Imidazopyridine derivative and application, pharmaceutical composition and pharmaceutical preparation

Abstract

The application discloses an imidazopyridine derivative and application, a pharmaceutical composition and a pharmaceutical preparation, and belongs to the technical field of medicinal chemistry. The technical problems to be solved are that existing P2X3 receptor inhibitors have poor safety, low selectivity for P2X3 or P2X2 / 3 and poor metabolic stability. The technical solution is that an imidazopyridine derivative is selected from the compounds shown in the formula I or racemates, stereoisomers, geometric isomers, tautomers, nitroxides, hydrates, isotopically labeled compounds, solvates, polymorphs, metabolites, esters, pharmaceutically acceptable salts or prodrugs.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical chemistry technology, specifically relating to an imidazopyridine derivative and its application, pharmaceutical compositions and pharmaceutical preparations. Background Technology

[0002] P2X receptors, also known as P2X purinoreceptors, are a family of cation-osmotic ATP-gated ion channels that bind to extracellular ATP. P2X receptors have seven subunits and exist as homotrimers or heterotrimers, primarily expressed at nerve endings (presynaptic and postsynaptic) in the nervous system, regulating synaptic transmission. They are also widely expressed in the immune, cardiovascular, skeletal, gastrointestinal, respiratory, and endocrine systems to participate in various biological functions and physiological processes, such as regulation of heart rate and contractility, regulation of vascular tone, nociception, especially chronic pain, vas deferens contraction during ejaculation, bladder contraction during urination, platelet aggregation, macrophage activation and apoptosis, and neuron-glial interactions.

[0003] P2X3 has been reported to be involved in the afferent pathway that controls the bladder capacity reflex. Inhibition of P2X3 can treat storage and voiding-related conditions such as overactive bladder. P2X3 is also selectively expressed on nociceptive, small-diameter sensory neurons (i.e., neurons stimulated by pain or injury). Blocking P2X3 receptors has analgesic effects in animal models of chronic inflammatory and neuropathic pain.

[0004] P2X3 can also form the P2X213 heterodimer with P2X2, another member of the P2X purinergic ligand-gated ion channel family. P2X2 / 3 is highly expressed at the terminals (central and peripheral) of sensory neurons, primarily in bladder sensory neurons (more so than P2X3), and may play a role in bladder fullness and nociceptive sensation. Therefore, the development of P2X3 antagonists has significant clinical implications and great potential.

[0005] Relevant patent documents retrieved:

[0006] Patent application publication number WO2020074962A1, published on April 16, 2020, discloses a method for treating pruritus in mammals using a P2X3 antagonist. The P2X3 antagonist is preferably a compound of formula (I). The pruritus may be associated with inflammatory dermatitis, infectious dermatitis, autoimmune dermatitis, or pregnancy-related dermatitis. The P2X3 antagonist can be administered intravenously, subcutaneously, orally, by inhalation, nasal administration, topical administration, or ophthalmic administration, and can be used in combination with an NK-1 antagonist. The P2X3 antagonist works by inhibiting the release of pathological ATP associated with the hyperexcitability of afferent pruritus neurons, thereby inhibiting peripheral hypersensitivity to pruritus through a broad mechanism independent of pathological stimuli acting on pruritus receptors.

[0007] Another invention patent, CN113272301A, published on August 17, 2021, discloses a heterocyclic derivative inhibitor, its preparation method, and its application. This invention provides a heterocyclic compound as shown in Formula I, its stereoisomers, geometric isomers, tautomers, nitrides, hydrates, solvates, metabolites, esters, pharmaceutically acceptable salts, or prodrugs. This heterocyclic compound exhibits high P2X3 antagonistic activity, good selectivity, low toxicity, good metabolic stability, and minimal impact on taste.

[0008] Relevant non-patent literature retrieved:

[0009] The journal is titled "Life Sciences", the article is titled "Mechanism of Action and Latest Research Progress of P2X Receptor Modulators", volume number 36, published on February 15, 2024. This article clarifies that the regulatory mechanism can provide new directions for the rational drug design targeting P2X receptors, thereby improving the probability and effectiveness of drug discovery. It reviews the mechanism of action of P2X receptor modulators and summarizes the latest research progress of small molecule drugs targeting P2X receptors.

[0010] The prior art represented by the aforementioned documents has at least the following unresolved technical problems or defects:

[0011] Although they have good safety profiles, evidence suggests that P2X3 inhibitors are not highly selective for P2X3 or P2X2 / 3. This is supported by the following evidence: the IC50 of the compounds in the HEK293-hP2X3 receptor cell functional calcium flow assay. 50 The value is too high.

[0012] Therefore, the development of safe, non-addictive, and highly selective P2X3 receptor inhibitors for the treatment of diseases such as chronic cough without side effects such as loss of taste is promising. Summary of the Invention

[0013] The purpose of this invention is to provide:

[0014] An imidazopyridine derivative, and related technologies thereof, to address the technical problems of existing P2X3 receptor inhibitors, such as poor safety, low selectivity for P2X3 or P2X2 / 3, and poor metabolic stability, or a combination thereof.

[0015] Terminology Explanation:

[0016] Unless otherwise defined, all technical terms in this invention have the same meanings as commonly understood by one of ordinary skill in the art to which this subject matter pertains. Unless otherwise stated, all patents, patent inventions, and disclosures referenced in this invention are incorporated herein by reference in their entirety. If multiple definitions exist for terms in this invention, the definitions in this chapter shall prevail.

[0017] It should be understood that the above brief description and the following detailed description are exemplary and for illustrative purposes only, and do not limit the subject matter of the invention in any way. In this invention, the singular is used in conjunction with the plural unless otherwise specifically stated. It should also be noted that, unless otherwise stated, the use of “or” or “or” means “and / or”. Furthermore, the use of the term “comprising” and other forms such as “including,” “containing,” and “contains” are not limiting.

[0018] The definition of standard chemical terms can be found in the reference "Basic Organic Chemistry (Volumes 1 & 2), Xing Qiyi, Higher Education Press, June 2005, Third Edition".

[0019] Unless otherwise stated, conventional methods within the scope of the art, such as cell culture, cell plating, and dye incubation, shall be used.

[0020] Unless specifically defined, the use of various commercially available products in this invention employs standard techniques. For example, it may be carried out using the manufacturer's instructions for use with the reagent kit, or in accordance with methods known in the art or the description of this invention. The techniques and methods described herein can generally be implemented according to conventional methods well known in the art, based on the descriptions in the various summary and more specific documents cited and discussed in this specification.

[0021] The terms “optional / arbitrary” or “optionally / arbitrarily” mean that the event or situation described below may or may not occur, including both the occurrence and non-occurrence of the event or situation.

[0022] As used in this invention, the term "stereoisomer" refers to any of the various stereoisomeric configurations (e.g., enantiomers, diastereomers, and racemates) of an asymmetric compound (e.g., a compound having one or more asymmetricly substituted carbon atoms or an "asymmetric center"). Compounds of this disclosure containing an asymmetric center can be separated by optical activity (enantiomers or diastereomers) or optical inactivation (racemate). The term "enantiomer" includes stereoisomer pairs that are not mirror images of each other. A 1:1 mixture of a pair of enantiomers is a "racemate mixture". The term "diastereomer" refers to a stereoisomer having at least two asymmetric atoms that are not mirror images of each other. Certain compounds containing one or more asymmetric centers can produce enantiomers, diastereomers, or other stereoisomers, which can be defined with respect to absolute configuration at each asymmetric center as (R)- or (S)- according to the Cahn-Ingold-Prelog RS system. The term "or" can be used to indicate the absolute configuration of a resolved compound at the asymmetric center. Methods for preparing optically active forms from racemic mixtures are known in the art, such as by HPLC resolution or stereoselective synthesis.

[0023] The term "geometric isomer" as used in this invention refers to compounds that have the same formula but whose functional groups are rotated to different orientations in three-dimensional space.

[0024] As used in this invention, the term "tautomer" refers to a proton transfer tautomer comprising a proton-transfer tautomer in an isomeric protonated state of a compound having the same formula and total charge. Examples of proton transfer tautomers include, but are not limited to, keto-enol-amide-imine pairs, lactam-lactamimide pairs, enamine-imine pairs, and cyclic forms in which a proton may occupy two or more positions in a heterocyclic system, such as 1H-imidazolium and 3H-imidazolium, 1H-1,2,4-triazole, 2H-1,2,4-triazole and 4H-1,2,4-triazole, 1H-isoindole and 2H-isoindole, and 1H-pyrazole and 2H-pyrazole. Tautomers may be in equilibrium or spatially locked into one form by appropriate substitution. Unless otherwise stated, the compounds of this disclosure identified by name or structure as a particular tautomer form are intended to include other tautomer forms.

[0025] As used in this invention, the term "prodrug" refers to any compound or conjugate that releases an active parent drug when administered to an animal or human subject. A prodrug can be prepared by modifying the functional groups present in a compound in a manner that allows the modification to be cleaved by the parent compound under normal operating conditions or in vivo. Prodrugs include compounds in which a hydroxyl, amino, thioglycolic, or carboxyl group is bonded to any group such that when administered to a mammalian subject, it can be cleaved to form a free hydroxyl, amino, thioglycolic, or carboxyl group, respectively. Examples of prodrugs include, but are not limited to, acetate, formate, and benzoate derivatives of compounds containing alcohol and amine functional groups.

[0026] The term "polymorph" as used in this invention refers to the crystal form of the compound as described. It should be understood that the compound as described can exist in many different crystal forms or polymorphs, or can be made into an amorphous form (i.e., a solid form without any definite crystal structure).

[0027] In a first aspect, the present invention provides:

[0028] An imidazopyridine derivative selected from compounds of Formula I or their racemates, stereoisomers, geometric isomers, tautomers, nitrides, hydrates, isotope labels, solvates, polymorphs, metabolites, esters, pharmaceutically acceptable salts, or prodrugs:

[0029]

[0030] Y is either O or CR5;

[0031] R1 is selected from -C(=O)R6, -C(=O)N(R7)2, and -S(=O). q R6, S(=O)(=NH)R7, -S(=O) q N(R7)2, -NR7C(=O)R6, -NR7S(=O) q R6 or -N=S(=O)(R7)2, or R1 and the carbon adjacent to its parent nucleus R4 form a 4 to 7 saturated or unsaturated heterocycle;

[0032] R2 is selected from -C(=O)SR6, -C(=S)SR6, -C(=S)OR6, -C(=S)N(R7)2, -C(=S)C(R7)2, -C(=NH)N(R7)2, -C(=NH)OR6, -C(=NH)SR6, -C(=O)C(=O)OR6, -C(=O)C(=O)NHR6, -C(=O)C(=O)N(R7)2 or -C(=O)C(=O)R6;

[0033] R3 is selected from hydrogen, deuterium, halogen, -CN, C1-C6 non-substituted alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 haloalkyl, C3-C6 cycloalkyl, C1-C6 alkoxyC1-C6 alkyl, -OR8 or -N(R8)2;

[0034] R4 is selected from hydrogen, deuterium, halogen, -CN, C1-C6 non-substituted alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 haloalkyl, C3-C6 cycloalkyl, C1-C6 alkoxyC1-C6 alkyl, -OR8 or -N(R8)2;

[0035] R5 is selected from hydrogen, deuterium, halogen, -CN, C1-C6 unsubstituted alkyl or C1-C6 haloalkyl, or two R5s form a spirocycle or bridged heterocycle;

[0036] R6 is independently selected from hydrogen, deuterium, hydroxyl, C1-C6 unsubstituted alkyl, C1-C6 haloalkyl, C3-C6 cycloalkyl, C1-C6 alkoxyC1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, phenyl, C3-C9 saturated ring or unsaturated heterocyclic group; or R6 and R7 form a 5- to 6-membered heterocycle;

[0037] R7 is independently selected from hydrogen, deuterium, C1-C6 unsubstituted alkyl, C1-C6 haloalkyl, C3-C6 cycloalkyl, C1-C6 alkoxyC1-C6 alkyl or phenyl; or two R7 form a 5- to 6-membered heterocycle;

[0038] R8 is independently selected from hydrogen, deuterium, C1-C6 unsubstituted alkyl, C1-C6 haloalkyl, C3-C6 cycloalkyl, C1-C6 alkoxyC1-C6 alkyl, phenyl or benzyl;

[0039] p is 0, 1, or 2;

[0040] q is 1 or 2.

[0041] Preferably, as shown in Formula II:

[0042]

[0043] R1 is selected from -C(=O)N(R7)2, -S(=O)(=NH)R7, -S(=O) q N(R7)2, -NR7C(=O)R6, -NR7S(=O) q R6 or -N = S(=O)(R7)2;

[0044] R2 is selected from -C(=O)SR6, -C(=S)SR6, -C(=S)OR6, -C(=S)N(R7)2, -C(=S)C(R7)2, -C(=NH)N(R7)2, -C(=NH)OR6, -C(=NH)SR6, -C(=O)C(=O)OR6, -C(=O)C(=O)NHR6, -C(=O)C(=O)N(R7)2 or -C(=O)C(=O)R6;

[0045] R3 is selected from halogens, C1-C3 non-substituted alkyl groups, or C1-C3 haloalkyl groups;

[0046] R4 is selected from hydrogen, deuterium, or halogen;

[0047] R6 is independently selected from hydrogen, deuterium, hydroxyl, C1-C6 unsubstituted alkyl, C1-C6 haloalkyl, C3-C6 cycloalkyl, C1-C6 alkoxyC1-C6 alkyl, phenyl, C3-C9 saturated ring or unsaturated heterocyclic group; or R6 and R7 form a 5- to 6-membered heterocycle;

[0048] R7 is independently selected from hydrogen, deuterium, C1-C6 unsubstituted alkyl, C1-C6 haloalkyl, C3-C6 cycloalkyl, C1-C6 alkoxyC1-C6 alkyl or phenyl; or two R7 form a 5- to 6-membered heterocycle;

[0049] q is 1 or 2.

[0050] Most preferably, it is selected from at least one of the following compounds:

[0051]

[0052] Based on further solutions to the technical problems of the present invention, or simultaneous solutions to multiple technical problems, the preferred solution in the technical solution provided in the first aspect of the present invention includes:

[0053] The first preferred option is as shown in Equation II:

[0054]

[0055] R1 is selected from -C(=O)N(R7)2, S(=O)(=NH)R7, and -S(=O). q N(R7)2, -NR7C(=O)R6, -NR7S(=O) q R6 or -N = S(=O)(R7)2;

[0056] R2 is selected from -C(=O)SR6, -C(=S)SR6, -C(=S)OR6, -C(=S)N(R7)2, -C(=S)C(R7)2, -C(=NH)N(R7)2, -C(=NH)OR6, -C(=NH)SR6, -C(=O)C(=O)OR6, -C(=O)C(=O)NHR6, -C(=O)C(=O)N(R7)2 or -C(=O)C(=O)R6;

[0057] R3 is selected from halogens, C1-C3 non-substituted alkyl groups, or C1-C3 haloalkyl groups;

[0058] R4 is selected from hydrogen, deuterium, or halogen;

[0059] R6 is independently selected from hydrogen, deuterium, hydroxyl, C1-C6 unsubstituted alkyl, C1-C6 haloalkyl, C3-C6 cycloalkyl, C1-C6 alkoxyC1-C6 alkyl, phenyl, C3-C9 saturated ring or unsaturated heterocyclic group; or R6 and R7 form a 5- to 6-membered heterocycle;

[0060] R7 is independently selected from hydrogen, deuterium, C1-C6 unsubstituted alkyl, C1-C6 haloalkyl, C3-C6 cycloalkyl, C1-C6 alkoxyC1-C6 alkyl or phenyl; or two R7 form a 5- to 6-membered heterocycle;

[0061] q is 1 or 2. This technical solution not only solves the technical problem of "existing P2X3 receptor inhibitors having poor safety and low selectivity for P2X3 or P2X2 / 3", but also solves the technical problem of "existing P2X3 receptor inhibitors having poor metabolic stability".

[0062] The second preferred option is selected from at least one of the following compounds:

[0063]

[0064] This technical solution not only addresses the technical problem of "poor safety and low selectivity for P2X3 or P2X2 / 3" of existing P2X3 receptor inhibitors, but also further solves the technical problem of "poor metabolic stability of existing P2X3 receptor inhibitors".

[0065] Secondly, the present invention provides:

[0066] A pharmaceutical composition comprising the above-mentioned imidazopyridine derivatives.

[0067] Thirdly, the present invention provides:

[0068] A pharmaceutical preparation comprising the above-described pharmaceutical composition.

[0069] The dosage form of the preparation is selected from: tablets, pills, granules, powders, capsules, emulsions, or suspensions.

[0070] The "route of administration of the preparation" is selected from: intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, or oral.

[0071] Fourthly, the present invention provides:

[0072] The use of the above-mentioned imidazopyridine derivatives, or the above-mentioned pharmaceutical compositions, in the preparation of medicaments for the prevention and / or treatment of diseases mediated by P2X3 receptors.

[0073] The "diseases" are selected from: asthma, chronic obstructive pulmonary disease, pulmonary fibrosis, pulmonary hypertension, cough, pain, urinary incontinence, difficulty urinating, endometriosis, or pruritus.

[0074] Among them, "cough" includes: chronic recurrent cough, chronic refractory cough, and chronic cough of unknown cause.

[0075] Among them, "pain" includes: migraines, pain from endometriosis.

[0076] The beneficial effects of this invention are as follows:

[0077] This invention provides a novel imidazopyridine derivative. Test results show that, compared with positive control drug 1 and positive control drug 2, the compound of this invention has good safety against P2X3 receptor inhibitors, higher selectivity for P2X3 or P2X2 / 3, and better metabolic stability in liver microsomes. Detailed Implementation

[0078] The following non-limiting embodiments are intended to enable those skilled in the art to gain a more comprehensive understanding of the present invention, but do not limit the invention in any way. The following content is merely an exemplary description of the scope of protection claimed by the present invention, and those skilled in the art can make various changes and modifications to the present invention based on the disclosed content, and such changes should also fall within the scope of protection claimed by the present invention.

[0079] The present invention will be further described below by way of specific embodiments. Unless otherwise specified, all instruments, devices, equipment, reagents, products, etc., used in the embodiments of the present invention are obtained through conventional commercial means.

[0080] The structural formula of positive control drug 1 (i.e., Camlipixant) is as follows, and its synthesis method is based on invention patent application WO2020074962A1, with a purity of 99%.

[0081] .

[0082] The structural formula of positive drug 2 is as follows, and its synthesis method is based on invention patent CN113272301A, with a purity of 99%:

[0083] .

[0084] The synthesis methods of the intermediates are respectively referred to invention patent application WO2022161462A1, invention patent CN113549068A, and invention patent CN113272301A.

[0085] Example 1

[0086] Synthesis of Compound 2: (S)-2-((2-(2,6-difluoro-4-aminosulfonylphenyl)-7-methylimidazo[1,2-a]pyridin-3-yl)methyl)morpholine-4-thiocarboxylate

[0087]

[0088] At 25°C, intermediate II (60 mg, 0.115 mmol) and MeOH / HCl (4 M, 4 mL) were added sequentially to a 50 mL single-necked flask. The mixture was stirred at 25°C for 1 h, and the reaction was determined to be complete by TLC (petroleum ether: ethyl acetate = 1:1, v / v). The mixture was concentrated and dried using dichloromethane (DCM) (5 mL × 2). DCM (5 mL) and triethylamine Et3N (38.4 mg, 0.345 mmol) were added to dissolve the mixture. Carbon disulfide CS2 (9.6 mg, 0.126 mmol) was then added, and the mixture was stirred at 0°C for 1.0 h under N2 protection. Iodomethane CH3I (5 mg, 10% wt) was then added, and the mixture was stirred at 0–25°C for 12 h. The reaction was determined to be complete by TLC (petroleum ether: ethyl acetate = 1:1). Finally, 10 mL of H2O was added. Extracted by DCM (10 mL × 2), the DCM phase was retained, dried with anhydrous sodium sulfate, concentrated under reduced pressure, and purified by preparative separation under high pressure (chromatographic column: AZZOTA-C18, 10 μm; mobile phase: acetonitrile-water; gradient: 5-45%), yielding 25 mg of white solid compound 2, yield 42%.

[0089] [M+2H] + :514.

[0090] 1 H NMR (400 MHz, Methanol-d4) δ 8.43 (d, J = 7.0 Hz, 1H), 7.66 (d, J =6.8 Hz, 2H), 7.37 (d, J= 1.4 Hz, 1H), 6.91 (dd, J = 7.1, 1.7 Hz, 1H), 3.90 –3.84 (m, 1H), 3.79 – 3.71 (m, 3H), 3.63 (q, J = 7.1 Hz, 1H), 3.45 (td, J =11.8, 2.6 Hz, 1H), 3.18 (d, J = 5.8 Hz, 2H), 2.73 (q, J = 7.3 Hz, 1H), 2.61 (s, 3H), 2.48 (d, J = 1.1 Hz, 3H).

[0091] Example 2

[0092] Synthesis of Compound 9: (S)-2-(2-(2-(2,6-difluoro-4-aminosulfonylphenyl)-7-methylimidazo[1,2-a]pyridin-3-yl)methyl)morpholino)-2-oxoacetic acid methyl ester

[0093]

[0094] At 25°C, intermediate II (100 mg, 0.193 mmol) and HCl / MeOH (4 M, 3 mL) were added sequentially to a 25 mL single-necked flask. The mixture was stirred at 25°C for 1 h, and the reaction solution was concentrated under reduced pressure. The solution was dried using DCM (5 mL). N,N-dimethylformamide DMF (3 mL) and Et3N (77.7 mg, 0.768 mmol) were added sequentially to the residue under N2 protection. Methyl 2-chloro-2-oxoacetate (24.6 mg, 0.201 mmol) was added, and the mixture was stirred at 25°C for 2 h. LC-MS monitoring showed that the reaction proceeded completely. The reaction solution was diluted with water (10 mL), extracted with DCM (10 mL × 2), and the organic phases were combined, dried over anhydrous Na2SO4, and concentrated under reduced pressure. High-pressure chromatography was performed (column: AZZOTA-C18, 10 μm; mobile phase: acetonitrile-water; gradient: 5-45%). 5 mg of white solid compound 9 was obtained, with a yield of 5.4%.

[0095] [M+2H] + :510.

[0096] 1 H NMR (400 MHz, Chloroform- d ) δ 8.18 (dd, J = 14.4, 7.1 Hz, 1H), 7.58(d,J = 4.8 Hz, 1H), 7.53 – 7.49 (m, 2H), 7.03 (s, 1H), 6.87 (s, 1H), 6.80 –6.74 (m, 1H), 4.28 (dd, J = 13.5, 6.6 Hz, 1H), 3.87 (s, 3H), 3.63 (d, J = 7.3 Hz,1H), 3.57 – 3.47 (m, 1H), 3.46 – 3.40 (m, 1H), 3.28 – 3.19 (m, 1H), 3.04 –2.95 (m, 2H), 2.93 – 2.81 (m, 1H), 2.56 (dd, J = 13.3, 10.7 Hz, 1H), 2.49 (dd, J = 4.0, 1.1 Hz, 3H).

[0097] Example 3

[0098] Synthesis of Compound 10: (S)-3,5-difluoro-N-methyl-4-(7-methyl-3-(4-(2-(methylamino)-2-oxoacetyl)morpholin-2-yl)methyl)imidazo[1,2-a]pyridin-2-yl)benzamide

[0099]

[0100] At 25°C, intermediate I (70 mg, 0.14 mmol) and EA / HCl (4 mL, 2.0 M) were added sequentially to a 50 mL single-necked flask. The mixture was stirred at 25°C for 1 h. The reaction was monitored by TLC (petroleum ether: ethyl acetate = 1:1, v / v) until complete. The mixture was concentrated under reduced pressure and dried twice with toluene (5 mL × 2). DMF (3 mL) and triethylamine (1 mL) were added sequentially to the residue, followed by the slow addition of 2-(methylamino)-2-oxoacetic acid (14 mg, 0.14 mmol) and HATU (80 mg, 0.21 mmol). ll The mixture was stirred at 25°C for 1.0 h, and the reaction was determined to be complete by TLC. The reaction was quenched by adding saturated ammonium chloride solution (10 mL), extracted with ethyl acetate (EA) (10 mL × 2), and the organic phases were combined. The organic phases were washed twice with saturated sodium chloride aqueous solution (10 mL × 2), dried over anhydrous sodium sulfate, concentrated under reduced pressure, and prepared by reversed-phase high-pressure chromatography (column: AZZOTA-C18, 10 μm; mobile phase: acetonitrile-water; gradient: 5-45%) to give 5 mg of white solid compound 10, yield 7.35%.

[0101] [M+H] + :487.

[0102] 1 H NMR (400 MHz, Chloroform- d ) δ 8.20 (dd, J = 8.0, 4.0 Hz, 1H), 7.44– 7.41 (m, 3H), 7.24 (s, 1H), 6.70 (dd, J = 8.0, 8.0 Hz, 1H), 6.46 (d, J =36.0Hz, 1H), 4.31 (s, 1H), 4.13 (d, J = 16.0 Hz, 1H), 3.94 (d, J = 12.0 Hz,1H), 3.89 – 3.82 (m, 2H), 3.65 (s, 2H) , 3.48 – 3.39 (m, 1H) , 3.24 – 3.17(m, 1H) , 3.07 – 3.04 (m, 3H), 2.86 (d, J = 4.0 Hz, 3H), 2.44 (s, 3H).

[0103] Example 4

[0104] Synthesis of Compound 13: (S)-2-(2-(2-(2,6-difluoro-4-aminosulfonylphenyl)-7-methylimidazo[1,2-a]pyridin-3-yl)methyl)morpholino)-N-methyl-2-oxoacetamide

[0105]

[0106] At 25°C, intermediate IV (125 mg, 0.2 mmol) and EA / HCl (2 M, 5 mL) were added sequentially to a 50 mL single-necked flask. The mixture was stirred at 25°C for 1 h. The reaction was determined to be complete by TLC (petroleum ether: ethyl acetate = 1:1, v / v). The mixture was concentrated and dried twice with DCM (5 mL × 2). DCM (10 mL) and Et3N (29 mg, 0.3 mmol) were added to dissolve the mixture. 2-(methylamino)-2-oxoacetic acid (22.7 mg, 0.22 mmol) and HATU (57 mg, 0.15 mmol) were then added. Under N2 protection, the mixture was stirred at 25°C for 12.0 h. The reaction was determined to be complete by TLC. Add saturated ammonium chloride solution (10 mL), extract with DCM (10 mL × 2), retain the DCM phase, dry with anhydrous sodium sulfate, concentrate under reduced pressure, and prepare under high pressure (chromatographic column: AZZOTA-C18, 10 μm; mobile phase: acetonitrile-water; gradient: 5-45%) to give 25 mg of white solid compound 13, yield 24.6%.

[0107] [M+H] + :509.

[0108] 1H NMR (400 MHz, Chloroform-d) δ 8.19 (dd, J = 7.2, 4.9 Hz, 1H), 7.57(s, 1H), 7.50 (dd, J = 8.0, 6.4 Hz, 2H), 7.35 – 7.30 (m, 1H), 7.02 (s, 2H), 6.76 (dd, J = 7.2, 1.7 Hz, 1H), 4.26 (d, J = 13.4 Hz, 1H), 4.03 (dd, J = 30.3, 13.4Hz, 1H), 3.92 – 3.72 (m, 1H), 3.61 (d, J = 4.3 Hz, 1H), 3.42 (ddd, J = 26.5,12.4, 9.8 Hz, 1H), 3.29 – 3.14 (m, 1H), 3.08 – 2.93 (m, 2H), 2.93 – 2.79 (m,4H), 2.51 – 2.43 (m, 3H).

[0109] Example 5

[0110] Synthesis of Compound 14: (S)-2-(2-(2-(2,6-difluoro-4-aminosulfonylphenyl)-7-methylimidazo[1,2-a]pyridin-3-yl)methyl)morpholino)-N,N-dimethyl-2-oxoacetamide

[0111]

[0112] At 25°C, intermediate IV (125 mg, 0.2 mmol) and EA / HCl (2 M, 5 mL) were added sequentially to a 50 mL single-necked flask. The mixture was stirred at 25°C for 1 h. The reaction was monitored by TLC (petroleum ether: ethyl acetate = 1:1, v / v) until complete. The mixture was concentrated and dried twice with DCM (5 mL × 2). DCM (10 mL) and Et3N (29 mg, 0.3 mmol) were added to dissolve the residue. HATU (98.8 mg, 0.26 mmol) and 2-(methylamino)-2-oxoacetic acid (25.7 mg, 0.22 mmol) were added. The mixture was protected with N2 and stirred at 25°C for 12.0 h. The reaction was determined to be complete by TLC. Add saturated ammonium chloride solution (10 mL), DCM (10 mL × 2), extract, retain the DCM phase, dry with anhydrous sodium sulfate, concentrate under reduced pressure, and prepare under high pressure (chromatographic column: AZZOTA-C18, 10 μm; mobile phase: acetonitrile-water; gradient: 5-45%) to give 5 mg of white solid compound 14, yield 5%.

[0113] [M+H] + :522.

[0114] 1 H NMR (400 MHz, Chloroform-d) δ 8.21 (d, J = 7.1 Hz, 1H), 7.68 (d, J =6.7 Hz, 3H), 7.38 (s, 2H), 6.69 (dd, J = 7.1, 1.7 Hz, 1H), 3.81 (d, J = 11.7 Hz,1H), 3.67 (s, 1H), 3.55 – 3.48 (m, 1H), 3.03 – 2.97 (m, 8H), 2.93 (d, J = 5.0Hz, 1H), 2.81 – 2.74 (m, 2H), 2.50 (d, J = 11.1 Hz, 1H), 2.43 (d, J = 1.1 Hz, 3H).

[0115] Example 6

[0116] Synthesis of Compound 16: (S)-4-(3-((4-(2-(dimethylamino)-2-oxoacetyl)morpholin-2-yl)methyl)-7-methylimidazo[1,2-a]pyridin-2-yl)-3,5-difluoro-N-methylbenzamide

[0117]

[0118] At 25°C, intermediate I (140 mg, 0.28 mmol) and MeOH / HCl (3 mL, 2.0 M) were added sequentially to a 50 mL single-necked flask. The mixture was stirred at 25°C for 1 h. The reaction was monitored by TLC (petroleum ether: ethyl acetate = 1:1, v / v) until completion. The mixture was concentrated and dried twice using DCM (5 mL × 2). DMF (4 mL) and N,N-diisopropylethylamine (DIPEA) (1 mL) were added sequentially, and the mixture was slowly added... N,N Dimethyl oxalic acid (36 mg, 0.31 mmol) and HATU (57 mg, 0.15 mmol) were stirred at 0 °C for 1.0 h, and the reaction was determined to be complete by TLC. The reaction was quenched by adding saturated ammonium chloride solution (10 mL), extracted with EA (10 mL × 2), washed with saturated brine (10 mL), the organic phases were combined, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and prepared by reversed-phase high-pressure chromatography (column: AZZOTA-C18, 10 μm; mobile phase: acetonitrile-water; gradient: 5-45%) to give 6 mg of white solid compound 16, yield 4.3%.

[0119] [M+2H] + :501.

[0120] 1 H NMR (400 MHz, Chloroform- d ) d 8.19 (t, J = 7.8 Hz, 1H), 7.56 – 7.38(m, 3H), 6.87 – 6.69 (m, 2H), 4.29 (d, J = 13.1 Hz, 1H), 4.02 – 3.75 (m, 2H), 3.78 – 3.52 (m, 2H), 3.51 – 3.33 (m, 2H), 3.23 (d, J = 11.8 Hz, 1H), 3.06 (dd, J = 4.8, 2.3 Hz, 3H), 3.01 (d, J = 5.2 Hz, 1H), 2.99 (d,J = 2.6 Hz, 3H), 2.92 (s,3H), 2.45 (dd, J = 3.8, 1.1 Hz, 3H).

[0121] Example 7

[0122] Synthesis of Compound 38: (S)-4-(3-(4-(2-(3,3-difluoroazacyclobutane-1-yl)-2-oxoacetyl)morpholin-2-yl)methyl)-7-methylimidazo[1,2-a]pyridin-2-yl)-3,5-difluorobenzenesulfonamide

[0123]

[0124] Step 1: Synthesize XY2302-68-3

[0125] At 25°C, intermediate III (200 mg, 0.302 mmol), DCM (2 mL), and triethylamine (92 mg, 0.906 mmol) were added sequentially to a 25 mL single-necked flask. After cooling the reaction solution to 0°C, methyl oxaloyl chloride (37 mg, 0.302 mmol) was slowly added dropwise. The mixture was stirred at 25°C for 1 h, and the reaction was monitored for completion by TLC (petroleum ether: ethyl acetate = 1:1, v / v). The reaction was quenched with saturated ammonium chloride aqueous solution (10 mL), extracted with DCM (15 mL × 2), washed with saturated brine (10 mL), and the organic phases were combined, dried over anhydrous Na₂SO₄, concentrated under reduced pressure, and purified by silica gel column chromatography (petroleum ether: ethyl acetate = 3:1, v / v) to give 200 mg of white solid XY₂3O₂-68-3, [M + 2H]. + : 750.

[0126] Step 2: Synthesize XY2302-68-4

[0127] At 25°C, XY2302-68-3 (200 mg, 0.267 mmol), methanol (2 mL), and water (0.2 mL) were added sequentially to a 25 mL single-necked flask, followed by lithium hydroxide monohydrate (LiOH) (29 mg, 0.534 mmol). The mixture was stirred at 25°C for 1 h. The reaction was monitored for completeness by TLC (petroleum ether:ethyl acetate = 1:1, v / v). The reaction solution was quenched with water (10 mL), concentrated under reduced pressure to remove methanol, and the pH was adjusted to weakly acidic with dilute hydrochloric acid aqueous solution (3 mL, 1.0 M). The solution was then extracted with EA (15 mL × 2), and the organic phase was collected. The organic phase was dried over anhydrous Na2SO4 and concentrated under reduced pressure to obtain 150 mg of yellow solid XY2302-68-4, [M + 2H+]. + :736.

[0128] Step 3: Synthesize XY2302-68-5

[0129] At 25°C, XY2302-68-4 (50 mg, 0.068 mmol), 3,3-difluorozacriane hydrochloride (13 mg, 0.102 mmol), HATU (39 mg, 0.102 mmol), DIPEA (26 mg, 0.204 mmol), and DMF (2 mL) were added sequentially to a 25 mL single-necked flask. The mixture was stirred at 25°C for 1 h. The reaction was monitored for completeness by TLC (petroleum ether:ethyl acetate = 1:1, v / v). The mixture was quenched with water (5 mL), extracted with EA (10 mL × 2), washed with saturated brine (10 mL), and the organic phases were combined, dried over anhydrous Na2SO4, concentrated under reduced pressure, and purified by silica gel column chromatography (petroleum ether:ethyl acetate = 1:1, v / v). 30 mg of white solid XY2302-68-5, [M + 2H], was obtained. + :811.

[0130] Step 4: Synthesize compound 38

[0131] At 25°C, XY2302-68-5 (30 mg, 0.037 mmol), DCM (1 mL), and trifluoroacetic acid (TFA) (1 mL) were added sequentially to a 25 mL single-necked flask. The mixture was stirred at 25°C for 4 h, and the reaction was monitored for completeness by TLC (petroleum ether: ethyl acetate = 1:1, v / v). The mixture was concentrated under reduced pressure and prepared by high-pressure reversed-phase chromatography (column: AZZOTA-C18, 10 μm; mobile phase: acetonitrile-water; gradient: 5-45%) to obtain 10 mg of white solid compound 38.

[0132] [M+2H] + :571.

[0133] 1 H NMR (400 MHz, CDCl3) d 8.18 (d, J = 4.0 Hz, 1H), 7.57 (s, 1H), 7.53 –7.51 (m, 2H), 6.82 (d, J = 8.0 Hz, 2H), 6.78 – 6.76 (m, 1H), 4.70 – 4.62 (m,2H), 4.43 – 4.36 (m, 2H), 4.24 (d, J = 12.0 Hz, 1H), 4.06 (d, J= 16.0 Hz, 1H),2.89 – 3.83 (m, 1H), 3.66 – 3.59 (m, 2H), 3.47 – 3.38 (m, 1H), 3.23 – 3.17(m, 1H), 3.02 – 2.96 (m, 2H), 2.49 (s, 3H).

[0134] Example 8

[0135] Synthesis of Compound 39: (S)-N-cyclopropyl-2-(2-(2,6-difluoro-4-aminosulfonylphenyl)-7-methylimidazo[1,2-a]pyridin-3-yl)methyl)morpholino)-2-oxoacetamide

[0136]

[0137] Step 1: Synthesize XY2302-69-2

[0138] At 25°C, XY2302-68-4 (50 mg, 0.068 mmol), cyclopropylamine (12 mg, 0.204 mmol), HATU (39 mg, 0.102 mmol), DIPEA (26 mg, 0.204 mmol), and DMF (2 mL) were added sequentially to a 25 mL single-necked flask, and the mixture was stirred at 25°C for 1 h. The reaction was monitored for completeness by TLC (petroleum ether:ethyl acetate = 1:1, v / v). The mixture was quenched with water (5 mL), extracted with EA (10 mL × 2), washed with saturated brine (10 mL), and the organic phases were combined, dried over anhydrous Na2SO4, filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by column chromatography (petroleum ether:ethyl acetate = 1:1, v / v). 20 mg of a white solid XY2302-69-2, [M + 2H], was obtained. + :775.

[0139] Step 2: Synthesize compound 39

[0140] At 25°C, XY2302-69-2 (20 mg, 0.026 mmol), DCM (1 mL), and trifluoroacetic acid (1 mL) were added sequentially to a 25 mL single-necked flask. The mixture was stirred at 25°C for 4 h, and the reaction was monitored by TLC (ethyl acetate) to indicate completion. The mixture was concentrated under reduced pressure, and the crude product was prepared by high-pressure reversed-phase chromatography (column: AZZOTA-C18, 10 μm; mobile phase: acetonitrile-water; gradient: 5-45%) to give 5 mg of white solid compound 39, with a yield of 36.2%.

[0141] [M+2H] + :535.

[0142] 1 H NMR (400 MHz, Chloroform- d ) δ 8.19 (t, J = 8.0 Hz, 1H), 7.56 – 7.51(m, 3H), 7.40 – 7.36 (m, 1H), 6.77 – 6.74 (m, 2H), 6.57 (s, 1H), 5.05 – 4.88(m, 1H), 4.26 (d, J = 13.2 Hz, 1H), 3.87 – 3.81 (m, 1H), 3.62 (s, 1H), 3.47 –3.40 (m, 1H), 3.24 – 3.17 (m, 1H), 3.04 – 2.96 (m, 2H), 2.82 – 2.67 (m, 2H),2.48 (s, 3H), 0.85 – 0.80 (m, 2H), 0.61 – 0.53 (m, 2H).

[0143] Example 9

[0144] Synthesis of Compound 40: (S)-2-(2-(2-(2,6-difluoro-4-aminosulfonylphenyl)-7-methylimidazo[1,2-a]pyridin-3-yl)methyl)morpholino)-2-oxoacetic acid

[0145]

[0146] At 25°C, XY2302-68-4 (50 mg, 0.068 mmol), DCM (1 mL), and trifluoroacetic acid (1 mL) were added sequentially to a 25 mL single-necked flask, and the mixture was stirred at 25°C for 4 h. The mixture was concentrated under reduced pressure, and the residue was adjusted to neutral pH with saturated sodium bicarbonate aqueous solution (5 mL). Then, EA (10 mL) was added for extraction. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain the crude product. The crude product was purified by high-pressure reversed-phase chromatography (column: AZZOTA-C18, 10 μm; mobile phase: acetonitrile-water; gradient: 5-45%) to obtain 10 mg of white solid compound 40, with a yield of 29.7%.

[0147] [M+2H] + :496.

[0148] 1 H NMR (400 MHz, DMSO-d6) d8.46 – 8.38 (m, 1H), 7.82 (s, 1H), 7.75 (s,1H), 7.65 – 7.62 (m, 2H), 7.38 (s, 1H), 6.87 – 6.84 (m, 1H), 4.05 (d, J = 12.0Hz, 1H), 3.93 (d, J = 16.0 Hz, 1H), 3.65 (d, J = 12.0 Hz, 1H), 3.21 – 3.15 (m,1H), 3.14 – 3.07 (m, 2H), 3.04 – 2.98 (m, 1H), 2.95 – 2.89 (m, 1H), 2.55 (s,1H), 2.39 (s, 3H).

[0149] Example 10

[0150] Synthesis of Compound 41: (S)-3,5-difluoro-N-methyl-4-(7-methyl-3-((4-(2-oxopropionyl)morpholin-2-yl)methyl)imidazo[1,2-a]pyridin-2-yl)benzamide

[0151]

[0152] At 25°C, intermediate I (200 mg, 0.40 mmol) and MeOH / HCl (4 mL, 2.0 M) were added sequentially to a 50 mL single-necked flask. The mixture was stirred at 25°C for 1 h. The reaction was monitored by TLC (ethyl acetate) until complete. The mixture was concentrated and dried twice with methanol (5 mL × 2) / ethyl acetate (5 mL × 2). It was then redissolved in DMF (6 mL), and DIPEA (1 mL), pyruvate (40 mg, 0.46 mmol), and HATU (197 mg, 0.52 mmol) were added sequentially. The mixture was stirred at 25°C for 3.0 h, and the reaction was determined to be complete by TLC (petroleum ether:ethyl acetate = 1:1, v / v). The reaction was quenched by adding saturated ammonium chloride solution (10 mL), extracted with EA (10 mL × 2), washed with saturated brine (10 mL), the organic phases were combined, dried with anhydrous sodium sulfate, concentrated under reduced pressure, and prepared by reversed-phase high-pressure chromatography (column: AZZOTA-C18, 10 μm; mobile phase: acetonitrile-water; gradient: 5-45%), to give 35 mg of white solid compound 41, yield 12.0%.

[0153] [M+2H] + :472.

[0154] 1H NMR (400 MHz, Chloroform- d ) d 8.19 (dd, J = 7.2, 4.1 Hz, 1H), 7.48 –7.37 (m, 3H), 6.84 (dd, J = 12.4, 5.6 Hz, 1H), 6.72 (td, J = 7.3, 1.7 Hz, 1H),4.30 – 4.21 (m, 1H), 3.94 – 3.80 (m, 1H), 3.70 – 3.56 (m, 2H), 3.50 – 3.41(m, 1H), 3.28 – 3.11 (m, 1H), 3.06 (dd, J = 4.9, 1.6 Hz, 3H), 3.01 – 2.78 (m,2H), 2.55 (dd, J = 13.2, 10.8 Hz, 1H), 2.49 – 2.31 (m, 6H).

[0155] Example 11

[0156] Synthesis of Compound 19: 2-[(2S)-2-({2-[4-(aminodioxane-λ6-thio)-2,6-difluorophenyl]-7-chloroimidozono[3,2-a]pyridin-3-yl}methyl)-1,4-oxazacyclohex-4-yl]-N-methyl-2-oxanediacetamide

[0157]

[0158] At 25°C, intermediate VI (90 mg, 0.16 mmol), DMF (3 mL), and triethylamine (1 mL) were added sequentially to a 50 mL single-necked flask. Then, 2-(methylamino)-2-oxomethyleneacetic acid (14 mg, 0.14 mmol) was slowly added, followed by HATU (80 mg, 0.21 mmol). The mixture was stirred at 25°C for 1.0 h, and the reaction was determined to be complete by TLC (petroleum ether: ethyl acetate = 1:1, v / v). The reaction was quenched by adding saturated ammonium chloride solution (10 mL), extracted with EA (10 mL × 2), and the organic phases were combined, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and prepared by reversed-phase high-pressure chromatography (column: AZZOTA-C18, 10 μm; mobile phase: acetonitrile-water; gradient: 5-45%) to give 3 mg of white solid compound 19, yield 3.4%.

[0159] [M+2H] + :529.

[0160] 1 H NMR (400 MHz, Chloroform- d ) d 8.26 – 8.08 (m, 1H), 7.56 (s, 1H),7.51 (dd, J = 8.0, 6.4 Hz, 2H), 7.35 – 7.31 (m, 1H), 7.22 – 6.91 (m, 2H), 6.77(dd, J = 7.2, 1.7 Hz, 1H), 4.94 (dd, J = 12, 13.4 Hz, 1H), 4.27 (d, J = 13.4 Hz,1H), 3.92 – 3.73 (m, 1H), 3.61 (d, J = 4.3 Hz, 1H), 3.55 – 3.36 (m, 1H), 3.30 –3.09 (m, 1H), 3.08 – 2.93 (m, 2H), 2.93 – 2.88 (m, 1H), 2.83 (d, J = 8.0, 5.1Hz, 3H).

[0161] Example 12

[0162] Synthesis of Compound 42: 2-[(2S)-2-({2-[2,6-difluoro-4-(2-oxoylidene-1-yl)phenyl]-7-methylimidazo[3,2-a]pyridin-3-yl}methyl)-1,4-oxazacyclohex-4-yl]-N-methyl-2-oxoylideneacetamide

[0163]

[0164] At 25°C, intermediate IV (100 mg, 0.19 mmol), DMF (3 mL), and triethylamine (1 mL) were added sequentially to a 20 mL single-necked flask. Then, 2-(methylamino)-2-oxomethyleneacetic acid (14 mg, 0.14 mmol) was slowly added, followed by HATU (80 mg, 0.21 mmol). The mixture was stirred at 25°C for 1.0 h, and the reaction was determined to be complete by TLC (petroleum ether: ethyl acetate = 1:1, v / v). The reaction was quenched by adding saturated ammonium chloride solution (10 mL), extracted with EA (10 mL × 2), and the organic phases were combined, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and prepared by reversed-phase high-pressure chromatography (column: AZZOTA-C18, 10 μm; mobile phase: acetonitrile-water; gradient: 5-45%) to give 3 mg of white solid compound 42, yield 3.5%.

[0165] [M+2H] + :513.

[0166] 1 H NMR (400 MHz, Chloroform-d) d 7.84 – 7.70 (m, 1H), 7.66 (d, J = 10.8Hz, 2H), 7.39 (dd, J = 8.3, 5.1 Hz, 1H), 7.21 (dd, J = 13.7, 8.3 Hz, 1H), 4.35 –4.31 (m, 1H), 4.15 – 4.07 (m, 1H), 3.91– 3.83 (m, 3H), 3.71 – 3.55 (m, 1H), 3.45 – 3.28 (m, 1H), 3.26 – 2.87(m, 3H), 2.84(d, J = 8.0, 5.1 Hz, 3H), 2.80-2.71(m, 2H), 2.69- 2.65(m, 1H), 2.57- 2.53(m, 1H), 2.48(s, 3H), 2.28- 2.21(m,2H).

[0167] Detection Example 1

[0168] 1. Experimental Objective

[0169] The inhibitory activities of positive drug 1, positive drug 2, and compounds prepared in Examples 1-12 on hP2X3 and hP2X2 / 3 receptors were determined using a fluorescence imaging plate reader (FLIPR).

[0170] 2. Experimental instruments and reagents / consumables

[0171] 2.1 Experimental instruments: Biosafety cabinet (ESCO, AC2-6S1-TC); CO2 cell incubator (ESCO, CCl-170B-8); inverted microscope (Olympus, CKX53); low-speed centrifuge (Xiangzhi, TD5B); five-channel fluorescence imaging microplate reader FLIPR® Penta (Molecular Devices, 5).

[0172] 2.2 Experimental reagents and consumables: DMEM medium (Hyclone, SH30243.01); high-channel intracellular calcium flow assay kit (Molecular Devices, R8191); fetal bovine serum (Avantor, 76294-180); hygromycin B (Solarbio, H8080); HBSS buffer (Gibco, 14025-092); DMSO (Sigma, D4540); 384-well conical bottom polypropylene plate (Nunc, 264573); 96-well polypropylene plate (Biosen, P-0.36-BSA-96); Corning® 384-well transparent flat-bottom black polystyrene microplate (Corning, 3764); HEK293 cells (ATCC).

[0173] 3. Experimental Procedure

[0174] 3.1 Cell Culture: Stable transfected hP2X3 or hP2X2 / 3 HEK293 cells were cultured in DMEM medium containing 10% fetal bovine serum at 37°C and 5% carbon dioxide.

[0175] 3.2 Buffer preparation: Prepare 1× buffer according to the instructions of the high-channel intracellular calcium flow detection kit, and then use 1× buffer to prepare 2× dye for later use.

[0176] 3.3 Cell Plating: HEK293 cells stably transfected with hP2X3 or hP2X2 / 3 receptors were digested and collected. After resuspending and counting, the cells were seeded into 384-well transparent flat-bottomed black polystyrene microplates at a seeding density of 8000 cells / 25 μL / well. The plates were incubated in a 5% CO2, 37°C incubator until the cells reached approximately 80% confluence. The plates were then centrifuged upside down to remove the culture medium from the 384-well plates, and 18 μL of 1× buffer was added.

[0177] 3.4 Dye incubation: Add another 18 μL of dye and incubate at 37°C in the dark for 2 h.

[0178] 3.5 Prepare 10× serially diluted intermediate solutions of positive and test compounds using DMSO, and transfer the prepared 10× test intermediate solutions to the corresponding 384-well conical bottom polypropylene plates.

[0179] 3.6 Prepare 5× agonist intermediate solution according to the instructions of the high-channel intracellular calcium flow assay kit, and transfer the intermediate solution to the corresponding 384-well conical bottom polypropylene plate.

[0180] 3.7 FLIPR Instrument Detection: After cell dye incubation, 4 μL of the test sample prepared in step 3.5 was added to the test well using a FLIPR instrument, and data was collected and recorded for 5 min, followed by incubation for 20 min. Then, 10 μL of the agonist intermediate solution prepared in step 3.6 was added, and data was collected and recorded for 5 min. Calcium flux detection was performed using excitation light at 470-515 nm and emission light at 515-575 nm.

[0181] 4. Experimental Data Processing Methods

[0182] Calcium flow signal values ​​were read using FLIPR. The highest concentration of the positive control drug was taken as the 100% inhibition rate, and 0.1% DMSO was taken as the 0% inhibition rate. The percentage inhibition rate was calculated using the following formula:

[0183] Inhibition% = (Test substance - Ave 0.1% DMSO) ÷ (Ave 50 μM Positive agent - Ave 0.1% DMSO).

[0184] The percentage inhibition rate and compound concentration were fitted using GraphPad Prism to generate a compound inhibition effect curve, and the IC50 was calculated. 50 value.

[0185] hP2X3 and hP2X2 / 3 selectivity factor = hP2X2 / 3 IC 50 Value / hP2X3 IC 50 value.

[0186] Verification of technical effectiveness and / or analysis of solutions to technical problems:

[0187] The inhibitory activity of the compounds of this invention against hP2X3 and hP2X2 / 3 receptors is shown in Table 1. It can be seen that the compounds of this invention exhibit strong inhibitory activity in the HEK293-hP2X3 receptor cell functional calcium flow assay, while showing weaker inhibitory activity in the HEK293-hP2X2 / 3 receptor cell functional calcium flow assay. Considering both the HEK293-hP2X3 and HEK293-p2X2 / 3 receptor cell functional calcium flow assays, the compounds of this invention exhibit good selectivity for both hP2X3 and hP2X2 / 3.

[0188] Table 1 Test Results

[0189]

[0190] Detection Example 2

[0191] 1. Experimental Objective

[0192] The inhibitory effect of the compounds of this invention on the hERG potassium ion channel was tested.

[0193] 2. Experimental cell lines

[0194] The HEK-293 cell line, which stably expresses the hERG potassium channel, was used.

[0195] 3. Experimental instruments and reagents / consumables

[0196] 3.1 Experimental instruments: Qpatch fully automated patch clamp system (Sophion, QPatch 48X); temperature control module (JULABO, FPE50-HE); data acquisition and analysis software (Sophion & GraphPad, Sophion Analyzer 6.5 & GraphPad Prism).

[0197] 3.2 Experimental reagents: DMEM cell culture medium (Gibco), fetal bovine serum (avantor), G418 (GPC), cell digestion solution TrypLE™ Express (Gibco), cisapride (Sigma), potassium aspartate (K-aspartic acid) (Sigma), sodium chloride (NaCl) (Sigma), potassium chloride (KCl) (Sigma), ethylene glycol bis(2-aminoethyl ether)tetraacetic acid (EGTA) (Sigma), 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES) (Santa Cruz), magnesium chloride (MgCl2•6H2O) (Sigma), glucose (D-Glucose) (Sigma), calcium chloride (CaCl2•2H2O) (Sigma), sodium dihydrogen phosphate (NaH2PO4•2H2O) (GENERAL-REAGENT), sodium adenosine triphosphate (Na2-ATP) (Sigma); HEK-293 cells (Creacell).

[0198] 4. Test Procedure

[0199] 4.1 Cell Culture: The HEK293 cell line, stably expressing potassium channels, was cultured in DMEM medium containing 10% fetal bovine serum and a final concentration of 0.8 mg / mL G418 at 37°C and 5% carbon dioxide. To maintain cell electrophysiological activity, the cell density must not exceed 80%. Before patch-clamp assay, cells were separated using TrypLE™ Express cell digestion solution, and after adding culture medium to terminate digestion, the cells were centrifuged, resuspended, and counted. The cell density was then adjusted to 2 × 10⁶ cells / mL. 6 -3×10 6 Cells / mL were collected, and then the cells were gently mixed on a balanced shaker for 15-20 min at room temperature before being analyzed.

[0200] 4.2 Patch clamp testing: Electrophysiological testing was performed using a fully automated patch clamp QPatch 48X (Sophion) device.

[0201] First, the prepared cells are placed on a centrifuge in the Qpatch workbench and washed using multiple centrifugation / resuspension methods to replace the cell culture medium with extracellular fluid. An MTP-96 plate is removed and placed in the MTP source position, followed by the QPlate chip, which is then placed in the QPlate source position. A robotic arm scans the barcodes on both the MTP-96 plate and the QPlate chip and picks them up to the measurement station. Intracellular and extracellular fluids are aspirated from the liquid pools and added to the intracellular fluid pool and cell and compound pool of the QPlate chip, respectively. At the measurement station, all measurement sites on the QPlate undergo initial quality control. The quality control process includes aspirating the cell suspension from the centrifuge's cell container and positioning the cells onto the chip wells using a pressure controller to establish a high-resistance seal, forming a whole-cell recording mode. Once a stable control current baseline is obtained, the test substances are sequentially aspirated from the MTP-96 plate according to their concentration and applied to the cells.

[0202] The voltage stimulation protocol for whole-cell patch-clamp recording of potassium channel currents is as follows: After whole-cell occlusion, the cell membrane voltage is clamped at -80 mV. The clamping voltage is depolarized from -80 mV to -50 mV and maintained for 0.5 s (as a leakage current detection), then stepped to 30 mV and maintained for 2.5 s, and then rapidly restored to -50 mV and maintained for 4 s to observe the tail current. Data is collected every 10 s to observe the effect of the drug on the potassium channel tail current. Experimental data are collected by a QPatch screening workstation and stored in a database server.

[0203] Each drug concentration was administered twice, with a time interval of at least 5 minutes. The current detected in each cell in an external solution free of the compound served as its own control group, and two cells were independently measured repeatedly. All electrophysiological experiments were performed at room temperature.

[0204] 5. Experimental data processing methods:

[0205] The second dose of the two administrations was used for data analysis. For each drug concentration, the average of the last three data points before the next administration concentration was taken to represent the current value after that concentration. The current after each drug concentration and the blank control current were normalized, and then the inhibition rate corresponding to each drug concentration was calculated. For each concentration inhibition rate, the mean, standard deviation (SD), and standard error (SE) were calculated.

[0206] Y = Lowest point + (Highest point - Lowest point) / (1 + 10^(LogIC)) 50 -X)×Hill slope))

[0207] Calculate the IC for each compound using the above equations. 50 The values ​​were calculated, and a nonlinear fit was performed on the dose-dependent effect, where IC 50 This is the half-inhibitory concentration (IC50). 50 The calculations and curve fitting were performed using GraphPad Prism software.

[0208] Verification of technical effectiveness and / or analysis of solutions to technical problems:

[0209] The results of the test on the inhibitory effect of the compounds of this invention on hERG potassium ion channels are shown in Table 2. It can be seen that the compounds of this invention have no inhibitory effect on hERG potassium ion channels and have a low risk of cardiotoxicity.

[0210] Table 2 Inhibitory effect on hERG potassium ion channels

[0211]

[0212] Detection Example 3

[0213] 1. Experimental Objective

[0214] The stability of the compounds of this invention in mouse, guinea pig and human liver microsomes was tested.

[0215] 2. Experimental reagents and instruments

[0216] Human liver microsomes (XenoTech, 2110263); mouse liver microsomes (RILD, HYXD); guinea pig liver microsomes (RILD, GPLM); LC / MS / MS (API4500 ABSciex ExionAC: SH-LCMS-003).

[0217] 3. Experimental Procedure

[0218] 3.1 Preparation of liver microsomes and working solutions of the compound: Transfer 0.800 mL of microsome stock solution and dilute with 15.200 mL of phosphate buffer to prepare a 1.00 mg / mL microsome working solution. Transfer 4.00 μL of 10.0 mM compound stock solution and dilute with 396 μL of DMSO to prepare a 100 μM compound working solution.

[0219] 3.2 Sample Addition: Prepare eight 96-well sample plates, named T0, T5, T15, T30, T60, T120, NCF120, and Blank, respectively. Add 100 μL of liver microsome working solution to the corresponding wells of the sample plates. For the Blank and T0 sample plates, first add 600 μL of stop solution (acetonitrile solution containing 0.1% formic acid and 50.0 ng / mL tolbutamide, labetalol, ethoxybenzamide, and buspirone); second, add 2.00 μL of the solvent and working solution used for the compound; finally, add 98.0 μL of NADPH (reduced nicotinamide adenine dinucleotide phosphate) coenzyme factor and shake the sample plate well. For T5, T15, T30, T60, T120, and NCF120 sample plates, first add 2.00 μL of the compound working solution to the corresponding well of the sample plate; second, pre-incubate the sample plate in a 37°C water bath for about 10 min; finally, for T5, T15, T30, T60, and T120 sample plates, add 98.0 μL of coenzyme factor to the corresponding well of the sample plate to start the reaction; for NCF120 sample plates, add 98.0 μL of phosphate buffer to the corresponding well of the sample plate to start the reaction.

[0220] The final contents of NADPH cofactor and other components are shown in Table 3 below:

[0221] Table 3 Final concentrations of each component

[0222]

[0223] 3.3 Adding the stop solution: After 5, 15, 30, 60 and 120 min of reaction, add 600 μL of the stop solution (acetonitrile solution containing 0.1% formic acid and 50.0 ng / mL tosylate, labetalol, ethoxybenzamide and buspirone) to the corresponding wells of the sample plate to terminate the reaction.

[0224] 3.4 Sample Detection: Mix all sample plates thoroughly and centrifuge at 3200 g for 20 min. Add 100 μL of supernatant to 100 μL of ultrapure water, mix well, and then perform LC-MS / MS analysis.

[0225] 4. Experimental Data Processing Methods

[0226] The remaining percentage (%Remaining) is calculated using the following formula:

[0227] .

[0228] T 1 / 2 The half-life is calculated using the following formula:

[0229] ;

[0230] k The values ​​were obtained by fitting the remaining percentage at each time point and the slope of the time curve using Excel.

[0231] CL invitro (In vitro clearance rate) is calculated using the following formula:

[0232] .

[0233] Verification of technical effectiveness and / or analysis of solutions to technical problems:

[0234] The test results of the compounds of this invention are shown in Table 4. It can be seen that the compounds of this invention exhibit good metabolic stability in the liver microsomes of mice, guinea pigs, and humans, with some compounds showing even better metabolic stability in liver microsomes compared to positive control drug 1 and positive control drug 2.

[0235] Table 4. Effects on stability in liver microsomes of mice, guinea pigs, and humans.

[0236]

[0237] Detection Example 4

[0238] 1. Experimental Objective

[0239] The inhibitory effect of the compounds of this invention on CYP enzymes was tested.

[0240] 2. Experimental instruments and reagents

[0241] LC / MS / MS (API4500 ABSciex ExionAC: SH-LCMS-003); Human liver microsomes (XenoTech, 2110263); Phenacetin (MCE, 17022); Diclofenac sodium (STANDARD, 21J119-G9); S-Mexenotoin (MCE, 133124); Dextromethorphan ambroxol (STANDARD, 22Z129-C2); Midazolam (Sigma, FE04082004).

[0242] 3. Experimental Methods

[0243] Mixed human liver microsomes were used to assess representative substrate metabolism responses of the five major human CYP subtypes (CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 / 5). The effects of different concentrations of analytes on the metabolic responses of phenacetin (CYP1A2), diclofenac sodium (CYP2C9), S-metphenytoin (CYP2C19), dextromethorphan ambroxol (CYP2D6), and midazolam (CYP3A4 / 5) were determined by liquid chromatography-tandem mass spectrometry (LC / MS / MS).

[0244] 3.1 Prepare the working solution of human liver microsomes: containing 20.0 mg / mL human liver microsomes and phosphate buffer solution.

[0245] 3.2 Preparation of NADPH cofactor:

[0246] The final content of each component is shown in Table 5 below:

[0247] Table 5 Final concentrations of each component

[0248]

[0249] 3.3 The reaction system of substrate 10 μM phenacetin, 5 μM diclofenac sodium, 30 μM S-methionine, 5 μM dextromethorphan ambroxol, 2 μM midazolam and the test compound (concentrations of 0.1, 0.3, 1, 3, 10, 30 and 50 μmol / L, respectively) or positive control 1 or blank control with mixed human liver microsomes (0.1 mg / mL) working solution was pre-incubated at 37 °C for 10 min.

[0250] Add 20.0 μL of NADPH cofactor to all culture wells, mix and incubate in a 37°C water bath for 10 min; add 200 μL of acetonitrile solution containing 50 ng / mL tolbutamide, labetalol, ethoxybenzamide and buspirone, shake the plate at 800 rpm for 10 min, and then centrifuge at 3200×g for 20 min;

[0251] Transfer 100 μL of the supernatant and mix it with 100 μL of ultrapure water. Shake at 800 rpm for 10 min, and then perform LC-MS / MS analysis on the sample.

[0252] 4. Experimental Data Processing Methods

[0253] Peak area is calculated based on chromatogram.

[0254] Activity (%) is calculated using the following formula:

[0255] Peak area ratio = Metabolite peak area ÷ Internal standard peak area

[0256] Activity (%) = Peak area ratio of the test compound group ÷ Peak area ratio of the blank group.

[0257] CYP half-maximal inhibitory concentration (IC50) 50 The values ​​were calculated by fitting Activity (%) and concentration data using Excel.

[0258] Verification of technical effectiveness and / or analysis of solutions to technical problems:

[0259] The inhibitory effects of the compounds of this invention on CYP enzymes are shown in Table 6. It can be seen that, according to the standard IC50... 50 <1 μM is a strong inhibitor, 1 μM <IC 50 <10 μM is a moderate inhibitor, IC50 50 ≥10 μM is a weak inhibitor, so positive drug 1 has a moderate inhibitory effect on CYP2C9. The compounds of this invention have a low risk of inhibiting the five enzymes CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A.

[0260] Table 6. Inhibitory effect on CYP enzymes

[0261]

[0262] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. An imidazopyridine derivative, characterized in that, Selected from at least one of the following compounds, or their racemic mixtures, stereoisomers, or pharmaceutically acceptable salts: 。 2. A pharmaceutical composition, characterized in that, It includes the imidazopyridine derivatives as described in claim 1.

3. A pharmaceutical preparation, characterized in that, The pharmaceutical composition comprising claim 2 is wherein the dosage form of the pharmaceutical preparation is tablet, pill, granule, powder, capsule, emulsion or suspension.

4. The pharmaceutical preparation according to claim 3, characterized in that, The administration routes of the preparation are intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, or oral.

5. Use of an imidazopyridine derivative as claimed in claim 1 or a pharmaceutical composition as claimed in claim 2 in the preparation of a medicament for the prevention and / or treatment of diseases mediated by the P2X3 receptor.

6. The use according to claim 5, characterized in that, The diseases mentioned include asthma, chronic obstructive pulmonary disease, pulmonary fibrosis, pulmonary hypertension, cough, pain, urinary incontinence, difficulty urinating, endometriosis, or pruritus.

7. The use according to claim 6, characterized in that, The pain includes migraines and pain from endometriosis; the cough includes chronic recurrent cough.

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