Novel allosteric trka inhibitor, and composition containing same as active component for preventing or treating pain disorders

Abstract

The present invention relates to a novel allosteric TrkA inhibitor and a composition for preventing or treating pain, wherein the composition contains the novel allosteric TrkA inhibitor as an active ingredient. The novel allosteric TrkA inhibitor has excellent selective binding ability to TrkA, and can bind to TrkA to block NGF signaling and inhibit TRPV1 activity, and thus can be used as an effective therapeutic agent for pain disorders or inflammatory diseases.

Description

Novel allosteric TrkA inhibitor, and a composition for the prevention or treatment of pain diseases containing the same as an active ingredient

[0001] The present invention relates to a treatment for pain diseases, and more specifically, to a novel allosteric TrkA inhibitor compound and a composition for preventing or treating pain diseases containing said compound as an active ingredient.

[0002] Nerve Growth Factor (NGF) is an important factor in acute, transient nociceptive responses and long-term chronic pain, as it is not only a significant factor in causing inflammation-related diseases, particularly arthritis, but also an essential component of arthritis pain. NGF, Interleukin (IL), and Tumor Necrosis Factor-α (TNF-α) are secreted by inflammatory cells in damaged tissues and Schwann cells in damaged nerves, and are involved in acute and chronic pain, with NGF expression increasing at the site of tissue damage.

[0003] NGF secreted by inflammatory cells acts on Tropomyosin receptor kinase A (TrkA), located on the cell membrane of sensory nerve endings, to phosphorylate other pain-related proteins, induce structural changes, and increase the expression of these proteins, subsequently causing peripheral sensitization in peripheral nerves and central sensitization in the spinal cord, thereby inducing hyperalgesia, allodynia, and painful responses to stimuli.

[0004] Tropomyosin receptor kinase A (TrkA) is expressed in various organs and tissues such as the peripheral nervous system, central nervous system, immune tissues, digestive tract, adrenal cortex, prostate, uterus, kidney, and skin. NGF binds to TrkA on the cell membrane, which has tyrosine kinase activity that phosphorylates tyrosine at amino acid residues, and the activation loop of TrkA is located at the center of the enzyme's active site in an inactive state, thereby preventing ATP from entering the active site and consequently inhibiting tyrosine kinase activity.

[0005] When an NGF dimer binds to a TrkA dimer, an activation loop is released from the center of the enzyme's active site, and subsequently, TrkA autophosphorylates tyrosine residues (Y676, Y680, Y681) on the ATP-containing symmetric activation loop (pY). This activated form of TrkA phosphorylates other intracellular substrate proteins, triggering the intracellular signaling system of NGF / TrkA signaling to transmit the signal to the nucleus. In particular, NGF acting on peripheral nociceptive neuron terminals binds to TrkA on the cell membrane, is subsequently absorbed by endosomes, transported via axons to dorsal root ganglion (DRG) cells, where downstream intracellular signaling systems are activated to produce various types of proteins. TrkA-NGF inhibition has been proven to reduce pain in humans and animals, and the recognition that TrkA-NGF plays a crucial role in the pain mechanism in adults provides an opportunity to develop a completely new class of drugs for the treatment of pain diseases.

[0006] TrkA is a therapeutic target for numerous types of tumors or chronic pain and is a crucial target for the development of treatments for inflammatory chronic pain diseases currently being developed by multinational pharmaceutical companies; however, most TrkA inhibitors to date are ATP-competitive Pan-Trk inhibitors lacking selectivity, and notably, several acquired resistance mutations have been identified in the Pan-TrkA kinase domain, which have been revealed to be major factors in drug resistance and side effects.

[0007] Therefore, the allosteric TrkA inhibition mechanism is ultimately expected to provide distinct differentiation from existing pain treatments as a target with a clear mechanism for chronic pain conditions; by selectively inhibiting TrkA, it is anticipated to reduce the side effects of treatments for chronic pain and anti-inflammatory effects while demonstrating equivalent or higher efficacy.

[0008] In this regard, although Array BioPharma, Merck, and Pfizer have reported various allosteric TrkA inhibitors, no new drugs have been developed to date due to the poor drug-like properties of the currently reported allosteric TrkA inhibitors or the lack of proof of concept targeting TrkA-induced signaling pathways. Consequently, there is a situation where treatments to replace non-steroidal anti-inflammatory drugs (NSAIDs) and opioid analgesics for the treatment of chronic pain diseases have not been developed.

[0009] Accordingly, there is a need to develop novel treatments that reduce conventional side effects and possess excellent therapeutic efficacy for pain disorders.

[0010] The objective of the present invention is to provide a novel allosteric TrkA inhibitor that exhibits excellent efficacy in treating pain and inflammation through selective inhibition of TrkA and has almost no side effects, in order to solve the problems of conventional allosteric TrkA inhibitors, such as low selectivity for TrkA, poor drug-like properties, or side effects such as neurological disorders.

[0011] Another objective of the present invention is to provide a composition comprising the novel allosteric TrkA inhibitor described above.

[0012] Another objective of the present invention is to provide a pharmaceutical composition for the prevention or treatment of pain or inflammatory diseases comprising the novel allosteric TrkA inhibitor described above.

[0013] Another objective of the present invention is to provide a health functional food for the prevention or improvement of pain or inflammatory diseases comprising the novel allosteric TrkA inhibitor described above.

[0014] To achieve the above objective, the present invention provides a compound represented by the following chemical formula 1 or a pharmaceutically acceptable salt thereof:

[0015] <Chemical Formula 1>

[0016]

[0017] In the above chemical formula 1, R 1 C 1-6 Alkyl or CONR a R b and, the above R a or R b may be the same or different, and hydrogen, C 1-6 Alkyl, C 1-6 Alkenyl, C 1-6 Alkainyl, C 3-10 Cycloalkyl, C 4-10 Heterocycloalkyl and C 5-10 Selected from the group consisting of heteroaryls, or Ra and R b C is connected 4-10 Heterocycloalkyl or C 5-10 Forming a heteroaryl, R 2 , R 3 or R 4 Each may be the same or different and may be selected from the group consisting of hydrogen, halogens, and alkyl halides.

[0018] The present invention provides a pharmaceutical composition comprising the above-mentioned compound or a pharmaceutically acceptable salt thereof.

[0019] The present invention provides a pharmaceutical composition for the prevention or treatment of pain diseases, comprising the above-mentioned compound or a pharmaceutically acceptable salt thereof.

[0020] The present invention provides a pharmaceutical composition for the prevention or treatment of inflammatory diseases, comprising the above-mentioned compound or a pharmaceutically acceptable salt thereof.

[0021] The present invention provides a health functional food composition for the prevention or improvement of pain diseases, comprising the above-mentioned compound or a food-grade acceptable salt thereof.

[0022] The present invention provides a health functional food composition for the prevention or improvement of inflammatory diseases, comprising the above-mentioned compound or a food-grade acceptable salt thereof.

[0023] The novel allosteric TrkA inhibitor according to the present invention has excellent selective binding affinity to TrkA and can block NGF signaling and inhibit TRPV1 activity by binding to TrkA.

[0024] In addition, the novel allosteric TrkA inhibitor according to the present invention has the advantage of exhibiting significantly superior pain-inhibiting effects in various animal pain model experiments, being free from cytotoxicity and genotoxicity, and lacking the side effects of conventional TrkA inhibitors such as neurological disorders.

[0025] In addition, the novel allosteric TrkA inhibitor according to the present invention exhibits superior effects in blocking NGF signaling in cells and suppressing pain in animal models of pain compared to the previously reported allosteric TrkA inhibitor of Pfizer, and can be utilized as an effective therapeutic agent for pain or inflammatory diseases.

[0026] Figure 1 is a figure showing the core framework for the synthesis of the compound of the present invention.

[0027] Figure 2 is a figure showing the results of the cytotoxicity evaluation of the compounds MTS-CP-0093 and MTS-CP-0154 of the present invention in HepG2 cell lines.

[0028] Figure 3 is a figure showing the results of the cytotoxicity evaluation of the MTS-CP-0093 compound of the present invention in PC12 cell lines.

[0029] Figure 4 is a figure showing the capsaicin response of DRG cells with and without nerve growth factor (NGF) and the blocking action of the MTS-CP-093 compound of the present invention.

[0030] Figure 5 is a figure showing the Hargreaves test results of the MTS-CP-093 compound of the present invention.

[0031] Figures 6a and 6b are figures showing the results of the neurite outgrowth assay of the MTS-CP-093 compound of the present invention.

[0032] Figures 7a and 7b are figures showing the results of the neurite outgrowth assay of the MTS-CP-154 compound of the present invention.

[0033] Figure 8 is a figure showing the results of evaluating the analgesic efficacy of some compounds of the present invention in an animal model of MIA-induced osteoarthritis pain (rat, before administration on day 10).

[0034] Figure 9 is a figure showing the results of evaluating the analgesic efficacy of some compounds of the present invention in an animal model of MIA-induced osteoarthritis pain (rat, after administration on day 10).

[0035] Figure 10 is a figure showing the results of evaluating the analgesic efficacy of some compounds of the present invention in an animal model of MIA-induced osteoarthritis pain (rat, after administration on day 12).

[0036] Figure 11 is a figure showing the results of evaluating the analgesic efficacy of some compounds of the present invention in an animal model of MIA-induced osteoarthritis pain (rat, after administration on day 14).

[0037] Figure 12 shows the results of the von Frey test and weight bearing test in an animal model of MIA-induced osteoarthritis pain (C57BL / 6N wild type mouse) using the MTS-CP-093 compound of the present invention.

[0038] Figure 13 is a figure showing the results of the von Frey test and weight bearing test in an animal model of MIA-induced osteoarthritis pain (TRPV1 gene deletion mouse) using the MTS-CP-093 compound of the present invention.

[0039] Figure 14 is a figure showing the results of the evaluation of the analgesic efficacy of the MTS-CP-093 compound of the present invention in a Carrageenan-induced osteoarthritis pain animal model (rat).

[0040] Figure 15 is a figure showing the results of a hyperthermia test of the MTS-CP-093 compound of the present invention in wild-type C57BL / 6N mice and wild-type Sprague-Dawley (SD) rats.

[0041] To design novel allosteric TrkA inhibitors, the inventors analyzed and reviewed the structure-activity relationship regarding TrkA action activity and classified the core framework of the allosteric TrkA inhibitor into three types: an amide linker, a pocket binding region, and a solvent-exposed region. They then synthesized novel derivatives with diversified pocket binding and solvent-exposed regions. The present invention was completed by confirming that some of these novel derivatives exhibit excellent selective binding affinity to TrkA, inhibit TRPV1 activity by blocking NGF signaling through binding to TrkA, demonstrate significantly superior pain-inhibiting efficacy in various animal pain models, are non-cytotoxic, and do not have the side effects associated with conventional TrkA inhibitors.

[0042]

[0043] The present invention will be described in detail below. In describing the present invention, detailed descriptions of related known components or functions may be omitted.

[0044] Terms and words used in this specification and claims are not to be interpreted as being limited to their ordinary or dictionary meanings, but should be interpreted in a sense and concept consistent with the technical details of the present invention.

[0045] The embodiments described in this specification and the configurations illustrated in the drawings are preferred embodiments of the present invention and do not represent all technical aspects of the present invention; therefore, various equivalents and modifications that can replace them may exist at the time of filing this application.

[0046]

[0047] The present invention provides a compound represented by the following chemical formula 1 or a pharmaceutically acceptable salt thereof:

[0048] <Chemical Formula 1>

[0049]

[0050] In the above chemical formula 1,

[0051] R 1 C 1-6 Alkyl or CONR a R b and, the above R a or R b may be the same or different, and hydrogen, C 1-6 Alkyl, C 1-6 Alkenyl, C 1-6 Alkainyl, C 3-10 Cycloalkyl, C 4-10 Heterocycloalkyl and C 5-10 Selected from the group consisting of heteroaryls, or R a and R b C is connected 4-10 Heterocycloalkyl or C 5-10 Forming a heteroaryl,

[0052] R 2 , R 3 or R 4 Each may be the same or different and may be selected from the group consisting of hydrogen, halogens, and alkyl halides.

[0053]

[0054] The above term "alkyl" means a saturated straight-chain or branched-chain hydrocarbon radical having a specified number of carbon atoms (e.g., C 1-6 Alkyl refers to an alkyl group having 1 to 6 carbon atoms). An alkyl group preferred for use in the present invention is a C group having 1 to 6 atoms. 1-6 It is an alkyl group. Exemplary alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, pentyl, hexyl, etc.

[0055] The term "alkenyl" above refers to a straight-chain or branched-chain hydrocarbon radical having a single carbon-carbon double bond and a specified number of carbon atoms (e.g., C 2-6Alkenyl refers to an alkenyl group having 2 to 6 carbon atoms). An alkenyl group preferred for use in the present invention is a C having 1 to 6 carbon atoms. 1-6 It is an alkenyl group. Exemplary alkenyl groups include ethenyl, propenyl, and butenyl.

[0056] The above term "alkynyl" refers to a straight-chain or branched-chain hydrocarbon radical having a single carbon-carbon triple bond and a specified number of carbon atoms (e.g., C 3-6 Alkenyl refers to an alkynyl group having 3 to 6 carbon atoms). An alkynyl group preferred for use in the present invention is a C having 1 to 6 carbon atoms. 1-6 It is an alkynyl group. Exemplary alkynyl groups include etynyl, propynyl, and butynyl.

[0057] The above term "cycloalkyl" means a non-aromatic saturated hydrocarbon group forming at least one ring essentially composed of 3 to 10 carbon atoms and a corresponding number of hydrogen atoms. The cycloalkyl group may be monocyclic or multicyclic. In addition to covalent substitution, the individual rings of a multicyclic cycloalkyl group may have different connectivitys, e.g., fusion, crosslinking, spiro, etc. Exemplary C 3-10 The cycloalkyl group includes cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, norbornanyl, bicyclo-octanyl, octahydro-pentalenyl, spiro-decanyl, cyclopropyl substituted with cyclobutyl, cyclobutyl substituted with cyclopentyl, cyclohexyl substituted with cyclopropyl, etc.

[0058] The above term "heterocycloalkyl" refers to a non-aromatic group having 4 to 10 atoms forming at least one ring, derived from a heterocycle comprising one or more atoms other than carbon (heteroatoms) among the atoms forming the ring structure, and a group that is bonded to another structure through an alkyl bond, wherein the heteroatoms may be one or more selected from nitrogen, oxygen, and sulfur. The heterocycloalkyl may be monocyclic or multicyclic. In addition to covalent substitution, the individual rings of such multicyclic heterocycloalkyl groups may have different bonding properties, e.g., fusion, crosslinking, spiro, etc. Exemplary C 4-10 Heterocycloalkyls include pyrrolidinyl, piperidinyl, morpholino, piperazinyl, azetidinyl, tetrahydrofuranil, tetrahydropyranil, thiomophorinyl, tetrahydroazefinil, and their bicyclic or spiro-type derivatives.

[0059] The above term "heteroaryl" means an aromatic monocyclic or polycyclic ring comprising 5 to 10 ring atoms, wherein at least one of the ring atoms is an atom other than carbon, and the atom other than carbon comprises one or more heteroatoms selected from the group consisting of nitrogen, oxygen, and sulfur, representing a monocyclic or polycyclic heteroaryl group. Exemplary C 5-10 Heteroaryls include pyrrolyl, pyridinyl, pyrimidinyl, pyrazinyl, imidazolyl, triazolyl, thiazolyl, furanyl, thiophenyl, indolyl, benzimidazolyl, quinolyl, etc.

[0060] The above term "halogen" includes fluoro(F), chloro(Cl), bromo(Br), and iodo(I).

[0061] The above term "alkyl halide" is a compound in which 1 to 3 hydrogen atoms of a saturated hydrocarbon are substituted with halogen atoms, wherein the halogen includes fluoro, chloro, bromo, and iodo.

[0062]

[0063] Preferably, the compound is R in Formula 1. 1 This C 1-6 Alkyl or CONR a R b and, the above R a or R b may be the same or different, and hydrogen, C 1-4 Alkyl and C 3-7 Selected from the group consisting of cycloalkyl, or R a and R b C is connected 4-10 It can form heterocycloalkyl groups.

[0064] The above compound is R of Chemical Formula 1. 2 , R 3 or R 4 Each may be the same or different and may be selected from the group consisting of hydrogen, Cl, F, and CF3.

[0065] The above C 4-10 Heterocycloalkyl or C 5-10 Heteroaryls may be selected from the group consisting of pyrrolyl, furanyl, thiophenyl, imidazolyl, pyridinyl, morpholino, piperidinyl, and piperazinyl, but are not limited thereto.

[0066] Specifically, the above C 4-10 The heterocycloalkyl group can be selected from the group consisting of morpholino, piperidinyl, and piperazinyl, and the above C 5-10 Heteroaryls may be selected from the group consisting of pyrrolyl, furanyl, thiophenyl, imidazolyl, and pyridinyl, but are not limited thereto.

[0067] More preferably, the compound may be any one compound selected from the group consisting of compounds represented by the following chemical formulas 2 to 10:

[0068] <Chemical Formula 2>

[0069] ;

[0070] <Chemical Formula 3>

[0071] ;

[0072] <Chemical Formula 4>

[0073] ;

[0074] <Chemical Formula 5>

[0075] ;

[0076] <Chemical Formula 6>

[0077] ;

[0078] <Chemical Formula 7>

[0079] ;

[0080] <Chemical Formula 8>

[0081] ;

[0082] <Chemical Formula 9>

[0083] ; and

[0084] <Chemical Formula 10>

[0085]

[0086]

[0087] The above compound may be used in the form of a pharmaceutically acceptable salt or a food-grade acceptable salt within a range having the same efficacy.

[0088] In this specification, "pharmaceuticalally acceptable salt or food-acceptable salt" means a salt having a safety and efficacy profile suitable for administration to humans, having no toxicity to cells or humans exposed thereto.

[0089] The above salt refers to a salt prepared from a pharmaceutically or food-acceptable non-toxic base or acid, including an inorganic or organic base and an inorganic or organic acid. The compound of the present invention may be a mono, di, or tri salt depending on the number of acid functional groups present in the free base form of the compound. Free bases and salts derived from inorganic bases may include aluminum, ammonium, calcium, copper, ferric, ferrous, lithium, magnesium, manganese(II) salt, manganese(I) salt, potassium, sodium, zinc, etc.

[0090] Salts in solid form may exist in more than one type of crystal structure and may also be in the form of hydrates. Salts derived from pharmaceutically or food-grade acceptable organic non-toxic bases may include primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, and basic ion exchange resins, such as arginine, betaine, caffeine, choline, N,N'-dibenzylethylene-diamine, diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, ethylenediamine, N-ethylpiperidine, N-ethylmorpholine, glucamine, glucosamine, histidine, hydravamin, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperidine, polyamine resins, procaine, purine, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine, etc.

[0091] When the compound of the present invention is basic, the salt may be prepared from pharmaceutically or food-acceptable non-toxic acids, including inorganic and organic acids. Such acids may include acetic acid, trifluoroacetic acid, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, citric acid, ethanesulfonic acid, fumaric acid, gluconic acid, glutamic acid, hydrobromide, hydrochloric acid, isethionic acid, lactic acid, maleic acid, malic acid, mandelic acid, methanesulfonic acid, mucinic acid, nitric acid, paromolic acid, pantothenic acid, phosphoric acid, succinic acid, sulfuric acid, tartaric acid, para-toluenesulfonic acid, etc.

[0092] In addition, the above-mentioned compound may include not only the above-mentioned salt, but also all salts, hydrates, solvates, derivatives, etc. that can be prepared according to conventional methods. The addition salt can be prepared by conventional methods, and can be prepared by dissolving it in a water-miscible organic solvent, such as acetone, methanol, ethanol, or acetonitrile, adding an excess amount of organic base or an aqueous solution of an inorganic base, and then precipitating or crystallizing it. Alternatively, the addition salt can be obtained by evaporating the solvent or the excess base from the mixture and then drying it, or by suction filtration of the precipitated salt.

[0093]

[0094] The compound according to the present invention or a pharmaceutically acceptable salt thereof can selectively bind to Tropomyosin receptor kinase A (TrkA), and by binding to TrkA, can block Nerve Growth Factor (NGF) signaling, thereby inhibiting the activity of TRPV1 (transient receptor potential vanilloid 1).

[0095] Through this mechanism, pain or inflammatory diseases can be prevented, improved, or treated.

[0096]

[0097] The present invention provides a pharmaceutical composition comprising the above-mentioned compound or a pharmaceutically acceptable salt thereof.

[0098] In this specification, the term “composition” encompasses a product comprising a predetermined amount or ratio, as well as any product generated directly or indirectly from a combination of specified amounts of specified components. In relation to pharmaceutical compositions, this term encompasses a product comprising one or more active components and any carrier comprising an inactive component, as well as any product generated directly or indirectly from a combination of any two or more components, complexation or aggregation, or dissociation of one or more components, or other types of reactions or interactions of one or more components.

[0099] In this specification, the term "pharmaceutical composition" means a composition administered for the purpose of preventing or treating a specific disease, and for the purposes of the present invention, means administered for the treatment of pain or inflammatory diseases, or diseases or complications caused by such diseases.

[0100] The above pharmaceutical composition is prepared by uniformly and densely associating an active ingredient with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, forming the product into a target formulation. In the above pharmaceutical composition, the active compound, which is a compound represented by Formulas 1 to 10, is included in an amount sufficient to produce a desired effect depending on the course or state of the disease. Accordingly, the above pharmaceutical composition encompasses any composition prepared by mixing the compound according to the present invention with a pharmaceutically acceptable carrier.

[0101] The above carrier may take on a wide variety of forms depending on the formulation form preferred for oral or parenteral (including intravenous) administration. Accordingly, the pharmaceutical composition may be presented as discrete units suitable for oral administration, such as capsules, cases, or tablets containing a predetermined amount of active ingredient. Additionally, the pharmaceutical composition may be provided as a powder, as granules, as a solution, as a suspension in an aqueous liquid, as a non-aqueous liquid, as an oil-in-water emulsion, or as a liquid-in-oil emulsion. In addition to the conventional administration forms presented above, the pharmaceutical composition may be administered by a controlled release means and / or a delivery device.

[0102] Pharmaceutical compositions intended for oral use may be prepared according to any method known in the field of pharmaceutical manufacturing technology, and such pharmaceutical compositions may contain one or more agents selected from the group consisting of sweeteners, flavoring agents, coloring agents, and preservatives. Tablets may contain an active ingredient mixed with non-toxic, pharmaceutically acceptable excipients suitable for the manufacture of tablets. These excipients may be, for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate, or sodium phosphate; granulating agents and disintegrating agents, for example, corn starch, or alginic acid; binders, for example, starch, gelatin, or acacia; and lubricants, for example, magnesium stearate, stearic acid, or talc. Tablets may not be coated, or they may be coated by known technology to delay disintegration and absorption in the gastrointestinal tract, thereby providing sustained action over a longer period.

[0103] Pharmaceutical compositions intended for parenteral use may be in the form of injectable aqueous or oily suspensions, or in the form of sterile powders for the immediate preparation of such sterile injectable solutions or dispersions. In all cases, the final injectable form must be sterile and possess effective fluidity for easy syringeability. The pharmaceutical composition must be stable under manufacturing and storage conditions; thus, preferably, it must be preserved against the action of microorganisms, such as bacteria and fungi. The pharmaceutical composition may be in a form suitable for topical use, e.g., aerosols, creams, ointments, lotions, sprays, etc. The pharmaceutical composition may be in a form suitable for use in transdermal devices. These formulations may be prepared through conventional processing methods. For example, a cream or ointment may be prepared by mixing a hydrophilic substance and water with a compound of about 5% by weight to about 10% by weight to produce a cream or ointment having the desired viscosity.

[0104]

[0105] The present invention provides a pharmaceutical composition for the prevention or treatment of pain diseases, comprising the above-mentioned compound or a pharmaceutically acceptable salt thereof.

[0106] The above pain conditions may include acute or chronic pain, inflammatory pain, neuropathic pain, or pain conditions associated with cancer, surgery, and fractures.

[0107] More specifically, the above pain conditions include neuropathic pain (e.g., postherpetic neuralgia, nerve injury, vulvar pain, phantom limb pain, nerve root expulsion, painful diabetic neuropathy, painful traumatic mononeuropathy, painful polyneuropathy, anticancer drug-induced neuropathy, etc.), central pain syndrome (e.g., potentially caused by any substantial lesion at any level of the nervous system), postoperative pain syndrome (e.g., post-mastectomy syndrome, post-thoracotomy syndrome, stump pain), bone and joint pain (e.g., osteoarthritis), repetitive motion pain, toothache, cancer pain, myofascial pain (e.g., muscle injury, fibromyalgia), perioperative pain, chronic pain, pain associated with angina, and inflammatory pain of various origin (e.g., osteoarthritis, rheumatoid arthritis, rheumatic disease, tenosynovitis, and gout), headache, migraine and cluster headache, headache, primary hyperalgesia, secondary hyperalgesia, primary allodynia, It may include, but is not limited to, secondary allodynia or other pain caused by central sensitization.

[0108] The above prevention or treatment includes suppressing the disease in animals that are suffering from or exhibiting the pathology or symptomaticity of the disease (e.g., stopping further development of the pathology and / or symptomaticity), or reversing the disease in animals that are suffering from or exhibiting the pathology or symptomaticity of the disease (e.g., reversing the pathology and / or symptomaticity).

[0109] The above pharmaceutical composition may be conveniently provided in a unit dosage form and may be manufactured by any method widely known in the field of pharmaceutical technology. The term “unit dosage form” means a single dosage in which all active and inactive ingredients are combined in a suitable system so that the patient or the person administering the drug to the patient can open a single container or package containing the entire dosage and there is no need to mix any ingredients from two or more containers or packages together. Typical examples of a unit dosage form may be tablets or capsules for oral administration, single-dose vials for injection, or suppositories for rectal administration.

[0110] The above pharmaceutical composition may be conveniently provided as a kit containing two or more components, which may be active or inactive components, carriers, diluents, etc., along with instructions for preparing an actual dosage form by a patient or a person administering the drug to the patient. Such a kit may be provided containing all necessary substances and components, or it may contain instructions for the use or preparation of substances or components that must be independently obtained by the patient or a person administering the drug to the patient.

[0111] With respect to the treatment or improvement of a disorder or disease, the above pharmaceutical composition generally yields satisfactory results when the compound of the present invention is administered at a daily dose of about 0.001 mg to about 1000 mg per kg of animal body weight, which is preferably given as a single daily dose, as divided doses 2 to 6 times daily, or in a sustained-release form. The total daily dose is about 0.1 mg to about 100 mg per kg of body weight, preferably about 0.1 mg to about 10 mg. For a 70 kg adult human, the total daily dose may generally be about 1 mg to about 1000 mg. This administration regimen may be adjusted to provide an optimal therapeutic response. The compound may be administered 1 to 4 times daily, preferably as a regimen of 1 or 2 times daily. However, it will be understood that specific dose levels and frequency of administration for any particular patient may vary and depend on various factors including the activity of the specific compound used, the metabolic stability and duration of action of said compound, age, body weight, overall health, gender, diet, mode and time of administration, elimination rate, drug combination, severity of the specific condition, and the host receiving the therapy.

[0112] The above pharmaceutical composition may be used in combination with one or more other drugs in the treatment of a disease or condition in which the compound according to the present invention is useful, where combining the drugs together is safer or more effective than each drug alone. Additionally, it may be used in combination with one or more other drugs that treat, prevent, control, improve, or reduce the risk thereof of side effects or toxicity of the compound according to the present invention. These other drugs may be administered simultaneously with or sequentially with the compound according to the present invention via the routes and amounts typically used for them. Accordingly, the above pharmaceutical composition comprises those containing one or more other active ingredients in addition to the compound according to the present invention. The combination may be administered as part of a combination product in a unit dosage form, or as a kit or treatment protocol in which one or more additional drugs are administered in individual dosage forms as part of a therapeutic regimen.

[0113]

[0114] The present invention provides a pharmaceutical composition for the prevention or treatment of inflammatory diseases comprising the above-mentioned compound or a pharmaceutically acceptable salt thereof.

[0115] The description of the components, dosage, and method of administration of the above pharmaceutical composition is the same as described above.

[0116] The above inflammatory disease may be at least one selected from the group consisting of osteoarthritis, rheumatoid arthritis, spondyloarthritis, ankylosing spondylitis, psoriatic arthropathy, juvenile arthropathy, juvenile ankylosing spondylitis, polyarteritis nodularis, hypersensitivity vasculitis, polymyalgia rheumatica, non-articular rheumatism, tenosynovitis, epicondylitis (tennis elbow), neuropathic joint disease (charco and joint), multiple sclerosis, and systemic lupus erythematosus, but is not limited thereto.

[0117]

[0118] The present invention provides a health functional food composition for the prevention or improvement of pain diseases comprising the above-mentioned compound or a food-grade acceptable salt thereof.

[0119] The above pain conditions may include acute or chronic pain, inflammatory pain, neuropathic pain, or pain associated with cancer, surgery, and fractures.

[0120] More specifically, the above pain conditions include neuropathic pain (e.g., postherpetic neuralgia, nerve injury, vulvar pain, phantom limb pain, nerve root expulsion, painful diabetic neuropathy, painful traumatic mononeuropathy, painful polyneuropathy, anticancer drug-induced neuropathy, etc.), central pain syndrome (e.g., potentially caused by any substantial lesion at any level of the nervous system), postoperative pain syndrome (e.g., post-mastectomy syndrome, post-thoracotomy syndrome, stump pain), bone and joint pain (e.g., osteoarthritis), repetitive motion pain, toothache, cancer pain, myofascial pain (e.g., muscle injury, fibromyalgia), perioperative pain, chronic pain, pain associated with angina, and inflammatory pain of various origin (e.g., osteoarthritis, rheumatoid arthritis, rheumatic disease, tenosynovitis, and gout), headache, migraine and cluster headache, headache, primary hyperalgesia, secondary hyperalgesia, primary allodynia, It may include, but is not limited to, secondary allodynia or other pain caused by central sensitization.

[0121] In this specification, "health functional food" means a food manufactured and processed using raw materials or ingredients that have functional properties useful to the human body in accordance with the Health Functional Foods Act, and the term "functional properties" means consuming for the purpose of obtaining useful effects for health purposes, such as regulating nutrients or physiological actions on the structure and function of the human body.

[0122] The above-mentioned health functional food may contain ordinary food additives, and unless otherwise specified, suitability as a "food additive" is determined by the specifications and standards for the relevant item in accordance with the general provisions and general test methods of the Food Additives Codex approved by the Ministry of Food and Drug Safety. Items listed in the above-mentioned "Food Additives Codex" may include, for example, chemically synthesized products such as ketones, glycine, potassium citrate, nicotinic acid, and cinnamon acid; natural additives such as persimmon dye, licorice extract, crystalline cellulose, sorghum dye, and guar gum; and mixed preparations such as L-sodium glutamate preparations, alkaline noodle additives, preservative preparations, and tar dye preparations.

[0123] The above health functional food may contain a compound according to the present invention in an amount of 0.01 to 95%, preferably 1 to 80% by weight percentage, based on the total weight of the composition.

[0124] The above health functional food can be manufactured and processed in the form of tablets, capsules, powder, granules, liquid, pills, etc.

[0125] For example, the above-described health functional food in tablet form may be produced by granulating a mixture of the compound according to the present invention, an excipient, a binder, a disintegrant, and other additives by a conventional method, and then adding a lubricant or the like and compression molding, or by directly compression molding the mixture. In addition, the above-described health functional food in tablet form may contain a binder or the like as needed, and may be coated with a suitable coating agent as needed.

[0126] Among health functional foods in capsule form, hard capsules can be manufactured by filling a conventional hard capsule with a mixture of the compound according to the present invention and additives such as excipients, or the granules thereof, or coated granules thereof, and soft capsules can be manufactured by filling a capsule base such as gelatin with a mixture of the compound according to the present invention and additives such as excipients. The soft capsules may contain plasticizers such as glycerin or sorbitol, coloring agents, preservatives, etc., as needed.

[0127] A granular health functional food can be prepared in granular form by a suitable method using a mixture of compounds, excipients, binders, disintegrants, etc. according to the present invention, and may contain flavoring agents, stimulating agents, etc. as needed.

[0128] The definitions of terms for the above excipients, binders, disintegrants, lubricants, synergists, and flavorings, etc., include those described in literature known in the art and those with identical or similar functions.

[0129]

[0130] In addition, the present invention provides a health functional food composition for preventing or improving inflammatory diseases, comprising a compound according to the present invention or a food-grade acceptable salt thereof.

[0131] The description of the additives, content, and formulation of the above-mentioned health functional food is the same as described above.

[0132] The above inflammatory disease may be at least one selected from the group consisting of osteoarthritis, rheumatoid arthritis, spondyloarthritis, ankylosing spondylitis, psoriatic arthropathy, juvenile arthropathy, juvenile ankylosing spondylitis, polyarteritis nodularis, hypersensitivity vasculitis, polymyalgia rheumatica, non-articular rheumatism, tenosynovitis, epicondylitis (tennis elbow), neuropathic joint disease (charco and joint), multiple sclerosis, and systemic lupus erythematosus, but is not limited thereto.

[0133] The present invention will be explained in more detail below through examples.

[0134] These examples are solely for the purpose of illustrating the invention more specifically, and it is obvious to those skilled in the art that the scope of the invention is not limited by these examples according to the gist of the invention.

[0135]

[0136] <Example 1> Synthesis of a Novel Allosteric TrkA Inhibitor

[0137] To design novel derivatives, the structure-activity relationship regarding TrkA action activity was analyzed and reviewed based on the results of prior research. Structural analysis of the derivatives derived from prior research revealed that the core framework could be classified into three types: an amide linker, a pocket binding region, and a solvent-exposed region. A strategy was established to immobilize the core structure, phenyl pyrazole carboxamide, and to diversify the solvent-exposed and pocket binding regions.

[0138] Novel derivatives were designed and synthesized by diversifying substituents in the solvent-exposed region and pocket-binding region using the previously known structure of TrkA activity (Sharan K Bagal, et al., J Med Chem. 2019 Jan 10;62(1):247-265) (Fig. 1).

[0139] The linker of the novel derivative is a key structure that interacts with Asp668 in the activation loop of TrkA and Leu486 in the JM region; the solvent-exposed region is the part exposed outside the active site of TrkA and can form hydrogen bonds with Lys544; and while the pocket binding region plays an important role in non-polar interactions with the hydrophobic pocket of TrkA, halogen interactions with His648 were also considered.

[0140] As a result, nine types of compounds were designed and synthesized as follows and named as MTS-CP compounds [MTS-CP1-058 (Formula 2), MTS-CP1-060 (Formula 3), MTS-CP1-072 (Formula 4), MTS-CP1-073 (Formula 5), ​​MTS-CP1-093 (Formula 6), MTS-CP1-117 (Formula 7), MTS-CP1-125 (Formula 8), MTS-CP1-146 (Formula 9), and MTS-CP1-154 (Formula 10), respectively].

[0141]

[0142] <1-1> Synthesis of Intermediates A~E

[0143]

[0144] (5-amino-1-phenyl-1H-pyrazole-3-yl)(morpholino)methanone (A)

[0145] Morpolino(5-nitro-1-phenyl-1H-pyrazole-3-yl)methanone was added to MeOH (0.1 M) and stirred. To the stirred solution, 10% Pd / C was added and stirred under a hydrogen atmosphere. After stirring at room temperature for 5 hours, the reaction mixture was filtered with EtOAc. Then, (5-amino-1-phenyl-1H-pyrazole-3-yl)(morpholino)methanone was purified by silica gel flash column chromatography (MeOH : DCM = 1 : 10).

[0146] 3-methyl-1-phenyl-1H-pyrazol-5-amine hydrate (B)

[0147] I purchased a commercially sold item.

[0148] 5-amino-N-methyl-1-phenyl-1H-pyrazol-3-carboxamide(C)

[0149] Methylamine hydrochloride (0.8 equivalents), EDC hydrochloride (3.0 equivalents), HOBt monohydrate (1.5 equivalents), DMAP (0.1 equivalents), and DIPEA (3.0 equivalents) were added to a stirred solution of 5-amino-1-phenyl-1H-pyrazole-3-carboxylic acid in DMF (0.4 M). After stirring overnight at room temperature, the reaction mixture was treated with H2O. The reaction mixture was diluted with EtOAc, and the organic layer was washed with H2O. The organic layer was dried with MgSO4 and concentrated under vacuum. 5-amino-N-methyl-1-phenyl-1H-pyrazole-3-carboxyamide was purified by flash column chromatography on silica gel (EtOAc : n-hexane = 1 : 1):

[0150] 1 H NMR (CDCl3, 500 MHz) δ 7.55 (dd, 2H,J= 8.4, 1.0 Hz), 7.50 (t, 2H,J= 7.8 Hz), 7.40 (t, 1H,J= 7.3 Hz), 6.13 (s, 1H), 3.85 (s, 2H), 2.92 (d, 2H,J= 5.0 Hz); 13 C NMR (CDCl3, 125 MHz) δ 163.0, 146.9, 146.3, 138.1, 129.8, 128.4, 124.4, 90.8, 25.8.

[0151] 5-amino-N-cyclopropyl-1-phenyl-1H-pyrazole-3-carboxamide(D)

[0152] Cyclopropylamine (0.8 equivalents), EDC hydrochloride (3.0 equivalents), HOBt monohydrate (1.5 equivalents), DMAP (0.1 equivalents), and DIPEA (3.0 equivalents) were added to a stirred solution of 5-amino-1-phenyl-1H-pyrazole-3-carboxylic acid in DMF (0.4 M). After stirring overnight at room temperature, the reaction mixture was quenched with H2O. The reaction mixture was diluted with EtOAc, and the organic layer was washed with H2O. The organic layer was dried with MgSO4 and concentrated under vacuum. 5-amino-N-cyclopropyl-1-phenyl-1H-pyrazole-3-carboxamide was purified by flash column chromatography on silica gel (EtOAc : n-hexane = 4 : 1):

[0153] 1 H NMR (DMSO-d6, 500 MHz) δ 7.93 (d, 2H,J= 4.3 Hz), 7.61 (dd, 2H,J= 8.4, 1.0 Hz), 7.50 (t, 2H,J= 7.9 Hz), 7.38 (t, 1H,J= 7.4 Hz), 5.82 (s, 1H), 5.46 (s, 2H), 2.78 (m, 1H), 0.63 (m, 2H), 0.58 (m, 2H); 13 C NMR (DMSO-d6, 125 MHz) δ 163.0, 148.2, 146.5, 138.7, 129.2, 127.16, 123.7, 89.4, 22.4, 5.7.

[0154] 5-amino-N-cyclobutyl-1-phenyl-1H-pyrazol-3-carboxamide(E)

[0155] Cyclobutylamine (0.8 equivalents), EDC hydrochloride (3.0 equivalents), HOBt monohydrate (1.5 equivalents), DMAP (0.1 equivalents), and DIPEA (3.0 equivalents) were added to a stirred solution of 5-amino-1-phenyl-1H-pyrazole-3-carboxylic acid in DMF (0.4 M). After stirring overnight at room temperature, the reaction mixture was quenched with H2O. The reaction mixture was diluted with EtOAc, and the organic layer was washed with H2O. The organic layer was dried with MgSO4 and concentrated under vacuum. 5-amino-N-cyclobutyl-1-phenyl-1H-pyrazole-3-carboxamide was purified by flash column chromatography on silica gel (EtOAc : n-hexane = 1 : 1):

[0156] 1 H NMR (DMSO-d6, 500 MHz) δ 8.09 (d, 2H,J= 8.4 Hz), 7.63 (d, 2H,J= 7.4 Hz), 7.52 (t, 2H,J= 7.9 Hz), 7.39 (t, 1H,J= 7.4 Hz), 5.80 (s, 1H), 5.46 (s, 2H), 4.38 (m, 1H), 2.14 (m, 2H), 2.08 (m, 2H), 1.60 (m, 2H); 13 C NMR (DMSO-d6, 125 MHz) δ 160.8, 148.2, 146.5, 138.7, 129.2, 127.1, 123.7, 89.5, 43.6, 30.1, 14.6.

[0157]

[0158] <1-2> Synthesis of the final derivative

[0159] (Chemical Formula 2) MTS-CP1-058

[0160] (COCl)2 (3.0 equivalents) and 2 drops of DMF were added to a stirred solution of 2-fluoro-4-(trifluoromethyl)benzoic acid (1.1 equivalents) dissolved in DCM (0.12 M). After stirring at room temperature for 1 hour, the reaction mixture was vacuum concentrated. A solution of (5-amino-1-phenyl-1H-pyrazole-3-yl)(morpholino)methanone (1.0 equivalent) dissolved in DCM (0.2 M) was added to the reaction mixture solution dissolved in DCM (0.2 M). After stirring at room temperature for 13 hours, the reaction mixture was treated with H2O and then extracted with DCM. The organic layer was dried with MgSO4 and vacuum concentrated. MTS-CP1-058 was purified by flash column chromatography on silica gel (MeOH : DCM = 1 : 40):

[0161] 1 H NMR (CDCl3, 500 MHz) δ 9.05 (d, 1H,J= 13.3 Hz), 8.32 (t, 1H,J= 7.8 Hz), 7.61 (d, 1H,J= 8.2 Hz), 7.48 (m, 5H), 7.40 (t, 1H,J= 7.0 Hz), 7.21 (s, 1H), 3.69 (s, 2H), 3.60 (s, 2H), 3.23 (s, 2H), 3.20 (s, 2H); 13 C NMR (CDCl3, 125 MHz) δ 161.2, 160.9, 159.4, 159.4, 159.2, 147.3, 139.1, 136.5, 133.5, 133.4, 129.7, 128.4, 123.8, 123.7, 123.1, 122.2, 122.2, 122.1, 122.1, 114.4, 114.3, 114.3, 114.1, 101.0, 66.4, 66.4, 47.2, 42.5.

[0162] (Chemical Formula 3) MTS-CP1-060

[0163] (COCl)2 (3.0 equivalents) and 2 drops of DMF were added to a stirred solution of 2,4-dichlorobenzoic acid (1.1 equivalents) dissolved in DCM (0.12 M). After stirring at room temperature for 1 hour, the reaction mixture was concentrated under vacuum. A solution of (5-amino-1-phenyl-1H-pyrazole-3-yl)(morpholino)methanone (1.0 equivalent) dissolved in DCM (0.2 M) was added to the reaction mixture solution dissolved in DCM (0.2 M). After stirring at room temperature for 16 hours, the reaction mixture was heated to 30°C. After stirring at 30°C for 17 hours, TEA (1.0 equivalent) was added to the reaction mixture. After stirring for 17.5 hours, the reaction mixture was quenched with H2O and then extracted with DCM. The organic layer was dried with MgSO4 and concentrated under vacuum. MTS-CP1-060 was purified by flash column chromatography in silica gel (MeOH : DCM = 1 : 30):

[0164] 1 H NMR (CDCl3, 500 MHz) δ 8.71 (s, 1H), 7.76 (d, 1H,J= 8.3 Hz), 7.47 (m, 5H), 7.38 (t, 2H,J= 8.0 Hz), 7.20 (s, 1H), 3.69 (s, 2H), 3.60 (s, 2H), 3.24 (s, 2H), 3.20 (s, 2H); 13 C NMR (CDCl3, 125 MHz) δ 162.8, 160.9, 147.4, 139.1, 138.0, 132.4, 131.9, 130.6, 129.7, 129.63, 128.34, 127.97, 123.07, 100.67, 66.45, 66.39, 47.23, 42.52.

[0165] (Chemical Formula 4) MTS-CP1-072

[0166] (COCl)2 (3.0 equivalents) and 1 drop of DMF were added to a stirred solution of 2-fluoro-4-(trifluoromethyl)benzoic acid (1.1 equivalents) in DCM (0.12 M). After stirring at room temperature for 1 hour, the reaction mixture was concentrated under vacuum. A solution of 5-amino-N-methyl-1-phenyl-1H-pyrazole-3-carboxamide (1.0 equivalent) dissolved in DCM (0.2 M) was added to the reaction mixture solution dissolved in DCM (0.2 M). After stirring at room temperature for 1.5 hours, the reaction mixture was treated with H2O and then extracted with DCM. The organic layer was dried with MgSO4 and concentrated under vacuum. MTS-CP1-072 was purified by silica gel (EtOAc : n-hexane = 1 : 2) flash column chromatography:

[0167] 1 H NMR (CDCl3, 500 MHz) δ 8.77 (d, 1H,J= 15.3 Hz), 8.31 (t, 1H,J= 7.9 Hz), 7.58 (d, 1H,J= 8.2 Hz), 7.54 (m, 2H), 7.50 (d, 2H,J= 7.2 Hz), 7.46 (t, 1H,J= 7.3 Hz), 7.39 (d, 1H,J= 11.8 Hz), 6.75 (s, 1H), 2.37 (s, 3H); 13 C NMR (CDCl3, 125 MHz) δ 161.00, 159.02, 157.94, 157.91, 150.02, 137.57, 135.75, 133.73, 133.72, 130.06, 128.8, 125.0, 123.8, 123.3, 123.2, 122.3, 122.3, 122.3, 121.6, 114.2, 114.2, 114.1, 113.9, 113.9, 98.6, 14.1.

[0168] (Chemical Formula 5) MTS-CP1-073

[0169] 3,5-dichlorobenzoic acid (0.8 equivalents) was added to DCM (0.12 M) and stirred, to which (COCl)2 (3.0 equivalents) and 1 drop of DMF were added. After stirring at room temperature for 1 hour, the reaction mixture was concentrated under vacuum. A solution of 5-amino-N-methyl-1-phenyl-1H-pyrazole-3-carboxamide (1.0 equivalent) in DCM (0.2 M) was added to the reaction mixture solution in DCM (0.2 M). After stirring at room temperature for 17 hours, the reaction mixture was treated with H2O and then extracted with DCM. The organic layer was dried with MgSO4 and concentrated under vacuum. MTS-CP1-073 was purified by silica gel (EtOAc : n-hexane = 1 : 2) flash column chromatography:

[0170] 1 H NMR (CDCl3, 500 MHz) δ 8.03 (s, 1H), 7.57 (s, 2H), 7.51 (t, 3H,J= 7.7 Hz), 7.46 (s, 1H), 7.43 (t, 2H,J= 8.3 Hz), 6.55 (s, 1H), 2.32 (s, 3H); 13 C NMR (CDCl3, 125 MHz) δ 149.8, 137.7, 136.1, 135.9, 135.4, 132.4, 130.0, 128.7, 125.9, 124.6, 99.8, 14.0.

[0171] (Chemical Formula 6) MTS-CP1-093

[0172] 2,4-dichlorobenzoic acid (1.5 equivalents), DIPEA (3.0 equivalents), and T3P (2.0 equivalents, 60% of EtOAc) were added to a stirred solution of 5-amino-N-methyl-1-phenyl-1H-pyrazole-3-carboxamide (1.0 equivalent) in THF (0.12 M). After stirring at room temperature for 1 hour, the reaction mixture was heated to 45°C. After stirring at 45°C for 2 hours, the reaction mixture was heated to 60°C. After stirring at 60°C for 15 hours, the reaction mixture was stopped by treating with aq. NH4Cl and then extracted with EtOAc. The organic layer was dried with MgSO4 and concentrated under vacuum. MTS-CP1-093 was purified by silica gel (EtOAc : n-hexane = 3 : 1) flash column chromatography:

[0173] 1 H NMR (CDCl3, 500 MHz) δ 8.43 (s, 1H), 7.85 (d, 1H,J= 8.3 Hz), 7.53 (m, 5H), 7.39 (s, 1H), 7.36 (d, 1H,J= 8.5 Hz), 7.32 (s, 1H), 6.87 (s, 1H), 2.97 (d, 3H,J= 5.0 Hz); 13 C NMR (CDCl3, 125 MHz) δ 162.2, 161.4, 147.4, 138.5, 137.1, 136.9, 133.0, 131.2, 131.0, 130.4, 130.1, 129.8, 128.2, 125.7, 99.4, 25.9.

[0174] (Chemical Formula 7) MTS-CP1-117

[0175] 2,4-dichlorobenzoic acid (1.5 equivalents), DIPEA (3.0 equivalents), and T3P (2.0 equivalents, 60% of EtOAc) were added at 80°C to a stirred solution of 5-amino-N-cyclopropyl-1-phenyl-1H-pyrazole-3-carboxamide (1.0 equivalent) in DMF (0.12 M). After stirring at 80°C for 15 hours, the reaction mixture was treated with aqueous NH4Cl. The reaction mixture was diluted with EtOAc, and the organic layer was washed with H2O. The organic layer was dried with MgSO4 and concentrated under vacuum. MTS-CP1-117 was purified by flash column chromatography on silica gel (EtOAc : n-hexane = 1 : 1):

[0176] 1 H NMR (DMSO-d6, 500 MHz) δ 10.74 (s, 1H), 8.29 (d, 1H,J= 4.4 Hz), 7.73 (s, 1H), 7.59 (d, 2H,J= 7.8 Hz), 7.54 (m, 4H), 7.47 (t, 1H,J= 7.2 Hz), 6.90 (s, 1H), 2.85 (m, 1H), 0.67 (m, 2H), 0.61 (m, 2H); 13 C NMR (DMSO-d6, 125 MHz) δ 164.6, 162.1, 146.5, 137.9, 136.4, 135.3, 134.2, 131.3, 130.3, 129.3, 129.2, 128.5, 127.4, 124.7, 102.3, 22.5, 5.7.

[0177] (Chemical Formula 8) MTS-CP1-125

[0178] 2,4-dichlorobenzoic acid (1.0 equivalent), DIPEA (2.5 equivalents), and T3P (1.8 equivalents, 60% of EtOAc) were added under reflux to a stirred solution of 5-amino-N-cyclobutyl-1-phenyl-1H-pyrazole-3-carboxamide (1.0 equivalent) in THF (0.2 M). After stirring at 85°C for 22 hours, the reaction mixture was quenched with aq. NaHCO3. The reaction mixture was diluted with EtOAc and then extracted with EtOAc. The organic layer was dried with MgSO4 and concentrated under vacuum. MTS-CP1-125 was purified by flash column chromatography on silica gel (EtOAc : DCM : n-hexane = 3 : 2 : 2):

[0179] 1 H NMR (DMSO-d6, 500 MHz) δ 10.74 (s, 1H), 8.46 (d, 1H,J= 8.2 Hz), 7.73 (d, 1H,J= 1.5 Hz), 7.61 (dd, 2H,J= 7.8, 1.6 Hz), 7.55 (m, 4H), 7.48 (m, 1H), 6.90 (s, 1H), 4.42 (p, 1H,J= 8.4 Hz), 2.15 (m, 4H), 1.64 (m, 2H); 13 C NMR (DMSO-d6, 125 MHz) δ 164.6, 159.9, 146.5, 137.9, 136.5, 135.3, 134.2, 131.3, 130.3, 129.3, 129.2, 128.5, 127.4, 124.7, 102.4, 43.8, 30.0, 14.6.

[0180] (Chemical Formula 9) MTS-CP1-146

[0181] 2,4-dichlorobenzoic acid (1.0 equivalent), DIPEA (2.5 equivalents), and T3P (1.8 equivalents, 60% of EtOAc) were added under reflux to a stirred solution of 5-amino-N-methyl-1-phenyl-1H-pyrazole-3-carboxamide (1.0 equivalent) in THF (0.2 M). After stirring under reflux for 20 hours, the reaction mixture was treated with aqueous NaHCO3. The reaction mixture was diluted with EtOAc and then extracted with EtOAc. The organic layer was dried with MgSO4 and then concentrated under vacuum. MTS-CP1-146 was purified by flash column chromatography on silica gel (EtOAc : DCM : n-hexane = 3 : 1 : 1):

[0182] 1 H NMR (DMSO-d6, 125 MHz) δ 10.75 (s, 1H), 8.27 (q, 1H,J= 4.6 Hz), 7.90 (d, 1H,J= 1.95 Hz), 7.86 (d, 2H,J= 2.0 Hz), 7.61 (dd, 2H,J= 7.9, 1.7 Hz), 7.52 (dd, 2H,J= 8.6, 7.1 Hz), 7.43 (t, 1H,J= 7.3 Hz), 6.82 (s, 1H), 2.77 (d, 3H,J= 4.7 Hz); 13 C NMR (DMSO-d6, 125 MHz) δ 163.3, 161.4, 146.6, 138.2, 136.6, 136.1, 134.4, 131.6, 129.2, 128.9, 126.5, 123.8, 103.4, 25.6.

[0183] (Chemical Formula 10) MTS-CP1-154

[0184] 2-fluoro-4-(trifluoromethyl)benzoic acid (1.0 equivalent), DIPEA (2.5 equivalents), and T3P (1.8 equivalents, 60% of EtOAc) were added under reflux to a stirred solution of 5-amino-N-methyl-1-phenyl-1H-pyrazole-3-carboxamide (1.0 equivalent) in THF (0.2 M). After stirring under reflux for 16 hours, the reaction mixture was treated with aqueous NaHCO3. The reaction mixture was diluted with EtOAc and then extracted with EtOAc. The organic layer was dried with Na2SO4 and concentrated under vacuum. MTS-CP1-154 was purified by flash column chromatography on silica gel (EtOAc : DCM = 1 : 10):

[0185] 1 H NMR (DMSO-d6, 500 MHz) δ 10.8 (s, 1H), 8.28 (q, 1H,J= 4.8 Hz), 7.87 (d, 1H,J= 9.3 Hz), 7.84 (d, 1H,J= 7.5 Hz), 7.72 (d, 1H,J= 8.1 Hz), 7.62 (d, 2H,J= 7.9 Hz), 7.56 (t, 2H,J= 7.7 Hz), 7.48 (t, 1H,J= 7.4 Hz), 6.92 (s, 1H), 2.77 (d, 3H,J= 4.7 Hz); 13 C NMR (DMSO-d6, 125 MHz) δ 162.0, 161.4, 159.9, 157.9, 146.7, 138.0, 136.4, 131.5, 131.4, 129.3, 128.5, 127.1, 126.9, 124.4, 121.7, 121.6, 121.6, 114.2, 114.1, 114.0, 113.9, 102.2, 25.7.

[0186]

[0187] <Experimental Example 1> Analysis of in vitro TrkA selectivity of TrkA inhibitors

[0188] The pain signaling inhibitory effect of the MTS-CP compounds synthesized in <Example 1> above was verified in human osteosarcoma cells, U2OS.

[0189] The β-galactosidase enzyme exhibits activity only when its α-fragment and ω-fragment bind, a process known as alpha complementation. The Path-Hunter system consists of U2OS osteosarcoma cells injected with a protein combining the TrkA receptor with an α-fragment (Prolink, PF) and a protein combining SHC1 with an ω-fragment (Enzyme Fragment, EF). When U2OS cells expressing TrkA-PF and SHC1-EF are treated with NGF, PF-EF binding occurs via TrkA-SHC1 binding, leading to the manifestation of β-galactosidase enzymatic activity. The enzymatic activity of β-galactosidase can be quantified by measuring the 540 nm fluorescence emitted upon the degradation of the substrate galacton star (Emerald II). Accordingly, the extent to which MTS-CP compounds inhibit NGF-induced enzyme activity can be measured.

[0190] U2OS cells of the Path-Hunter system were treated with NGF, BDNF, and NT3 at various concentrations (TrkA treated with NGF, TrkB with BDNF, and TrkC with NT3), respectively, to determine the concentrations of NGF, BDNF, and NT3 and EC that maintain maximum enzymatic activity. 50 The values ​​were measured. The above U2OS cells were seeded at a density of 10,000 / well and cultured for 48 hours at 37°C under 5% CO2 conditions. The above NGF, BDNF, and NT3 were treated at various concentrations (2000, 400, 80, 16, 3.2, 0.64, 0.128, 0.025, 0.005, 0.001, 0.0002 ng / mL), respectively, at 23°C for 3 hours, luminescence was measured using a plate reader, and EC was calculated.50 The concentrations of NGF, BDNF, and NT3 exhibiting maximum effects were determined by calculating the values. After treating with NGF, BDNF, and NT3 (1 μg / ml) at concentrations that induce maximum enzymatic activity, respectively, and then treating with MTS-CP compounds at various concentrations (3.2 nM, 16 nM, 80 nM, 400 nM, 2 μM, 10 μM, 50 μM), the IC50 values ​​for each MTS-CP compound were calculated. 50 The value was calculated.

[0191] As a result, it was confirmed that MTS-CP compounds have selectivity for TrkA.

[0192] Compound IC 50 (μM)TrkATrkBTrkCMTS-CP1-0720.48>100>100MTS-CP1-0930.54>100>100MTS-CP1-1170.56>100>100MTS-CP1-1252.52>100>100MTS-CP1-1540.36> 100> 100

[0193]

[0194] <Experimental Example 2> Evaluation of Cytotoxicity of TrkA Inhibitors

[0195] To evaluate the cytotoxicity of MTS-CP compounds, measurements were taken using DoGen’s Ez-Cytox product, which has the same mechanism as the MTT assay.

[0196] EZ-Cytox's WST is reduced by succinate-tetrazolium reductase, a dehydrogenase present in the mitochondrial respiratory chain that is active only in living cells, to produce a chromogenic substance called formazan. Therefore, as the number of viable cells in the sample increases, the activity of mitochondrial dehydrogenase also increases, leading to increased formazan production and consequently, an increase in absorbance. In other words, the production of formazan has a linear correlation with the number of living cells, which can be determined by measuring absorbance. Generally, a substance exhibiting a cell viability of 80% or less compared to the absorbance of a control group can be considered to possess cytotoxicity.

[0197] First, cytotoxicity experiments were conducted on the human liver cell line HepG2. HepG2 cells were seeded into 96-well cell culture plates and cultured overnight. After treatment with the compounds at various concentrations, cell viability was confirmed by measuring absorbance at 450 nm after 48 hours. 800 μM was set as the final concentration, and experimental groups at lower concentrations of 400, 200, 100, and 50 μM were established through serial dilution. DMSO was set as the negative control.

[0198] As a result of absorbance measurements, all MTS-CP compounds showed cell viability of more than 80% compared to the control group up to 400 μM, indicating no cytotoxicity (Fig. 2).

[0199] Next, cytotoxicity experiments were conducted on PC12 cells derived from the adrenal glands of rats against the compound MTS-CP-093. The PC12 cell line is known to possess the ability to differentiate into neurofibrils upon NGF treatment and to co-express TrkA and TRPV1. The final concentration was set at 30 μM, and experimental groups with concentrations of 10, 3, and 1 μM were established below this level. Additionally, a negative control group was established, and the negative control was defined as the excipient DMSO.

[0200] As a result of absorbance measurement, the MTS-CP-093 compound showed a cell viability of over 80% compared to the control group up to 10 μM, indicating no cytotoxicity (Fig. 3).

[0201]

[0202] <Experimental Example 3> Target Verification and Mechanism of Action Study of TrkA Inhibitor

[0203] <3-1> Verification of whether NGF blocks the TRPV1 response

[0204] To investigate how TRPV1, a well-known factor related to pain, is affected by the MTS-CP compound, the mechanism of action of the MTS-CP compound was confirmed through the calcium response after administering capsaicin, a representative activator of TRPV1, to DRGs, which are sensory nerve cells.

[0205] When the neurotransmitter NGF binds to TrkA expressed on the surface of DRG cells, membrane expression of sodium channels (Nav1.8) increases, generating action potentials that transmit pain signals to the brain; additionally, pain-inducing substances such as capsaicin are presented to ensure the generation of a definite action potential. In this process, the binding of TrkA to NGF increases intracellular calcium ion flow, thereby achieving membrane expression of sodium channels. The increase in intracellular calcium induced by NGF is caused by calcium entering from outside the cell and calcium released from the smooth endoplasmic reticulum, an intracellular calcium reservoir; thus, NGF is known to act on both intracellular and extracellular mechanisms through TrkA.

[0206] As a result of treating DRG cells with MTS-CP compounds under NGF-free conditions in calcium imaging, the MTS-CP compounds were each used to [induce] capsaicin-induced Ca 2+ TRPV1 activity was inhibited by blocking inflow.

[0207] Furthermore, even in the presence of NGF, MTS-CP compounds [relative to] capsaicin-induced Ca 2+ It was found that blocking the inflow inhibited not only the activity of TRPV1 but also the increase in TRPV1 activity caused by TrkA (Fig. 4).

[0208]

[0209] <3-2> Heat Response Test (43℃)

[0210] TRPV1 is activated by capsaicin, but it is also activated by heat stimulation (> 42°C). The TRPV1 blockers known to date have a heat-blocking effect, and their use can lead to the side effect of hyperthermia.

[0211] We confirmed in animals using the Hargreaves test whether the original function of TRPV1, heat sensing above 43°C, is inhibited by the MTS-CP-093 compound. After administering the MTS-CP-093 compound at i) sole injection (1 μM, 194.62 npk), ii) intraperitoneal injection (10 mpk), and iii) oral administration (10 mpk), the avoidance reflex was analyzed after applying a heat stimulus of 43–45°C in the Hargreaves test.

[0212] As a result, the MTS-CP-093 compound did not inhibit responsiveness to heat stimulation in animals. Therefore, it was confirmed that the MTS-CP-093 compound did not inhibit the function of TRPV1, which detects heat stimulation at 43°C. This indicates that the MTS-CP-093 compound acts on TrkA to secondarily block TRPV1, and thus does not exhibit side effects similar to those of existing direct TRPV1 blockers (Fig. 5).

[0213]

[0214] <Experimental Example 4> Analysis of Neurote Outgrowth by TrkA Inhibitors

[0215] The inhibitory effect of the MTS-CP compound on TrkA signaling was verified through the inhibition of neurite formation in pheochromocytoma (PC12) cells.

[0216] Nerve Growth Factor (NGF) acts on pain receptors to amplify pain signals; specifically, NGF binds to the receptor TrkA to activate neurons and enhance the transmission of pain signals. Signal transduction mediated by TrkA is initiated by the binding of the SHC1 protein through the autophosphorylation of the receptor. The phenomenon in which the activity of SHC1 bound by NGF is inhibited by the MTS-CP compound was verified through the presence or absence of neurite formation in PC12 cells.

[0217] PC12 cells were cultured in RPMI-1640 medium supplemented with 10% DHS, 5% FBS, 1% amphotericin B, and gentamycin. Neurodegeneration of PC12 cells was induced by treating with NGF while culturing in RPMI-1640 medium supplemented with 1% DHS, 1% amphotericin B, and gentamycin. For the neurogenesis of PC12 cells, culture vessels coated with poly-D-lysine and laminin were used. PC12 cells were treated with MTS-CP compounds MTS-CP-000, MTS-CP-093, and MTS-CP-154 at concentrations of 0.5 μM, 1 μM, 10 μM, and 30 μM, respectively. After 1 hour, NGF was added at a concentration of 100 ng / ml, and the cells were cultured for 9 days (replaced with culture medium containing the same concentrations of NGF and MTS-CP compounds every 2 days). The inhibitory effect of MTS-CP compounds on neurite formation was then measured. The presence or absence of neurites was confirmed by staining with Tuj1 (neuron-specific class III beta-tubulin), a neurite marker, and the length of the neurites was measured using the Neuron J program. When statistical significance (p < 0.05) was confirmed in one-way ANOVA, a post hoc test using Tukey's HSD (Honestly Significant Difference) analysis was performed to analyze the statistical significance between each concentration group.

[0218] As a result, it was found that the MTS-CP compounds statistically significantly inhibited NGF-induced neurite formation at all concentrations, effectively blocking NGF signaling induced by TrkA (Figs. 6a, 6b, 7a and 7b).

[0219]

[0220] <Experimental Example 5> Evaluation of TrkA Inhibitors in an Osteoarthritis Pain Animal Model

[0221] <5-1> Verification of Analgesic Efficacy in an MIA-Induced Osteoarthritis Model in SD (Sprague Dawley) Rat

[0222] The evaluation of analgesic efficacy using animals was analyzed using an osteoarthritis model induced by MIA (monosodium iodoacetate).

[0223] The MIA-induced osteoarthritis model is the most frequently performed animal model for osteoarthritis pain. It induces chondrocyte necrosis by inhibiting glycolysis in chondrocytes that constitute cartilage, and this process induces various inflammatory cytokines that cause damage to the cartilage matrix; thus, it is an animal model that most closely replicates the mechanism of human osteoarthritis.

[0224] Sprague-Dawley (SD) rats, which are commonly used in animal experiments, were used as experimental animals. All animal experiments were approved by the Mothers Pharma Institutional Animal Care and Use Committee (IACUC) in accordance with the National Institutes of Health (NIH) guidelines (IACUC approval number: MTS-IACUC-24002). Six-week-old SD rats (approximately 180 g body weight) were used as experimental animals. After a one-week stabilization period following introduction, osteoarthritis was induced by injecting MIA when the animals reached a body weight of approximately 200 g at seven weeks of age.

[0225] The method for inducing an osteoarthritis model using MIA injection was performed as follows. An MIA solution was prepared at a concentration of 1 mg / 50 μL / joint, and physiological saline was used as the solvent. Seven-week-old SD rats (body weight 200–230 g) were anesthetized with isoflurane (Oxygen: 1, ISO: 2–3). The right leg was then disinfected by wiping it with 70% ethanol, and an intra-articular injection was administered using a 500 μL insulin syringe. The injection was made by penetrating the patellar tendon; the injection site was pressed firmly for 30 seconds to stop bleeding, and the animals were checked to ensure they safely recovered from anesthesia. The osteoarthritis induction period was set to 10 days after the MIA injection. The experimental groups were configured as shown in Table 2 below.

[0226] Experimental group animals Sham 5MIA 1 mg injection MIA only (vehicle control) 5Naproxen, 10 mpk 5Pfizer(MTS-CP-000), 10 mpk 5MTS-CP-0931 mpk 55 mpk 510 mpk 5MTS-CP-1541 mpk 55 mpk 510 mpk 5

[0227]

[0228] To evaluate mechanical pain sensitivity, the paw withdrawal threshold (PWT) was measured using the von Frey test. A von Frey assay was performed before administration of the MTS-CP compound to determine whether pain due to MIA was induced.

[0229] The von Frey assay was performed as follows. Experimental animals were placed individually in von Frey cages, and the center of the animal's hind paw was stimulated using von Frey filaments of increasing thickness, starting from 2 g (2, 4, 6, 8, 10, 15, 26, 60, and 100 g). The filament was pricked for at least 1 second per stimulation, and the value was recorded if the animal shook its paw, licked it, or exhibited avoidance behavior. Once one measurement was completed for each animal moving from left to right, stimulation was repeated starting from the left with the previously stimulated filament. This process was repeated three times to calculate the values, and the average was determined. The PWT values ​​before and after the administration of the compound were compared.

[0230] Differences in PWT after administration of 1, 5, and 10 mpk of naproxen, MTS-CP-000, or MTS-CP-093 and MTS-CP-154 compounds were analyzed using the Kruskal-Wallis test. When statistical significance of differences in PWT caused by CP compounds and naproxen was confirmed in the Kruskal-Wallis test (p < 0.05), post-hoc comparisons were performed using Dunn's multiple comparisons test to analyze statistical significance between the vehicle control (MIA only) group, the compound administration group, and the naproxen administration group.

[0231] As a result, PWT values ​​decreased significantly in all experimental groups except the Sham group, confirming pain induction caused by MIA. When the pain-inhibiting effects of MTS-CP-093 and MTS-CP-154 compounds were confirmed through an MIA-induced pain model, a dose-dependent analgesic effect was observed. When comparing the effects of both MTS-CP-093 and MTS-CP-154 compounds with those of naproxen and CP-000 (Pfizer candidate compound), they showed similar or superior trends at the same dose (10 mpk) (Figs. 8 to 11).

[0232]

[0233] <5-2> Verification of Analgesic Efficacy in MIA-Induced Osteoarthritis Model in C57BL / 6N Mice

[0234] The analgesic effect of the MTS-CP-093 compound was confirmed in a pain model created by injecting MIA into the left knee cartilage of wild-type C57BL / 6N mice. Pain induction and the analgesic efficacy of the drug were confirmed by measuring weight-bearing asymmetry using the von Frey test and an incapacitance meter.

[0235] C57BL / 6N mice (Coatech Co., Ltd.) were housed in groups (5–6 mice / cage) in polycarbonate cages. The temperature was maintained at 19–25℃, humidity at 40–60%, the light-dark cycle / illumination at 12 hours / day and 150–300 Lux. Sterile water was provided free of charge, and laboratory solid feed (Envigo 18% Protein Rodent Diet 2018SC) was provided free of charge. This study was approved by the Animal Ethics Committee of Mothers Pharma Co., Ltd. in accordance with the Animal Protection Act (Approval No.: MTS-IACUC-24002).

[0236] The experimental group was set as shown in Table 3 below.

[0237] Experimental group animals Sham 6MIA 1 mg injection MIA only (vehicle control) 5MTS-CP-09310 mpk5

[0238]

[0239] Since mice have a faster metabolism than rats, the osteoarthritis induction period after MIA injection was shortened to 3 days. The von Frey test was performed on day 3 after MIA injection, and the weight-bearing test was performed on day 4 after MIA injection.

[0240] For the von Frey test, experimental animals were placed on a von Frey system made of 3 mm mesh, their vision was blocked, and they were stabilized for 30 minutes. Then, a 0.4 g von Frey filament was used to puncture the center of the sole of the leg injected with MIA or saline, and it was confirmed whether the animal avoided it. For the weight-bearing asymmetry test, experimental animals were placed in a cage equipped with an incapacitance meter and stabilized for 5 minutes. Afterward, the floor was placed against the same part of the left and right scales, and once the animal assumed a stable posture, measurements were taken for 3 to 5 seconds; the measurement was repeated 5 times per individual.

[0241] The difference in PWT after administration of Sham, vehicle control (MIA only), and CP-093 (10 mpk) was analyzed using the Kruskal-Wallis test. When statistical significance of the difference in PWT was confirmed in the Kruskal-Wallis test (p < 0.05), a post-hoc comparison test was performed using Dunn's multiple comparisons test to analyze the statistical significance between the vehicle control (MIA only) group and the CP-093 (10 mpk) administration group.

[0242] As a result, pain caused by MIA was successfully induced in wild-type C57BL / 6N mice, and it was confirmed that the pain-induced paw withdrawal threshold (PWT) and the balance of weight distribution between the left and right legs improved after administration of MTS-CP-093 (Fig. 12).

[0243]

[0244] <5-3> Verification of Analgesic Efficacy in an MIA-Induced Osteoarthritis Model in TRPV1 Gene-Deficient Mice

[0245] The analgesic effect of MTS-CP-093 was confirmed in a pain model created by injecting MIA into the left knee cartilage of TRPV1 gene-deficient mice. Pain induction and the analgesic efficacy of the drug were confirmed by measuring weight-bearing asymmetry using the von Frey test and an incapacitance meter.

[0246] The experimental animals were B6.129X1-Trpv1 tm1Jul The subjects were 6 males (21–29 g) and 5 females (9–21 g) aged 10 weeks (*J*, The Jackson Laboratory, USA). They were housed in groups (5–6 animals / cage) in polycarbonate cages, with a temperature of 19–25°C, humidity of 40–60%, a light-dark cycle / illumination of 12 hours / day and 150–300 Lux. Sterile water was provided free of charge, and laboratory solid feed (Envigo 18% Protein Rodent Diet 2018SC) was provided free of charge. This study was approved by the Animal Ethics Committee of Mothers Pharma Co., Ltd. and the Animal Ethics Committee of Hanyang University College of Medicine in accordance with the Animal Protection Act (Approval Nos.: MTS-IACUC-24002, HY-IACUC-2024-0045A).

[0247] The experimental group was set as shown in Table 4 below.

[0248] Experimental Group Individual Number Age Male(g) Female(g) Sham Non-hole 1024R1 1027R2 1020R3 1020 Vehicle L1 1024 108 1023 L1 (Torn Ear) 1020 CP-093,10 mpk L2 (Torn Ear) 1021R1 L1 1029 113 1021R2 L2 1019

[0249]

[0250] The administration was done orally, once a day for two days, and was forcibly administered into the stomach using a disposable syringe with an oral delivery device attached, and no fasting was performed prior to administration.

[0251] For the von Frey test, experimental animals were placed on a von Frey system made of 3 mm mesh, their vision was blocked, and they were stabilized for 30 minutes. Then, a 0.4 g von Frey filament was used to puncture the center of the sole of the leg injected with MIA or saline, and it was confirmed whether the animal avoided it. For the weight-bearing asymmetry test, experimental animals were placed in a cage equipped with an incapacitance meter and stabilized for 5 minutes. Afterward, the floor was placed against the same part of the left and right scales, and once the animal assumed a stable posture, measurements were taken for 3 to 5 seconds; the measurement was repeated 5 times per individual.

[0252] The difference in PWT after administration of Sham, vehicle control (MIA only), and CP-093 (10 mpk) was analyzed using the Kruskal-Wallis test. When statistical significance of the difference in PWT was confirmed in the Kruskal-Wallis test (p < 0.05), a post-hoc comparison test was performed using Dunn's multiple comparisons test to analyze the statistical significance between the vehicle control (MIA only) group and the CP-093 (10 mpk) administration group.

[0253] As a result, it was confirmed that pain caused by MIA was not induced in TRPV1 gene-deficient mice, and no changes were observed even after administration of MTS-CP-093. Therefore, it was found that pain caused by MIA is induced through TRPV1, and accordingly, the analgesic effect of the developed substance acts through TRPV1 (Fig. 13).

[0254]

[0255] <5-4> Evaluation of Carrageenan-Induced Osteoarthritis Pain in Animal Model

[0256] The analgesic efficacy was evaluated using a carrageenan-induced osteoarthritis animal model.

[0257] The carrageenan-induced osteoarthritis pain animal model is a frequently used animal model for reproducing acute pain. It induces osteoarthritis through a mechanism in which pain is induced by directly injecting a thickening agent into the tissue, which triggers the release of histamine and serotonin from mast cells, thereby inducing an initial inflammatory response and pain signals, followed by inflammatory cytokines such as TNF-α, IL-1β, COX2, and PEG2 increasing the inflammatory and pain responses.

[0258] Osteoarthritis was induced using SD rats, which are commonly used in animal experiments. The experimental groups were set as shown in Table 5 below.

[0259] Experimental group animals Sham 6 2% Carrageenan injection Carrageenan (vehicle control) 6 Naproxen, 10 mpk 6 Pfizer (MTS-CP-000), 10 mpk 6 MTS-CP-09 31 mpk 6 5 mpk 6 10 mpk 6

[0260]

[0261] The paw withdrawal threshold (PWT) was measured as the mechanical pain sensitivity of experimental animals that reproduced osteoarthritis-like pain with carrageenan.

[0262] As a result, it was confirmed that osteoarthritis was effectively induced, as the PWT values ​​of the carrageenan (= vehicle control) group decreased significantly compared to the sham group. After administration of the test substance, a trend of improvement in PWT was observed in the positive control substances naproxen and MTS-CP-000 (Pfizer substance), and MTS-CP-093 showed a dose-dependent trend of improvement, showing a statistically significant improvement at a dose of 10 mpk (Fig. 14).

[0263]

[0264] <Experimental Example 6> Evaluation of Hyperthermia Induction

[0265] TRPV1 channels are ion channels present in c fibers that transmit pain signals; they are known to amplify pain stimuli by increasing cell membrane ion permeability in response to pain stimulation caused by capsaicin and heat stimulation, thereby inducing depolarization. Clinical studies on new drugs for pain treatment have shown that hyperthermia is a representative side effect of new TRPV1 antagonist drugs.

[0266] Accordingly, to determine whether hyperthermia occurs upon administration of the MTS-CP compound, the body temperature of wild-type C57BL / 6N mice and wild-type Sprague-Dawley (SD) rats was measured at 15-minute intervals for 30 minutes prior to the administration of the compound, and body temperature was measured at 0, 15, 30, 45, 60, and 90 minutes after oral administration of the MTS-CP-093 compound to check for changes in body temperature before and after administration.

[0267] As a result, no increase in body temperature caused by MTS-CP-093 was observed in both mice and rats (Fig. 15).

[0268]

[0269] <Experimental Example 7> Off-target profiling (scanMAX) analysis of TrkA inhibitors

[0270] Eurofins’ scanMAX is an analytical platform designed to evaluate the activity of compounds in the early stages of drug development, used to assess target specificity and mechanisms of action. Utilizing a target-based analytical platform, scanMAX generates comprehensive data that can evaluate the binding affinity, selectivity, efficacy, and off-target effects of compounds. This enables the identification of pharmacological properties and the efficient selection of lead compounds, particularly allowing for a quantitative understanding of interactions with targets. Furthermore, scanMAX can be utilized to analyze the action of specific signaling pathways as well as enzyme and receptor-based targets.

[0271] To evaluate the selectivity of MTS-CP compounds for kinases, two compounds, MTS-CP-093 and MTS-CP-154, were analyzed at 10 μM using Eurofins’ scanMAX, a 468-type kinase assay.

[0272] As a result, TrkA was the only kinase that exhibited an inhibition rate of 65% or higher, which is the evaluation criterion for MTS-CP-093 and MTS-CP-154 compounds, confirming that these MTS-CP compounds have excellent selectivity for kinases.

[0273]

[0274] <Experimental Example 8> Chromosomal Abnormality Test of TrkA Inhibitors

[0275] To evaluate the genotoxicity of the MTS-CP compound, a chromosomal aberration test was performed using Chinese Hamster Lung (CHL) cells.

[0276] CHL cells are a suitable cell line for detecting chromosomal abnormalities and are primarily used in tests to determine whether compounds or drugs cause DNA damage.

[0277] Chromosomal structural abnormalities were measured by exposing mammalian cell line CHL cells to compounds MTS-CP-093 and MTS-CP-154. Precipitation and cytotoxicity caused by the test substances were observed by setting a total of 12 dose steps with a common ratio of 2, starting with 2,000 μg / mL as the maximum dose of the test substance. A negative control (excipient) and a positive control were added.

[0278] As a result, it was confirmed that both MTS-CP-093 and MTS-CP-154 compounds do not induce chromosomal abnormalities.

[0279]

[0280] <Experimental Example 9> Reversion mutation test using bacterial strains of TrkA inhibitor

[0281] To evaluate the genotoxicity of the MTS-CP compound, an Ames test using bacterial strains was performed.

[0282] The Ames test allows for the early assessment of the potential to induce DNA mutations, and since mutations are directly linked to the development of cancer, it plays an important role in predicting the carcinogenic potential of a substance.

[0283] Reverse mutation tests were conducted on the compounds MTS-CP-093 and MTS-CP-154 using histidine-requiring Salmonella typhimurium strains (TA98, TA100, TA1535, TA1537) and tryptophan-requiring Escherichia coli strains (WP2uvrA (pKM101)). The maximum dose was set at 5,000 μg / plate, and precipitation and growth inhibition by the test substances were observed by treating with a total of 9 dose levels, including the maximum dose and a zero-reference ratio of 2.

[0284] As a result, it was confirmed that both MTS-CP-093 and MTS-CP-154 compounds did not induce reverse mutations in bacterial strains.

[0285]

[0286] <Experimental Example 10> In vivo micronucleus test using rats with TrkA inhibitor

[0287] To evaluate whether the MTS-CP compound induces genetic damage, an in vivo micronucleus test was performed using rats.

[0288] The micronucleus test verifies whether a compound induces chromosomal damage (chromosome breakage or aneuploidy) leading to the formation of abnormal micronuclei during cell division; as chromosomal damage is one of the major causes of carcinogenesis and mutation, this test is essential for predicting the carcinogenic potential of a substance. Additionally, the in vivo test is a method that considers metabolic processes and physiological interactions within the body, allowing for the evaluation of the potential for genotoxicity caused by metabolites activated by hepatic metabolism.

[0289] To evaluate the induction of micronuclei in SD rat bone marrow cells, the compound MTS-CP-093 was administered orally to rats twice at 24-hour intervals. The maximum dose was set at 1,000 mg / kg / day, and the lower doses were set at 300 and 100 mg / kg / day. A negative control group was established, and the test was conducted on male rats known to be highly susceptible to micronucleus induction.

[0290] As a result, it was confirmed that the MTS-CP-093 compound was not micronucleus-inducing at a maximum dose of 1000 mpk.

[0291]

[0292] <Experimental Example 11> Evaluation of the Effects of a Single Oral Dose of a TrkA Inhibitor on the Central Nervous System of Rats

[0293] Central nervous system (CNS) impact assessment tests are designed to identify the potential toxicity of pharmaceuticals, chemicals, and other substances to the central nervous system. Since the CNS plays a critical role in maintaining life and regulating behavior, substances that induce toxicity in the brain and spinal cord can lead to severe behavioral abnormalities, neurological disorders, or fatal consequences. Substances that exhibit CNS toxicity from a single exposure are highly likely to cause more severe effects upon long-term or repeated exposure; therefore, potential risks to humans can be managed in advance by identifying the substance's potential CNS toxicity through animal models (such as rats).

[0294] The effects on the central nervous system were evaluated by conducting a functional observational battery (FOB) after administering a single oral dose of the test substance, compound MTS-CP-093, to 6-week-old male Sprague-Dawley rats. A total of four groups were established, consisting of a control group and groups administered 100, 300, and 1000 mpk of MTS-CP-093 compound, with six rats in each group. The control group was administered a 0.5% methylcellulose 1500 cP aqueous solution, which served as an excipient. For the functional observational battery, observations were conducted in the home cage, open field, and under hand holding conditions. Sensory and motor function tests and body temperature measurements were performed before administration (hour 0) and at 0.5, 1, 3, 6, and 24 hours after administration.

[0295] As a result, it was confirmed that no changes caused by the administration of the MTS-CP-093 compound were observed in all test items, including observation in the home cage, observation in the open field, observation under hand holding, sensory and motor function tests, body temperature measurement, general symptoms, and changes in body weight.

[0296]

[0297] Foregoing, specific parts of the present invention have been described in detail. It is evident to those skilled in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the invention. That is, the actual scope of the invention is defined by the appended claims and their equivalents.

Claims

Compound represented by the following chemical formula 1 or a pharmaceutically acceptable salt thereof: <Chemical Formula 1> In the above chemical formula 1, R 1 C 1-6 Alkyl or CONR a R b And, The above R a or R b may be the same or different, and hydrogen, C 1-6 Alkyl, C 1-6 Alkenyl, C 1-6 Alkainyl, C 3-10 Cycloalkyl, C 4-10 Heterocycloalkyl and C 5-10 Selected from the group consisting of heteroaryls, or R a and R b C is connected 4-10 Heterocycloalkyl or C 5-10 Forming a heteroaryl, R 2 , R 3 or R 4 Each may be the same or different and is selected from the group consisting of hydrogen, halogens, and alkyl halides. In Article 1, The above compound is, The above R 1 C 1-6 Alkyl or CONR a R b and, the above R a or R b may be the same or different, and hydrogen, C 1-4 Alkyl and C 3-7 Selected from the group consisting of cycloalkyl, or R a and R b C is connected 4-10 A compound or a pharmaceutically acceptable salt thereof characterized by forming a heterocycloalkyl group. In Article 1, The above compound is, The above R 2 , R 3 or R 4 A compound or a pharmaceutically acceptable salt thereof, characterized in that each may be the same or different and is selected from the group consisting of hydrogen, Cl, F, and CF3. In Article 1, The above C 4-10 Heterocycloalkyl or C 5-10 Heteroaryls are, A compound or a pharmaceutically acceptable salt thereof, characterized by being selected from the group consisting of pyrrolyl, furanyl, thiophenyl, imidazolyl, pyridinyl, morpholino, piperidinyl, and piperazinyl. In Article 1, The above compound is, A compound or a pharmaceutically acceptable salt thereof, characterized as being any one compound selected from the group consisting of compounds represented by the following chemical formulas 2 to 10: <Chemical Formula 2> ; <Chemical Formula 3> ; <Chemical Formula 4> ; <Chemical Formula 5> ; <Chemical Formula 6> ; <Chemical Formula 7> ; <Chemical Formula 8> ; <Chemical Formula 9> ; and <Chemical Formula 10> In Article 1, The above compound is, A compound or a pharmaceutically acceptable salt thereof characterized by selectively binding to tropomyosin receptor kinase A (TrkA). In Article 1, The above compound is, A compound or a pharmaceutically acceptable salt thereof characterized by binding to TrkA and blocking Nerve Growth Factor (NGF) signaling. In Article 1, The above compound is, A compound or a pharmaceutically acceptable salt thereof characterized by inhibiting the activity of TRPV1 (transient receptor potential vanilloid 1). A pharmaceutical composition comprising a compound according to claim 1 or a pharmaceutically acceptable salt thereof. A pharmaceutical composition for the prevention or treatment of pain diseases, comprising a compound according to claim 1 or a pharmaceutically acceptable salt thereof. In Article 10, The above pain condition is, A pharmaceutical composition for the prevention or treatment of pain diseases, characterized by being osteoarthritis pain, diabetic neuropathy, neuropathic pain, or anticancer agent-induced neuropathy. A pharmaceutical composition for the prevention or treatment of inflammatory diseases, comprising a compound according to claim 1 or a pharmaceutically acceptable salt thereof. A health functional food composition for preventing or improving pain disorders, comprising a compound according to claim 1 or a food-grade acceptable salt thereof. In Article 13, The above pain condition is, A health functional food composition for preventing or improving pain diseases, characterized by being osteoarthritis pain, diabetic neuropathy, neuropathic pain, or anticancer drug-induced neuropathy. A health functional food composition for preventing or improving inflammatory diseases, comprising a compound according to claim 1 or a food-grade acceptable salt thereof.

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