WO2024205157A1 - Novel pyrrolidinium compounds having antagonistic activity against muscarinic receptors and use thereof - Google Patents

Abstract

The present invention relates to a novel pyrrolidinium compound, a stereoisomer, hydrate, or solvate thereof, which exhibits excellent binding ability and antagonistic activity to muscarinic receptors, especially muscarinic M3 receptors. The compound of the present invention is useful in the treatment of a muscarinic acetylcholine receptor-mediated disease, such as hyperhidrosis, hypersalivation, chronic obstructive pulmonary disease, chronic bronchitis, asthma, rhinitis, urinary incontinence, overactive bladder syndrome, gastroesophogeal reflux disease, and irritable bowel syndrome, etc.

Description

NOVEL PYRROLIDINIUM COMPOUNDS HAVING ANTAGONISTIC ACTIVITY AGAINST MUSCARINIC RECEPTORS AND USE THEREOF

The present invention relates to a novel pyrrolidinium compound, a stereoisomer, hydrate, or solvate thereof, which has antagonistic activity against muscarinic receptors, a pharmaceutical composition comprising the same as an active ingredient, and a medicinal use thereof.

Muscarinic acetylcholine receptors (mAChRs) belong to the superfamily of G-protein-coupled receptors, with seven transmembrane domains. There are five subtypes of mAChR, referred to as M1 to M5, each of which exhibits unique pharmacological properties. For example, stimulation of M3 receptors in the airways causes contraction of airway smooth muscles, resulting in bronchoconstriction, and stimulation of M3 receptors in salivary and sweat glands increases fluid and mucous membrane secretion, resulting in increased secretion of saliva and sweat.

Muscarinic receptor antagonists act by inhibiting the binding of acetylcholine to muscarinic cholinergic receptors at various points of action in the body, such as smooth muscle, myocardium, peripheral ganglia, and central nervous system. Therefore, muscarinic receptor antagonists are known to be useful in the treatment of various diseases, such as chronic obstructive pulmonary disease (COPD), chronic bronchitis, asthma, rhinitis, hypersalivation, hyperhidrosis, urinary incontinence, overactive bladder syndrome, gastroesophogeal reflux disease, irritable bowel syndrome, and the like.

Accordingly, the present inventors completed the present invention by preparing a novel pyrrolidinium compound that exhibits excellent antagonistic activity against muscarinic receptors, especially muscarinic M3 receptors.

An object of the present invention is to provide a compound represented by Formula I, a stereoisomer, hydrate, or solvate thereof.

Another object of the present invention is to provide a pharmaceutical composition for preventing or treating a muscarinic acetylcholine receptor-mediated disease, comprising a compound represented by Formula I, a stereoisomer, hydrate, or solvate thereof, and a pharmaceutically acceptable carrier.

Another object of the present invention is to provide a method for preventing or treating a muscarinic acetylcholine receptor-mediated disease, comprising the step of administering or topically applying a compound represented by Formula I, a stereoisomer, hydrate, or solvate thereof to a subject.

Each description and embodiment disclosed herein can also be applied to each of other descriptions and embodiments. That is, all combinations of the various elements disclosed herein fall within the scope of the present application. In addition, the scope of the present application is not to be construed as being limited by the specific descriptions set forth below.

The present invention provides a compound represented by Formula I below, a stereoisomer, hydrate, or solvate thereof:

[Formula I]

in Formula I,

R¹ and R² are each independently selected from the group consisting of hydrogen, halogen, hydroxy, cyano, amino, nitro, C₁-C₆ alkyl, C₁-C₆ haloalkyl, C₁-C₆ hydroxyalkyl_, C₁-C₆ alkoxy, C₁-C₆ haloalkoxy, C₁-C₆ alkylamino, dialkylamino, and aminocarbonyl;

R³ is hydrogen or C₁-C₆ alkyl;

R⁴ is absent, A, -OC(O)-A, -O-A, or -C(O)-A;

A is 5- to 6-membered heteroaryl or 5- to 6-membered heterocycloalkyl containing 1 to 3 heteroatoms selected from N, O, or S, wherein the 5- to 6-membered heteroaryl and 5- to 6-membered heterocycloalkyl may be optionally substituted with oxo, halogen, hydroxy, cyano, amino, C₁-C₆ alkyl, or C₁-C₆ alkoxy;

n and m are each independently integers from 0 to 3;

k is an integer from 1 to 3,

p is an integer from 1 to 5, and

X^- is a monovalent anion.

In Formula I of the present invention, R¹ and R² may be each independently selected from the group consisting of hydrogen, halogen, hydroxy, cyano, amino, nitro, C₁-C₆ alkyl, C₁-C₆ haloalkyl, C₁-C₆ hydroxyalkyl_, C₁-C₆ alkoxy, C₁-C₆ haloalkoxy, C₁-C₆ alkylamino, dialkylamino, and aminocarbonyl. In some embodiments, R¹ and R² may be each independently hydrogen, halogen, hydroxy, cyano, or C₁-C₃ alkoxy. In one embodiment, R¹ and R² may be each independently hydrogen or halogen. In this case, the halogen may be F, Cl, Br, or I. For example, the halogen may be F or Cl. In one embodiment, R¹ is halogen (e.g., F, Cl, Br, or I), and R² may be hydrogen. In one embodiment, R¹ may be hydrogen, and R² may be halogen (e.g., F, Cl, Br, or I). In one embodiment, R¹ and R² may both be hydrogen.

In Formula I above, n and m are each independently 0, 1, 2, or 3. In some embodiments, n and m may be each independently 0, 1, or 2. When n and m are each independently 2 or 3, a plurality of R¹ and R² may be the same or different from each other.

In Formula I of the present invention, R³ is hydrogen or C₁-C₆ alkyl. In one embodiment, R³ may be C₁-C₆ alkyl. For example, R³ may be C₁-C₃ alkyl. For example, R³ may be methyl, ethyl, propyl, or isopropyl.

In Formula I of the present invention, p is an integer from 1 to 5. In some embodiments, p may be 1, 2, or 3.

In Formula I of the present invention, R⁴ is absent, -A, -OC(O)-A, -O-A, or -C(O)-A. In this case, A is 5- to 6-membered heteroaryl or 5- to 6-membered heterocycloalkyl. The 5- to 6-membered heteroaryl or 5- to 6-membered heterocycloalkyl may contain 1 to 3 heteroatoms selected from N, O, or S. For example, the 5- to 6-membered heteroaryl may include pyridine, pyrimidine, pyrrolyl, pyrazolyl, furanyl, thiophenyl, and the like, but is not limited thereto. For example, the 5- to 6-membered heterocycloalkyl may include tetrahydropyranyl, dihydropyranyl, 1,3-dioxolyl, 1,3-dioxolanyl, tetrahydrofuranyl, pyrrolidinyl, piperidinyl, and the like, but is not limited thereto. Said A may be optionally substituted with one or more substituents selected from the group consisting of oxo, halogen, hydroxy, cyano, amino, C₁-C₆ alkyl, and C₁-C₆ alkoxy.

In some embodiments, R⁴ may be absent, -A, or -OC(O)-A. In this case, A may be pyridinyl, or 2-oxo-1,3-dioxol-4-yl (

) or 1,3-dioxol-4-yl (

). Said A may be optionally substituted with halogen, hydroxy, cyano, amino, C₁-C₃ alkyl, or C₁-C₃ alkoxy. In one embodiment, said A may be optionally substituted with C₁-C₃ alkyl, such as methyl or ethyl. In one embodiment, R⁴ may be absent. In this case, in Formula I,

may be a structure of methyl ester, ethyl ester, or propyl ester.

In Formula I of the present invention, X^- is a monovalent anion. X^- is not particularly limited as long as it can form a salt with N⁺ of pyrrolidinium. In one embodiment, X^- may be chloride, bromide, iodide, sulfate, phosphate, methanesulfonate, nitrate, maleate, acetate, citrate, fumarate, tartrate, oxalate, succinate, benzoate, or p-toluenesulfonate. For example, X^- may be chloride, bromide, 4-toluenesulfonate, or methanesulfonate. Preferably, X^- may be bromide.

In one embodiment, the compound of the present invention may be selected from:

Definition

All technical and scientific terms used herein have meanings commonly understood by those of ordinary skill in the art and, unless otherwise stated, conventional measurement methods, manufacturing methods, and conventional ingredients or substances are used based on conventional techniques such as pharmacology, pharmaceutical chemistry, mass spectrometry, NMR, HPLC, and biochemistry.

Individual features and components of each implementation described and illustrated herein may be combined with features and components of any other implementation without departing from the scope or spirit of the present disclosure.

Unless otherwise specified, in the present specification and the appended claims, "or" and "and" mean "and/or". The terms "include" and "included" are open-ended and mean that a compound, composition or method may include additional features or ingredients in addition to the specific features or ingredients listed.

In the present specification, the numerical range indicated using the term "to" refers to a range that includes the numerical values described before and after the term "to" as the lower limit and the upper limit, respectively.

As used herein, the term "optional" or "optionally" means that a subsequently described event or circumstance may or may not occur, and that the description includes instances in which said event or circumstance occurs and instances in which it does not occur. For example, the term "optionally substituted" means both substitution and unsubstitution with the specified substituents.

Compound

As used herein, unless otherwise stated, the term "alkyl", whether used alone or as a part of a substituent, refers to a saturated straight chain and branched carbon chain having 1 to 6 carbon atoms, 1 to 4 carbon atoms, or 1 to 3 carbon atoms. Non-limiting examples of the alkyl group include methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, iso-butyl, tert-butyl, and the like.

The term "alkoxy" refers to the -O-alkyl group. Non-limiting examples of the alkoxy group include methoxy, ethoxy, n-propoxy, iso-propoxy, n-butoxy, sec-butoxy, iso-butoxy, tert-butoxy, and the like.

The term "heteroatom" means nitrogen, oxygen, or sulfur, and includes any oxidized form of nitrogen, such as N(O) (N⁺-O^-) and sulfur, such as S(O) and S(O)₂, and any basic quaternized forms of nitrogen.

The term "heteroaryl" refers to a heteroaromatic group containing one or more heteroatoms and the remaining ring atom being carbon. The heteroaryl group may contain, for example, 1 to 3, 1, or 2 heteroatoms. The heteroaryl group may contain 5 to 10 ring elements, 5 to 7 ring elements, or 5 or 6 ring elements. The "heteroaryl" may be, for example, pyrrolyl, furanyl, thiophenyl, pyrazolyl, imidazolyl, isoxazolyl, oxazolyl, thiazolyl, isothiazolyl, pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl, indolyl, benzofuranyl, benzothiophenyl, benzopyrazolyl, benzoimidazolyl, benzooxazolyl, benzoisoxazolyl, benzothiazolyl, or benzoisothiazolyl, etc.

The term "heterocycloalkyl", "heterocyclic", or "heterocycle" refer to a saturated or partially unsaturated cyclic group containing one or more heteroatoms and the remaining ring atoms being carbon. The heterocycloalkyl group may contain, for example, 1 to 3, 1, or 2 heteroatoms. The heterocycloalkyl group may contain 5 to 10 ring elements, 5 to 7 ring elements, or 5 or 6 ring elements. The heterocycloalkyl group includes, for example, tetrahydropyranyl, dihydropyranyl, 1,3-dioxolyl, 1,3-dioxolanyl, tetrahydrofuranyl, pyrrolidinyl, piperidinyl, piperazinyl, 2-pyrrolidon-1-yl, morpholinyl, and the like, but is not limited thereto.

The term "oxo" refers to the (=O) group.

As used herein, the term "halogen" refers to an atom belonging to group 17 of the periodic table. The halogen atom includes fluorine, chlorine, bromine, and iodine, etc., and may be used interchangeably with the term "halo", which refers to a monovalent functional group composed of halogen.

As used herein, the term "hydroxy" refers to the -OH functional group (hydroxyl group).

As used herein, the term "cyano" is -CN, which refers to a functional group consisting of a triple bond between a carbon atom and a nitrogen atom.

As used herein, the term "amino" refers to -NH₂.

As used herein, the term "alkylamino" refers to a group in which one of two Hs in an amino group is substituted with an alkyl group. Examples of the alkylamino group include, but are not limited thereto, methylamino, ethylamino, and propylamino.

As used herein, the term "dialkylamino" refers to -N(alkyl)₂. In this case, the two alkyls may be the same or different from each other. Examples of the dialkylamino substituent include, but are not limited thereto, dimethylamino, diethylamino, ethylmethylamino, and dipropylamino.

As used herein, the term "nitro" refers to -NO₂.

As used herein, the term "haloalkyl" refers to an alkyl group substituted by one or more halogen atoms. The halogens may be the same (e.g., CHF₂, -CF₃) or different (e.g., CF₂Cl). Where specified, the haloalkyl group may be optionally substituted with one or more substituents other than halogen. Examples of haloalkyl groups may include fluoromethyl, dichloroethyl, trifluoromethyl, trichloromethyl, pentafluoroethyl, and pentachloroethyl groups, but are not limited thereto.

As used herein, the term "haloalkoxy" refers to an alkoxy group substituted with one or more halogen atoms, where alkoxy is as defined above. Non-limiting examples of the haloalkoxy group may include fluoromethoxy, dichloroethoxy, trifluoromethoxy, trichloromethoxy, and the like.

As used herein, the term "hydroxyalkyl" refers to an alkyl group substituted with one or more -OH groups, where alkyl is as defined above.

As used herein, the term "aminocarbonyl" refers to amino attached to a carbonyl group through a nitrogen atom.

In the present specification, "

", "*", or "-" is used to indicate a position at which a substituent is bonded to the remaining moieties of the compound. For example, if - is indicated at the end of a substituent, it means that the end is attached to the remaining moieties of the compound. In addition, when two or more substituents are linked by "-," it means that the substituent immediately before "-" is bonded to a substitutable atom of the substituent immediately after "-."

As used herein, the term "solvate" may refer to a compound of the present invention or a salt thereof comprising a stoichiometric or non-stoichiometric amount of a solvent bound by non-covalent intermolecular forces. Preferred solvents therefor may be solvents that are volatile, non-toxic, and/or suitable for administration to humans. The solvent may be water, in which case the "solvate" is referred to as "hydrate".

As used herein, the term "stereoisomer" may refer to a compound of the present invention or a salt thereof that has the same chemical formula or molecular formula but is optically or sterically different, and specifically, may be a diastereomer, an enantiomer, or a geometric isomer.

In some embodiments, the compound of the present invention contains one or more asymmetric centers and may be in the form of a racemate, a single enantiomer, a mixture of enantiomers, a single diastereomer, a mixture of diastereomers, etc. In one embodiment, due to the nature of the asymmetric center or limited rotation, the compound of the present invention may exist in the form of an enantiomer or a diastereomer.

When two or more asymmetric centers are present in the compound of the present invention, multiple diastereomers and enantiomers of the chemical structures disclosed herein may exist. Pure isomers, separated isomers, partially pure isomers, or racemic mixtures are all intended to be within the scope of the present invention.

Purification of the isomers and separation of isomer mixtures can be achived by standard techniques known in the art. For example, a diastereomeric mixture can be separated into its respective diastereomers by a chromatographic process or crystallization, and a racemate can be separated into its respective enantiomers by a chromatographic process or resolution of the chiral phase.

The compound represented by Formula I according to the present invention is a quaternary ammonium salt and may be converted into various salt forms by ion exchange chromatography. The compound may be obtained in the form of a hydrate or solvate. The compound represented by Formula I may be recovered from the reaction mixture and purified by known methods. When the compound represented by Formula I contains a chiral carbon, it may be used as a diastereomeric mixture or as a single enantiomer or diastereomer.

In addition, the compound of the present invention may be used in the form of a pharmaceutically acceptable salt derived from an inorganic acid or an organic acid, and for example, the salt may be a salt derived from hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid, acetic acid, glycolic acid, lactic acid, pyruvic acid, malonic acid, succinic acid, glutaric acid, fumaric acid, malic acid, mandelic acid, tartaric acid, citric acid, ascorbic acid, palmitic acid, maleic acid, hydroxymaleic acid, benzoic acid, hydroxybenzoic acid, phenylacetic acid, cinnamic acid, salicylic acid, methanesulfonic acid, benzenesulfonic acid, toluenesulfonic acid, or the like.

A pharmaceutically acceptable salt of the compound may be prepared by dissolving the compound of formula I in a water-miscible organic solvent, such as acetone, methanol, ethanol, acetonitrile, or the like, and adding an excess of an organic acid or adding an aqueous acid solution of an inorganic acid, and then precipitating or crystallizing. Subsequently, after evaporating the solvent or an excess of acid from this mixture, it may be prepared by drying to obtain an addition salt or by suction filtration of the precipitated salt.

General method for preparing compound

The compound according to the present invention can be easily prepared from commercially available starting materials, compounds known in the literature, or intermediates easily prepared therefrom through standard synthetic methods and procedures in the related field.

The methods described herein can be monitored according to any suitable method known in the art. For example, the formation of a product can be monitored by spectroscopic means, such as nuclear magnetic resonance spectroscopy (e.g., ¹H or ¹³C), infrared spectroscopy, spectrophotometry (e.g., UV-visible rays), mass spectrometry, or chromatography, such as high performance liquid chromatography (HPLC), gas chromatography (GC), gel permeation chromatography (GPC), or thin layer chromatography (TLC).

The following general reaction scheme generally illustrates a representative method for preparing a compound represented by Formula I. Those of ordinary skill in the art can easily prepare a compound represented by Formula I by appropriately selecting starting materials, reaction temperature, reaction conditions, catalyst, solvent, processing method, and the like, suitable for the desired compound based on the preparation method specifically disclosed in the examples herein. Hereinafter, unless otherwise limited, the designation of each substituent in Formula I in the reaction scheme is the same as that of the substituent at the corresponding position in Formula I.

In one aspect, a compound represented by Formula I can be prepared by reacting intermediate A1 represented by Formula A1 below with a bromoacetate compound represented by Formula B1 below:

[Formula A1]

[Formula B1]

BrCH₂COO-(CH₂)_p-R⁴

The bromoacetate compound represented by Formula B1 can be appropriately selected depending on R⁴ of the final target product, and may be, for example, ethyl 2-bromoacetate, methyl 2-bromoacetate, (5-methyl-2-oxo-1,3-dioxol-4-yl) methyl 2-bromoacetate, and the like.

The bromoacetate compound represented by Formula B1 can be changed to other halogenated acetate compounds if necessary. In this case, a halide (e.g., I^-, Cl^-) where X^- is a halogen anion other than bromide can be obtained.

In one aspect, a compound represented by Formula I can be prepared by reacting intermediate A2 represented by Formula A2 below with a hydroxyalkyl ester compound represented by Formula B2 below:

[Formula A2]

[Formula B2]

HO-(CH₂)_p-O-C(O)-A

The hydroxyalkyl ester compound represented by Formula B2 can be appropriately selected depending on R⁴ of the final target product, and may be, for example, 3-hydroxypropylnicotinate, and the like.

Medicinal use, pharmaceutical composition, and administration method

In another aspect, there is provided a pharmaceutical composition for preventing or treating a muscarinic acetylcholine receptor-mediated disease, comprising the compound represented by Formula I, stereoisomer, hydrate, or solvate thereof. The compound represented by Formula I, stereoisomer, hydrate, and solvate are as described above.

As used herein, the term "preventing" or "prevention" refers to preventing a disease, for example, preventing a disease, condition or disorder in a subject who may be predisposed to the disease, condition or disorder but has not yet experienced or exhibited the pathology or signs of the disease.

As used herein, the term "treating" or "treatment" refers to inhibiting a disease, for example, inhibiting a disease, condition or disorder in a subject who experiences or exhibits the pathology or signs of the disease, condition or disorder, i.e., preventing further development of the pathology and/or signs, or ameliorating the disease, for example, ameliorating the disease, condition or disorder in a subject who experiences or exhibits the pathology or signs of the disease, condition or disorder, i.e., reversing the pathology and/or signs, for example, reducing the severity of the disease.

The muscarinic acetylcholine receptor-mediated disease refers to a disease that can be prevented or treated by inhibiting the binding of acetylcholine to muscarinic receptors, especially muscarinic M3 receptors. The compound of the present invention has excellent antagonistic activity against muscarinic acetylcholine receptors, especially muscarinic M3 receptors. Specifically, the compound of the present invention was confirmed to have an excellent IC₅₀ value at the nM level when measuring its antagonistic activity against the human muscarinic M3 receptor according to the method described in Experimental Example 1 herein.

Therefore, the compound of the present invention has excellent antagonistic activity against muscarinic acetylcholine receptors, especially muscarinic M3 receptors, and is useful in the prevention or treatment of various muscarinic acetylcholine receptor-mediated diseases. In some embodiments, the compound of the present invention can be effectively used for the prevention or treatment of hyperhidrosis, hypersalivation, chronic obstructive pulmonary disease, chronic bronchitis, asthma, rhinitis, urinary incontinence, overactive bladder syndrome, gastroesophogeal reflux disease, or irritable bowel syndrome.

In one embodiment, the pharmaceutical composition may comprise conventional pharmaceutically acceptable carriers, excipients or additives. The pharmaceutical composition may be formulated according to a conventional method, and may be prepared as various oral formulations such as tablets, pills, powders, capsules, syrups, emulsions, and microemulsions, or parenteral formulations such as intramuscular, intravenous or subcutaneous formulation, or formulations for topical application to the skin. The pharmaceutical composition may be a single composition or separate compositions. The pharmaceutical composition comprises a compound, stereoisomer, hydrate, or solvate according to one aspect as an active ingredient of the pharmaceutical composition.

When the pharmaceutical composition is prepared in the form of an oral formulation, examples of additives or carriers used may include cellulose, calcium silicate, corn starch, lactose, sucrose, dextrose, calcium phosphate, stearic acid, magnesium stearate, calcium stearate, gelatin, talc, surfactant, suspending agent, emulsifying agent, diluent, and the like. When the pharmaceutical composition of the present invention is prepared in the form of an injection, the additive or carrier may include water, saline, aqueous glucose solution, similar aqueous sugar solution, alcohol, glycol, ether (for example, polyethylene glycol 400), oil, fatty acid, fatty acid ester, glyceride, surfactant, suspending agent, emulsifying agent, and the like.

The dosage of the pharmaceutical composition is an amount effective for treatment or prevention of a subject or patient, and may be administered orally or parenterally as desired. It may be administered in one to several divided doses to be administered in an amount of 0.01 to 1000 mg, more specifically 0.1 to 300 mg per kg of body weight daily based on the active ingredient when administered orally, or in an amount of 0.01 to 100 mg, more specifically 0.1 to 50 mg per kg of body weight daily based on the active ingredient when administered parenterally. The dose to be administered to a specific subject or patient should be determined in light of several related factors such as body weight, age, sex, and health condition of the patient, the diet, the administration time, the administration method, the severity of the disease, and the like, and it should be understood that it may be appropriately increased or decreased by a specialist. The above dosage is not intended to limit the scope of the present invention in any way. A physician or veterinarian of ordinary skill in the art can readily determine and prescribe the required effective amount of the pharmaceutical composition. For example, by a physician or veterinarian, a dose of the compound of the present invention used in a pharmaceutical composition may start at a level lower than that required to achieve the desired therapeutic effect, and may gradually increase until the desired effect is achieved.

In one embodiment, the pharmaceutical composition includes within its scope a pharmaceutical composition comprising, as an active ingredient, a therapeutically effective amount of at least one of the compounds according to one embodiment, alone or in combination with a pharmaceutically acceptable carrier. The term "therapeutically effective amount" or "effective amount" refers to an amount sufficient to produce a beneficial or desired clinical result, for example, an amount sufficient to alleviate, ameliorate, stabilize, reverse, slow or delay the progression of a disease.

When the composition of the present invention is used for the prevention or treatment of hyperhidrosis, the composition of the present invention may be formulated into formulations for topical application to the skin, such as a solid, semi-solid, powder, gel, ointment, cream, lotion, foam, solution, suspension, aerosol, patch, and emulsion. In one embodiment, the formulations for topical application of the present invention may be a gel, cream, emulsion, lotion, or spray.

In one embodiment, a gel, ointment, and cream may be formulated using, for example, suitable thickening agents and/or gelling agents and/or aqueous or oily bases. For example, water and/or oils such as liquid paraffin, or vegetable oils such as arachis oil or castor oil, or glycol solvents such as propylene glycol or 1,3-butanediol may be included. For example, thickening agents include soft paraffin, aluminum stearate, cetostearyl alcohol, polyethylene glycol, wool fat, hydrogenated lanolin and beeswax and/or glyceryl monostearate and/or nonionic emulsifiers. In one embodiment, lotions may be formulated using an aqueous or oily base and may also include one or more of emulsifiers, dispersants, suspending agents, thickening agents, solvents, colorants, and flavoring agents. The powder may be formed with the aid of any suitable powder base, such as talc, lactose or starch. Spray compositions may, for example, be formulated as aerosols using a suitable propellant, such as dichlorodifluoromethane or trichlorofluoromethane.

The proportion of an active ingredient in the formulation for topical application according to the present invention will vary depending on the compound used, the type of formulation, and the specific condition for which the composition is to be administered. For example, the formulation for topical application may comprise from about 0.1% to about 50%, from about 1% to about 30%, or from about 5% to about 20% of the compound of the present invention.

When used for the treatment of hyperhidrosis, the formulation for topical application can be applied topically as needed to the area of the subject's skin in need of reduced sweating, such as the subject's palms, soles, groin, armpits, or face. The formulation for topical application may be applied to the subject as needed, for example, more than once a week, 3 to 4 times a week, once a day, twice a day, or three times a day.

In another aspect, there is provided a method for preventing or treating a muscarinic acetylcholine receptor-mediated disease, comprising the step of administering or topically applying a compound represented by Formula I, a stereoisomer, hydrate, or solvate thereof to a subject.

Among the terms or elements mentioned in the description of the method, those that are the same as those mentioned above are as described above.

The administration may be oral or parenteral. It may be administered in one to several divided doses to be administered in an amount of 0.01 to 1000 mg, more specifically 0.1 to 300 mg per kg of body weight daily based on the active ingredient when administered orally, or in an amount of 0.01 to 100 mg, more specifically 0.1 to 50 mg per kg of body weight daily based on the active ingredient when administered parenterally.

The dose to be administered to a specific subject or patient should be determined in light of several related factors such as body weight, age, sex, and health condition of the patient, the diet, the administration time, the administration method, the severity of the disease, and the like, and it may be appropriately increased or decreased by a specialist.

In some embodiments, when the method of the present invention is performed to treat hyperhidrosis in a subject, the formulation of the present invention may be applied topically to the area of the subject's skin in need of reduced sweating. In one embodiment, the area of the subject's skin may include the subject's palms, soles, groin, armpits, or face. The compound of the present invention may be applied to the subject as needed, for example, more than once a week, 3 to 4 times a week, once a day, twice a day, or three times a day. Depending on the method of the present invention, sweating can be reduced by about 10% to about 99%, by about 30% to about 80%, or by at least 50%.

As used herein, the term "subject" refers to a subject in need of treatment for a disease, and more specifically means a mammal such as a human or non-human primate, a mouse, a dog, a cat, a horse, and a cow.

In another aspect, there is provided a medicinal use of the compound represented by Formula I, stereoisomer, hydrate, or solvate thereof, for the prevention or treatment of a muscarinic acetylcholine receptor-mediated disease; or a use of the compound represented by Formula I, stereoisomer, hydrate, or solvate thereof, for manufacturing a medicament for preventing or treating a muscarinic acetylcholine receptor-mediated disease. Among the terms or elements mentioned in the description of the use, those that are the same as those mentioned above are as described above.

A compound represented by Formula I, a stereoisomer, hydrate, or solvate thereof exhibits excellent antagonistic activity against muscarinic M3 receptors, and is useful in the prevention or treatment of various diseases such as hyperhidrosis, hypersalivation, chronic obstructive pulmonary disease, asthma, irritable bowel syndrome, urinary incontinence, rhinitis, glaucoma, and cardiac arrhythmias, especially hyperhidrosis.

Figure 1 is a graph showing a result obtained by measuring the number of sweating spots per hind footpad in the normal group (vehicle), the hyperhidrosis-induced group, the positive control group administered with 20% sofpironium, and the experimental group administered with 20 % Compound 1 and 20% Compound 3, respectively. ## p<0.01 vs normal group; ** p<0.01 vs induced group (one-way ANOVA, LSD post hoc test)

Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are only for illustrating the present invention, and the scope of the present invention is not limited to the following examples.

Preparation Example

Preparation Example 1-1: Synthesis of (R)-(1-methylpyrrolidin-3-yl)methyl(3'-chloro-4'-fluoro-[1,1'-biphenyl]-2-yl)carbamate

3'-chloro-4'-fluoro-[1,1'-biphenyl]-2-carboxylic acid (0.80 g, 3.19 mmol) and (R)-(1-methylpyrrolidin-3-yl)methanol (0.40 g, 3.51 mmol) were dissolved in toluene (8 mL), and then triethylamine (0.97 g, 9.57 mmol) and diphenylphosphorylazide (1.32 g, 4.79 mmol) were added at room temperature, and then the mixture was stirred for 12 hours at an external temperature of 120 °C under nitrogen atmosphere. After completion of the reaction, the temperature of the reactant was cooled to room temperature, and then 1N hydrochloric acid solution (30 mL) was added. This mixed solution was extracted with ethyl acetate and then washed with sodium bicarbonate. The organic layer was dehydrated with anhydrous sodium sulfate, filtered, and then concentrated under reduced pressure. The resulting residue was purified by column chromatography to obtain the title compound (0.50 g, 43%).

MS: m/z [M-H]^- = 362.8

¹HNMR (400 MHz, CDCl3): δ 7.89 (d, J = 7.6 Hz, 1H), 7.44-7.39 (m, 1H), 7.38-7.33 (m, 1H), 7.25-7.12 (m, 4H), 6.97 (t, J = 6.8 Hz, 1H), 4.11-3.97 (m, 2H), 2.92-2.85 (m, 1H), 2.83-2.58 (m, 4H), 2.50 (s, 3H), 2.10-2.03 (m, 1H), 1.71-1.63 (m, 1H)

Preparation Example 1-2: Synthesis of (R)-(1-methylpyrrolidin-3-yl)methyl(3'-chloro-4'-fluoro-[1,1'-biphenyl]-2-yl)carbamate (alternative method)

Step 1: Synthesis of 3'-chloro-4'-fluoro-[1,1'-biphenyl]-2-amine

(3-chloro-4-fluoro)phenylboronic acid (5.0 g, 28.7 mmol), sodium carbonate (6.6 g, 63.8 mmol), and tetrakistriphenylphosphinepalladium (1.8 g, 1.56 mmol) were dissolved in a mixed solvent of toluene (40 mL) and water (20 mL) in a reactor, and then 2-bromo-4-fluoroaniline (3.8 g, 22.1 mmol) was added, and then the mixture was stirred for 18 hours at an external temperature of 95 °C. After completion of the reaction, a saturated ammonium solution (50 mL) and dichloromethane (50 mL) were added and stirred, and then the aqueous layer and the organic layer were separated. The aqueous layer was extracted with dichloromethane (2 x 50 mL), and then the organic layer was collected, dehydrated with anhydrous sodium sulfate, filtered, and then concentrated under reduced pressure. The resulting residue was purified by column chromatography to obtain the title compound (2.5 g, 51%).

Step 2: Synthesis of (R)-(1-methylpyrrolidin-3-yl)methyl(3'-chloro-4'-fluoro-[1,1'-biphenyl]-2-yl)carbamate

Triphosgene (1.2 g, 4.0 mmol) was dissolved in dichloromethane (30 mL), and then 3'-chloro-4'-fluoro-[1,1'-biphenyl]-2-amine (2.5 g, 11.3 mmol) and triethylamine (3.4 g, 33.9 mmol) were added at 0 °C. After reaction at 0 °C for 1 hour, (R)-(1-methylpyrrolidin-3-yl)methanol (1.2 g, 10.7 mmol) was added dropwise and then stirred for 1 hour at room temperature. After completion of the reaction, the mixture was extracted with water and dichloromethane, then dehydrated with anhydrous sodium sulfate, filtered, and then concentrated under reduced pressure. The resulting residue was purified by column chromatography to obtain the title compound (1.2 g, 31%).

MS: m/z [M+H]⁺ = 363.2

Preparation Example 2: Synthesis of (S)-(1-methylpyrrolidin-2-yl)methyl(3'-chloro-4'-fluoro-[1,1'-biphenyl]-2-yl)carbamate

The title compound (0.50 g, 43%) was obtained in the same manner as in Preparation Example 1-1 by reacting 3'-chloro-4'-fluoro-[1,1'-biphenyl]-2-carboxylic acid as a starting material with (S)-(1-methylpyrrolidin-2-yl)methanol.

MS: m/z [M-H]^- = 362.8

¹H NMR (400 MHz, DMSO-d6): δ 8.06 (d, J = 7.6 Hz, 1H), 7.47-7.36 (m, 2H), 7.30-7.22 (m, 2H), 7.21-7.11 (m, 2H), 6.56 (s, 1H), 4.27-4.08 (m, 2H), 3.13-3.07 (m, 1H), 2.56-2.46 (m, 1H), 2.42 (s, 3H), 2.32-2.22 (m, 1H), 1.99-1.59 (m, 4H)

Preparation Example 3: Synthesis of 2-(1-methylpyrrolidin-2-yl)ethyl(3'-chloro-4'-fluoro-[1,1'-biphenyl]-2-yl)carbamate

The title compound (0.67 g, 45%) was obtained in the same manner as in Preparation Example 1-1 by reacting 3'-chloro-4'-fluoro-[1,1'-biphenyl]-2-carboxylic acid as a starting material with 2-(1-methylpyrrolidin-2-yl)ethan-1-ol.

MS: m/z [M-H]^- = 376.8

¹H NMR (400 MHz, CDCl3): δ 7.84 (d, J = 7.6 Hz, 1H), 7.58-7.38 (m, 1H), 7.32-7.28 (m, 1H), 7.24-7.19 (m 1H), 7.14-7.03 (m, 1H), 6.95 (t, J = 6.4 Hz, 2H), 6.88 (s, 1H), 4.17-4.00 (m, 2H), 3.55-3.51 (m, 1H), 2.79-2.49 (m, 5H), 2.16-2.05 (m, 2H), 1.95-1.54 (m, 4H)

Preparation Example 4: Synthesis of 2-(1-methylpyrrolidin-2-yl)ethyl[1,1'-biphenyl]-2-ylcarbamate

2-isocyanato-1,1'-biphenyl (0.50 g, 2.96 mmol) was dissolved in tetrahydrofuran (10 mL), and then 2-(1-methylpyrrolidin-2-yl)ethanol (0.38 g, 2.96 mmol) was added dropwise. The reaction was stirred at room temperature for 14 hours. After completion of the reaction, the mixture was concentrated under reduced pressure and then purified by column chromatography to obtain the title compound (0.70 g, 72%).

MS: m/z [M+H]⁺ = 325.3

Preparation Example 5: Synthesis of (S)-(1-methylpyrrolidin-2-yl)methyl[1,1'-biphenyl]-2-ylcarbamate

The title compound (0.40 g, 84%) was obtained in the same manner as in Preparation Example 4 using 2-isocyanato-1,1'-biphenyl as a starting material and (S)-(1-methylpyrrolidin-2-yl)methanol as a reactant.

MS: m/z [M+H]⁺ = 311.1

Preparation Example 6: Synthesis of (R)-(1-methylpyrrolidin-2-yl)methyl[1,1'-biphenyl]-2-ylcarbamate

The title compound (0.45 g, 94%) was obtained in the same manner as in Preparation Example 4 using 2-isocyanato-1,1'-biphenyl as a starting material and (R)-(1-methylpyrrolidin-3-yl)methanol as a reactant.

MS: m/z [M+H]⁺ = 311.0

Preparation Example 7: Synthesis of 2-(1-methylpyrrolidin-2-yl)ethyl(5-fluoro-[1,1'-biphenyl]-2-yl)carbamate

Step 1: Synthesis of 5-fluoro-[1,1'-biphenyl]-2-amine

Phenylboronic acid (3.9 g, 31.9 mmol), potassium carbonate (8.8 g, 63.8 mmol), and tetrakistriphenylphosphinepalladium (1.8 g, 1.56 mmol) were dissolved in a mixed solvent of toluene (40 mL), water (20 mL), and ethanol (10 mL) in a reactor, and then 2-bromo-4-fluoroaniline (3.0 g, 15.9 mmol) was added, and then the mixture was stirred for 16 hours at an external temperature of 95 °C. After completion of the reaction, a saturated ammonium solution (50 mL) and dichloromethane (50 mL) were added and stirred, and then the aqueous layer and the organic layer were separated. The aqueous layer was extracted with dichloromethane (2 * 50 mL), and then the organic layer was collected, dehydrated with anhydrous sodium sulfate, filtered, and then concentrated under reduced pressure. The resulting residue was purified by column chromatography to obtain the title compound (2.9 g, 97%).

Step 2: Synthesis of 2-(1-methylpyrrolidin-2-yl)ethyl(5-fluoro-[1,1'-biphenyl]-2-yl)carbamate

The title compound (2.15 g, 50%) was obtained in the same manner as in Step 2 of Preparation Example 1-2 by reacting 5-fluoro-[1,1'-biphenyl]-2-amine as a starting material with 2-(1-methylpyrrolidin-2-yl)ethanol.

MS: m/z [M+H]⁺ = 343.2

Preparation Example 8: Synthesis of (5-methyl-2-oxo-1,3-dioxol-4-yl)methyl 2-bromoacetate

4-(hydroxymethyl)-5-methyl-1,3-dioxol-2-one (1.0 g, 7.7 mmol) was dissolved in dichloromethane (10 mL) in a reactor, and then 2-bromoacetyl bromide (1.5 g, 7.7 mmol) and pyridine (1.8 g, 23.1 mmol) were added dropwise at 0 °C. The reaction mixture was stirred for 18 hours at room temperature and then extracted with a saturated ammonium solution and dichloromethane. The organic layer was dehydrated with anhydrous sodium sulfate, filtered, and then concentrated under reduced pressure. The resulting residue was purified by column chromatography to obtain the title compound (1.0 g, 53%).

Preparation Example 9: Synthesis of (3R)-1-(carboxymethyl)-3-((((3'-chloro-4'-fluoro-[1,1'-biphenyl]-2-yl-carbamoyl)oxy)methyl)-1-methylpyrrolidin-1-ium bromide

(3R)-3-((((3'-chloro-4'-fluoro-[1,1'-biphenyl]-2-yl-carbamoyl)oxy)methyl)-1-(2-ethoxy-2-oxomethyl)-1-methylpyrrolidin-1-ium bromide (0.30 g, 0.69 mmol) was dissolved in methanol (10 mL) in a reactor, and then a sodium hydroxide solution (0.69 mL, 2N) was slowly added dropwise at 0 °C. The reaction mixture was stirred for 2 hours at room temperature and then concentrated under reduced pressure. The concentrated residue was purified by prep-HPLC with acetonitrile and water containing 0.1% hydrogen bromide to obtain the title compound (0.20 g, 69%).

Example

Example 1: (3R)-3-((((3'-chloro-4'-fluoro-[1,1'-biphenyl]-2-yl)carbamoyl)oxy)methyl)-1-(2-ethoxy-2-oxoethyl)-1-methylpyrrolidin-1-ium bromide (Compound 1)

(R)-(1-methylpyrrolidin-3-yl)methyl(3'-chloro-4'-fluoro-[1,1'-biphenyl]-2-yl)carbamate (0.50 g, 1.38 mmol) was dissolved in acetonitrile (10 mL), and then ethyl 2-bromoacetate (0.46 g, 2.76 mmol) was added dropwise. The mixture was stirred for 12 hours under nitrogen atmosphere at 25 °C. After completion of the reaction, the reaction mixture was concentrated under reduced pressure. The concentrated residue was purified by column chromatography to obtain Compound 1 (0.38 g, 52%).

MS: m/z [M-Br]^- = 449.2

1H NMR (400 MHz, DMSO-d6): δ 9.00 (d, J = 5.6 Hz, 1H), 7.57 (d, J = 6.4 Hz, 1H), 7.52-7.46 (m, 1H), 7.41-7.33 (m, 3H), 7.31-6.95 (m, 2H), 4.56 (d, J= 21.2 Hz, 2H), 4.29-4.18 (m, 2H), 4.14-3.42 (m, 6H), 3.23-3.19 (m, 3H), 2.88-2.81 (m, 1H), 2.28-2.21 (m, 1H), 1.90-1.75 (m, 1H), 1.25 (t, J = 6.8 Hz, 3H)

Example 2: 2-(2-(([1,1'-biphenyl]-2-ylcarbamoyl)oxy)ethyl)-1-(2-ethoxy-2-oxoethyl)-1-methylpyrrolidin-1-ium bromide (Compound 2)

Compound 2 (0.33 g, 65%) was obtained in the same manner as in Example 1 using 2-(1-methylpyrrolidin-2-yl)ethyl[1,1'-biphenyl]-2-ylcarbamate (0.40 g, 1.23 mmol).

MS: m/z [M-Br]^- = 411.3

¹H NMR (400 MHz, DMSO-d6): δ 8.70 (s, 1H), 7.50-7.27 (m, 9H), 4.67-4.27 (m, 1H), 4.26-4.18 (m, 3H), 4.11-3.83 (m, 3H), 3.77-3.46 (m, 2H), 3.10 (d, J = 112.4 Hz, 3H), 2.32-2.13 (m, 2H), 2.08-1.99 (m, 2H), 1.92-1.69 (m, 2H), 1.25 (t, J = 7.2 Hz, 3H)

Example 3: (2S)-2-((((3'-chloro-4'-fluoro-[1,1'-biphenyl]-2-yl)carbamoyl)oxy)methyl)-1-(2-ethoxy-2-oxoethyl)-1-methylpyrrolidin-1-ium bromide (Compound 3)

Compound 3 (0.40 g, 69%) was obtained in the same manner as in Example 1 using (S)-(1-methylpyrrolidin-2-yl)methyl(3'-chloro-4'-fluoro-[1,1'-biphenyl]-2-yl)carbamate (0.40 g, 3.51 mmol).

MS: m/z [M-Br]^- = 448.0

¹H NMR (400 MHz, DMSO-d6): δ 7.63-7.57 (m, 1H), 7.50-7.29 (m, 6H), 4.48-4.31 (m, 3H), 4.26-4.09 (m, 3H), 3.95-3.65 (m, 2H), 3.43-3.41 (m, 2H), 3.09 (s, 2H), 2.30-1.99 (m, 3H), 1.95-1.82 (m, 1H), 1.27-1.22 (m, 3H)

Example 4: 1-(2-ethoxy-2-oxoethyl)-2-(2-(((5-fluoro-[1,1'-biphenyl]-2-yl)carbamoyl)oxy)ethyl)-1-methylpyrrolidin-1-ium bromide (Compound 4)

Compound 4 (0.53 g, 70%) was obtained in the same manner as in Example 1 using 2-(1-methylpyrrolidin-2-yl)ethyl(5-fluoro-[1,1'-biphenyl]-2-yl)carbamate (0.50 g, 1.46 mmol).

MS: m/z [M-Br]^- = 429.2

¹H NMR (400 MHz, CDCl3): δ 7.84 (dd, J = 15.6, 10.4 Hz, 1H), 7.55-7.30 (m, 5H), 7.19-6.79 (m, 3H), 5.40-4.80 (m, 1H), 4.68 (dd, J = 20.0, 17.2 Hz, 1H), 4.43 (m, 1H), 4.33-4.05 (m, 6H), 3.60 (s, 2H), 3.13 (s, 1H), 2.60-1.78 (m, 6H), 1.29 (td, J = 7.2, 1.6 Hz, 3H)

Example 5: (2S)-2-((([1,1'-biphenyl]-2-ylcarbamoyl)oxy)methyl)-1-(2-ethoxy-2-oxoethyl)-1-methylpyrrolidin-1-ium bromide (Compound 5)

Compound 5 (0.35 g, 57%) was obtained in the same manner as in Example 1 using (S)-(1-methylpyrrolidin-2-yl)methyl[1,1'-biphenyl]-2-ylcarbamate (0.32 g, 1.93 mmol).

MS: m/z [M-Br]^- = 397.3

¹H NMR (400 MHz, DMSO-d6): δ 9.06-8.94 (m, 1H), 7.65-7.12 (m, 9H), 4.69-4.04 (m, 7H), 3.96-3.57 (m, 2H), 3.28-2.98 (m, 3H), 2.36-1.72 (m, 4H), 1.28-1.22 (m, 3H)

Example 6: (3R)-3-((([1,1'-biphenyl]-2-ylcarbamoyl)oxy)methyl)-1-(2-ethoxy-2-oxoethyl)-1-methylpyrrolidin-1-ium bromide (Compound 6)

Compound 6 (0.30 g, 52%) was obtained in the same manner as in Example 1 using (R)-(1-methylpyrrolidin-2-yl)methyl[1,1'-biphenyl]-2-ylcarbamate (0.45 g, 1.45 mmol).

MS: m/z [M-Br]^- = 397.2

¹H NMR (400 MHz, CDCl3) δ 8.81-8.78 (m, 1H), 7.46-7.33 (m, 9H), 4.52-4.49 (m, 2H), 4.26-4.21 (m, 2H), 4.02-3.98 (m, 2H), 3.88-3.61 (m, 3H), 3.31-3.26 (m, 1H), 3.19-3.17 (m, 3H), 2.85-2.78 (m, 1H), 2.28-2.14 (m, 1H), 1.94-1.74 (m, 1H), 1.27-1.24 (m, 3H).

Example 7: 2-(2-(((3'-chloro-4'-fluoro-[1,1'-biphenyl]-2-yl)carbamoyl)oxy)ethyl)-1-(2-ethoxy-2-oxoethyl)-1-methylpyrrolidin-1-ium bromide (Compound 7)

Compound 7 (0.45 g, 47%) was obtained in the same manner as in Example 1 using 2-(1-methylpyrrolidin-2-yl)ethyl(3'-chloro-4'-fluoro-[1,1'-biphenyl]-2-yl)carbamate (0.67 g, 1.78 mmol).

MS: m/z [M-Br]^- = 463.2

¹H NMR (400 MHz, DMSO-d6): δ 8.94 (s, 1H), 7.58 (d, J = 6.8 Hz, 1H), 7.51-7.30 (m, 6H), 4.68-4.31 (m, 1H), 4.30-4.20 (m, 3H), 4.15-3.90 (m, 3H), 3.75-3.64 (m, 1H), 3.63-3.51 (m, 1H), 3.26-2.97 (m, 3H), 2.28-2.20 (m, 2H), 2.08-2.00 (m, 2H), 1.93-1.75 (m, 2H), 1.25 (t, J = 6.8 Hz, 3H).

Example 8: (3R)-3-((((3'-chloro-4'-fluoro-[1,1'-biphenyl]-2-yl)carbamoyl)oxy)methyl)-1-(2-methoxy-2-oxoethyl)-1-methylpyrrolidin-1-ium bromide (Compound 8)

(R)-(1-methylpyrrolidin-3-yl)methyl(3'-chloro-4'-fluoro-[1,1'-biphenyl]-2-yl)carbamate (0.30 g, 0.83 mmol) was dissolved in acetonitrile (10 mL), and then methyl 2-bromoacetate (0.19 g, 1.25 mmol) was added dropwise. The mixture was stirred for 12 hours under nitrogen atmosphere at 25 °C. After completion of the reaction, the reaction mixture was concentrated under reduced pressure. The concentrated residue was purified by column chromatography to obtain Compound 8 (66 mg, 16%).

MS: m/z [M-Br]^- = 435.2

¹H NMR (400 MHz, DMSO): δ 8.99 (d, J = 6.0 Hz, 1H), 7.58 (dd, J = 7.2, 1.2 Hz, 1H), 7.52-7.46 (m, 1H), 7.44-7.30 (m, 5H), 4.58 (d, J = 21.6 Hz, 2H), 4.15-3.82 (m, 3H), 3.76-3.59 (m, 2H), 3.48-3.35 (m, 1H), 3.27-3.12 (m, 3H), 2.99-2.78 (m, 1H), 2.37-2.11 (m, 1H), 2.01-1.66 (m, 1H)

Example 9: (3R)-3-((((3'-chloro-4'-fluoro-[1,1'-biphenyl]-2-yl)carbamoyl)oxy)methyl)-1-methyl-1-(2-((5-methyl-2-oxo-1,3-dioxol-4-yl)methoxy)2-oxoethyl)pyrrolidin-1-ium bromide (Compound 9)

(R)-(1-methylpyrrolidin-3-yl)methyl(3'-chloro-4'-fluoro-[1,1'-biphenyl]-2-yl)carbamate (0.30 g, 0.83 mmol) was dissolved in acetonitrile (10 mL), and then (5-methyl-2-oxo-1,3-dioxol-4-yl)methyl 2-bromoacetate (0.31 g, 1.25 mmol) was added dropwise. The mixture was stirred for 12 hours under nitrogen atmosphere at 25 °C. After completion of the reaction, the reaction mixture was concentrated under reduced pressure. The concentrated residue was purified by prep-HPLC with acetonitrile and water containing 0.1% hydrogen bromide to obtain Compound 9 (104 mg, 20%).

MS: m/z [M-Br]^- = 533.2

¹H NMR (400 MHz, DMSO): δ 8.96 (d, J = 8.4 Hz, 1H), 7.84-7.16 (m, 7H), 5.14 (s, 2H), 4.57 (d, J = 15.6 Hz, 2H), 4.17-3.56 (m, 5H), 3.45-3.34 (m, 1H), 3.26-3.11 (m, 3H), 2.90-2.73 (m, 1H), 2.34-2.12 (m, 4H), 1.98-1.78 (m, 1H)

Example 10: (3R)-3-((((3'-chloro-4'-fluoro-[1,1'-biphenyl]-2-yl)carbamoyl)oxy)methyl)-1-methyl-1-(1-(2-(3-(nicotinoyloxy)propoxy)-2-oxoethyl)pyrrolidin-1-ium bromide (Compound 10)

(3R)-1-(carboxymethyl)-3-((((3'-chloro-4'-fluoro-[1,1'-biphenyl]-2-yl-carbamoyl)oxy)methyl)-1-methylpyrrolidin-1-ium bromide (0.20 g, 0.48 mmol), 1-hydroxybenzotriazole (71 mg, 0.53 mmol), and 3-hydroxypropylnicotinate (96 mg, 0.53 mmol) were dissolved in dichloromethane (20 mL), and then 3-(((ethylimino)methylene)amino)-N,N-dimethylpropan-1-amine hydrochloride (101 mg, 0.53 mmol) was added, and then the mixture was stirred for 12 hours at room temperature. After completion of the reaction, the reaction mixture was concentrated under reduced pressure, and then the concentrated residue was purified by prep-HPLC with acetonitrile and water containing 0.1% hydrogen bromide to obtain Compound 10 (50 mg, 16%).

MS: m/z [M-Br]^- = 583.8

¹H NMR (400 MHz, DMSO): δ 9.20-9.09 (m, 1H), 9.01-8.90 (m, 1H), 8.87-8.79 (m, 1H), 8.51-8.32 (m, 1H), 7.68-7.60 (m, 1H), 7.57-7.50 (m, 1H), 7.50-7.42 (m, 1H), 7.41-7.24 (m, 5H), 4.75-4.45 (m, 3H), 4.45-4.29 (m, 5H), 4.11-3.83 (m, 8H), 3.38-3.27 (m, 2H), 3.24-3.08 (m, 3H), 2.90-2.76 (m, 1H), 2.32-2.04 (m, 3H), 1.92-1.76 (m, 1H).

Experimental Example

Experimental Example 1: In vitro muscarinic M3 receptor antagonism

1. Human muscarinic M3 receptor binding ability test

The affinity (Ki) of the compounds of the present invention for the human muscarinic acetylcholine M3 receptor was measured by a competitive filtration binding assay using the radio-labeled M3 receptor antagonist, [3H]-methylscopolamine.

Cell membrane protein derived from CHO-K1 overexpressing human muscarinic M3 receptor (PerkinElmer), [3H]-methylscopolamine, and test substances at various concentrations were cultured in 0.1 mL of Tris-HCl buffer at 25 °C for 120 minutes, and suction filtered through a glass filter (Whatman GF/B), and then the filter was washed six times with 0.5 mL of cold Tris-HCl buffer. The radioactivity of [3H]-methylscopolamine adsorbed on the filter was measured by TopCount^TM after 50 uL of Microscint 20 (Packard) was added to each well and then incubated in an orbital shaker for 15 minutes. Non-specific binding was evaluated in the presence of N-methylscopolamine at a 200- to 300-fold concentration. In order to evaluate the binding ability of the example compounds to the muscarinic M3 receptor, According to the method of Cheng and Prusoff [Cheng and Prusoff, Biochem. Pharmacol., 22, 3099, 1973], the dissociation constant (Ki) was calculated from the concentration (IC₅₀) of the test substance that inhibits the binding of the labeled ligand, [3H]-methylscopolamine, by 50%. The lower the dissociation constant (Ki), the stronger the compound's binding ability to the human muscarinic M3 receptor.

The binding ability of each test substance to the M3 receptor is shown in Table 1 below.

Binding ability to human muscarinic M3 receptor

Compound	IC50 (nM)	M3 receptor binding ability Ki (nM)
Compound 1	46.6	5.17
Compound 3	100	11.1
Compound 5	173	19.3

As can be seen from Table 1 above, the compounds of the present invention were confirmed to have excellent IC₅₀ values at the nM level for the human muscarinic M3 receptor.

2. Human muscarinic M3 receptor antagonism test

The antagonism against the M3 receptor was evaluated using CHO-K1 cells (CHO-K1 mt aequorin, PerkinElmer) transfected with the human muscarinic M3 receptor.

The recombinant cells grown in antibiotic-free medium 18 hours before testing were separated by gentle flushing with PBS-EDTA (5mM EDTA), and recovered by centrifugation, and then resuspended in buffer (DMEM/HAM's F12 containing HEPES + 0.1% BSA protease). The cells were cultured with Coelenterazine h (Molecular Probes) at room temperature for at least 4 hours. A dose response curve was obtained using a reference compound (4-DAMP) prior to the evaluation of compounds.

For evaluation, 50 μL of cell suspension was injected into 50 μL of a test substance or a reference compound plated in a 96-well plate. Luminescence was recorded using a Hamamatsu Functional Drug Screening System 6000 (FDSS 6000).

After the first injection, incubation was performed for 15 minutes, and then 100 μL of a reference agonist (acetylcholine) at a concentration corresponding to EC₈₀ was injected into 100 μL of the mixture of cell suspension and the test compound for antagonist testing. Luminescence was recorded using FDSS6000.

The antagonist activity of the test compound was calculated as the percentage of inhibition of the reference activity at EC₈₀ concentration of the reference agonist (acetylcholine), and then the IC₅₀ value was calculated. The M3 receptor antagonistic ability for each test substance is shown in the table below.

Antagonistic ability against human muscarinic M3 receptor

Compound	IC50 (nM)
Compound 1	49.3
Compound 2	221
Compound 3	91.7
Compound 4	>250
Compound 5	46.8
Compound 6	152
Compound 7	160

Experimental Example 2: Evaluation of drug efficacy in pilocarpine-induced mouse hyperhidrosis model

An iodine-starch sweat test was performed to confirm the efficacy of the test substance in suppressing sweating in the pilocarpine-induced hyperhidrosis model.

6-week-old male ICR mice (ORIENT BIO INC.) were purchased, acclimatized for one week, and then grouped using body weight. The test group is shown in Table 3 below.

Test group composition

Test group	Pilocarpine 5 mg/kg, ip	Administration dose (%)	Administration volume (μL/hind footpad)	Number of subjects
Normal group (vehicle)	-	-	10	8
Induced group (vehicle)	+	-	10	8
Positive control group (Sofpironium)	+	20	10	8
Compound 1	+	20	10	8
Compound 3	+	20	10	8

Vehicle: absolute ethanol

Four hours before pilocarpine administration, the test substance was prepared at 20% in absolute ethanol (Daejung) and applied at 10 μL to both hind footpads of the mouse. Four hours after application of the test substance, an anesthetic (rompum:ketamine = 1:4) was administered intraperitoneally, and then pilocarpine (5 mg/kg/5 mL) was administered into the abdominal cavity of the anesthetized animal. Iodine (Sigma-Aldrich) was dissolved in ethanol at 3.5% and applied to the right and left hind footpads using a brush. After drying for about 1 minute, starch (Milipore) was suspended in castor oil (Sigma-Aldrich) at a concentration of 10% and applied to the same location using a brush. 10 minutes after pilocarpine administration, photos of the footpad skin were taken, and then sweating spots per foodpad of each subject (sweating spots/foodpad) were analyzed. Sofpironium bromide (JHCHEM) was used as a positive control group.

All data obtained from the experiment are expressed as mean ± standard error, and all results were analyzed by one-way ANOVA using SPSS (version 20, IBM SPSS Statistics, USA), and significance was verified by post hoc test using LSD test.

The test results are shown in Table 4 below.

Number of sweating spots on left and right hind footpads

Test group	Number of sweating spots (mean ± standard error)
Normal group (vehicle)	17.00 ± 4.21
Induced group (vehicle)	116.88 ± 17.51##
Positive control group (20% sofpironium)	42.13 ± 8.35**
20% Compound 1	32.25 ± 6.14**
20% Compound 3	37.50 ± 13.02**

## p<0.01 vs normal group; ** p<0.01 vs induced group (one-way ANOVA, LSD post hoc test)

The number of sweating spots was 17.00 ±11.90 in the normal group (vehicle) that was not administered with pilocarpine, and 116.88 ± 49.54 in the sweating induced group that was administered with pilocarpine, indicating the induction of hyperhidrosis (p<0.01).

The number of sweating spots in the group administered with Compound 1 and the group administered with Compound 3 was 32.25 ± 6.14 and 37.50 ± 13.02, respectively, which was significantly reduced compared to the sweating induced group (p<0.01; see Table 4 and Figure 1).

In addition, the number of sweating spots in the positive control group (sofpironium) at the same concentration was confirmed to be 42.13 ± 8.35, indicating that the compounds of the present invention exhibit at least equal or better sweating inhibition ability compared to the positive control group (sofpironium).

The present invention has been described herein based on exemplary embodiments. However, it should be understood that the present invention is not limited to the described embodiments, and all changes, modifications, alterations, and alternatives that do not depart from the spirit and essential characteristics of the present invention are within the scope of the present invention.

Claims (11)

1. A compound represented by Formula I below, a stereoisomer, hydrate, or solvate thereof:

   [Formula I]

   

   in Formula I,

   R¹ and R² are each independently selected from the group consisting of hydrogen, halogen, hydroxy, cyano, amino, nitro, C₁-C₆ alkyl, C₁-C₆ haloalkyl, C₁-C₆ hydroxyalkyl_, C₁-C₆ alkoxy, C₁-C₆ haloalkoxy, C₁-C₆ alkylamino, C₁-C₆ dialkylamino, and aminocarbonyl;

   R³ is hydrogen or C₁-C₆ alkyl;

   R⁴ is absent, -A, -OC(O)-A, -O-A, or -C(O)-A;

   A is 5- to 6-membered heteroaryl or 5- to 6-membered heterocycloalkyl containing 1 to 3 heteroatoms selected from N, O, or S, wherein the 5- to 6-membered heteroaryl and 5- to 6-membered heterocycloalkyl may be optionally substituted with oxo, halogen, hydroxy, cyano, amino, C₁-C₆ alkyl, or C₁-C₆ alkoxy;

   n and m are each independently integers from 0 to 3;

   k is an integer from 1 to 3,

   p is an integer from 1 to 5, and

   X^- is a monovalent anion.
2. The compound, stereoisomer, hydrate, or solvate thereof according to claim 1, wherein

   R¹ and R² are each independently hydrogen, halogen, hydroxy, cyano, or C₁-C₃ alkoxy; and

   n and m are each independently 0, 1, or 2.
3. The compound, stereoisomer, hydrate, or solvate thereof according to claim 1, wherein R³ is C₁-C₃ alkyl.
4. The compound, stereoisomer, hydrate, or solvate thereof according to claim 1, wherein

   p is an integer from 1 to 3;

   R⁴ is absent, -A or -OC(O)-A; and

   A is pyridinyl, 2-oxo-1,3-dioxol-4-yl, or 1,3-dioxol-4-yl, which may be optionally substituted with halogen, hydroxy, cyano, amino, C₁-C₃ alkyl, or C₁-C₃ alkoxy.
5. The compound, stereoisomer, hydrate, or solvate thereof according to claim 1, wherein X^- is selected from the group consisting of chloride, bromide, iodide, sulfate, phosphate, methanesulfonate, nitrate, maleate, acetate, citrate, fumarate, tartrate, oxalate, succinate, benzoate, and p-toluenesulfonate.
6. The compound, stereoisomer, hydrate, or solvate thereof according to claim 5, wherein X^- is bromide.
7. The compound, stereoisomer, hydrate, or solvate thereof according to claim 1, wherein the compound is selected from the following group:

   

   
8. A pharmaceutical composition for preventing or treating a muscarinic acetylcholine receptor-mediated disease, comprising the compound, stereoisomer, hydrate, or solvate thereof according to any one of claims 1 to 7, and a pharmaceutically acceptable carrier.
9. The pharmaceutical composition according to claim 8, wherein the muscarinic acetylcholine receptor-mediated disease is selected from the group consisting of hyperhidrosis, hypersalivation, chronic obstructive pulmonary disease, chronic bronchitis, asthma, rhinitis, urinary incontinence, overactive bladder syndrome, gastroesophogeal reflux disease, and irritable bowel syndrome.
10. The pharmaceutical composition according to claim 9, wherein the muscarinic acetylcholine receptor-mediated disease is hyperhidrosis.
11. A method for preventing or treating a muscarinic acetylcholine receptor-mediated disease, comprising the step of administering or topically applying the compound, stereoisomer, hydrate, or solvate thereof according to any one of claims 1 to 7 to a subject.

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