetamide, and benzyl bromide according to the procedure described for the synthesis of compound 54A. The product was purified by normal-phase flash chromatography or reverse-phase preparative HPLC.

[0644]

[0645]

[0646]

[0647] Synthesis of 1-benzyl-1-(2-((2,6-dimethylphenyl)amino)-2-oxoethyl)-4-(propoxycarbonyl)-1,4-diazepan-1-um bromide

[0648]

[0649] Compound 68A

[0650] · Synthesis of 1-(tert-butyl)4-propyl 1,4-diazepan-1,4-dicarboxylate

[0651] Carbonyl diimidazole (1.21 g, 7.488 mmol, 3.0 equivalents) and TEA (0.758 g, 7.488 mmol), followed by 1-propanol (0.449 g, 7.488 mmol), were added to a stirred solution of tert-butyl 1,4-diazepan-1-carboxylate (0.5 g, 2.496 mmol) in THF (5 ml) at 0°C. The resulting reaction mixture was stirred at 80°C for 16 hours while monitoring the reaction progress by TLC (30% EtOAc-hexane, visualization: PMA). The reaction mixture was concentrated under reduced pressure to obtain an unpurified product, which was diluted with EtOAc (150 ml) and washed with water (3 x 30 ml). The organic phase was dried over Na2SO4 and concentrated under reduced pressure to obtain 1-(tert-butyl)4-propyl 1,4-diazepan-1,4-dicarboxylate (0.655 g). MS (ESI): m / z 287.19 [M+H] + .

[0652] · Synthesis of Propyl 1,4-Diazepan-1-Carboxylate

[0653] A solution of 4M dioxane·HCl (10 ml) was added to 1-(tert-butyl)4-propyl 1,4-diazepan-1,4-dicarboxylate (0.65 g, 2.269 mmol, 1.0 equivalent) at 0°C, and the resulting reaction mixture was stirred at room temperature for 16 hours while monitoring the progress of the reaction by TLC (10% methanol in DCM, visualization: PMA). The reaction mixture was concentrated under reduced pressure to obtain propyl 1,4-diazepan-1-carboxylate hydrochloride salt (0.41 g) as a pale yellow rubbery solid. MS (ESI): m / z 187.12 [M+H] + .

[0654] · Synthesis of Propyl 4-(2-((2,6-Dimethylphenyl)amino)-2-oxoethyl)-1,4-diazepan-1-carboxylate

[0655] DIPEA (0.522 g, 4.046 mmol) and propyl 1,4-diazepan-1-carboxylate hydrochloride salt (0.376 g, 2.023 mmol) were added to a stirred solution of 2-chloro-N-(2,6-dimethylphenyl)acetamide (0.4 g, 2.023 mmol) in ACN (10 ml) at 0°C. The resulting reaction mixture was stirred at 90°C for 16 hours while monitoring the reaction progress by TLC (50% EtOAc-hexane, visualization: UV). The reaction mixture was concentrated under reduced pressure to obtain the residue, which was diluted with EtOAc (100 ml), washed with water (60 ml), dried over Na2SO4, and concentrated under reduced pressure. The resulting crude product was purified by normal-phase flash chromatography (elution with 10%–50% EtOAc in petroleum ether) to obtain propyl 4-(2-((2,6-dimethylphenyl)amino)-2-oxoethyl)-1,4-diazepan-1-carboxylate (0.25 g) as a white solid. MS (ESI): m / z 348.69 [M+H] + . 1 ¹H NMR (400 MHz, DMSO- d6) δ ppm 9.19 (s, 1 H), 7.00 (s, 3 H), 3.96-3.91 (m, 2 H), 3.50-3.43 (m, 4 H), 3.25 (s, 2 H), 2.80-2.65 (m, 4 H), 2.13 (s, 6 H), 1.84-1.81 (m, 2 H), 1.60-1.53 ​​(m, 2 H), 0.90-0.85 (m, 3 H).

[0656] · Synthesis of 1-benzyl-1-(2-((2,6-dimethylphenyl)amino)-2-oxoethyl)-4-(propoxycarbonyl)-1,4-diazepan-1-um bromide

[0657] Benzyl bromide (0.419 g, 2.876 mmol) was added to a stirred solution of propyl 4-(2-((2,6-dimethylphenyl)amino)-2-oxoethyl)-1,4-diazepan-1-carboxylate (0.25 g, 0.719 mmol) in ACN (5 ml) at room temperature, and the resulting reaction mixture was stirred at 90°C for 16 hours while monitoring the progress of the reaction by TLC (10% methanol in DCM, visualization: UV). The reaction mixture was concentrated under reduced pressure to obtain an unpurified product, which was ground with ethyl acetate (40 ml) to obtain 1-benzyl-1-(2-((2,6-dimethylphenyl)amino)-2-oxoethyl)-4-(propoxycarbonyl)-1,4-diazepan-1-um bromide (122.1 mg) as a white solid. Mass (ESI): m / z 438.2 [M] + . 1 ¹H NMR (400 MHz, DMSO- d 6) δ ppm 9.99 (s, 1 H), 7.61-7.53 (m, 5 H), 7.18-7.11 (m, 3 H), 5.00 (s, 2 H), 4.29-4.16 (m, 2 H), 4.01-3.56 (m, 10 H), 2.32-2.30 (m, 2 H), 2.21 (s, 6 H), 1.62-1.57 (m, 2 H), 0.92-0.88 (m, 3 H).

[0658] Synthesis of 1-benzyl-1-(2-((2-fluoro-6-methylphenyl)amino)-2-oxoethyl)azepan-1-um bromide

[0659]

[0660] Compound 69A

[0661] · Synthesis of 2-Bromo-N-(2-fluoro-6-methylphenyl)acetamide

[0662] A solution of 2-fluoro-6-methylaniline (3.0 g, 23.980 mmol) in water (30.0 ml) was cooled to 0°C, and bromoacetyl bromide (29 g, 143.884 mmol) was added. The reaction mixture was stirred at room temperature for 16 hours while monitoring the progress of the reaction by TLC (50% EtOAc in petroleum ether, visualization: UV). The reaction mixture was basicized with NaCO3, stirred for 20 minutes, filtered, washed with water (100 ml) followed by petroleum ether (50 mL), and dried to obtain the crude compound 2-bromo-N-(2-fluoro-6-methylphenyl)acetamide (2.8 g) as an off-white solid. MS (ESI): m / z 246.08 [M] + . 1 ¹H NMR (400 MHz, DMSO- d6 ) δ ppm 9.95 (s, 1 H), 7.18 (d, 3 H), 4.15 (s, 2 H), 2.20 (s, 3 H).

[0663] · Synthesis of 2-(azepan-1-yl)-N-(2-fluoro-6-methylphenyl)acetamide

[0664] Potassium carbonate (2.243 g, 48.762 mmol) was added at room temperature to a stirred solution of 2-bromo-N-(2-fluoro-6-methylphenyl)acetamide (4.0 g, 16.254 mmol) in ACN (40 ml), and the mixture was stirred for 10 minutes. Next, azepan (3.225 g, 32.509 mmol) was added, and the reaction mixture was stirred at room temperature for 16 hours while monitoring the progress of the reaction by TLC (50% EtOAc in petroleum ether, visualization: UV). The reaction mixture was diluted with ethyl acetate (100 ml), washed with water (2 x 50 ml), dried over sodium sulfate, filtered, and concentrated under reduced pressure. The crude compound was ground with n-pentane (3 x 30 ml) to obtain 2-(azepan-1-yl)-N-(2-fluoro-6-methylphenyl)acetamide (2.2 g) as a grayish-white solid. MS (ESI): m / z 264.34 [M] + . 1 1H NMR (400 MHz, CHLOROFORM- d ) δ ppm 8.88 (s, 1 H), 7.26 (s, 1 H), 7.10-7.16 (m, 1 H), 6.93-7.03 (m, 1 H), 3.31 (s, 2 H), 2.82 (t, 4 H), 2.27 (s, 3 H), 1.58-1.73 (m, 8 H).

[0665] · Synthesis of 1-benzyl-1-(2-((2-fluoro-6-methylphenyl)amino)-2-oxoethyl)azepan-1-um bromide

[0666] Benzyl bromide (1.9411 g, 11.35 mmol) was added at room temperature to a stirred solution of 2-(azepan-1-yl)-N-(2-fluoro-6-methylphenyl)acetamide (0.50 g, 1.89 mmol) in ACN (5.0 mL), and the resulting reaction mixture was heated at 80°C for 16 hours while monitoring the progress of the reaction by TLC (10% MeOH in DCM, visualization: UV). The reaction mixture was cooled to room temperature and concentrated under reduced pressure to obtain an unpurified compound, which was ground with a 1:1 solution of EtOAc / petroleum ether (3 x 10 mL) to obtain the product 1-benzyl-1-(2-((2-fluoro-6-methylphenyl)amino)-2-oxoethyl)azepan-1-ium bromide (200 mg). MS (ESI): m / z 355.2 [M] + . 1 ¹H NMR (400 MHz, DMSO- d6 ) δ ppm 10.19 (s, 1 H), 7.64 (t, 2 H), 7.51-7.57 (m, 3 H), 7.27-7.32 (m, 1 H), 7.17 (t, 2 H), 4.88 (s, 2 H), 4.11 (s, 2 H), 3.78 (q, 2 H), 3.51-3.56 (m, 2 H), 2.27 (s, 3 H), 1.96 (s, 4 H), 1.66 (s, 4 H).

[0667] The following examples were prepared from bromoacetyl bromide, benzyl bromide, azepan, and suitable aniline according to the procedure described for the synthesis of compound 69A.

[0668]

[0669]

[0670] Synthesis of 1-benzyl-1-(2-((2-hydroxy-6-methylphenyl)amino)-2-oxoethyl)azepan-1-um formate.

[0671]

[0672] Compound 79A

[0673] Synthesis of 1-benzyl-1-(2-((2-hydroxy-6-methylphenyl)amino)-2-oxoethyl)azepan-1-um

[0674] BBr3 (2.239 g, 8.94 mmol) was added at 0°C to a stirred solution of 1-benzyl-1-(2-((2-methoxy-6-methylphenyl)amino)-2-oxoethyl)azepan-1-um bromide (1 g, 2.235 mmol) in DCM (25 ml). The resulting reaction mixture was stirred at room temperature for 16 hours while monitoring the progress of the reaction mixture by TLC (10% MeOH in DCM, visualization: UV). The reaction mixture was concentrated under reduced pressure to obtain an unpurified product, which was purified by normal-phase flash chromatography (eluting with 6% MeOH in DCM) gradient. The pure fraction was concentrated under reduced pressure to obtain 1-benzyl-1-(2-((2-hydroxy-6-methylphenyl)amino)-2-oxoethyl)azepan-1-um bromide (0.9 g) as a grayish-white solid. MS (ESI): m / z 353.21 [M] + . 1 H NMR (300 MHz, DMSO-d6) δ ppm 9.82 (s, 1 H), 9.59 (s, 1 H), 7.73-7.71 (m, 2 H), 7.59-7.49 (m, 3 H), 7.07-7.02 (t, 1 H), 6.77-6.70 (m, 2 H), 4.88 (s, 2 H), 4.06 (s, 2 H), 3.77-3.72 (m, 2 H), 3.58-3.53 (m, 2 H), 2.17 (s, 3 H), 2.05-1.9 (m, 4 H), 1.66-1.45 (m, 4 H).

[0675] Synthesis of 1-benzyl-1-(2-((2-methyl-6-(propionyloxy)phenyl)amino)-2-oxoethyl)azepan-1-um bromide

[0676]

[0677] Compound 80A

[0678] Synthesis of 1-benzyl-1-(2-((2-methyl-6-(propionyloxy)phenyl)amino)-2-oxoethyl)azepan-1-um bromide

[0679] Propionic anhydride (5 ml) was added to 1-benzyl-1-(2-((2-hydroxy-6-methylphenyl)amino)-2-oxoethyl)azepan-1-um bromide (0.4 g, 0.922 mmol) at room temperature, and the resulting reaction mixture was heated to 80°C for 16 hours while monitoring the progress of the reaction mixture by TLC (10% MeOH in DCM, visualization: UV). The reaction mixture was concentrated under reduced pressure to obtain an unpurified product, which was purified by normal-phase flash chromatography (eluting with 15% MeOH in DCM). The pure fraction was concentrated under reduced pressure to obtain the product, which was then ground again with diethyl ether (45 ml) to obtain 1-benzyl-1-(2-((2-methyl-6-(propionyloxy)phenyl)amino)-2-oxyethyl)azepan-1-um bromide (95.6 mg) as a grayish-white solid. MS (ESI): m / z 409.2 [M] + . 1 ¹H NMR (400 MHz, DMSO- d 6) δ ppm 10.16 (s, 1 H), 7.66-7.64 (m, 2 H), 7.57-7.51 (m, 3 H), 7.31-7.27 (t, 1 H), 7.22-7.21 (d, 1 H), 7.09-7.07 (m, 1 H), 4.89 (s, 2 H), 4.08 (s, 2 H), 3.75-3.71 (m, 2 H), 3.51-3.47 (m, 2 H), 2.58 (q, 2 H), 2.27 (s, 3 H), 2.05-1.85 (m, 4 H), 1.75-1.55 (m, 4) H), 1.12 (t, 3 H).

[0680] Synthesis of 1-benzyl-1-(2-((2-carbamoyl-6-methylphenyl)amino)-2-oxoethyl)azepan-1-um bromide.

[0681]

[0682] Compound 80A

[0683] Synthesis of ·N,N,3-trimethyl-2-nitrobenzamide:

[0684] Dimethylamine (2M in THF) (16.56 ml, 33.12 mmol) was added to a stirred solution of 3-methyl-2-nitrobenzoic acid (3.0 g, 16.56 mmol) in DMF (30 ml), followed by DIPEA (6.42 g, 49.68 mmol) and HATU (9.44 g, 24.84 mmol). The resulting reaction mixture was stirred at room temperature for 16 hours while monitoring the progress of the reaction by TLC (30% EtOAc / petroleum ether, visualization: UV). The reaction mixture was poured into ice water and extracted with EtOAc (2 x 50 ml). The combined extract was washed with brine (30 ml), dried over Na2SO4, and concentrated under reduced pressure to obtain an unpurified product, which was purified by normal-phase flash chromatography (eluting with 20%–80% EtOAc in petroleum ether) to obtain N,N,3-trimethyl-2-nitrobenzamide (2.2 g) as a black liquid. Mass (ESI): m / z 209.09 [M+H] + . 1 H NMR (400 MHz, CDCl3) δ ppm 7.47-7.43 (m, 1 H), 7.36-7.33 (m, 1 H), 7.22-7.19 (m, 1 H), 3.09 (s, 3 H), 2.93 (s, 3 H), 2.80 (s, 6 H), 2.33 (s, 3 H).

[0685] Synthesis of ·2-amino-N,N,3-trimethylbenzamide:

[0686] Fe (3.75 g, 67.235 mmol) and NH4Cl (3.59 g, 67.235 mmol) were added at room temperature to a stirred solution of N,N,3-trimethyl-2-nitrobenzamide (2 g, 9.605 mmol) in ethanol (20 ml) and H2O (20 ml). The resulting reaction mixture was stirred at 80°C for 8 hours while monitoring the progress of the reaction by TLC (10% EtOAc in petroleum ether, visualization: UV). The reaction mixture was filtered through a Celite pad and washed twice with EtOH (2 x 50 ml). The filtrate was concentrated under reduced pressure to obtain the residue, which was diluted with water (50 ml) and extracted with EtOAc (2 x 50 ml). The combined organic extract was washed with brine (50 ml), dried over Na2SO4, and concentrated under reduced pressure to obtain an unpurified product, which was purified by column chromatography (eluting with 10%–40% EtOAc in petroleum) to obtain 2-amino-N,N,3-trimethylbenzamide (600 mg). LCMS purity: 98.02%, mass (ESI): m / z 179.12 [M+H] + . 1 H NMR (400 MHz, CDCl3) δ ppm 7.26 (s, 1 H), 7.06 (s, 1 H), 6.97 (d, 1 H), 6.66-6.63 (m, 1 H), 4.33 (bs, 2 H), 3.05 (s, 6 H), 2.17 (s, 3 H).

[0687] Synthesis of ·2-(2-bromoacetamido)-N,N,3-trimethylbenzamide:

[0688] 2-bromoacetyl bromide (5.34 g, 26.928 mmol) was added at 0°C to a stirred solution of isopropyl 2-amino-N,N,3-trimethylbenzamide (0.6 g, 3.366 mmol) in H2O (6 ml). The resulting reaction mixture was stirred at room temperature for 16 hours while monitoring the progress of the reaction by TLC (10% MeOH in DCM, visualization: UV). The reaction mixture was basicized with saturated Na2CO3 at 0°C to obtain a precipitated solid, which was filtered, washed with water, and dried to obtain (2-(2-bromoacetamido)-N,N,3-trimethylbenzamide) (650 mg) as a white solid. LCMS purity: 93.98%, mass (ESI): m / z 301.12 [M+2] +. 1 H NMR (400 MHz, CDCl3) δ ppm 8.84 (s, 1 H), 7.26-7.07 (m, 3 H), 3.93 (s, 2 H) 3.09 (s, 3 H), 2.93 (s, 3 H), 2.21 (s, 3 H).

[0689] Synthesis of 2-(2-(azepan-1-yl)acetamide)-N,N,3-trimethylbenzamide

[0690] K2CO3 (0.692 g, 5.012 mmol) and azepan (0.397 g, 4.01 mmol) were added at room temperature to a stirred solution of 2-(2-bromoacetamido)-N,N,3-trimethylbenzamide (0.6 g, 2.005 mmol) in acetonitrile (6 ml). The resulting reaction mixture was stirred at 80°C for 16 hours while monitoring the progress of the reaction by TLC (10% MeOH in DCM, visualization: UV). After the reaction was complete on the TLC, the reaction mixture was concentrated under reduced pressure to obtain the residue, poured into ice water, and extracted with EtOAc (2 x 25 ml). The combined organic extract was washed with brine (50 ml), dried over Na2SO4, and concentrated under reduced pressure to obtain (2-(2-(azepan-1-yl)acetamido)-N,N,3-trimethylbenzamide (600 mg). MS (ESI): m / z 318.27 [M+H] + . 1 H NMR (400 MHz, CDCl3) δ ppm 9.19 (s, 1 H) 7.27-7.25 (m, 1 H), 7.20-7.17 (m, 1 H), 7.09-7.07 (m, 1 H), 3.24 (s, 2 H) 3.04 (s, 3 H), 2.91 (s, 3 H), 2.79-2.76 (t, 4 H), 2.26 (s, 3 H) 1.71 - 1.62 (m, 8 H).

[0691] Synthesis of 1-benzyl-1-(2-((2-dimethylcarbamoyl-6-methylphenyl)amino)-2-oxoethyl)azepan-1-um bromide

[0692] Benzyl bromide (0.538 g, 3.15 mmol) was added at room temperature to a stirred solution of 2-(2-(azepan-1-yl)acetamido)-N,N,3-trimethylbenzamide (0.5 g, 1.575 mmol) in acetonitrile (5 ml). The resulting reaction mixture was stirred at 80°C for 48 hours while monitoring the progress of the reaction by TLC (mobile phase: 10% MeOH in DCM, Rf: 0.47, visualization: UV). After the consumption of the starting material on TLC, the reaction mixture was concentrated under reduced pressure to obtain an unpurified product, which was purified by column chromatography (eluting with 4%–8% MeOH in DCM) to obtain (1-benzyl-1-(2-((2-(dimethylcarbamoyl)-6-methylphenyl)amino)-2-oxoethyl)azepan-1-ium bromide (100 mg) as a brown solid. Mass (ESI): m / z 408.2 [M] + . 1 H NMR (400 MHz, DMSO-d6) δ ppm 10.31 (s, 1 H), 7.70-7.68 (m, 2 H), 7.56 - 7.51 (m, 3 H), 7.38-7.36 (m, 1 H), 7.32-7.28 (m, 1 H), 7.19-7.16 (m, 1 H), 4.85 (s, 2 H), 4.03 (s, 2 H), 3.70-3.66 (m, 2 H), 3.47-3.43 (m, 2 H), 2.95 (s, 3 H), 2.87 (s, 3 H), 2.27 (s, 3 H), 1.94-1.90 (s, 4 H), 1.70-1.65 (s, 4 H).

[0693] Synthesis of 1-Benzyl-1-(2-((2,6-Dimethylbenzoyl)oxy)ethyl)azepan-1-um bromide

[0694]

[0695] Compound 82A

[0696] · Synthesis of the intermediate 2-bromoethyl 2,6-dimethylbenzoate:

[0697] 2-bromoethanol-1-ol (4.16 g, 33.29 mmol) and a catalyst amount of concentrated H2SO4 (0.2 ml) were added to a stirred solution of 2,6-dimethylbenzoic acid (5 g, 33.29 mmol) in toluene (40 mL). The reaction mixture was refluxed for 16 hours using a Dean-Stark condenser while monitoring the progress of the reaction by TLC (30% EtOAc in petroleum ether, visualization: UV). After the reaction was complete, the mixture was cooled to room temperature and concentrated under reduced pressure to obtain the residue, which was diluted with ice water (250 ml) and extracted with EtOAc (3 x 150 ml). The combined organic extract was washed with a saturated sodium bicarbonate solution (50 ml) and brine (100 ml), dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain 2-bromoethyl 2,6-dimethylbenzoate (5.52 g). MS (ESI): m / z 258.82 [M+H+2] +. 1 H NMR (400 MHz, CDCl3) δ ppm 7.22-7.18 (m, 1 H), 7.0-7.02 (m, 2 H), 4.64 (t, 2 H), 3.63 (t, 2 H), 2.33 (s, 6 H).

[0698] · Synthesis of the intermediate 2-(azepan-1-yl)ethyl 2,6-dimethylbenzoate:

[0699] K2CO3 (3.224 g, 23.334 mmol) and azepan (1.157 g, 11.667 mmol) were added to a stirred solution of 2-bromoethyl 2,6-dimethylbenzoate (2.0 g, 7.778 mmol) in ACN (30 ml). The resulting mixture was stirred at 90°C for 16 hours while monitoring the progress of the reaction by TLC (30% EtOAc in petroleum ether, visualization: UV). The reaction mixture was cooled to room temperature and concentrated under reduced pressure to obtain the residue, which was diluted with cold water (100 ml) and extracted with EtOAc (3 x 50 ml). The combined organic extract was washed with brine (50 ml), dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain 2-(azepan-1-yl)ethyl 2,6-dimethylbenzoate (1.80 g) as a brown liquid. MS (ESI): m / z 276.35 [M + H] +. 1 H NMR (400 MHz, CDCl3) δ ppm 7.19-7.15 (m, 1 H), 7.03-7.01 (m, 2 H), 4.41 (t, 2 H), 2.86 (t, 2 H), 2.72-2.69 (mt, 4 H), 2.33 (s, 6 H), 1.65-1.56 (m, 8 H).

[0700] Synthesis of 1-benzyl-1-(2-((2,6-dimethylbenzoyl)oxy)ethyl)azepan-1-um bromide:

[0701] Benzyl bromide (279.3 mg, 1.633 mmol) was added to a stirred solution of 2-(azepan-1-yl)ethyl 2,6-dimethylbenzoate (300 mg, 1.089 mmol) in ACN (5 ml), and the resulting mixture was stirred at 90°C for 16 hours while monitoring the progress of the reaction by TLC (MeOH in DCM, visualization: UV). After consuming the starting materials, the reaction mixture was cooled to room temperature and then concentrated under reduced pressure to obtain an unpurified product, which was ground with a 1:2 mixture of Et2O and EtOAc (60 ml) to obtain 1-benzyl-1-(2-((2,6-dimethylbenzoyl)oxy)ethyl)azepan-1-um bromide (117.4 mg) as a grayish-white solid. Mass (ESI): m / z 366.2 [M] + . 1 H NMR (400 MHz, DMSO-d6) δ ppm 7.61-7.59 (m, 2 H), 7.54-7.48 (m, 3 H), 7.29-7.26 (m, 1 H), 7.13-7.11 (d, 2 H), 4.89 (t, 2 H), 4.67 (s, 2 H), 3.65-3.45 (m, 6 H), 2.28 (s, 6 H), 1.83-.1.77 (d, 4 H), 1.6-1.5 (m, 4 H).

[0702] Synthesis of 1-Benzyl-1-(2-(2,6-Dimethylphenoxy)-2-oxoethyl)azepan-1-um 2,2,2-trifluoroacetate

[0703]

[0704] Compound 83A

[0705] · Synthesis of the intermediate 2,6-dimethylphenyl 2-bromoacetate:

[0706] Pyridine (0.647 g, 8.184 mmol) and 2-bromoacetyl bromide (1.23 g, 6.138 mmol) were added to a stirred solution of 2,6-dimethylphenol (0.5 g, 4.092 mmol) in ACN (5 ml) at 0°C, and the resulting reaction mixture was stirred at 0°C for 15 minutes while monitoring the progress of the reaction by TLC (10% EtOAc in petroleum ether, visualization: UV). After the starting material was consumed, the reaction mixture was diluted with water (20 mL) and extracted with EtOAc (2 x 25 mL). The combined organic extract was washed with brine (30 ml), dried over Na2SO4, and concentrated under reduced pressure to obtain the product 2,6-dimethylphenyl 2-bromoacetate (500 mg) as a pale yellow liquid. 1 H NMR (400 MHz, CDCl3) δ ppm 7.07 (s, 3 H), 4.07 (s, 2 H), 2.18 (s, 6 H).

[0707] · Synthesis of the intermediate 2,6-dimethylphenyl 2-(azepan-1-yl)acetate:

[0708] K2CO3 (0.71 g, 5.14 mmol) and azepan (0.407 g, 4.112 mmol) were added at room temperature to a stirred solution of 2,6-dimethylphenyl 2-bromoacetate (0.5 g, 2.056 mmol) in ACN (5 ml). The resulting reaction mixture was stirred at 90°C for 16 hours while monitoring the reaction progress by TLC (mobile phase: ethyl acetate, visualization: UV). After the reaction was complete, the mixture was concentrated under reduced pressure to obtain the residue, which was diluted with ice water (25 ml) and extracted with ethyl acetate (2 x 100 ml). The combined organic extract was washed with brine (50 ml), dried over Na2SO4, and concentrated under reduced pressure to obtain 2,6-dimethylphenyl 2-(azepan-1-yl)acetate (500 mg) as a red liquid. Mass (ESI): m / z 262.09 [M+H] + .

[0709] Synthesis of 1-benzyl-1-(2-(2,6-dimethylphenoxy)-2-oxoethyl)azepan-1-ium 2,2,2-trifluoroacetate

[0710] Benzyl bromide (0.588 g, 3.442 mmol) was added to a stirred solution of 2,6-dimethylphenyl 2-(azepan-1-yl)acetate (0.45 g, 1.721 mmol) in ACN (4.5 ml), and the resulting reaction mixture was stirred at 90°C for 16 hours while monitoring the reaction progress by TLC (mobile phase: 10% MeOH in DCM, visualization: UV). After the reaction was complete, the reaction mixture was concentrated under reduced pressure to obtain an unpurified product, which was purified by reverse-phase preparative HPLC (Column: X-select phenylhexyl C18 (19*250), 5 µm; Mobile phase A: 0.1% TFA (Aq); Mobile phase B-acetonitrile, Flow rate: 15 ml / min, Method: 0 / 20, 2 / 20, 10 / 50, 15 / 75, 15.2 / 98, 19 / 98, 19.2 / 20, 23 / 20; Solubility: ACN + water + THF, Temperature: ambient).

[0711] The collected pure fraction was freeze-dried to obtain 1-benzyl-1-(2-(2,6-dimethylphenoxy)-2-oxoethyl)azepan-1-ium 2,2,2-trifluoroacetate (56 mg) as a pale yellow solid. MS (ESI): m / z 352.2 [M] + . 1 H NMR (400 MHz, DMSO-d6) δ ppm 7.60-7.53 (m, 5 H), 7.21-7.16 (m, 3 H), 4.87 (s, 2 H), 4.71 (s, 2 H), 3.90-3.84 (m, 2 H), 3.66-3.60 (m, 3 H), 2.19 (s, 6 H), 2.07-1.91 (m, 4 H), 1.40-1.20 (s, 4 H).

[0712] Synthesis of 1-Benzyl-1-(2-((2,6-Dimethylphenyl)amino)ethyl)azepan-1-ium Trifluoroacetate

[0713]

[0714] Compound 84A

[0715] · Synthesis of intermediate 1-(azepan-1-yl)-2-chloroethane-1-one:

[0716] 2-chloroacetyl chloride (13.6 g, 120.98 mmol) was added to a cooled solution of azepan (10 g, 100.826 mmol) (0°C) in ACN (100 mL), followed by TEA (30.6 g, 302.478 mmol). The reaction mixture was stirred at room temperature for 3 hours while monitoring the progress of the reaction by TLC (mobile phase: EtOAc, visualization by UV and ninhydrin). The reaction was quenched with saturated aqueous NaHCO3 and extracted with EtOAc (2 x 250 mL). The combined organic extract was washed with brine (200 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to obtain an unpurified product, which was purified by column chromatography (eluting with 30% EtOAc in petroleum ether) to obtain 1-(azepan-1-yl)-2-chloroethane-1-one (8.5 g) as a brown liquid. Mass (ESI): m / z 175.89 [M+1] + . 1 H NMR (400 MHz, CDCl3) δ ppm 4.08 (s, 2 H), 3.49-3.55 (m, 4 H), 1.72-1.79 (m, 4 H), 1.58-1.63 (d, 4 H).

[0717] · Synthesis of intermediate 1-(azepan-1-yl)-2-((2,6-dimethylphenyl)amino)ethane-1-one:

[0718] To a stirred solution of 1-(azepan-1-yl)-2-chloroethane-1-one (5 g, 28.464 mmol) in ACN (40 mL), 2,6-dimethylaniline (5.17 g, 42.696 mmol) was added after DIPEA (11.03 g, 85.392 mmol), and the resulting reaction mixture was stirred at 90°C for 3 days while monitoring the reaction progress by TLC (mobile phase: 30% EtOAc in petroleum ether, visualization: UV). The reaction mixture was quenched with saturated aqueous NaHCO3 (20 mL) and extracted with EtOAc (2 x 250 mL). The combined organic extract was washed with brine (200 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to obtain an unpurified product (8.5 g), which was purified by column chromatography (eluting with 30% EtOAc in petroleum ether) to obtain 1-(azepan-1-yl)-2-((2,6-dimethylphenyl)amino)ethane-1-one (2.7 g, 36%) as a brown solid. Mass (ESI): m / z 261.18 [M+1] + . 1 H NMR (400 MHz, cdcl3) δ ppm 6.97 - 7.99 (m, 2 H), 6.76 - 6.80 (m, 1 H), 4.78 (s, 1 H), 3.81 (s, 2 H), 3.33 - 3.58 (m, 2 H), 3.30 - 3.32 (m, 2 H), 2.34 (s, 6 H), 1.72 - 1.73 (m, 4 H), 1.55-1.59 (m, 5 H).

[0719] · Synthesis of the intermediate N-(2-(azepan-1-yl)ethyl)-2,6-dimethylaniline

[0720] A solution of 1-(azepan-1-yl)-2-((2,6-dimethylphenyl)amino)ethane-1-one (1.5 g, 5.760 mmol) in THF (15 mL) was cooled to 0°C, and LAH (2 M in THF, 5.76 mL, 11.521 mmol) was added dropwise while stirring. Subsequently, the resulting mixture was stirred at room temperature for 15 hours while monitoring the progress of the reaction by TLC (mobile phase: 30% EtOAc in petroleum ether, visualization: UV). The reaction mixture was quenched with a saturated aqueous NH4Cl solution at 0°C and then stirred at room temperature for 1 hour. The precipitated solid was removed by filtration, and the filtrate was concentrated under reduced pressure to obtain N-(2-(azepan-1-yl)ethyl)-2,6-dimethylaniline (1.4 g) as a pale yellow liquid. Mass (ESI): m / z 247.23 [M+1] + .

[0721] · Synthesis of the intermediate t-butyl(2-(azepan-1-yl)ethyl)(2,6-dimethylphenyl)carbamate:

[0722] To a stirred solution of N-(2-(azepan-1-yl)ethyl)-2,6-dimethylaniline (800 mg, 3.247 mmol) in a 1:1 mixture of 1,4-dioxane and H2O (10 ml), NaOH (260 mg, 6.494 mmol) was followed by (Boc)2O (2.12 g, 9.741 mmol), and the resulting mixture was stirred at room temperature for 24 hours while monitoring the reaction progress by TLC (mobile phase: 30% EtOAc in petroleum ether, detector: ninhydrin). The reaction mixture was diluted with water (75 mL), extracted with EtOAc (2 x 75 mL), and the combined organic extract was washed with brine (70 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The crude product was purified by column chromatography (elution with 15% EtOAc in petroleum ether) to obtain tert-butyl(2-(azepan-1-yl)ethyl)(2,6-dimethylphenyl)carbamate (600 mg) as a colorless liquid. Mass (ESI): m / z 347.24 [M+1] + . 1H NMR (400 MHz, DMSO-d6) δ ppm 7.00-7.26 (m, 3 H), 3.47-3.57 (m, 2 H), 2.60-2.73 (m, 6 H), 2.30 (s, 6 H), 2.22 (t, 11 H), 1.57 (s, 6 H).

[0723] · Synthesis of the intermediate 1-benzyl-1-(2-((tert-butoxycarbonyl)(2,6-dimethylphenyl)amino)ethyl)azepan-1-ium chloride:

[0724] Benzyl chloride (328 mg, 2.595 mmol) was added to a stirred solution of tert-butyl(2-(azepan-1-yl)ethyl)(2,6-dimethylphenyl)carbamate (300 mg, 0.865 mmol) in ACN (4 mL), and the resulting reaction mixture was stirred at 90°C for 15 hours while monitoring the reaction progress by TLC (mobile phase: 10% MeOH in DCM, visualization: UV). The reaction mixture was concentrated under reduced pressure to obtain crude 1-benzyl-1-(2-((tert-butoxycarbonyl)(2,6-dimethylphenyl)amino)ethyl)azepan-1-ium chloride salt (400 mg) as a colorless gum. Mass (ESI): m / z 437.30 [M+1] + .

[0725] Synthesis of 1-benzyl-1-(2-((2,6-dimethylphenyl)amino)ethyl)azepan-1-ium trifluoroacetate

[0726] 4 M HCl in 1,4-dioxane (3.17 mL, 12.68 OZ) was added to a stirred solution of 1-benzyl-1-(2-((tert-butoxycarbonyl)(2,6-dimethylphenyl)amino)ethyl)azepan-1-ium chloride (360 mg, 0.634 mmol) at 0°C. The resulting reaction mixture was stirred at room temperature for 15 hours while monitoring the reaction progress by TLC (mobile phase: 10% MeOH in DCM, visualization: UV). The reaction mixture was concentrated under reduced pressure to obtain an unpurified product, which was purified by reverse-phase preparative HPLC (Column: X-Bridge C18 (150*25) mm, 10 u; Mobile phase A: 0.1% TFA in water (aqueous); Mobile phase B: Acetonitrile, Flow rate: 14 ml / min, Method (T / %) of B: 0 / 20, 2 / 30, 10 / 40, 19 / 40, 19.2 / 98, 22 / 98, 22.2 / 20, 26 / 20, Solubility: ACN + WATER + THF, Temperature: Ambient). The pure fraction was collected and freeze-dried to obtain 1-benzyl-1-(2-((2,6-dimethylphenyl)amino)ethyl)azepan-1-ium trifluoroacetate (165 mg) as a light brown solid. Mass (ESI): m / z 337.30 [M] + . LCMS: 99.72%. 1 H NMR (400 MHz, DMSO-d6) δ ppm 7.41-7.53 (m, 5 H), 6.96-7.21 (m, 2 H), 6.83 (t, 1 H), 4.56 (s, 2 H), 3.31-3.53 (m, 8 H), 2.27 (s, 6 H), 1.82 - 1.83 (m, 4 H), 1.58 - 1.59 (m, 4 H).

[0727] Synthesis of 1-Benzyl-1-(2-((2,6-Dimethylphenoxy)ethyl)azepan-1-um Bromide

[0728]

[0729] Compound 85A

[0730] Synthesis of ·2-(2-bromoethoxy)-1,3-dimethylbenzene:

[0731] Potassium carbonate (1.698 g, 12.288 mmol) and 1,2-dibromoethane (3.847 g, 20.480 mmol) were added to a stirred solution of 2,6-dimethylphenol (0.500 g, 4.096 mmol) in acetonitrile (8.0 mL). The resulting reaction mixture was stirred at 90°C for 16 hours while monitoring the progress of the reaction by TLC (mobile phase: 5% EtOAc in petroleum ether, visualization: UV). The reaction mixture was cooled to room temperature, concentrated under reduced pressure, diluted with water (100 mL), and extracted with dichloromethane (3 x 50 mL). The combined organic extract was washed with a brine solution (1 x 50 ml), dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain an unpurified substance, which was purified by silica gel chromatography (eluted with petroleum ether) to obtain pure 2-(2-bromoethoxy)-1,3-dimethylbenzene (0.20 g) as a colorless liquid. 1 H NMR (400 MHz, CDCl3) δ ppm 7.00 - 7.25 (m, 2 H), 6.91 - 6.95 (m, 1 H), 4.01 - 4.10 (m, 2 H), 3.65 - 3.68 (m, 2 H), 2.30 (s, 6 H).

[0732] · Synthesis of the intermediate 1-(2-(2,6-dimethylphenoxy)ethyl)azepan:

[0733] DIPEA (451.2 mg, 3.49 mmol) and azepan (120.2 mg, 1.22 mmol) were added to a solution of 2-(2-bromoethoxy)-1,3-dimethylbenzene (200 mg, 0.87 mmol) in acetonitrile (3.0 mL). The resulting reaction mixture was stirred at 90°C for 16 hours while monitoring the reaction progress by TLC (mobile phase: 10% EtOAc in petroleum ether, visualization: UV). The reaction mixture was cooled to room temperature, concentrated under reduced pressure, diluted with water (20 mL), and extracted with dichloromethane (3 x 20 mL). The combined organic extract was washed with a brine solution (1 x 20 ml), dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain 1-(2-(2,6-dimethylphenoxy)ethyl)azepan (210 mg). 1 H NMR (400 MHz, CDCl3) δ ppm 6.98 - 7.00 (m, 2 H), 6.88 - 6.92 (m, 1 H), 3.85 - 3.88 (m, 2 H), 2.93 - 2.96 (m, 2 H), 2.75 - 2.77 (m, 4 H), 2.28 (s, 6 H), 1.59-1.68 (m, 8 H).

[0734] Synthesis of 1-benzyl-1-(2-((2,6-dimethylphenoxy)ethyl)azepan-1-um bromide:

[0735] Benzyl bromide (0.072 ml, 0.606 mmol) was added to a solution of 1-(2-(2,6-dimethylphenoxy)ethyl)azepan (100 mg, 0.404 mmol) in acetonitrile (1.5 mL), and the resulting reaction mixture was stirred in a sealed tube at 90°C for 16 hours while monitoring the progress of the reaction by TLC (mobile phase: 50% EtOAc in petroleum ether, visualization: UV). The reaction mixture was cooled to room temperature and concentrated under reduced pressure to obtain crude rubber, which was ground with ethyl acetate (3 x 5 ml) to obtain 1-benzyl-1-(2-(2,6-dimethylphenoxy)ethyl)azepan-1-ium bromide (80.5 mg) as an off-white solid. MS (ESI): m / z 338.41 [M] + . 1 H NMR (400 MHz, DMSO-d6) δ ppm 7.63 - 7.65 (m, 2 H), 7.49-7.57 (m, 3 H), 7.04 - 7.08 (m, 2 H), 6.96 - 6.98 (m, 1 H), 4.72 (s, 2 H), 4.29 (t, 2 H), 3.52-3.69 (m, 6 H), 2.29 (s, 6 H), 1.88 - 1.92 (m, 4 H), 1.59 - 1.62 (m, 4 H).

[0736] Synthesis of N-(2-((2,6-dimethylphenyl)amino)-2-oxoethyl)-N,N-diethyl-3-phenylpropane-1-amium bromide

[0737]

[0738] Compound 86A

[0739] · Synthesis of the intermediate N,N-diethyl-3-phenylpropane-1-amine:

[0740] Diethylamine (1.46 g, 19.96 mmol) was added at room temperature to a stirred solution of (3-bromopropyl)benzene (2 g, 10.04 mmol) in acetonitrile (5 ml). The resulting reaction mixture was stirred at 75°C for 16 hours while monitoring the progress of the reaction by TLC (mobile phase: 50% EtOAc in petroleum ether, UV visualization). The reaction mixture was allowed to cool to room temperature, quenched with water, and extracted with ethyl acetate (2 x 15 ml). The combined organic extract was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain N,N-diethyl-3-phenylpropane-1-amine (1.3 g). MS (ESI): m / z 192.25 [M+H] + . 1 H NMR (400 MHz, CDCl3) δ ppm 7.3 - 7.25 (m, 2 H), 7.22 - 7.15 (m, 3 H), 2.6 - 2.25 (m, 2 H), 2.55 - 2.42 (m, 6 H), 1.81 - 1.75 (m, 2 H), 1.0 (t, 6 H).

[0741] Synthesis of ·N-(2-((2,6-dimethylphenyl)amino)-2-oxoethyl)-N,N-diethyl-3-phenylpropane-1-amium bromide:

[0742] 2-bromo-N-(2,6-dimethylphenyl)acetamide (0.328 g, 1.354 mmol) was added to a solution of N,N-diethyl-3-phenylpropane-1-amine (0.2 g, 1.045 mmol) in toluene (5 ml), and the resulting mixture was heated and refluxed for 16 hours while monitoring the reaction progress by TLC (mobile phase: 10% MeOH in DCM, visualization: UV). The reaction mixture was cooled to room temperature and concentrated under reduced pressure to obtain an unpurified product, which was ground with EtOAc (20 ml) to obtain N-(2-((2,6-dimethylphenyl)amino)-2-oxoethyl)-N,N-diethyl-3-phenylpropane-1-aminium bromide (95 mg) as an off-white solid. MS (ESI): m / z 353.45 [M] + . 1H NMR (400 MHz, DMSO-d6) δ ppm 9.9 (s, 1 H), 7.45 - 7.3 (m, 5 H), 7.15 - 7.09 (m, 3 H), 4.28 (s, 2 H), 3.58 (q, 4 H), 3.51 - 3.45 (m, 2 H), 2.68 - 2.51 (m, 2 H), 2.15 (s, 6 H), 2.08 - 2.0 (m, 2 H), 1.28 (t, 6 H).

[0743] Synthesis of N-benzyl-2-((2,6-dimethylphenyl)sulfonyl)-N,N-diethylethane-1-amulium bromide

[0744]

[0745] Compound 87A

[0746] · Synthesis of the intermediate (2-chloroethyl)(2,6-dimethylphenyl)sulfan:

[0747] NaOH (1.736 g, 43.405 mmol) and 1-bromo-2-chloroethane (4.149 g, 28.937 mmol) were added at 0°C to a stirred solution of 2,6-dimethylbenzenethiol (2 g, 14.468 mmol, 1 equivalent) in ethanol (30 ml). The resulting reaction mixture was stirred at room temperature for 16 hours while monitoring the progress of the reaction mixture by TLC (mobile phase: 100% petroleum ether, visualization: UV). The reaction mixture was concentrated under reduced pressure to obtain an unpurified residue, which was diluted with ethyl acetate (80 ml), washed with water (50 ml x 2) and brine, dried over anhydrous Na2SO4, and concentrated under reduced pressure to obtain an unpurified product. The unrefined product was purified by flash chromatography (elution with petroleum ether). The collected pure fraction was concentrated under reduced pressure to obtain (2-chloroethyl)(2,6-dimethylphenyl)sulfan (0.500 g) as a colorless liquid. 1 H NMR (400 MHz, CDCl3) δ ppm 7.085-7.163 (m, 3 H), 3.502-3.542 (m, 2 H), 2.963-3.003 (m, 2 H), 2.544 (s, 6 H).

[0748] · Synthesis of the intermediate 2-((2-chloroethyl)sulfonyl)-1,3-dimethylbenzene:

[0749] Hydrogen peroxide (33% in H2O) (0.847 g, 24.910 mmol) was added at room temperature to a stirred solution of (2-chloroethyl)(2,6-dimethylphenyl)sulfan (0.500 g, 2.491 mmol) in acetic acid (10 ml), and the reaction mixture was heated to 110°C for 16 hours while monitoring the progress of the reaction by TLC (mobile phase: 20% ethyl acetate in petroleum ether, visualization: UV). The reaction mixture was diluted with water (60 ml) and extracted with ethyl acetate (70 ml x 3). The combined organic extracts were washed with aqueous sodium thiosulfate (50 ml x 2) and a brine solution (50 ml), then concentrated under reduced pressure to obtain an unpurified product, which was purified by flash chromatography (eluting with 10% ethyl acetate in petroleum). The collected pure fraction was concentrated under reduced pressure to obtain 2-((2-chloroethyl)sulfonyl)-1,3-dimethylbenzene (0.350 g) as a grayish-white solid. MS (ESI): m / z 232 [M] + . 1 H NMR (400 MHz, CDCl3) δ ppm 7.345-7.383 (m, 1 H), 7.176-7.195 (m, 2 H), 3.815-3.854 (m, 2 H), 3.531-3.569 (m, 2 H), 2.714 (s, 6 H).

[0750] · Synthesis of the intermediate 2-((2,6-dimethylphenyl)sulfonyl)-N,N-diethylethane-1-amine:

[0751] Potassium iodide (0.428 g, 2.578 mmol) and diethylamine (0.283 g, 3.867 mmol) were added at room temperature to a stirred solution of 2-((2-chloroethyl)sulfonyl)-1,3-dimethylbenzene (0.300 g, 1.289 mmol) in acetonitrile (10 ml). The resulting reaction mixture was heated at 90°C in a sealed tube for 16 hours while monitoring the progress of the reaction mixture by TLC (mobile phase: 50% ethyl acetate in petroleum ether, visualization: UV). The reaction mixture was concentrated under reduced pressure to obtain an unpurified product, which was diluted with ethyl acetate (60 ml), washed with water (30 ml x 3) and brine (30 ml), dried over anhydrous Na2SO4, concentrated under reduced pressure to obtain an unpurified product, and purified by flash chromatography (eluted with 40% ethyl acetate in petroleum ether). The collected pure fraction was concentrated under reduced pressure to obtain the target product 2-((2,6-dimethylphenyl)sulfonyl)-N,N-diethylethane-1-amine (0.200 g) as an off-white solid. MS (ESI): m / z 270.16 [M+H] + . 1 H NMR (400 MHz, CDCl3) δ ppm 7.308-7.346 (m, 1 H), 7.153-7.172 (m, 2 H), 3.244-3.283 (m, 2 H), 2.947-2.985 (m, 2 H), 2.716 (s, 6 H), 2.437-2.490 (m, 4 H), 0.937-0.973 (m, 6 H).

[0752] Synthesis of N-Benzyl-2-((2,6-Dimethylphenyl)sulfonyl)-N,N-Diethylethane-1-Amium Bromide:

[0753] Benzyl bromide (0.127 g, 0.742 mmol) was added to a stirred solution of 2-((2,6-dimethylphenyl)sulfonyl)-N,N-diethylethane-1-amine (0.100 g, 0.371 mmol) in acetonitrile (5 ml), and the resulting reaction mixture was heated at 90°C for 16 hours while monitoring the progress of the reaction mixture by TLC (mobile phase: 10% MeOH in DCM, visualization: UV). The reaction mixture was concentrated under reduced pressure to obtain a crude product, which was ground with EtOAc (20 ml) and n-pentane (10 ml) to obtain N-benzyl-2-((2,6-dimethylphenyl)sulfonyl)-N,N-diethylethane-1-aluminum bromide (0.060 mg) as a grayish-white solid. MS (ESI): m / z 360.0 [M] + . 1 H NMR (400 MHz, DMSO-d6) δ ppm 7.475-7.552 (m, 6 H), 7.347-7.366 (d, 2 H), 4.604 (s, 2 H), 3.925-3.965 (m, 2 H), 3.520-3.560 (m, 2 H), 3.243-3.288 (m, 4 H), 2.676 (s, 6 H), 1.235-1.312 (m, 6 H).

[0754] Synthesis of 1-Benzyl-1-(2-((2,6-Dimethylphenyl)sulfonyl)ethyl)azepan-1-um bromide

[0755]

[0756] Compound 88A

[0757] · Synthesis of the intermediate (2-((2,6-dimethylphenyl)sulfonyl)ethyl):

[0758] Azepan (0.639 g, 6.4455 mmol) and potassium iodide (1.426 g, 8.5940 mmol) were added to a stirred solution of 2-((2-chloroethyl)sulfonyl)-1,3-dimethylbenzene (1.0 g, 4.2970 mmol) in ACN (10.0 ml). The resulting reaction mixture was heated and refluxed for 16 hours while monitoring the progress of the reaction by TLC (mobile phase: 30% EtOAc in petroleum ether, visualization: UV). The reaction mixture was diluted with ethyl acetate (50 mL) and washed with water (2 x 50 ml) and brine (1 x 50 mL). Subsequently, the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain an unpurified compound, which was ground with 2 x 10 ml of n-pentane to obtain (2-((2,6-dimethylphenyl)sulfonyl)ethyl)azepan (180 mg) as a grayish-white solid. MS (ESI): m / z 295.44 [M+H]. 1 H NMR (400 MHz, CDCl3) δ ppm 7.34 (t, 1 H), 7.17 (d, 2 H), 3.1-3.8 (m. 6H), 2.71 (d, 6 H), 1.57-1.82 (m, 10 H), 2.71 (d, 6 H).

[0759] Synthesis of 1-benzyl-1-(2-((2,6-dimethylphenyl)sulfonyl)ethyl)azepan-1-um bromide:

[0760] Benzyl bromide (0.637 g, 3.728 mmol) was added to a stirred solution of 1-(2-((2,6-dimethylphenyl)sulfonyl)ethyl)azepan (0.550 g, 1.864 mmol) in ACN (6.0 ml), and the resulting reaction mixture was heated and refluxed for 48 hours while monitoring the progress of the reaction by TLC (mobile phase: 10% methanol in DCM, visualization: UV). The reaction mixture was cooled to room temperature and concentrated under reduced pressure to obtain an unpurified compound, which was ground with ethyl acetate (3 x 10 ml) to obtain 1-benzyl-1-(2-(2,6-dimethylphenoxy)sulfonyl)ethyl)azepan-1-ium bromide (80 mg) as an off-white solid. MS (ESI): m / z 386.40 [M] + . 1 H NMR (400 MHz, DMSO-d6) δ ppm 7.5-7.63 (m, 6 H), 7.44 (d, 2 H), 4.59-4.65 (d, 2 H), 4.06-4.15 (m, 2 H), 3.49-3.55 (m, 6 H), 2.696 (s, 6 H), 1.817-1.95 (m, 4 H), 1.569-1.65 (m, 4 H).

[0761] Synthesis of 1-Benzyl-1-(1-(2,6-Dimethylphenyl)-2-oxopyrrolidine-3-yl)piperidine-1-um bromide

[0762]

[0763] Compound 89A

[0764] · Synthesis of 3-Bromo-1-(2,6-Dimethylphenyl)pyrrolidin-2-one:

[0765] A stirred solution of 2,6-dimethylaniline (1.0 g, 8.252 mmol) in ACN (20 ml) was treated with K3PO4 (1.751 g, 8.252 mmol) and 2,4-dibromobutanoyl chloride (2.181 g, 8.252 mmol) at 0°C, and the reaction mixture was stirred at room temperature for 1 hour. After 1 hour, 50% NaOH aqueous solution (1.650 g in 3.32 mL of water) was added to the resulting reaction mixture, and the reaction mixture was stirred at room temperature for an additional 1 hour while monitoring the progress of the reaction mixture by TLC (mobile phase: 50% ethyl acetate in petroleum ether, visualization: UV). After completion, the reaction mixture was filtered to remove inorganic salts, the filtrate was dried over anhydrous Na2SO4, and concentrated under reduced pressure to obtain an unpurified product as colorless rubber. The crude product was ground with n-pentane (30 ml) to obtain 3-bromo-1-(2,6-dimethylphenyl)pyrrolidin-2-one (1.5 g). MS (ESI): m / z 268.03 [M+H] + . 1 H NMR (400 MHz, CDCl3) δ ppm 7.18-7.14 (m, 1 H), 7.10-7.08 (m, 2 H), 4.56-4.54 (m, 1 H), 3.92-3.86 (m, 1 H), 3.51-3.46 (m, 1 H), 2.84-2.78 (m, 1 H), 2.52-2.47 (m, 1 H), 2.29 (s, 3 H), 2.19 (s, 3 H).

[0766] · Synthesis of 1-(2,6-dimethylphenyl)-3-(piperidin-1-yl)pyrrolidin-2-one:

[0767] Piperidine (0.114 g, 1.342 mmol) and DIPEA (0.578 g, 4.475 mmol) were added at room temperature to a stirred solution of 3-bromo-1-(2,6-dimethylphenyl)pyrrolidin-2-one (0.300 g, 1.118 mmol) in ACN (10 ml), and the resulting mixture was heated at 90°C for 16 hours in a sealed tube while monitoring the progress of the reaction mixture by TLC (mobile phase: 10% methanol in DCM, visualization: UV). The reaction mixture was concentrated under reduced pressure to obtain an unpurified product, which was diluted with DCM (70 ml) and washed with water (40 ml x 3) and brine (40 ml). The organic layer was dried over anhydrous Na2SO4 and concentrated under reduced pressure to obtain 1-(2,6-dimethylphenyl)-3-(piperidin-1-yl)pyrrolidin-2-one (0.220 g). MS (ESI): m / z 273.35 [M+H] + . 1 H NMR (400 MHz, CDCl3) δ ppm 7.14-7.06 (m, 3 H), 3.70-3.48 (m, 3 H), 2.80-2.89 (m, 2 H), 2.60-2.65 (m, 2 H), 2.35-2.28 (m, 2 H), 2.21 (s, 3 H), 2.15 (s, 3 H), 1.67-1.60 (m, 4 H), 1.50-1.46 (m, 2 H).

[0768] · Synthesis of 1-Benzyl-1-(1-(2,6-Dimethylphenyl)-2-oxopyrrolidine-3-yl)piperidine-1-um bromide:

[0769] Benzyl bromide (0.276 g, 1.615 mmol) was added to a stirred solution of 1-(2,6-dimethylphenyl)-3-(piperidin-1-yl)pyrrolidin-2-one (0.220 g, 0.807 mmol) in ACN (5 ml), and the resulting mixture was heated in a sealed tube at 90°C for 16 hours while monitoring the progress of the reaction mixture by TLC (mobile phase: 10% methanol in DCM, visualization: UV). The reaction mixture was cooled to room temperature, diluted with ethyl acetate (20 ml), and stirred for 2 hours to obtain a solid, which was filtered, washed with ethyl acetate (40 ml) followed by n-pentane (20 ml), and dried under high vacuum to obtain 1-benzyl-1-(1-(2,6-dimethylphenyl)-2-oxopyrrolidine-3-yl)piperidine-1-um bromide (0.090 g) as a grayish-white solid. MS (ESI): m / z 363.1 [M] + . 1 H NMR (400 MHz, DMSO-d6) δ ppm 7.60-7.52 (m, 5 H), 7.25-7.16 (m, 3 H), 5.58-4.02 (m, 4 H), 3.65-3.57 (m, 2 H), 3.13-2.92 (m, 3 H), 2.67-2.66 (m, 1 H), 2.21-1.94 (m, 10 H), 1.61-1.23 (m, 3 H).

[0770] The following example was prepared from 3-bromo-1-(2,6-dimethylphenyl)pyrrolidin-2-one, azepan, and benzyl bromide according to the procedure described for the synthesis of compound 89A.

[0771]

[0772] Synthesis of 1-Benzyl-1-(1-(2,6-Dimethylphenyl)-2-oxopiperidine-3-yl)pyrrolidin-1-um bromide:

[0773]

[0774] Compound 91A(493)

[0775] · Synthesis of 2,5-Dibromopentanoyl Chloride:

[0776] A stirred suspension of 5-bromopentanoyl chloride (5 g, 25.066 mmol) was treated with bromine (2.56 ml, 50.132 mmol) at room temperature, and the resulting reaction mixture was stirred at 100°C for 2 hours. After 2 hours, the reaction mixture was cooled to room temperature and concentrated under reduced pressure to obtain unpurified 2,5-dibromopentanoyl chloride (7.2 g), which was used in the next step without further purification.

[0777] · Synthesis of 3-bromo-1-(2,6-dimethylphenyl)piperidin-2-one:

[0778] A stirred solution of 2,6-dimethylaniline (2.0 g, 16.504 mmol) and potassium phosphate (3.503 g, 16.504 mmol) in acetonitrile (50 ml) was cooled to 0°C, and 2,5-dibromopentanoyl chloride (4.594 g, 16.504 mmol) was added dropwise. The resulting reaction mixture was stirred at room temperature for 1 hour. After 1 hour, a 50% aqueous NaOH solution (3.3 g, 6.6 ml water) was added to the reaction mixture, and the resulting reaction mixture was stirred at room temperature for 1 hour while monitoring the progress of the reaction by TLC (mobile phase: 50% EtOAc: petroleum ether, visualization: US). The reaction mixture was filtered to remove inorganic salts, the filtrate was dried over anhydrous Na2SO4, and concentrated under reduced pressure to obtain an unpurified product, which was purified by column chromatography (eluting with 25%–30% EtOAc in petroleum ether) to obtain 3-bromo-1-(2,6-dimethylphenyl)piperidin-2-one (2.2 g) as a grayish-white solid. MS (ESI): 282.25 m / z, [M+H] + . 1H NMR (400 MHz, CDCl3) δ ppm 7.15-7.08 (m, 3 H), 4.87 (t, 1 H), 3.54-3.49 (m, 1 H), 3.37-3.34 (m, 1 H), 2.51-2.45 (m, 1 H), 2.27-2.21 (m, 2 H), 2.22-2.10 (m, 6 H),1.94 (m, 1 H).

[0779] · Synthesis of 1-(2,6-dimethylphenyl)-3-(piperidin-1-yl)piperidin-2-one:

[0780] Pyrrolidine (0.0655 g, 0.921 mmol) was added to a solution of 3-bromo-1-(2,6-dimethylphenyl)piperidin-2-one (0.2 g, 0.709 mmol) and DIPEA (0.366 g, 2.83 mmol) in acetonitrile (10 mL), and the resulting reaction mixture was stirred in a sealed tube at 90°C for 16 hours while monitoring the reaction progress by TLC (mobile phase: 50% EtOAc in petroleum ether, visualization: UV). The reaction mixture was concentrated under reduced pressure to obtain an unpurified residue, which was diluted with water (20 ml) and extracted with ethyl acetate (2 x 50 ml). The combined organic extracts were dried and concentrated over anhydrous Na2SO4 to obtain an unpurified product, which was purified by normal-phase flash chromatography (eluting with 25%–30% EtOAc in petroleum ether) to obtain 1-(2,6-dimethylphenyl)-3-(pyrrolidin-1-yl)piperidin-2-one (350 mg) as a pale yellow gummy oil. LCMS purity: 75.17%, MS (ESI): 273.43 m / z, [M+H] + .

[0781] · Synthesis of 1-Benzyl-1-(1-(2,6-Dimethylphenyl)-2-oxopiperidine-3-yl)pyrrolidin-1-um bromide:

[0782] Benzyl bromide (0.2041 g, 1.1931 mmol) was added to a stirred solution of 1-(2,6-dimethylphenyl)-3-(pyrrolidin-1-yl)piperidin-2-one (250 mg, 0.9178 mmol) in acetonitrile (10 ml), and the resulting reaction mixture was stirred in a sealed tube at 90°C for 16 hours while monitoring the progress of the reaction by TLC (mobile phase: 10% methanol in DCM, visualization: UV). The reaction mixture was cooled to room temperature and concentrated under reduced pressure to obtain an unpurified product, which was ground with EtOAc (20 mL x 2) to obtain 1-benzyl-1-(1-(2,6-dimethylphenyl)-2-oxopiperidin-3-yl)pyrrolidin-1-um bromide (105 mg) as a grayish-white solid. MS (ESI): m / z 363.4 [M] + . 1 ¹H NMR (400 MHz, DMSO- d 6) δ ppm 7.64-7.62 (m, 2 H), 7.57-7.48 (m, 3 H), 7.20-7.13 (m, 3 H), 4.78 (m, 3 H), 4.03-4.01 (m, 2 H), 3.80 -3.72 (m, 2 H), 3.55-3.50 (m, 1 H), 3.41-3.61 (m, 1 H), 2.51-2.49 (m, 1 H), 2.39-2.35 (m, 1 H), 1.21-1.99 (m, 10 H), 1.97-1.90 (m, 2 H).

[0783] The following examples were prepared from 3-bromo-1-(2,6-dimethylphenyl)piperidin-2-one, benzyl bromide, and a suitable azacycloalkane according to the procedure described for the synthesis of compound 91A. The product was purified by grinding or reverse-phase preparative HPLC.

[0784]

[0785] The following example was prepared from bromoacetyl bromide, benzyl bromide, a suitable azacycloalkane, and substituted aniline according to the procedure for preparing compound 69A.

[0786]

[0787]

[0788]

[0789] Example 2 - Nav1.7 Current Suppression

[0790] Representative compounds of the present invention were synthesized according to the described method and tested for their ability to inhibit voltage-gated sodium channels.

[0791] Manual Patch Clamp:

[0792] cell culture

[0793] NaV1.7 was expressed in HEK293 upon tetracycline induction. Cells were treated with 10% dialyzed fetal bovine serum (VWR, Radnor, PA), 1% glutamex (VWR, Radnor, PA), 1% penicillin-streptomycin (VWR, Radnor, PA), 100 mg / L hygromycin (Thermo Fisher Scientific, Waltham, MA), and 5 mg / L blasticidin (Alfa Aesar, Haverhill, MACells were cultured in DMEM containing ). Cells were grown and maintained at 37°C in a humidified environment containing 10% CO2 in the air. Cells were detached from culture flasks for subculturing and harvested using 0.05% trypsin-EDTA (Thermo Fisher Scientific, Waltham, MA). To induce NaV1.7, cells were induced with tetracycline (0.1–1 μg / mL, IBI Scientific, Peosta, IA) the day before recording and plated in 24-well plates. Cells were washed with DPBS (VWR, Radnor, PA), and after trypsin treatment, cell aggregates were separated by pulverizing five times in 10 mL of growth medium. For one 24-well plate, 2 mL of cell suspension was mixed with 23 mL of fresh growth medium, and 0.1–1 μg / mL of tetracycline was added. Subsequently, 1 ml of medium mixed with cells was added to each well of a 24-well plate in which a 12 mm coverslip was already placed at the bottom of the wells. Then, the cells were incubated overnight at 37°C and 10% CO2.

[0794] Patch Clamp Solution & Drug

[0795] The intracellular solution contained 135 mg of CsCl, 10 mg of NaCl, 10 mg of EGTA, 10 mg of HEPES, and 22 mg of MgCl (in mM units) and was adjusted to pH 7.2 with CsOH. The external solution was normal Ringer's solution containing 155 mg of NaCl, 10 mg of HEPES, 10 mg of glucose, 3.5 mg of KCl, 1.5 mg of CaCl2, and 21 mg of MgCl2 (in mM units) and adjusted to pH 7.4 using NaOH. The CsCl was from Alfa Aesar, Haverhill, MA. All other chemicals were from Sigma-Aldrich, St. Louis, MO. To test the degree of internal blockade by the test compound, the compound was dissolved in the internal solution at the indicated test concentration. In the control experiment, the internal solution did not contain any compound. To test the degree of external blockade by the test compound, the compound was dissolved in the external solution at the indicated test concentration.

[0796] Whole Cell Patch Clamp Protocol

[0797] 18–24 hours after inducing cells with tetracycline, the coverslip was placed in a chamber filled with normal Ringer's solution at room temperature, and the chamber was placed under the microscope. The pipette was removed from the borosilicate glass of a P97 puller (Sutter Instrument, Novato, CA) and polished with an MF-830 Microforge (Narishige International USA, Inc, Amityville, NY) to have a resistance of 1.5–2.5 MΩ when filled with an internal CsCl solution at room temperature. Healthy cells (spherical, translucent cells without visible defects) were selected for seal formation. Once a seal was formed between the pipette and the cells, they were "broken in" using a short suction pulse, and the total cell composition was established. The membrane potential was maintained at -100 mV before the voltage protocol began. Only cells with a series resistance of 1.5 to 5 MΩ were retained for analysis. The voltage protocol was as follows: the cells were maintained at -100 mV for 12 ms, followed by a hyperpolarization step at -105 mV for 12 ms to monitor for leakage. Subsequently, the cells were reverted to -100 mV for 40 ms. Afterward, the cells were depolarized to -20 mV for 10 ms, followed by a return to -100 mV for 26 ms.

[0798] Internal blockage by test compound

[0799] When recording began, the voltage protocol was executed at 30-second intervals for 5 minutes to acquire a stable baseline. This was followed by four 30-second cycles of 5 Hz stimulation of the same voltage protocol, separated by a 1-minute rest, and then a 0.33 Hz stimulation after the last training. Current was recorded using PatchMaster software with the Heka EPC10 (HEKA Electronics, Lambrecht, Germany). Only cells with an internal current amplitude of -20 mV between 400 pA and 4 nA were allowed. Additionally, cells with a leakage current exceeding 10% of the current amplitude were discarded.

[0800] Data Analysis: Internal Blocking

[0801] The data were plotted using Patchmaster software (HEKA Electronics, Lambrecht, Germany), and the minimum current during voltage steps up to -20 mV (peak internal current) was plotted and analyzed as a function of time. To determine the degree of rundown throughout the experiment, the average peak internal current amplitude (2-3 points) prior to the 5 Hz stimulation was set as the baseline (I baseline It was designated as ). The average peak internal current during the last 2 seconds of the last 5 Hz train was measured (I test ). I test ul I baseline The remaining control fraction current was calculated by dividing by... Three cells were tested with the control internal solution on each recording day, and the average fraction of the remaining current was calculated (control fraction current).

[0802] To determine the % blocking generated by the internally applied test compound, the following was performed. The average peak internal current amplitude (2-3 points) before 5 Hz stimulation was 0% blocking (I 0%block It was designated as ). To correct for current changes under control group conditions, I 0%blockThe modified 0% cutoff current was obtained by multiplying it by the remaining average control fraction current. The average peak internal current during the last 2 seconds of the last 5 Hz train was the uncutoff current (I unblocked It was designated as ). The blocking % was calculated using the following equation: (1 - I unblocked / ( I 0%block * Remaining control fraction current) x 100).

[0803] Representative examples of the present invention were tested for intracellular inhibition of NaV 1.7. The range of activity is % inhibition at a test concentration of 10 μM: "++++" (>95%), "+++" 95-70%, "++" (70-40%), or "+" (< 40%). The results are presented in the table below.

[0804]

[0805] Representative examples of the present invention were tested for intracellular inhibition of NaV 1.7. The range of activity is % inhibition at 3 μM: "++++" (>90%), "+++" 95-70%, "++" (70-40%), or "+" (< 40%). The results are presented in the table below.

[0806]

[0807]

[0808] External blockage by the test compound

[0809] Once recording began, a voltage protocol was executed at 30-second intervals for 5 minutes to establish a stable baseline. A 5 Hz stimulus of the same voltage protocol was followed until the end of the experiment. The test compound was added during the 5 Hz stimulus train to allow cells to wait until they exhibited a stable current rundown rate before compound addition. After adding the test compound for 5 minutes, the cells were washed with standard Ringer's solution. Current was recorded using PatchMaster software with a Heka EPC10 (HEKA Electronics, Lambrecht, Germany). Only cells with an internal current amplitude of -20 mV between 400 pA and 4 nA were allowed. Additionally, cells with a leakage current exceeding 10% of the current amplitude were discarded.

[0810] Data Analysis: External Blocking

[0811] The data were plotted using Patchmaster software (HEKA Electronics, Lambrecht, Germany), and the minimum current during voltage steps up to -20 mV (peak internal current) was plotted and analyzed as a function of time. To determine the % cutoff generated by the externally applied test compound, the following was performed: After a stable current rundown rate was established during a 5 Hz excitation train, the rate was calculated by dividing the change in peak current amplitude by time. rundown was calculated. Using the average peak internal current amplitude (2-3 sec) before compound addition, 0% cutoff (I 0%block Decided on ). To modify the rundown, I 0%block (speed rundown The modified 0% cutoff current was obtained by subtracting * 5 min). The average peak internal current during the last 2-3 seconds of the 5-minute compound application time before washing was the uncutoff current (I unblocked ...is. Subsequently, the blocking % was calculated using the following equation: Fractional current blocking = 1 - I unblocked / ( I0%block - Speed rundown * 5 minutes).

[0812] Representative examples of the present invention were tested for extracellular inhibition of NaV 1.7. The range of activity is % inhibition at a test concentration of 10 μm: "++++" (>90%), "+++" 95-70%, "++" (70-40%), or "+" (< 40%). The results are presented below.

[0813]

[0814] Automated Patch Clamp:

[0815] cell culture

[0816] NaV1.7 was expressed in HEK293 cells upon tetracycline induction. Cells were cultured in DMEM containing 10% dialyzed fetal bovine serum (VWR, Radnor, PA), 1% glutamax (VWR, Radnor, PA), 1% penicillin-streptamicin (VWR, Radnor, PA), 100 mg / L hygromycin (Thermo Fisher Scientific, Waltham, MA), and 5 mg / L blasticidin (Alfa Aesar, Haverhill, MA). Cells were grown and maintained at 37°C in a humidified environment containing 10% CO2 in the air. Cells were detached from culture flasks for passage and harvested using 0.05% trypsin-EDTA (Thermo Fisher Scientific, Waltham, MA). To induce NaV1.7, cells were induced with tetracycline (0.1 - 1 μg / mL, IBI Scientific, Peosta, IA) the day before recording.

[0817] Before the experiment, cells were washed with DPBS (VWR, Radnor, PA), digested with Detachin, and then resuspended by grinding 10 times in CHO serum-free medium (VWR, Radnor, PA) to separate cell aggregates. Cells were counted, and the final concentration was set to 2 million to 5 million cells per mL.

[0818] Patch Clamp Solution & Drug

[0819] The intracellular solution contained the following: 140 mM CsF, 1 mM / 5 mM EGTA / CsOH, 10 mM HEPES, 10 mM NaCl, pH 7.3 using CsOH, and osmolal concentration 320 using OSM solution. The extracellular solution contained the following: 145 mM NaCl, 4 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 10 mM HEPES, 10 mM glucose, pH 7.4 using CsOH, and osmolal concentration 305 using OSM solution. The OSM solution was standard Ringer's solution containing 155% NaCl, 10% HEPES, sucrose, 3.5% KCl, 1.5% CaCl2, and 1% MgCl2, adjusted to pH 7.4 using NaOH. All chemicals were from Sigma-Aldrich (St. Louis, MO). To test the degree of internal blockade by the test compound, the compound was dissolved in the internal solution at the indicated test concentration. In the control experiment, the internal solution contained no compound. To test the degree of external blockade by the test compound, the compound was dissolved in the external solution at the indicated test concentration.

[0820] Automated Patch Clamp Protocol

[0821] Automated patch clamping was performed on a Qube 384 (Sophion Bioscience, Woburn MA) equipped with a multi-hole Qchip at a temperature setting of 22°C. The entire cell configuration was formed using default Qube sealing and break-in parameters. The membrane potential was maintained at -100 mV before the voltage protocol was initiated. Two voltage protocols were followed.

[0822] Step 1: Cells were maintained at -100 mV with a depolarization pulse of -20 mV for 10 ms, and the interval was set to 5 s. The current was modified by subtracting the default leakage from all pulses. The duration was set to 5 minutes.

[0823] Step 2: The cells were maintained at -100 mV with a depolarization pulse of -20 mV for 10 ms. The frequency was 5 Hz. The current was modified by the leakage deduction calculated prior to Step 2. The duration was set to 4 minutes.

[0824] Internal blockage by test compound

[0825] After Step 2, the Q-chip was removed from the recording chamber. The internal solution was changed to a solution containing the test compound. After replacing the Q-chip in the recording chamber, it was maintained at -100mV without pulses. The total solution switching time was 8 minutes. After the internal solution exchange, the cells were recorded, and Step 2 was repeated for 10 minutes.

[0826] Data analysis was performed using a Sophion Analyzer. Cells were filtered with a minimum sealing resistance of 50 MOhm and a minimum starting current of 5 nA. The current rundown was adjusted for control cells (non-drug). The remainder was calculated by averaging the last three points at the end of the experiment. The baseline was calculated as the average of the last three points of Step 2. IC 50The curve was plotted using the DR-plot / Hill function (dose-response plot with Hill fit). A representative example of the present invention was tested for the intracellular inhibition of NaV 1.7 in an automated patch clamp assay. The active range is IC50. 50 It is reported as: "++++" (< 0.3 μM), "+++" (1-3 μM), "++" (3-10 μM) or "+" (10-30 μM). The results are presented below.

[0827]

[0828] External blockage by the test compound

[0829] After Step 2, the external solution was changed to a solution containing the test compound. The Qchip was maintained at -100mV without pulses. The total solution switching time was 8 minutes. After the external solution exchange, cells were recorded for 10 minutes using the same procedure as in Step 2.

[0830] Data analysis was performed using Sophion Analyzer. The data was modified to control cells (non-drug). IC 50 The data was plotted using the DR-plot / Hill function (dose-response plot with Hill fit).

[0831] A representative example of the present invention was tested for the extracellular inhibition of NaV 1.7 in an automated patch clamp assay. The active range is IC50. 50 It is reported as: "++++" (<1 μM), "+++" (1-3 μM), "++" (3-10 μM) or "+" (>10 μM). The results are presented below.

[0832]

[0833] Example 3 - Membrane Permeability

[0834] The ability of the compound of the present invention to cross an artificial lipid membrane by passive diffusion was determined using the PAMPA assay (pION, Inc., Woburn MA). The test compound was dissolved in DMSO (10 mM) and diluted 200-fold in buffer (pION Inc., pH 7.4) to provide a 50 μM stock solution. Buffer (150 μL) was added to a UV blank plate, and the stock solution (150 μL) was transferred to a UV reference plate. The blank and reference spectra were read using a spectrophotometer. Stock solution (200 μL) was added to the donor plate of a PAMPA sandwich plate, and a receptor plate painted with GIT lipid (pION Inc., 5 μL) was placed on top. Buffer (200 μL) was added to the receptor plate, and the PAMPA sandwich plate was incubated for 4 hours. An aliquot (150 μL) from the receptor plate was added to a UV plate and read using the receptor spectrum. An aliquot (150 μL) of the donor solution was added to a UV analysis plate and read as a donor spectrum. Based on the AUC of the reference plate, donor plate, and acceptor plate, the penetration coefficient of the test compound was calculated using PAMPA Explorer™ software (version 3.5.0.4).

[0835] PAMPA penetration results of representative compounds (10 -6 cm / s) is "+" (< 0.1 10 -6 cm / s), "++"0.1-2.0 10 -6 cm / s), "+++" (2.0-10.0 10 -6 cm / s) or "++++" (>10.0 10 -6 It is reported as cm / s).

[0836]

[0837]

[0838] The patents and scientific literature cited herein establish the knowledge available to those skilled in the art. All U.S. patents and published or unpublished U.S. patent applications cited herein are incorporated by reference. All published foreign patents and patent applications cited herein are incorporated by reference. All other published references, literature, manuscripts, and scientific literature cited herein are incorporated by reference.

[0839] Although the present invention has been specifically presented and described with reference to preferred embodiments thereof, it will be understood by those skilled in the art that various variations in form and detail may be made within the scope of the invention as set forth in the appended claims without departing from the scope of the invention. Furthermore, it will be understood that none of the embodiments described herein are mutually exclusive and may be combined in various ways without departing from the scope of the invention as set forth in the appended claims.

[0840] Although the present invention has been specifically illustrated and described with reference to preferred embodiments thereof, it will be understood by those skilled in the art that various modifications of form and detail may be made without departing from the scope of the invention as set forth in the appended claims. Furthermore, it will be understood that none of the embodiments described herein are mutually exclusive and may be combined in various ways without departing from the scope of the invention as set forth in the appended claims.

Claims

Claim 1 Chemical formula ( I Compounds represented by ): In the above equation, Y - is a pharmaceutically acceptable anion; R A and R B Each is independently a C1-C6 alkyl, and R C is H, D, halogen, C1-C3 alkyl, OR I , CN, and NR J R K Selected independently from each;R I , R J , R K Each is independently selected from H, D, and C1-C3 alkyl; X 1 -NR Z C(O)-is; R Z is independently selected from H, D, and C1-C3 alkyl; R D and R E Each is independently selected from H, D, and C1-C4 alkyl; R F and R G is N to which they are attached + Together with, forming a substituted or unsubstituted 5, 6, 7, or 8-membered saturated monocyclic heterocyclic ring having zero or one or more nitrogen atoms in addition to N+ as a heteroatom; R H is a substituted or unsubstituted phenyl; where the substitution is a halogen, C1-C3 alkyl, OR I , and NR J R K Selected from. Claim 2 In paragraph 1, Y - A compound that is bromide, chloride, or iodide. Claim 3 In paragraph 1, X 1 This is a compound that is -NHC(O)-. Claim 4 delete Claim 5 delete Claim 6 delete Claim 7 delete Claim 8 In paragraph 1, R A and R B Each is CH3, and R C A compound selected from the group consisting of H, CH3, halogen, nitrile, methoxy, and ethoxy. Claim 9 delete Claim 10 In paragraph 1, R D and R E A compound in which both are hydrogen. Claim 11 In paragraph 1, R D is hydrogen, and R E Ga C 1-6 Alkyl compound. Claim 12 delete Claim 13 In paragraph 1, the compound is: Phosphorus, compound. Claim 14 In paragraph 1, R F and R G Compounds independently selected from each unsubstituted C1-C4 alkyl. Claim 15 In claim 1, the compound is a compound selected from the following table: . Claim 16 In claim 1, the compound is a compound selected from the following table: . Claim 17 A pharmaceutical composition for treating pain, cough, itching, or neuroinflammatory disorders in a patient, comprising the compound of claim 1 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient. Claim 18 In claim 17, the composition is formulated for oral, intravenous, intramuscular, rectal, skin, subcutaneous, topical, transdermal, sublingual, nasal, inhalation, vaginal, intradural, epidural, or ocular administration. Claim 19 A pharmaceutical composition for treating pain, cough, itching, or neuroinflammatory disorders in a patient, comprising the compound of claim 1. Claim 20 In paragraph 19, the above pain refers to lower back pain and neck pain, lumbago, cancer pain, gynecological pain and analgesia, fibromyalgia, arthritis, rheumatoid arthritis, osteoarthritis, rheumatic pain, orthopedic pain, acute and post-herpetic neuralgia and other neuropathic pain, sickle cell crisis, vulvodynia, perianal pain, irritable bowel disease, irritable bowel syndrome, inflammatory bowel disease, oral mucositis, esophagitis, interstitial cystitis, urethritis and other urological pain, toothache, headache, trigeminal neurodystrophic syndrome, erythematous limb pain, abdominal wall pain, chronic abdominal wall pain, allergic rhinitis, myalgia, rectal pain, levator ani syndrome, transient rectalgia, hemorrhoidal pain, stomach pain, skin ulcer, gastric ulcer, burn pain, ophthalmic irritation, conjunctivitis, eye hyperemia, dry eye, dry eye syndrome, chronic ocular pain, complex regional pain syndrome, postoperative ocular pain, surgery A pharmaceutical composition selected from the group consisting of pain due to post-operative pain, acute postoperative pain, and procedure pain, injection, abscess drainage, surgery, dental procedure, ophthalmic procedure, ophthalmic irritation, conjunctivitis, eye redness, dry eye, pain due to pain associated with the use of arthroscopy and other medical devices, cosmetic surgery procedures, dermatological procedures, bone setting, and biopsy. Claim 21 A pharmaceutical composition according to claim 19, wherein the cough is selected from the group consisting of asthma, COPD, asthma-COPD overlap syndrome (ACOS), interstitial pulmonary fibrosis (IPF), idiopathic pulmonary fibrosis, postviral cough, post-infectious cough, chronic idiopathic cough, and cough in patients with lung cancer. Claim 22 A pharmaceutical composition according to claim 19, wherein the above-mentioned itching is selected from the group consisting of pruritus, brachioradialis pruritus, chronic idiopathic pruritus, genital / anal pruritus, paresthetic back pain, scalp pruritus, allergic dermatitis, contact dermatitis, atopic dermatitis, hand eczema, poison ivy, infection, parasite, insect bite, pregnancy, metabolic disorder, hepatic or renal failure, drug reaction, allergic reaction, eczema, genital and anal itching, hemorrhoidal itching, and itching caused by cancer. Claim 23 A pharmaceutical composition according to claim 19, wherein the neuroinflammatory disorder is selected from the group consisting of allergic inflammation, asthma, chronic cough, conjunctivitis, rhinitis, psoriasis, inflammatory bowel disease, interstitial cystitis, arthritis, colitis, contact dermatitis, diabetes mellitus, eczema, cystitis, gastritis, migraine, rosacea, sunburn, pancreatitis, chronic rhinosinusitis, traumatic brain injury, polybacterial sepsis, tendinopathy, chronic urticaria, rheumatic disease, acute lung injury, and atopic dermatitis. Claim 24 In paragraph 19, chemical formula ( I A pharmaceutical composition in which a compound represented by ) is used in combination with one or more exogenous large porous receptor agonists.

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