Polycyclic system compounds, methods of making and using the same

Abstract

This invention relates to a polycyclic compound system, its preparation method, and its applications. The compound of this invention is shown in formula (I): wherein M, ring A, ring B, R1, R2, R3, R4, Z, and X... ‑ The definitions of Y are as described in the instruction manual.

Description

Technical Field

[0001] This invention relates to polycyclic compounds, their preparation methods, and applications. Background Technology

[0002] Muscarinic receptors are members of the G protein-coupled receptor (GPCR) family. Because they are involved in many physiological and pathological processes, GPCRs are among the most important targets for drug development. Muscarinic receptor antagonists are known to produce bronchial protective effects and are used to treat lung diseases including asthma, chronic obstructive pulmonary disease (COPD), and bronchopulmonary dysplasia (BPD). Muscarinic receptor antagonists can also treat urinary incontinence and, in combination with muscarinic acetylcholine receptor agonists, are used to treat schizophrenia.

[0003] Currently marketed long-acting bromide anticholinergic drugs (such as tiotropium bromide, adecyl bromide, fudecyl bromide, and glycopyrronium bromide) are mainly available in dry powder inhalation formulations. Dry powder inhalation requires patients to learn the inhalation technique and requires good lung function to inhale the drug. For some patients with severe COPD, their poor lung function may prevent them from reaching the required airflow for inhaling the powder, resulting in ineffective drug delivery to the lungs or insufficient inhalation doses, leading to decreased drug efficacy. In contrast, nebulized inhalation requires no special training, especially for patients with severe COPD, who can inhale sufficient doses using normal breathing techniques. Therefore, there is a need for long-acting anticholinergic drugs suitable for inhalation solutions to meet the clinical needs of such patients. Summary of the Invention

[0004] To overcome the shortcomings of existing technologies, this invention develops a polycyclic compound system, its preparation method, and its application. The polycyclic compound system of this invention is a muscarinic acetylcholine receptor inhibitor, which has stable ester bonds and can be suitable for preparation into multiple dosage forms, including inhalation solutions.

[0005] Therefore, one object of the present invention is to provide a polycyclic compound that is a muscarinic acetylcholine receptor inhibitor, which can be prepared into a potent, long-acting, nebulizable inhalation solution or dry powder inhalation formulation.

[0006] Another object of the present invention is to provide a method for preparing the above-mentioned polycyclic system compounds.

[0007] Another object of the present invention is to provide a pharmaceutical composition comprising the above-mentioned polycyclic system compounds.

[0008] Another object of the present invention is to provide a use for the above-mentioned polycyclic system compounds.

[0009] On the one hand, the present invention provides a compound of formula (I) or a stereoisomer, tautomer, isotope derivative, nitride, hydrate, solvate, metabolite, pharmaceutically acceptable salt or prodrug thereof,

[0010]

[0011] in,

[0012] M is independently selected from O, NH and S;

[0013] Ring A and ring B are independently selected from five- or six-membered aromatic heterocycles, benzene rings, and C3-C8 aliphatic rings (saturated or unsaturated) containing one or more heteroatoms selected from O, N, and S;

[0014] R1 and R2 are independently selected from hydrogen, deuterium, halogens, and C. 1～6 Alkyl, C 3～6 cycloalkyl, cyano, C 1～6 Alkoxy, C 3～6 Cycloalkoxy, halogenated C 1～6 Alkyl and Halogenated C 3～6 Cycloalkyl; In this invention, the number of substituents R1 and R2 can be multiple, for example, one, two, or three. These multiple substituents can be the same or different. For example, R2 can be a halogen, and there can be two of them, both of which can be F, or one can be F and the other can be Cl. As another example, one of R2 can be a halogen, such as F, and the other can be C. 1～6 alkyl.

[0015] R3 and R4 are independently selected from C 1～6 Alkyl, C 3～6 cycloalkyl, halogenated C 1～6 Alkyl and Halogenated C 3～6 Cycloalkyl; or R3 and R4 together with the N atom they are attached to form a five- or six-membered ring (saturated or unsaturated);

[0016] Z is selected from single bonds, -O-, -S-, and -(CH2). 1～5 -、-S(=O)-、-SO2-、-NR5-、R5 is selected from H、C 1～6 Alkyl, C 3～6 cycloalkyl, halogenated C 1～6 Alkyl and Halogenated C 3～6 cycloalkyl;

[0017] X -It represents an anion with a negative charge, selected from fluoride, chloride, bromide, iodide, formate, hydrogen sulfate, sulfate, phosphate, trifluoromethanesulfonate, methanesulfonate, nitrate, maleate, acetate, citrate, fumarate, tartrate, oxalate, succinate, benzoate, and p-toluenesulfonate.

[0018] Y is selected from:

[0019] Preferably, the compound represented by formula (I) can be a compound of formulas (II), (III), (IV), (V), (VI), (Va), (VI-a):

[0020]

[0021] Preferably, ring A and ring B are independently selected from:

[0022] More preferably, ring A and ring B are independently selected from:

[0023] More preferably, ring A is selected from: Ring B is selected from:

[0024] More preferably, ring A is selected from: Ring B is selected from:

[0025] Preferably, R1 and R2 are independently selected from hydrogen, deuterium, halogen, and C. 1～4 Alkyl, C 3～5 cycloalkyl, cyano, C 1～4 Alkoxy, C 3～5 Cycloalkoxy, halogenated C 1～4 Alkyl and Halogenated C 3～5 Cycloalkyl.

[0026] Preferably, R1 and R2 are independently selected from hydrogen, deuterium, halogens (e.g., F, Cl, Br), and C. 1～4 Alkyl (e.g., methyl, ethyl), halogenated C 1～4 Alkyl groups (e.g., trifluoromethyl groups).

[0027] Preferably, R1 and R2 are independently selected from hydrogen, deuterium, F, Cl, methyl, ethyl, and trifluoromethyl. For example, R1 and R2 can both be hydrogen, or R1 can be hydrogen and R2 can be one F or two F.

[0028] Preferably, R3 and R4 are independently selected from C 1～4 Alkyl, C 3～5 cycloalkyl, halogenated C 1～4 Alkyl and Halogenated C3～5 Cycloalkyl; or R3 and R4 together with the N atom attached to them to form a five- or six-membered ring;

[0029] Preferably, R3 and R4 are independently selected from methyl, ethyl, propyl, and isopropyl; or R3 and R4 together with the N atom they are attached to form a five- or six-membered ring;

[0030] Preferably, Z is selected from single bonds, -O-, -S-, and -(CH2). 1～2 -;

[0031] Preferably, Z is a single bond;

[0032] Preferably, X - Selected from chloride ions and bromide ions.

[0033] Preferably, Y is selected from

[0034] Preferably, Y is

[0035] In one specific implementation, in equations (I), (II), (III), (IV), (V), (Va), and (VI-a) above, if any, M is independently selected from O, NH, and S.

[0036] Ring A and ring B are selected independently from:

[0037]

[0038] R1 and R2 are independently selected from hydrogen, deuterium, halogens, and C. 1～4 Alkyl, C 3～5 cycloalkyl, cyano, C 1～4 Alkoxy, C 3～5 Cycloalkoxy, halogenated C 1～4 Alkyl and Halogenated C 3～5 cycloalkyl;

[0039] R3 and R4 are independently selected from C 1～4 Alkyl, C 3～5 cycloalkyl, halogenated C 1～4 Alkyl and Halogenated C 3～5 Cycloalkyl; or R3 and R4 together with the N atom attached to them to form a five- or six-membered ring;

[0040] Z is selected from single bonds, -O-, -S-, and -(CH2). 1～2 -;

[0041] X - Selected from chloride ions and bromide ions;

[0042] Y is selected from

[0043] In another specific implementation, in equations (I), (II), (III), (IV), (V), (Va), and (VI-a) above, M is independently selected from O, NH, and S, if any.

[0044] Ring A and ring B are selected independently from:

[0045]

[0046] R1 and R2 are independently selected from hydrogen, deuterium, halogens (e.g., F, Cl, Br), and C. 1～4 Alkyl (e.g., methyl, ethyl), halogenated C 1～4 Alkyl groups (e.g., trifluoromethyl);

[0047] R3 and R4 are independently selected from methyl, ethyl, propyl, and isopropyl; or R3 and R4 together with the N atom they are attached to form a five- or six-membered ring;

[0048] Z is selected from single bonds, -O-, -S-, and -(CH2). 1～2 -;

[0049] X - Selected from chloride ions and bromide ions;

[0050] Y is selected from

[0051] In another specific technical solution, in the above formulas (I), (II), (III), (IV), (V), (Va), (VI-a), if any, M is independently selected from O, NH, and S, and ring A is selected from: Ring B is selected from:

[0052] R1 and R2 are independently selected from hydrogen, deuterium, F, Cl, methyl, ethyl, and trifluoromethyl;

[0053] R3 and R4 are independently selected from methyl, ethyl, propyl, and isopropyl; or R3 and R4 together with the N atom they are attached to form a five- or six-membered ring;

[0054] Z represents a single bond;

[0055] X - Selected from chloride ions and bromide ions;

[0056] Y is

[0057] In yet another specific technical solution, in the above formulas (I), (II), (III), (IV), (V), (Va), (VI-a), if any, M is independently selected from O, NH, and S, and ring A is selected from: Ring B is selected from:

[0058] R1 and R2 are independently selected from hydrogen, deuterium, F, Cl, methyl, ethyl, and trifluoromethyl;

[0059] R3 and R4 are independently selected from methyl, ethyl, propyl, and isopropyl; or R3 and R4 together with the N atom they are attached to form a five- or six-membered ring;

[0060] Z represents a single bond;

[0061] X - Selected from chloride ions and bromide ions;

[0062] Y is

[0063] In a particularly specific technical solution, the compound is selected from the following preparation example compounds:

[0064]

[0065]

[0066]

[0067]

[0068]

[0069]

[0070] On the other hand, the present invention provides a method for preparing the compound (Va) represented by the above formula (I), the method comprising the following steps:

[0071]

[0072] (1) The substituted 2-thiophene boronic acid V-7 (or boronic ester) and the substituted 2-bromobenzoic acid ester V-8 undergo a coupling reaction under the first basic condition to generate intermediate V-1.

[0073] (2) Intermediate V-1 is hydrolyzed under the second alkaline condition and then acidified to generate intermediate V-2;

[0074] (3) Intermediate V-2 was treated with reagents such as SOCl2, oxalyl chloride or phosphorus oxychloride to obtain intermediate acyl chloride V-3. The crude product was directly used in the next step of the reaction.

[0075] (4) Intermediate V-3 undergoes a ring-closing reaction under Lewis acid conditions (aluminum trichloride, tin tetrachloride, etc.) to generate intermediate V-4;

[0076] (5) Intermediate V-4 reacts with trimethylsilyl cyanide in the presence of zinc iodide to generate intermediate V-5. The crude product is directly used in the next step of the reaction.

[0077] (6) Intermediate V-5 in alcohol (ROH(where R is C) 1-6 The reaction is carried out by passing hydrogen chloride gas into a solution of alkyl groups (such as methanol, ethanol, etc.) or in a commercially available solution of hydrogen chloride alcohol to generate V-6.

[0078] (7) Intermediates V-6 and Va-0 undergo transesterification under the third alkaline conditions to generate intermediate Va-1;

[0079] (8) Intermediate Va-1 undergoes a quaternization reaction with R4X (such as bromomethane) to obtain Va.

[0080] In the above reaction equation, R1, R2, R3, R4, X, and Y are as described above, and R is C. 1-6 Alkyl groups.

[0081] The present invention includes a chiral separation method: intermediate V-6 is separated under conventional chiral chromatographic column conditions (e.g., column type). Under the following conditions (IBN-5: 0.46cm ID×25cm L×5μm; mobile phase: n-Hexane / Isopropylalcohol / Diethylamine=50 / 50 / 0.1(v / v / v); flow rate: 0.6ml / min; wavelength: UV 220nm; column temperature: 35℃; run time: 20min), chiral monomers of V-6 with R and S configurations (chirality is indicated by *labeled carbon sites) can be obtained.

[0082] The R and S configuration chiral monomers of V-6 undergo transesterification reactions with Va-0 under alkaline conditions to generate the R and S configuration chiral monomers of intermediate Va-1.

[0083] The R and S configuration chiral monomers of Va-1 undergo quaternization reactions with R4X (such as bromomethane) to generate the R and S configuration chiral monomers of Va.

[0084] Preferably, in step (1), the base used in the first alkaline condition is an inorganic base or an organic base, such as sodium carbonate.

[0085] Preferably, in step (2), the base used in the second alkaline condition is an inorganic base or an organic base, such as sodium hydroxide.

[0086] Preferably, in step (2), the acid used for acidification is an inorganic acid or an organic acid, such as hydrochloric acid.

[0087] Preferably, in step (7), the base used in the third alkaline condition is an inorganic or organic base, such as 60% NaH, potassium carbonate, etc.

[0088] This invention provides a method for preparing the compound (VI-a) represented by formula (I) above, the method comprising the following steps:

[0089]

[0090] The preparation methods of compound (VI-a) and its chiral monomers are the same as those of compound (Va) mentioned above.

[0091] (1) The substituted 3-thiophene boronic acid VI-7 (or boronic ester) and the substituted 2-bromobenzoic acid ester VI-8 undergo a coupling reaction under the first basic condition to generate intermediate VI-1.

[0092] (2) Intermediate VI-1 is hydrolyzed under the second alkaline condition and then acidified to generate intermediate VI-2;

[0093] (3) Intermediate VI-2 was treated with reagents such as SOCl2, oxalyl chloride or phosphorus oxychloride to prepare intermediate acyl chloride VI-3. The crude product was directly used in the next step of the reaction.

[0094] (4) Intermediate VI-3 undergoes a ring-closing reaction under Lewis acid conditions (aluminum trichloride, tin tetrachloride, etc.) to generate intermediate VI-4;

[0095] (5) Intermediate VI-4 reacts with trimethylsilyl cyanide in the presence of zinc iodide to generate intermediate VI-5. The crude product is directly used in the next step of the reaction.

[0096] (6) Intermediate VI-5 in alcohol (ROH (R is C 1-6 Hydrogen chloride gas is passed into a solution of alkyl groups (such as methanol, ethanol, etc.), or a commercially available solution of hydrogen chloride alcohol is reacted to generate VI-6.

[0097] (7) Intermediates VI-6 and VI-a-0 undergo transesterification under the third alkaline conditions to generate intermediate VI-a-1;

[0098] (8) Intermediate VI-a-1 undergoes a quaternization reaction with R4X (such as bromomethane) to obtain VI-a.

[0099] In the above reaction equation, R1, R2, R3, R4, X, and Y are as described above, and R is C. 1-6Alkyl groups.

[0100] The present invention includes a chiral separation method: intermediate VI-6 is separated under conventional chiral chromatographic column conditions (e.g., column type). Under the following conditions (IBN-5: 0.46cm ID×25cm L×5μm; mobile phase: n-Hexane / Isopropylalcohol / Diethylamine=50 / 50 / 0.1(v / v / v); flow rate: 0.6ml / min; wavelength: UV 220nm; column temperature: 35℃; run time: 20min), chiral monomers of VI-6 with R and S configurations (chirality is indicated by *labeled carbon sites) can be obtained.

[0101] The R and S configuration chiral monomers of VI-6 undergo transesterification reactions with VI-a-0 under basic conditions to generate the R and S configuration chiral monomers of intermediate VI-a-1.

[0102] The R and S configuration chiral monomers of VI-a-1 undergo quaternization reactions with R4X (such as bromomethane) to generate the R and S configuration chiral monomers of VI-a.

[0103] Preferably, in step (1), the base used in the first alkaline condition is an inorganic base or an organic base, such as sodium carbonate.

[0104] Preferably, in step (2), the base used in the second alkaline condition is an inorganic base or an organic base, such as sodium hydroxide.

[0105] Preferably, in step (2), the acid used for acidification is an inorganic acid or an organic acid, such as hydrochloric acid.

[0106] Preferably, in step (7), the base used in the third alkaline condition is an inorganic base or an organic base, such as potassium carbonate.

[0107] In another aspect, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of the compound represented by the aforementioned formula (I) or its stereoisomers, tautomers, isotope derivatives, nitrides, hydrates, solvates, metabolites, pharmaceutically acceptable salts or prodrugs thereof, and a pharmaceutically acceptable carrier.

[0108] Representative examples of substances that can serve as pharmaceutically acceptable carriers include, but are not limited to, the following: (1) sugars, such as lactose, glucose, and sucrose; (2) starches, such as corn starch; (3) cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; (4) powdered tragacanth gum; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; (1 (0) Diols, such as propylene glycol; (11) Polyols, such as glycerol, sorbitol, mannitol and polyethylene glycol; (12) Esters, such as ethyl oleate and ethyl laurate; (13) Agar; (14) Buffers, such as magnesium hydroxide and aluminum hydroxide; (15) Alginate; (16) Pyrogen-free water; (17) Isotonic saline; (18) Ringer's solution; (19) Ethanol; (20) Phosphate buffer; (21) Compressed propellant gases, such as chlorofluorocarbons and hydrofluorocarbons; and (22) Other non-toxic and compatible substances used in pharmaceutical compositions.

[0109] In another aspect, the present invention provides the use of the compound represented by the aforementioned formula (I) or its stereoisomers, tautomers, isotope derivatives, nitrogen oxides, hydrates, solvates, metabolites, pharmaceutically acceptable salts or their prodrugs, or the aforementioned pharmaceutical compositions or combinations thereof, in the preparation of a medicament for the prevention and / or treatment of the following diseases: lung diseases including asthma, chronic obstructive pulmonary disease (COPD), bronchopulmonary dysplasia (BPD); urinary incontinence; and schizophrenia.

[0110] The polycyclic aromatic hydrocarbons of this invention are typically administered to patients in the form of pharmaceutical compositions or formulations. These pharmaceutical compositions can be administered to patients via any acceptable route of administration, including but not limited to inhalation, oral, nasal, topical (including transdermal), and parenteral administration.

[0111] In one embodiment, the pharmaceutical composition of the present invention is suitable for inhalation administration. Suitable pharmaceutical compositions for inhalation administration are typically in aerosol or powder form. Such compositions are typically administered using known delivery devices, such as nebulizer inhalers, metered-dose inhalers (MDIs), dry powder inhalers (DPIs), or similar delivery devices.

[0112] In a particular embodiment of the invention, the pharmaceutical composition comprising the active agent is administered by inhalation using a nebulizer. When formulated for use in a nebulizer, the active agent is typically dissolved in a suitable carrier to form a solution, such carriers as: 1) water for injection, disodium edetate, benzalkonium chloride, and hydrochloric acid; 2) water for injection, disodium edetate, benzalkonium chloride, 0.9% sodium chloride, and citric acid; 3) water for injection, disodium edetate, 0.9% sodium chloride, and hydrochloric acid.

[0113] In another aspect, the present invention provides a combination medicament comprising the aforementioned compound or its stereoisomers, tautomers, isotope derivatives, nitric oxides, hydrates, solvates, metabolites, pharmaceutically acceptable salts or prodrugs thereof, or the aforementioned pharmaceutical composition, and other therapeutic agents. This therapeutic agent is administered in combination with a compound of formula I, or its stereoisomers, tautomers, isotope derivatives, nitric oxides, hydrates, solvates, metabolites, pharmaceutically acceptable salts or prodrugs thereof. For example, the pharmaceutical composition of the present invention may further comprise one or more therapeutic agents selected from other bronchodilators (e.g., PDE3 / 4 inhibitors, β2-adrenergic receptor agonists, etc.); anti-inflammatory agents (e.g., corticosteroids and PDE4 inhibitors); other toxic alkaloid receptor antagonists (i.e., anticholinergics); afferent blockers (e.g., D2 agonists and neurokinin modulators); and toxic alkaloid receptor agonists. Other therapeutic agents may be applied in the form of pharmaceutically acceptable salts or solvates.

[0114] Compared with the prior art, the present invention has at least the following beneficial technical effects:

[0115] Currently available long-acting bromide anticholinergic drugs are mainly in dry powder inhalation formulations. Dry powder inhalation requires patients to learn the inhalation technique and requires good lung function to inhale the drug. For some patients with severe COPD, poor lung function may prevent them from achieving the required airflow for inhaling the powder, resulting in ineffective drug delivery to the lungs or insufficient inhalation dosage, leading to decreased drug efficacy. In contrast, nebulized inhalation requires no special training, especially for patients with severe COPD, who can inhale sufficient doses using normal breathing. Therefore, there is a significant clinical need to develop new, highly potent, long-acting muscarinic receptor antagonists in formulations such as dry powder and nebulized solutions.

[0116] Stability tests show that the compounds of this invention have good ester bond stability in aqueous solution and have the potential to be developed into nebulized inhalation formulations. Detailed Implementation

[0117] The technical solution of the present invention will be further described below in conjunction with specific implementation methods.

[0118] Preparation of 5-fluorothiophene-2-boronic acid pinacol ester

[0119]

[0120] Thiophene (12 g, 1 eqv) and anhydrous tetrahydrofuran (300 ml) were added to a reaction flask. Under nitrogen protection, the mixture was cooled to -78 °C, and 2.5 Mn-BuLi / n-hexane (60 ml, 1.05 eqv) was added dropwise. After the addition was complete, the reaction was maintained at this temperature for 1.5 h. At -78 °C, an anhydrous tetrahydrofuran (180 ml) solution of N-fluorobisbenzenesulfonamide (41.4 g, 1.05 eqv) was added dropwise. After the addition was complete, the temperature was raised to 20 °C and the reaction was maintained for 1.5 h. The mixture was then cooled to -78 °C, and 2.5 Mn-BuLi / n-hexane (60 ml, 1.05 eqv) was added dropwise. After the addition was complete, the reaction was maintained at this temperature for 1.5 h, and then 2- A solution of isopropoxy-4,4,5,5-tetramethyl-1,3,2-dioxoborane (31 ml, 1.07 eqv) in anhydrous tetrahydrofuran (120 ml) was added dropwise, and the mixture was heated to 20 °C overnight. The reaction solution was cooled to about 0 °C, and a saturated ammonium chloride aqueous solution (300 ml) was added to quench the reaction. The mixture was separated, and the aqueous phase was extracted with petroleum ether. The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography (100-200 mesh silica gel, PE / EA = 100:1 to 60:1) to obtain 20.4 g of 5-fluorothiophene-2-boronic acid pinacol ester.

[0121] 1 H-NMR (400MHz, CDCl3): δ7.25-7.18 (m, 1H), 6.48 (dd, J = 4.0, 0.8Hz, 1H), 1.25 (s, 12H).

[0122] Intermediate preparation example 1 (int-1):

[0123]

[0124] Step a: 4H-cyclopentano[2,1-B:3,4-B′]dithiophene-4-one (10 g, 1 eqv), DCM (200 ml), zinc iodide (1.66 g, 0.1 eqv), and trimethylcyanosilane (51.6 g, 10 eqv) were added to a reaction flask and refluxed under nitrogen protection for 2 hours. The reaction was found to be almost complete by TLC. The reaction solution was concentrated under reduced pressure and evaporated to dryness to obtain 20 g of brownish-red solid crude product int-1-1, which was used directly in the next step of the reaction without purification.

[0125] Step b: Add 20g of crude int-1-1 and 150ml of methanol to a reaction flask, stir and cool to 0℃, then bubble HCl (gas) into the reaction flask for 1h. Stop the reaction, pour the reaction solution into water, extract with EA, combine the organic phases, and evaporate to dryness. Purify the crude product by column chromatography (100-200 mesh silica gel, PE / EA = 50:1~5:1) to obtain int-1 product (7g, overall yield of two steps: 53.4%). ESI-MS: m / z: 253.1 [M+H] +

[0126] Intermediate preparation example 2 (int-2):

[0127]

[0128] 1 eqv), 2-thiopheneboronic acid (12.8 g, 1.08 eqv), sodium carbonate (34.8 g, 3 eqv), Pd(PPh3)4 (5.37 g, 0.05 eqv), THF (400 ml) and water (100 ml) were added to a reaction flask. Under nitrogen protection, the reaction was carried out at 70 °C for 16 h. TLC showed that the reaction was basically complete. The reaction solution was cooled, concentrated under reduced pressure, extracted with water and EA, and the organic phases were combined and evaporated to dryness to obtain crude int-2-1 (30 g). The crude product was used directly for the next reaction without purification.

[0129] Step b: Dissolve 30g of crude int-2-1 in ethanol (300ml), add 215ml of 4M sodium hydroxide aqueous solution, and reflux for 2h. Stop the reaction, place the reaction solution in an ice-water bath, adjust the pH to about 2 with 6M HCl aqueous solution, concentrate the mixture to remove ethanol by rotary evaporation, extract the residue with EA, and evaporate the organic phase to dryness; purify the crude product by column chromatography (DCM / MeOH = 100:1~50:1) to obtain 22g of int-2-2 product.

[0130] Step c: Add int-2-2 (22g), DMF (4ml), and DCM (600ml) to a reaction flask, cool to 0℃, add oxaloyl chloride (66ml) dropwise, maintain the temperature between 0 and 5℃, and allow the reaction to proceed overnight at room temperature. Concentrate the reaction solution to obtain crude int-2-3 (30g); the unpurified crude product is used directly in the next step.

[0131] Step d: Dissolve AlCl3 (33g, 2.3 eqv) in DCM (300ml), stir and cool to 0℃. Add 30g of crude int-2-3 in DCM (300ml) dropwise, maintaining the temperature at 0-5℃. After the addition is complete, react at room temperature for 1h. Quench the reaction solution in ice water, extract with DCM, evaporate the organic phase to dryness, and purify the residue by column chromatography (PE / EA = 20:1-10:1) to obtain int-2-4 product (12.8g, yield 68%).

[0132] Step e: Add int-2-4 (12.8 g, 1 eqv), ZnI2 (2.20 g, 0.1 eqv), TMSCN (68.16 g, 10 eqv), and DCM (250 ml) to a reaction flask, reflux overnight under nitrogen protection. After the reaction is complete, evaporate the reaction solution to dryness under reduced pressure to obtain crude int-2-5 (26.0 g). The unpurified crude product is used directly in the next step.

[0133] Step f: Add 26g of crude int-2-5 and 500ml of anhydrous ethanol to a reaction flask, stir and cool to 0°C, then bubble HCl (gas) into the reaction flask for 1 hour. Stop the reaction, pour the reaction solution into water, extract with EA, combine the organic phases, and evaporate the organic phase to dryness. Purify the crude product by column chromatography (100-200 mesh silica gel, PE / EA = 100:1~50:1) to obtain int-2 product (11.2g, overall yield of two steps: 62.6%).

[0134] ESI-MS: m / z: 261.1 [M+H] + 283.1 [M+Na] + 243.1 [M-OH] +

[0135] 1 H-NMR (400MHz, DMSO-d6): δ7.57-7.56(m,1H),7.47-7.42(m,2H),7.37-7.33(m,1H),7. 24-7.20(m,1H),7.13-7.12(m,1H),6.70(s,1H),4.09-4.01(m,2H),1.05(t,J=4Hz,3H).

[0136] Examples of intermediate preparation: 3-26, 28-29

[0137] Using intermediate 2 and the following steps with commercially available or homemade starting materials, int3~26, 28-29 are synthesized.

[0138]

[0139]

[0140]

[0141]

[0142]

[0143] Intermediate preparation example 27 (int-27):

[0144]

[0145] Step a: Add anhydrous tetrahydrofuran (250 ml) and LDA (98 ml, 2 M in THF) to the reaction flask. Under nitrogen protection, cool to -78 °C and add dropwise a solution of 3-bromothiophene (25 g, 1 eqv) in anhydrous tetrahydrofuran (50 ml). Incubate the reaction for 1 h. Then add dropwise a solution of 3-bromothiophene-2-carboxaldehyde (29 g, 1 eqv) in anhydrous tetrahydrofuran (60 ml). After the addition is complete, incubate the reaction at -78 °C for 2 h. Monitor the reaction for completeness by TLC. Add saturated ammonium chloride solution (100 ml) to the reaction solution to quench the reaction. Separate the solution and extract the aqueous phase with EA. Combine the organic phases. Concentrate the organic phase under reduced pressure and purify the residue by column chromatography (100-200 mesh silica gel, PE / EA = 50:1 to 20:1) to obtain int-27-1 product (4.9 g, yield 9.11%).

[0146] Step b: Add int-27-1 (4.9 g, 1 eqv), pyridinium chlorochromate (5.9 g, 2 eqv), and dichloromethane (100 ml) to a reaction flask and react at room temperature for 5 h. Monitor the reaction by TLC until complete. Add water to the reaction mixture, separate the layers, concentrate the organic phase under reduced pressure, and purify the residue by column chromatography (100-200 mesh silica gel, PE / EA = 50:1–20:1) to obtain int-27-2 product (1.2 g, yield 25%). ESI-MS: m / z = 353 [M+H] +

[0147] Step c: Add int-27-2 (1.2 g, 1 eqv), copper powder (0.22 g, 1 eqv), and DMF (12 ml) to a microwave-safe test tube and react at 145 °C for 2 h. Monitor the reaction by TLC until complete. Pour the reaction solution into a mixture of water (120 ml) and EA (60 ml), stir, separate the layers, extract the aqueous phase with EA, and combine the organic phases. Concentrate the organic phase under reduced pressure, and purify the residue by column chromatography (100-200 mesh silica gel, PE / EA = 50:1–20:1) to obtain a red solid int-27-3 product (600 mg, yield 90%). ESI-MS: m / z = 193 [M+H] +

[0148] Step d: Add int-27-3 (600 mg, 1 eqv), DCM (30 ml), zinc iodide (100 mg, 0.1 eqv), and trimethylcyanosilane (3 g, 10 eqv) to the reaction flask, reflux under nitrogen protection for 30 h, and TLC showed that the reaction was basically complete; concentrate the reaction solution under reduced pressure and evaporate to dryness to obtain 2 g of brownish-red solid crude int-27-4, which was used directly in the next step of the reaction without purification.

[0149] Step e: Add 2g of crude int-27-4 and 20ml of anhydrous ethanol to a reaction flask, stir and cool to 0°C, then bubble HCl (gas) into the flask for 1 hour to stop the reaction. Pour the reaction solution into water, extract with EA, combine the organic phases, and evaporate to dryness. Purify the crude product by column chromatography (100-200 mesh silica gel, PE / EA = 50:1 to 5:1) to obtain int-27 product (285mg, overall yield of two steps: 34%). ESI-MS: m / z: 267.1 [M+H] +

[0150] Intermediate preparation example A-1 (INT A-1)

[0151]

[0152] INT-2 (500 mg, 1 eqv), THF (5 mL), and H₂O (5 mL) were added to a reaction flask, and the mixture was cooled to 0–5 °C. Then, LiOH (146 mg, 3 eqv) was added, and the mixture was allowed to react at room temperature for 2 h. After the reaction was complete, the reaction solution was concentrated to remove most of the solvent. The pH was adjusted to 2 with 2 M HCl aqueous solution under an ice-water bath, and a solid precipitated. The solid was filtered, and the filter cake was washed with an appropriate amount of water. The filter cake was then dried under vacuum to obtain an off-white solid, INT A-1 (400 mg, yield: 84.8%). ESI-MS: m / z: 215.0 [M-OH] +

[0153] Using the steps of intermediate A-1, the corresponding intermediate acid fragments A-2 to A-7 were prepared.

[0154]

[0155]

[0156] Intermediate preparation example B-1:

[0157]

[0158] 10 g of int-2-5 and 200 ml of methanol were added to a reaction flask, stirred, and cooled to 0 °C. HCl (gas) was bubbled into the reaction flask for 1 h. The reaction was stopped, the reaction solution was poured into water, extracted with EA, and the organic phases were combined and evaporated to dryness. The crude product was purified by column chromatography (100-200 mesh silica gel, PE / EA = 100:1~50:1) to obtain 4 g of int-2B product; ESI-MS: m / z: 229.0 [M-OH] +

[0159] 1H-NMR (400MHz, DMSO-d6): δ7.58-7.57(m,1H),7.48-7.42(m,2H),7.38-7. 34(m,1H),7.25-7.21(m,1H),7.14-7.13(m,1H),6.75(s,1H),3.56(s,3H).

[0160] int-2B was separated by a chiral column (column type). IBN-5 0.46cm ID×25cm L×5μm; mobile phase: n-Hexane / Isopropyl alcohol / Diethylamine=50 / 50 / 0.1(v / v / v); flow rate: 0.6ml / min; wavelength: UV 220nm; column temperature: 35℃; run time: 20min) yielded int-2B-T1 (1.58g, retention time 7.644min); [α] 20 D = +3.65 (c = 1, MeOH). This yields int-2B-T2 (1.67 g, retention time 14.076 min);

[0161] [α] 20 D = -3.67 (c = 1, MeOH).

[0162] Using the same steps as intermediate B-1, the corresponding chiral intermediate B-2 was prepared.

[0163]

[0164]

[0165] Intermediate preparation example C-1 (INT-C-1)

[0166]

[0167] Step a: Scopolamine hydrobromic acid trihydrate (5g), zinc-copper coupling agent (1-3% copper, 10g), and anhydrous ethanol (50ml) were added to a reaction flask and refluxed for 6 hours. The reaction solution was cooled to room temperature, filtered through diatomaceous earth, and the filter cake was washed three times with ethanol. The filtrate was concentrated under reduced pressure. Purification was performed by column chromatography (100-200 mesh silica gel, DCM / MeOH = 10:1-3:1), and the solution was evaporated to dryness to obtain INT-C-1-1 (4.375g, yield 92.3%); ESI-MS: m / z = 287.94 [M+Na] +

[0168] Step b: Add INT-C-1-1 (4.375 g) and 2 M NaOH aqueous solution (43.75 ml) to the reaction flask and stir at room temperature for 1 h. Stop the reaction, extract the reaction solution three times with DCM, combine the organic phases, and evaporate to dryness to obtain INT-C-1 (1.312 g, yield 61.9%); ESI-MS: m / z = 139.89 [M+H] +

[0169] Intermediate preparation example D-1 (INT-D-1)

[0170]

[0171] Step a: Scopolamine (5 g, 1 eqv) and anhydrous tetrahydrofuran (50 ml) were added to a reaction flask and cooled to 0 °C. 60% NaH (2.58 g, 2 eqv) was added in portions, and the mixture was reacted at room temperature for 2 h. The mixture was then cooled to 0 °C, and a solution of p-toluenesulfonyl chloride (7.35 g, 1.2 eqv) in anhydrous tetrahydrofuran (50 ml) was added dropwise. After the addition was complete, the mixture was reacted at room temperature for 1.5 h, and the reaction was checked for completeness. The reaction solution was slowly poured into a saturated ammonium chloride solution to quench the reaction, extracted with EA, concentrated the organic phase, and purified by column chromatography (PE / EA = 100:1–20:1) to obtain INT-D-1-1, a white solid product (6 g, yield 60%). ESI-MS: m / z = 310.12 [M+H]. +

[0172] Step b: Add INT-D-1-1 (2g), isopropanol (20ml), and ammonia (20ml) to a reaction flask and react overnight at 70°C. After confirming the reaction is nearly complete, concentrate the reaction solution until a solid precipitates. Add pure water to dissolve the precipitate. Wash the aqueous phase with n-hexane / EA = 10:1 until no starting material is detected. Concentrate the aqueous phase and lyophilize to obtain INT-D-1 as a yellow solid product (1.6g); ESI-MS: m / z = 155.1 [M+H] +

[0173] Preparation Example 1 (Compound 1):

[0174]

[0175] Step a: Add int1 (4 g, 1 eqv), scopolamine (2.95 g, 1.25 eqv), anhydrous toluene (80 ml), and 60% sodium hydride (254 mg, 0.4 eqv) to a reaction flask, and reflux to remove water for 7 hours. Stop the reaction, cool the reaction solution to room temperature, pour it into a saturated ammonium chloride solution, extract with EA, combine the organic phases, evaporate to dryness, and purify the crude product by column chromatography (100-200 mesh silica gel, DCM / MeOH = 100:1~80:1) to obtain Compound1-1 (1.0 g, yield: 16.8%).

[0176] Step b: Compound 1-1 (1.0 g) and a saturated DCM solution (30 ml) of methyl bromide were reacted overnight at room temperature. The reaction was monitored by TLC until complete. The reaction was stopped, the reaction mixture was filtered, the filter cake was slurried with acetone, and dried under vacuum to obtain Compound 1 (500 mg, yield: 40%); ESI-MS: m / z = 390.1 [M-Br]. +

[0177] 1 H-NMR (400MHz, MeOH-d4): δ7.43-7.41(m,2H),7.16-7.15(m,2H),5.12-5.09(m,1H),3.98- 3.97(m,2H),3.31(s,3H),3.07(s,3H),3.02(s,2H),2.71-2.64(m,3H),1.69-1.64(m,2H).

[0178] Preparation Examples 2-4

[0179] Following the steps of Preparation Example 1, Compounds 2 to 4 were synthesized using intermediate int-1 and the following corresponding raw materials.

[0180]

[0181]

[0182] Preparation Example 5 (Compound 5):

[0183]

[0184] Step a: Add int2 (15.32 g, 1 eqv), scopolamine (9.80 g, 1.1 eqv), anhydrous toluene (100 ml), and 60% sodium hydride (910 mg, 0.4 eqv) to a reaction flask, and reflux to remove water for 9 hours. Stop the reaction, cool the reaction solution to room temperature, pour it into a saturated ammonium chloride solution, extract with EA, combine the organic phases, evaporate to dryness, and purify the crude product by column chromatography (100-200 mesh silica gel, DCM / MeOH = 100:1~20:1) to obtain Compound 5-1 (10.0 g, yield: 46.08%).

[0185] Step b: Compound 5-1 (10.0 g) and a saturated DCM solution (150 ml) of methyl bromide were reacted overnight at room temperature. The reaction was monitored by TLC until complete. The reaction was stopped, the reaction mixture was filtered, the filter cake was washed with DCM, and dried under vacuum to obtain Compound 5 (4.3 g, yield: 34.40%); ESI-MS: m / z = 384.3 [M-Br] +

[0186] 1 H-NMR (400MHz, MeOH-d4): δ7.56-7.52(m,1H),7.52-7.42(m,3H),7.34-7.29(m,1H),7.21-7.20(m,1H),5.10-5.07(m,1H),3.95-3.93(m,1H) ,3.90-3.88(m,1H),3.28(s,3H),3.04(s,3H),3.00-2.99(m,1H),2.73 -2.72(m,1H),2.68-2.58(m,2H),1.68-1.64(m,1H),1.56-1.52(m,1H).

[0187] Preparation Example 6 (Compound 6):

[0188]

[0189] Step a: Add INT-2 (740 mg, 1 eqv), DMF (5 ml), potassium carbonate (589 mg, 1.5 eqv), INT-C-1 (435 mg, 1.1 eqv), and n-heptane (15 ml) to a reaction flask and react at 108 °C for 5 h. Stop the reaction, cool the reaction solution to room temperature, add saturated brine, extract three times with EA, combine the organic phases, concentrate under reduced pressure and evaporate to dryness, and purify the residue by column chromatography (100-200 mesh silica gel, DCM / MeOH = 50:1~10:1) to obtain Compound 6-1 (370 mg, yield 36.8%).

[0190] Step b: Compound 6-1 (370 mg) and a saturated DCM solution (50 mL) of methyl bromide were reacted overnight at room temperature. The reaction was monitored by TLC until complete. The reaction was stopped, the reaction mixture was filtered, the filter cake was washed with DCM, and dried under vacuum to obtain Compound 6 (360 mg, yield: 76.7%); ESI-MS: m / z = 368.3 [M-Br]. +

[0191] 1 H-NMR (400MHz, DMSO-d6): δ7.61-7.60(m,1H),7.53-7.51(m,1H),7.44-7. 37(m,2H),7.27-7.23(m,1H),7.17-7.15(m,1H),6.75(s,1H),5.57-5.55( m,1H),5.38-5.36(m,1H),4.93-4.89(m,1H),4.16-4.07(m,2H),3.13(s,3 H),3.06(s,3H),2.50-2.41(m,2H),1.52-1.48(m,1H),1.43-1.39(m,1H).

[0192] Preparation Examples 7-8

[0193] Compounds 7-8 were synthesized using intermediate int-2 and the following commercially available raw materials, following the steps of Preparation Example 6.

[0194]

[0195]

[0196] Preparation Example 9 (Compound 9):

[0197]

[0198] Step a: Add int-3 (680 mg, 1 eqv), DMF (9 ml), potassium carbonate (541 mg, 1.5 eqv), scopolamine (447 mg, 1.1 eqv), and n-heptane (27 ml) to a reaction flask and react at 108 °C for 4 h. Stop the reaction, cool the reaction solution to room temperature, add saturated brine, extract three times with EA, combine the organic phases, concentrate under reduced pressure and evaporate to dryness, and purify the residue by column chromatography (100-200 mesh silica gel, DCM / MeOH = 100:1~20:1) to obtain Compound 9-1 (270 mg, yield 28%).

[0199] Step b: Compound 9-1 (270 mg) and a saturated DCM solution (30 mL) of methyl bromide were reacted overnight at room temperature. The reaction was monitored by TLC until complete. The reaction was stopped, the reaction mixture was filtered, the filter cake was washed with DCM, and dried under vacuum to obtain Compound 9 (300 mg, yield: 88.5%); ESI-MS: m / z = 384.3 [M-Br]. +

[0200] 1 ¹H-NMR (400MHz, DMSO-d6): δ 7.76–7.75 (m, 1H), 7.55–7.49 (m, 2H), 7.41–7.37 (m, 2H), 7.26–7.22 (m, 1H), 7.05 (s, 1H), 4.99–4.97 (m, 1H), 4.06–4.00 (m, 2H), 3.23–3.22 (m, 1H), 3.19 (s, 3H), 3.01–3.00 (m, 1H), 2.99 (s, 3H), 2.60–2.55 (m, 2H), 1.64–1.60 (m, 1H), 1.54–1.50 (m, 1H). Preparation Examples 10–12

[0201] Using the steps of Preparation Example 9, Compound 10 to 12 were synthesized with intermediate int-3 and the following corresponding raw materials.

[0202]

[0203]

[0204] Preparation Example 13 (Compound 13)

[0205]

[0206] Step a: Add int-4 (600 mg, 1 eqv), DMF (10 ml), potassium carbonate (447 mg, 1.5 eqv), scopolamine (368 mg, 1.1 eqv), and n-heptane (30 ml) to a reaction flask and react at 108 °C for 7 h. Stop the reaction, cool the reaction solution to room temperature, add saturated brine, extract three times with EA, combine the organic phases, concentrate under reduced pressure and evaporate to dryness, and purify the residue by column chromatography (100-200 mesh silica gel, DCM / MeOH = 100:1-50:1) to obtain Compound 13-1 (190 mg, yield 22.7%).

[0207] Step b: Compound 13-1 (190 mg) and a saturated DCM solution (10 mL) of methyl bromide were reacted overnight at room temperature. The reaction was monitored by TLC until complete. The reaction was stopped, the reaction mixture was filtered, the filter cake was washed with DCM, and dried under vacuum to obtain Compound 13 (120 mg, yield: 60.9%); ESI-MS: m / z = 402.2 [M-Br] +

[0208] 1 H-NMR (400MHz, DMSO-d6): δ7.70-7.68(m,1H),7.48-7.43(m,1H),7.38-7.36(m,1H),7.23-7.22(m,1H),7.09-7.04(m,1H),6.94(s,1H) ,5.06-5.02(m,1H),4.06-4.04(m,2H),3.20(s,3H),3.19(s,1H),3.11-3.10(m,1H),3.00(s,3H),2.63-2.55(m,2H),1.62-1.56(m,2H).

[0209] Preparation Examples 14–35, 50 and 69

[0210] Compounds 14–35, 50, and 69 were synthesized using the corresponding intermediates and scopolamine following the steps of Preparation Example 13.

[0211]

[0212]

[0213]

[0214]

[0215]

[0216]

[0217]

[0218]

[0219]

[0220]

[0221] Preparation Example 36 (Compound 36):

[0222]

[0223] Step a: Add 2 g of thioxanone (1 eqv) and 20 ml of tetrahydrofuran to the reaction flask, cool to 0 °C, add 15 ml of borane-tetrahydrofuran solution (1 M, 1.5 eqv), reflux for 3 h after addition, and monitor the reaction by TLC until completion; cool the reaction solution to room temperature, add 1 M hydrochloric acid aqueous solution until no gas is produced, add 100 ml of purified water, extract three times with EA, combine the organic phases, wash with saturated brine, dry with anhydrous sodium sulfate, filter, evaporate to dryness, and purify by column chromatography (200-300 mesh silica gel, PE flushing) to obtain Compound 36-1 (1.2 g, yield: 64.17%).

[0224] Step b: Add Compound 36-1 (1.2 g, 1 eqv) and tetrahydrofuran (20 ml) to a reaction flask, cool to -10 °C under nitrogen protection, and add LDA (4.54 ml, 2 M in THF, 1.5 eqv) dropwise. After the addition is complete, maintain the temperature for 30 min. Transfer to room temperature, add powdered dry ice (10 g) in portions to the reaction solution. After the addition is complete, react at room temperature for 1 h, and monitor the reaction for completion by TLC. Quench the reaction with hydrochloric acid (1 M), pour the reaction solution into 100 ml of purified water, extract three times with ethyl acetate, combine the organic phases, wash with saturated brine, dry with anhydrous sodium sulfate, filter and evaporate to dryness, and purify by column chromatography (200-300 mesh silica gel, DCM / MeOH = 100:1~20:1) to obtain Compound 36-2 (1 g, yield: 68.21%).

[0225] Step c: Add Compound 36-2 (1g, 1eqv), scopolamine (1.28g, 2.0eqv), DCC (1.02g, 1.5eqv), DMAP (50mg, 0.1eqv), and dichloromethane (50ml) to a reaction flask, reflux overnight, and monitor the reaction by TLC until it is complete; concentrate the reaction solution to dryness, and perform column chromatography on the crude product (200-300 mesh silica gel, PE / EA = 100:1 to 0:1) to obtain Compound 36-3 (1.9g, yield: 89.40%).

[0226] Step d: Add Compound 36-3 (1.9 g, 1 eqv) and tetrahydrofuran (40 ml) to a reaction flask, cool to 0 °C, add 60% NaH (300 mg, 1.5 eqv) in portions, replace oxygen three times, heat to 45 °C and react for 1 h, monitor the reaction by TLC until it ends; cool the reaction solution to room temperature, pour the reaction solution into 100 ml of purified water, extract three times with EA, combine the organic phases, wash with saturated brine, dry with anhydrous sodium sulfate, filter and concentrate, and purify by column chromatography (200-300 mesh silica gel, PE / EA = 100:1 to 0:1) to obtain Compound 36-4 (1.0 g, yield: 50.50%).

[0227] Step e: Add Compound 36-4 (1.0 g) and dichloromethane (100 ml) to a reaction flask, purge with bromomethane, and react overnight. LC-MS indicates the reaction is complete, with a large amount of solid produced. Filter, wash the filter cake with dichloromethane, add acetone (20 ml) to the filter cake, stir at room temperature for 1 h, filter, and vacuum dry to obtain Compound 36 (960 mg, yield: 77.42%).

[0228] ESI-MS: m / z = 410.2 [M-Br] +

[0229] 1 H-NMR (400MHz, DMSO-d6): δ7.91-7.88(m,2H),7.56-7.51(m,3H),7.43-7.34(m,4H),4.90-4.88( m,1H),4.03-4.02(m,2H),3.18(s,3H),2.97-2.96(m,5H),2.12-2.10(m,1H),1.65-1.60(m,2H).

[0230] Preparation Example 41 (Compound 41):

[0231]

[0232] Step a: Compound 5-1 (4.9 g), 2,2,2-trichloroethyl chloroformate (5.61 g, 2 eqv) and toluene (50 ml) were added to a reaction flask and refluxed overnight. The reaction was monitored by TLC until it was complete. The reaction solution was cooled to room temperature, concentrated to dryness under reduced pressure, and purified by column chromatography (200-300 mesh silica gel, PE / EA = 1:0 to 3:1) to obtain Compound 41-1 (4.9 g, yield: 70.05%).

[0233] Step b: Add Compound 41-1 (4.9 g, 1 eqv), zinc powder (6.06 g, 10 eqv) and acetic acid (50 ml) to a reaction flask, heat to 70 °C and react for 3 h. Monitor the reaction end by TLC. Cool the reaction solution to room temperature, filter through diatomaceous earth, concentrate, dissolve in dichloromethane, wash with saturated sodium carbonate aqueous solution, dry the organic phase with anhydrous sodium sulfate, filter and concentrate, slurry with EA, filter, and vacuum dry to obtain Compound 41-2 (2.1 g, yield: 62%).

[0234] Step c: Compound 41-2 (0.49 g, 1 eqv), 1,4-dibromobutane (2.97 g, 10 eqv), and acetonitrile (15 ml) were added to the reaction flask and refluxed for 48 h. The reaction was monitored by TLC until it ended. The reaction solution was cooled to room temperature and purified by direct silica gel column chromatography (100-200 mesh silica gel, DCM / MeOH = 1:0~15:1). The crude product was stirred in 10 ml of acetone at room temperature for 30 min, filtered, and the filter cake was washed with acetone and dried to obtain Compound 41 (180 mg, yield: 26.25%).

[0235] ESI-MS: m / z = 410.4 [M-Br] +

[0236] Preparation Examples 42–44 (Compound 42–44)

[0237] Compounds 42-44 were prepared using the steps and methods of Preparation Example 41, with the corresponding intermediates and commercially available raw materials.

[0238]

[0239] Preparation Example 46 (Compound 5-T1)

[0240]

[0241] Step a: Add int-2B-T1 (400 mg, 1 eqv), DMF (10 ml), potassium carbonate (337 mg, 1.5 eqv), scopolamine (277 mg, 1.1 eqv), and n-heptane (30 ml) to a reaction flask and react at 108 °C for 8 h. Stop the reaction, cool the reaction solution to room temperature, add saturated brine, extract three times with EA, combine the organic phases, concentrate under reduced pressure and evaporate to dryness, and purify the residue by column chromatography (100-200 mesh silica gel, DCM / MeOH = 100:1~30:1) to obtain Compound 5-1-T1 (300 mg, yield 50%).

[0242] Step b: Compound 5-1-T1 (300 mg) and a saturated DCM solution (30 mL) of methyl bromide were reacted overnight at room temperature. The reaction was monitored by TLC until complete. The reaction was stopped, the reaction mixture was filtered, the filter cake was washed with DCM, and dried under vacuum to obtain Compound 5-T1 (240 mg, yield: 62.7%). Chiral purity HPLC (99.56%), RT = 1.239 min; [α] 20 D =-16.9(c=1,MeOH); ESI-MS: m / z=384.1[M-Br] +

[0243] 1 H-NMR (400MHz, MeOH-d4): δ7.56-7.52(m,1H),7.52-7.42(m,3H),7.34-7.29(m,1H),7.21-7.20(m,1H),5.10-5.07(m,1H),3.95-3.93(m,1H) ,3.90-3.88(m,1H),3.28(s,3H),3.04(s,3H),3.00-2.99(m,1H),2.73 -2.72(m,1H),2.68-2.58(m,2H),1.68-1.64(m,1H),1.56-1.52(m,1H).

[0244] Preparation Example 47 (Compound 5-T2)

[0245]

[0246] Step a: Add int-2B-T2 (400 mg, 1 eqv), DMF (10 ml), potassium carbonate (337 mg, 1.5 eqv), scopolamine (277 mg, 1.1 eqv), and n-heptane (30 ml) to a reaction flask and react at 108 °C for 8 h. Stop the reaction, cool the reaction solution to room temperature, add saturated brine, extract three times with EA, combine the organic phases, concentrate under reduced pressure and evaporate to dryness, and purify the residue by column chromatography (100-200 mesh silica gel, DCM / MeOH = 100:1~30:1) to obtain Compound 5-1-T2 (330 mg, yield 55%).

[0247] Step b: Compound 5-1-T2 (330 mg) and a saturated DCM solution (30 mL) of methyl bromide were reacted overnight at room temperature. The reaction was monitored by TLC until complete. The reaction was stopped, the reaction mixture was filtered, the filter cake was washed with DCM, and dried under vacuum to obtain Compound 5-T2 (250 mg, yield: 60.4%). Chiral purity HPLC (99.96%), RT = 1.434 min; [α] 20 D = +17.01 (c = 1, MeOH);

[0248] ESI-MS: m / z = 384.1 [M-Br] +1 H-NMR (400MHz, MeOH-d4): δ7.55-7.54(m,1H),7.52-7.41(m,3H),7.33-7.29(m,1H),7.21-7.19(m,1H),5.10-5.07(m,1H),3.95-3.93(m,1H) ,3.89-3.88(m,1H),3.28(s,3H),3.04(s,3H),3.00-2.99(m,1H),2.73 -2.72(m,1H),2.69-2.58(m,2H),1.68-1.64(m,1H),1.56-1.52(m,1H).

[0249] Preparation Examples 48 and 49

[0250] Compounds 48-49 were prepared using the steps and methods of Preparation Examples 46 and 47, respectively, with the corresponding intermediates.

[0251]

[0252]

[0253] Preparation Example 51 (Compound 51)

[0254]

[0255] Compound 5 (200 mg) was dissolved in 10 ml of saturated sodium chloride solution and stirred at room temperature. The reaction solution gradually became turbid. Stirring was continued for 36 h. The reaction solution was filtered, and the filter cake was added to saturated sodium chloride solution and stirred for another 4 h. The filter cake was filtered, dried, and dissolved in 5 ml of methanol. The solution was heated to reflux, filtered while hot, and the filtrate was evaporated to dryness. The filtrate was then slurried with acetone to obtain a white solid, Compound 51 (100 mg). ESI-MS: m / z = 384.1 [M-Cl] +

[0256] 1H-NMR (400MHz, MeOH-d4): δ7.55-7.54(m,1H),7.52-7.41(m,3H),7.33-7.29(m,1H),7.21-7.20(m,1H),5.09(t,J=4Hz,1H),3.97-3.95(m,1H), 3.91-3.90(m,1H),3.28(s,3H),3.05(s,3H),3.00-2.99(m,1H),2.72-2 .70(m,1H),2.68-2.59(m,2H),1.66(d,J=8Hz,1H),1.53(d,J=8Hz,1H).

[0257] Preparation Example 52 (Compound 52)

[0258]

[0259] Compound 9 (75 mg) was dissolved in 10 ml of saturated sodium chloride solution and stirred at room temperature. The reaction solution gradually became turbid. Stirring was continued for 36 h. The reaction solution was filtered, and the filter cake was added to saturated sodium chloride solution and stirred for another 4 h. The filter cake was filtered, dried, and dissolved in 5 ml of methanol. The solution was heated to reflux, filtered while hot, and the filtrate was evaporated to dryness. The filtrate was then slurried with acetone to obtain a white solid, Compound 52 (35 mg). ESI-MS: m / z = 384.1 [M-Cl] +

[0260] 1 H-NMR (400MHz, MeOH-d4): δ7.69-7.67(m,1H),7.55-7.51(m,2H),7.45-7.41(m,1H),7.37-7.35(m,1H),7.32-7.28(m,1H),5.10-5.09(m,1H), 3.96-3.94(m,1H),3.89-3.88(m,1H),3.28(s,3H),3.05(s,3H),3.01- 3.00(m,1H),2.71-2.59(m,3H),1.70-1.606(m,1H),1.56-1.52(m,1H).

[0261] Preparation Example 67 (Compound 67)

[0262]

[0263] Step a: Add INT A-1 (300 mg, 1 eqv), INT-D-1 (398 mg, 2 eqv), DMAP (632 mg, 4 eqv), and DMF (10 ml) to a reaction flask, cool to 0°C, add EDCI (372 mg, 1.5 eqv) in portions, and react overnight at room temperature; extract the reaction solution with water and EA, wash the organic phase twice with saturated brine, concentrate, and purify by column chromatography (100-200 mesh silica gel, DCM / MeOH = 100:1~50:1), resulting in a pale yellow oily compound Compound 67-1 (102 mg, yield 16%); ESI-MS: m / z = 369.10 [M+H] +

[0264] Step b: Compound 67-1 (102 mg) and a saturated DCM solution (20 ml) of methyl bromide were reacted overnight at room temperature. The reaction was monitored by TLC until complete. The reaction was stopped, the reaction mixture was filtered, the filter cake was washed with DCM, and dried under vacuum to obtain Compound 67 (60 mg); ESI-MS: m / z = 383.2 [M-Br] +

[0265] Preparation Example 74 (Compound 74)

[0266]

[0267] Step a: 4-Bromothiophene-3-carboxylic acid methyl ester (3 g, 1 eqv), phenylboronic acid (1.82 g, 1.1 eqv), sodium carbonate (5.03 g, 3.5 eqv), Pd(PPh3)4 (1.57 g, 0.1 eqv), 1,4-dioxane (120 ml), and water (12 ml) were added to a reaction flask. Under nitrogen protection, the reaction was carried out at 100 °C for 6 h. TLC analysis showed that the reaction was basically complete. The reaction solution was cooled, concentrated under reduced pressure, extracted with water and EA, and the organic phases were combined, evaporated to dryness, and purified by column chromatography (PE / EA = 50:1~20:1) to obtain the oily compound Compound 74-1 (2.39 g, yield 82.4%).

[0268] Step b: Add trifluoromethanesulfonic acid (30 mL) to Compound 74-1 (2.39 g) and react overnight at room temperature; TLC showed no residue of the starting material, pour the reaction solution into ice water, add EA for extraction and separation three times, combine the organic phases, wash, concentrate, and purify by column chromatography (PE / EA = 100:1-50:1) to obtain Compound 74-2 yellow solid (1.79 g, yield 88.1%).

[0269] Step c: Add Compound 74-2 (1.69 g, 1 eqv), ZnI2 (0.59 g, 0.2 eqv), TMSCN (9 g, 10 eqv), and DCM (25 ml) to a reaction flask. Reflux overnight under nitrogen protection. After the reaction is complete, evaporate the reaction solution to dryness under reduced pressure to obtain crude Compound 74-3 (1.7 g). The unpurified crude product was used directly in the next step.

[0270] Step d: Add 1.7 g of crude Compound 74-3 and 50 ml of 10 M hydrogen chloride in ethanol to a reaction flask and react at room temperature for 3 h. After the reaction is complete as monitored by TLC, stop the reaction, pour the reaction mixture into water, extract with EA, combine the organic phases, and evaporate to dryness. Purify the crude product by column chromatography (100-200 mesh silica gel, PE / EA = 100:1–50:1) to obtain Compound 74-4 product (1.36 g, overall yield of both steps: 55%).

[0271] Step e: Compound 74-4 (1.36 g, 1 eqv), scopolamine (1.05 g, 1.3 eqv), anhydrous potassium carbonate (0.94 g, 1.3 eqv), DMF (10 ml), and n-heptane (60 ml) were added to a reaction flask, and the mixture was heated to 110 °C and reacted for 5 h. The reaction solution was extracted with water and EA, and the organic phase was washed twice with saturated brine. The solution was concentrated and purified by column chromatography (DCM / MeOH = 100:1 to 50:1) to obtain Compound 74-5 foamy solid (1.3 g, yield 69.9%).

[0272] Step f: Compound 74-5 (600 mg) and a saturated DCM solution (20 mL) of methyl bromide were reacted overnight at room temperature. The reaction was monitored by TLC until complete. The reaction was stopped, the reaction mixture was filtered, the filter cake was washed with DCM, and dried under vacuum to obtain Compound 74 (310 mg, yield 49.6%); ESI-MS: m / z = 384.5 [M-Br] +

[0273] 1H-NMR (400MHz, DMSO-d6): δ7.67-7.65(m,2H),7.57-7.56(m,1H),7.50-7.48(m,1H),7.42(t,J=8Hz,1H),6.83(s,1H),4.96(t,J=4Hz,1H),4. 04-3.99(m,2H),3.18(s,3H),3.16-3.15(m,1H),3.02-3.01(m,1H),2. 98(s,3H),2.58-2.55(m,2H),1.62(d,J=8Hz,1H),1.51(d,J=8Hz,1H).

[0274] Preparation Examples 75 & 76 (Compound 75 & Compound 76)

[0275]

[0276] Compound 74-5 (1.00 g) was separated by Chiral-HPLC (column: CHIRALPAK IM 20*250 5 μm; mobile phase: phase A: n-hexane; phase B: isopropanol (0.1% trifluoroacetic acid); mobile phase A: B = 40%:60% isocratic elution for 30 min, flow rate 9 mL / min; wavelength: 220 nm; solvent: anhydrous ethanol: n-hexane = 1:1, concentration 10 mg / mL), yielding Compound 74-5-T1 (RT1: 19.5 min, 283 mg, yield 28.3%), [α-hydroxylamine (RT1) and [α-hydroxylamine (RT2)). 20 D = -7.39 (c = 1, DMSO); and Compound 74-5-T2 (RT2: 24.1 min, 224 mg, yield 22.4%), [α] 20 D = +5.52 (c = 1, DMSO);

[0277] Following step f of the preparation example of compound 74, Compound 74-5-T1 (283 mg) and Compound 74-5-T2 (224 mg) were reacted separately with a DCM-saturated solution of methyl bromoethane to give Compound 75 (246 mg, yield 69%); ESI-MS: m / z = 384.5 [M-Br] +

[0278] 1H-NMR(400 MHz, D2O): δ 7.63 - 7.61 (m, 1H), 7.45 - 7.40 (m, 4H), 7.31 (t, J = 8 Hz, 1H), 4.99 (t, J = 4 Hz, 1H), 3.81 - 3.75 (m, 2H), 3.12 (s, 3H), 2.88 (s, 3H), 2.86 - 2.81 (m, 1H), 2.61 - 2.60 (m, 1H), 2.55 - 2.45 (m, 2H), 1.62 (d, J = 8 Hz, 1H), 1.51 (d, J = 8 Hz, 1H).

[0279] Compound 76 (211 mg, yield 75%); ESI-MS: m / z = 384.5 [M - Br] +

[0280] 1 H-NMR(400 MHz, D2O): δ 7.63 - 7.61 (m, 1H), 7.45 - 7.40 (m, 4H), 7.31 (t, J = 8 Hz, 1H), 4.99 (t, J = 4 Hz, 1H), 3.82 - 3.75 (m, 2H), 3.12 (s, 3H), 2.88 (s, 3H), 2.86 - 2.81 (m, 1H), 2.61 - 2.60 (m, 1H), 2.55 - 2.45 (m, 2H), 1.62 (d, J = 8 Hz, 1H), 1.51 (d, J = 8 Hz, 1H).

[0281] Preparation Examples 77 & 78 (Compound 77 & Compound 78)

[0282]

[0283] Step a: Add int-8 (3.00 g, 1 eqv), DMF (40 ml), potassium carbonate (2.09 g, 1.5 eqv), scopolamine (1.88 g, 1.2 eqv), and n-heptane (120 ml) to a reaction flask and react at 108 °C for 7 h. Stop the reaction, cool the reaction solution to room temperature, add saturated brine, extract three times with EA, combine the organic phases, concentrate under reduced pressure and evaporate to dryness, and purify the residue by column chromatography (100-200 mesh silica gel, DCM / MeOH = 100:1~50:1) to obtain Compound 17-1 (910 mg, yield 19.9%). Compound 17-1 (910 mg) was separated by Chiral-HPLC (column: CHIRALPAK IF 5cm×25cm, 10μm; mobile phase: phase A: n-hexane (0.5% 2mM NH3-MeOH); phase B: EtOH:DCM; mobile phase ratio A:B = 80:20; flow rate: 100 ml / min; wavelength: 220 nm; column temperature: 25℃; solvent: anhydrous ethanol: n-hexane = 1:1, concentration: 10 mg / ml) to obtain Compound 17-1-T1 (RT1: 14.3 min, 150 mg, yield 16.48%) and Compound 17-1-T2 (RT2: 16.5 min, 150 mg, yield 16.48%).

[0284] Step b: Following the method in step f of the preparation example of compound 74, Compound 17-1-T1 (150 mg) and Compound 17-1-T2 (150 mg) were reacted separately with a DCM-saturated solution of methyl bromoethane to obtain Compound 77 (110 mg, yield 59%); ESI-MS: m / z = 420.2 [M-Br] +

[0285] 1 H-NMR (400MHz, D2O): δ7.48-7.47(m,1H),7.40-7.31(m,2H),7.10-7.08(m,1H),5.01(t,J=4Hz ,1H),3.84-3.81(m,2H),3.15(s,3H),2.89-2.81(m,5H),2.58-2.48(m,2H),1.65-1.51(m,2H).

[0286] Compound 78 (138 mg, 74% yield) was obtained; ESI-MS: m / z = 420.2 [M-Br] +

[0287] 1H-NMR (400MHz, D2O): δ7.48-7.47(m,1H),7.40-7.31(m,2H),7.09-7.08(m,1H),5.01(t,J=4Hz ,1H),3.84-3.80(m,2H),3.15(s,3H),2.89-2.81(m,5H),2.58-2.48(m,2H),1.65-1.51(m,1H).

[0288] Preparation Examples 79 & 80 (Compound 79 & Compound 80)

[0289]

[0290] Step a: Add int-6 (1.30 g, 1 eqv), DMF (10 ml), potassium carbonate (967 mg, 1.5 eqv), scopolamine (869 mg, 1.2 eqv), and n-heptane (30 ml) to a reaction flask and react at 108 °C for 6 h. Stop the reaction, cool the reaction solution to room temperature, add saturated brine, extract three times with EA, combine the organic phases, concentrate under reduced pressure and evaporate to dryness, and purify the residue by column chromatography (100-200 mesh silica gel, DCM / MeOH = 100:1~50:1) to obtain Compound 15-1 (600 mg, yield 35.2%). Compound 15-1 (600 mg) was separated by Chiral-HPLC (column: CHIRALPAK IM 20*250 5 μm; mobile phase: phase A: n-hexane; phase B: isopropanol (0.1% trifluoroacetic acid); mobile phase A: B = 50%:50% isocratic elution for 50 min, flow rate 9 ml / min; wavelength: 220 nm; solvent: anhydrous ethanol: n-hexane = 1:1, concentration 10 mg / ml) to obtain Compound 15-1-T1 (RT1: 23.0 min, 180 mg, yield 30%) and Compound 15-1-T2 (RT2: 33.5 min, 180 mg, yield 30%).

[0291] Step b: Following the method in step f of the preparation example of compound 74, Compound 15-1-T1 (180 mg) and Compound 15-1-T2 (180 mg) were reacted separately with a DCM-saturated solution of methyl bromide to obtain Compound 79 (108 mg, yield 48.3%); ESI-MS: m / z = 402.4 [M-Br]+

[0292] 1H-NMR (400MHz, D2O): δ7.51-7.50(m,1H),7.38-7.35(m,1H),7.24-7.21(m,1H),7.10-7.09(m,1H),6.98-6.91(m,1H),4.99 (t,J=4Hz,1H),3.82-3.78(m,2H),3.13(s,3H),2.90-2.88(m,4H),2.76-2.75(m,1H),2.56-2.46(m,2H),1.64-1.46(m,2H).

[0293] Compound 80 (113 mg, yield 50.45%) was obtained; ESI-MS: m / z = 402.4 [M-Br]+

[0294] 1 H-NMR (400MHz, D2O): δ7.51-7.50(m,1H),7.38-7.35(m,1H),7.24-7.21(m,1H),7.10-7.09(m,1H),6.98-6.93(m,1H),4.99 (t,J=4Hz,1H),3.83-3.78(m,2H),3.13(s,3H),2.90-2.88(m,4H),2.76-2.75(m,1H),2.56-2.46(m,2H),1.64-1.49(m,2H).

[0295] Preparation Example 81 (Compound 81)

[0296]

[0297] Step a: Add 1-cyclopentenic acid (10 g, 1 eqv) and benzene (100 mL) to a reaction flask, cool to 0 °C, add aluminum trichloride (35.6 g, 3 eqv) in portions, and then raise the temperature to 70 °C and react for 3 h. Cool to 0 °C, quench the reaction with 2 M dilute hydrochloric acid dropwise, separate the liquid and aqueous phases, extract with EA, combine the organic phases, and purify by column chromatography (PE / EA = 100:1-10:1) to obtain Compound 81-1 orange-red oil (10 g, yield 66%).

[0298] Step b: Under nitrogen protection, Compound 81-1 (3.5 g, 1 eqv) was dissolved in DCM (35 mL), and 90% lithium ethylenediamine complex (3.7 g, 2 eqv) was added. The reaction was allowed to proceed overnight. The reaction was quenched with water, and the mixture was separated. The aqueous phase was extracted with DCM, the organic phases were combined, evaporated to dryness, and purified by column chromatography (PE / EA = 100:1-10:1) to obtain Compound 81-2 yellow-green oil (1.45 g, yield 36.3%).

[0299] Step c: Dissolve Compound 81-2 (700 mg, 1 eqv) in tert-butanol (7 mL), then add potassium permanganate alkaline solution (38 mL) and sodium periodate (4.57 g, 6 eqv). After the addition is complete, let the reaction proceed overnight at room temperature. Stop the reaction, add ethanol (20 mL) to the reaction solution, stir at room temperature for 30 min, filter, wash with tert-butanol, concentrate under reduced pressure to 50 mL, adjust the pH to 2-3 with 1 M dilute hydrochloric acid, extract with EA, combine the organic phases, evaporate to dryness, and purify by column chromatography (PE / EA = 100:1-4:1) to obtain Compound 81-3 yellowish-white solid (450 mg, yield 58.4%).

[0300] Step d: Compound 81-3 (400 mg, 1 eqv), scopolamine (341.3 mg, 1.2 eqv), and DIEA (592.16 mg, 2.5 eqv) were dissolved in DMF (10 mL) and stirred at room temperature for 10 min. The mixture was then cooled to 0 °C and HATU (836.26 mg, 1.2 eqv) was added. The reaction was allowed to proceed overnight. The reaction mixture was stopped, and the reaction solution was extracted with water and EA. The organic phases were combined, washed with sodium chloride solution, and evaporated to dryness. The mixture was purified twice by pre-TLC (DCM / MeOH = 20:1) to obtain Compound 81-4 (130 mg, yield 19.96%). ESI-MS: m / z = 356.2 [M+H] +

[0301] Step e: Compound 81-4 (130 mg) and a saturated DCM solution (10 mL) of methyl bromide were reacted overnight at room temperature. The reaction was monitored by TLC until complete. The reaction was stopped, the reaction solution was concentrated under reduced pressure, and the residue was slurried with 2-methyltetrahydrofuran and dried under vacuum to obtain Compound 81 (82 mg, yield 49.8%); ESI-MS: m / z = 370.3 [M-Br] +

[0302] Experimental Example 1: Detection of Intracytal IP1 Content

[0303] The antagonistic effect of the test compound on the M3 target was tested based on the intracytoplasmic IP1 content.

[0304] 1. Test materials

[0305] Cells: CHO-K1-M3-clone3

[0306] Consumables: 384-well plate (company: Greiner, part number: 784075), centrifuge tubes

[0307] Reagents: 0.25% trypsin, PBS, F12K medium, fetal bovine serum, penicillin and streptomycin antibiotics, deionized water, IP-One-Gq kit.

[0308] Instrument: EnVision (Company: PerkinElmer)

[0309] 2. Experimental Procedure:

[0310] 2.1 Before the experiment, preheat the culture medium and trypsin in a 37-degree water bath, turn on the laminar flow hood, and clean the laminar flow hood surface with an alcohol-soaked cotton swab.

[0311] 2.2 Observe the cell proliferation and adhesion under a microscope. When the cell adhesion rate / cell density reaches 80%-90%, start preparing for cell resuspension.

[0312] 2.3 Discard the supernatant culture medium, then gently wash the adherent cells once with 1 ml of PBS, then discard the supernatant and aspirate the remaining liquid with a pipette tip.

[0313] 2.4 Add 1 ml of trypsin solution with 0.25% EDTA, shake the culture dish until the liquid completely covers the walls of the flask where the cells are attached, and observe them under a microscope at any time.

[0314] 2.5. Immediately after digestion, add 2 ml of F12K medium containing 20% ​​serum, stop digestion, gently pipette several times, centrifuge at 1000 rpm for 10 min, discard the supernatant, add 1 ml of complete medium and mix by pipetting.

[0315] 2.6. Take 10 μl of cell suspension, add 10 μl of trypan blue and mix well, then take 10 μl for cell counting.

[0316] 2.7 After counting, seed 5000 cells per well into a 384 plate, with a seeding volume of 7 μL / well.

[0317] 2.8. Prepare a 4-fold final concentration gradient of carbacholine and the test compound using experimental buffer. Add 3.5 μl of the test compound to each well of a 384 plate containing cells. After pre-incubation at 37°C for 15 min, add 3.5 μl of carbacholine. The concentrations of the test compounds are 2.5 nM and 0.5 nM, and the carbacholine concentration is 2 μM. Seal the plates and incubate them at 37°C in a 5% CO2 incubator for 1 hour.

[0318] 2.9 After incubation, add 3 μl of IP1-d2 working solution and 3 μl of IP1 Tb crypate antibody working solution to each well, mix well and continue incubation.

[0319] 2.10 After incubation at 25°C for 1 hour, the experimental parameters of LANCE were read on the Envision instrument.

[0320] The concentration of inositol phosphate in the sample was calculated based on the standard curve. The data were fitted using the Gaddum equation, and the dose ratio was plotted according to Schild's equation.

[0321] The results are shown in Table 1.

[0322] Table 1: Results of the test for intracytoplasmic IP1 content

[0323]

[0324]

[0325] Experimental results show that the compound in this invention has good activity.

[0326] Example 2: Inhibitory effect of the compound on carbacholine-induced contraction of isolated guinea pig tracheal smooth muscle

[0327] The compounds involved in the following experiments were prepared from the previous preparation examples.

[0328] a) Method: Isolated guinea pig tracheal smooth muscle tracheal rings (5-7 mm) specimens were placed in Krebs-Henseleit solution, a load of 3.50 ± 0.05 g was applied, and the tension generated by the samples was measured and recorded at equal lengths.

[0329] b) Results: Carbacholine induced concentration-dependent contraction of tracheal smooth muscle. The compound in this patent inhibits carbacholine-induced contraction in a concentration-dependent manner, pK b The results are shown in Table 2.

[0330] Table 2: Inhibitory effects of compounds on carbacholine-induced contraction of isolated guinea pig tracheal smooth muscle

[0331]

[0332]

[0333] Where n: represents the number of experimental samples

[0334] Conclusion: The compound in this patent exhibits an anticholinergic effect on tracheal smooth muscle contraction induced by cholinergic agonists.

[0335] Example 3: PK test of intratracheal drug administration

[0336] 3.1 Experimental Objective: To evaluate the pharmacokinetic characteristics and lung tissue distribution of the compound in SD rats after intratracheal administration.

[0337] 3.2 Experimental animals:

[0338] Male SD rats, SPF grade, 6-8 weeks old, Hunan Slack Jingda Experimental Animal Co., Ltd.

[0339] 3.3 Drug Preparation:

[0340] The test compound was added to a solution of 0.01% benzalkonium chloride + 0.05% EDTA + 0.9% sodium chloride + 0.0084% citric acid-water solution, and the mixture was vortexed and sonicated to obtain a solution with a concentration of 22 mg / ml.

[0341] 3.4 Dosing regimen:

[0342]

[0343] 3.5 Test Operation

[0344] 3.5.1 Intratracheal administration to rats

[0345] Rats were administered the drug via tracheal nebulizer. Plasma was collected at the following time points: before administration (0 h), and 5 min, 15 min, 0.5 h, 1 h, 4 h, 8 h, and 24 h after administration. 0.25 mL of whole blood was collected and anticoagulated with EDTA-K2. The whole blood samples were placed on wet ice and centrifuged at 3000 g for 10 minutes within 1.5 h of wet ice. The resulting plasma was transferred to appropriately labeled test tubes and temporarily stored at -20°C or below. After sample collection, the samples were transferred to a -80°C freezer for later analysis. Lung tissue was collected at the following time points: 5 min, 15 min, 0.5 h, 1 h, 4 h, and 8 h after administration. Tissue samples were transferred to appropriately labeled test tubes and temporarily stored at -20°C or below for later analysis.

[0346] 3.5.2 Plasma processing and LC-MS / MS analysis

[0347] Take 20.0 μL of plasma sample, add 25.0 μL of internal standard working solution and 300 μL of acetonitrile, vortex mix for 5 min, centrifuge for 15 min (1700 g, 4 ℃), take 100 μL of supernatant into a 96-well plate, add 100 μL of ultrapure water and inject for analysis, inject 2.00 μL for LC-MS / MS analysis.

[0348] 3.5.3 Lung tissue processing

[0349] Accurately weigh an appropriate amount of lung tissue sample, place it in a homogenization tube, add methanol:water (1:1) equivalent to 10 times its weight, mix and homogenize, and sonicate for 5 minutes.

[0350] Take 20.0 μL of lung tissue homogenate sample, add 25.0 μL of internal standard working solution and 300 μL of acetonitrile, vortex mix for 5 min, centrifuge for 15 min (1700 g, 4 ℃), take 100 μL of supernatant into a 96-well plate, add 100 μL of ultrapure water, shake well (1700 g, RT), and inject 2.00 μL for LC-MS / MS analysis.

[0351] 3.6 Pharmacokinetic Parameter Results

[0352] The pharmacokinetic parameters are shown in Table 3, which displays the experimental results of the test substance in plasma and lung tissue.

[0353] Table 3

[0354]

[0355] Experimental conclusions: Compared with tiotropium bromide, the compound of this invention has better pharmacokinetic properties in lung tissue; and the compound of this invention is more easily metabolized in plasma; therefore, the compound of this invention better meets the pharmacokinetic characteristics required for pulmonary inhalation administration.

[0356] Example 4: Pharmacodynamic study of the compound in conscious guinea pigs

[0357] Experimental objective: To evaluate the efficacy of the compound in a guinea pig bronchoconstriction model induced by acetylcholine.

[0358] 4.1 Animal Information:

[0359] Species guinea pig strain Hartley (Guinea Pig) name Hartley Guinea Pig (SPF Grade) gender female weight Less than 300g Zhou Ling 3 weeks supplier Beijing Viton Lihua Hubei Branch Production License Number SCXK (Beijing) 2021-0011

[0360] 4.2 Key Instruments and Equipment:

[0361] Instrument Name Manufacturer Equipment Model Small animal oral and nasal inhalation exposure system Beijing Huironghe Technology Co., Ltd. HRH-2024043501-1 Ultrasonic atomizer Guangdong Yuehua Medical Equipment Factory WH-2000

[0362] 4.3 Test Methods:

[0363] 4.3.1 Grouping Method

[0364] After the adaptation period, 1% acetylcholine solution was nebulized for 25 seconds. The time from the start of the spray until the animal showed signs of loss of eye redness, difficulty breathing, and convulsions was recorded as the bronchoconstriction onset time. Animals with bronchoconstriction onset time of 40-60 seconds were divided into three groups according to the mean: solvent group, positive drug group, and test compound group, n=10. The remaining animals were culled.

[0365] 4.3.2 Administration method

[0366] A single dose was administered via nebulization using a small animal oral-nasal exposure system (dose 1 mg / ml, nebulization for 1 min). The solvent group received an equal volume of solvent using the same method.

[0367] Small animal mouth and nose exposure system parameters: aerosol flow rate: 12L / min, dilution flow rate: 0L / min, suction flow rate: 15L / min, preset concentration: 10000mg / m3. Other parameters are system defaults.

[0368] Ultrasonic atomizer settings: Set the airflow adjustment knob to medium, and leave all other settings as default.

[0369] 4.3.3 Recording of bronchial contraction time in guinea pigs

[0370] At 0, 1, 3, 6, 12, 24, and 30 hours after nebulized administration, 1% acetylcholine solution was nebulized for 25 seconds. The time from the start of spraying until the animals showed signs of loss of eye color, difficulty breathing, and convulsions was recorded as the time of bronchoconstriction onset.

[0371] 4.4 Test Results:

[0372] The results are shown in Table 4.

[0373] Table 4. Time of bronchoconstriction in guinea pigs during the experiment (s)

[0374]

[0375] Note: Compared with the solvent group, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001;

[0376] 4.5 Experimental Conclusions:

[0377] Based on general clinical observations, no apparent toxic side effects were observed in the animals under the administration conditions of this study, indicating good tolerability in the animals.

[0378] Based on the overall comparison of bronchial contraction time results, the bronchial contraction time of the compound group was significantly longer than that of the solvent group and the tiotropium bromide group throughout the entire testing period after drug administration, indicating significant long-acting efficacy in vivo.

[0379] Example 5: Single-dose intravenous administration toxicity test in mice

[0380] Experimental objective: To test the toxicity of a compound dissolved in physiological saline and administered via tail vein.

[0381] 5.1 Animal Information

[0382]

[0383] 5.2 Administration method: The test compound was dissolved in physiological saline and administered via tail vein injection.

[0384] 5.3 Test Results:

[0385] The experimental results are shown in Table 5.

[0386] Table 5 Results of single intravenous administration toxicity test in mice

[0387]

[0388] The results of this experiment show that, at the same dosage, the compound has lower toxicity than tiotropium bromide and is safer.

[0389] Example 6: Stability test of the formulation

[0390] 6.1 Preparation method of blank solvent:

[0391] Dissolve EDTA (2 mg) and sodium chloride (180 mg) in 20 ml of water for injection, adjust the pH to 3.0 with 0.1 mol / L hydrochloric acid, filter through a 0.22 μm filter membrane, and fill into a sealed container for later use.

[0392] 6.2 The compound of the present invention was dissolved in the above solvent to prepare an aqueous solution with a concentration of 5 μg / ml; a stability test of the aqueous solution formulation was conducted (stability test for 10 days at high temperatures of 40℃ / 60℃), and the results are shown in Table 6. The test results indicate that the ester bond of the compound has good stability in aqueous solution, and it has the potential to be developed into an nebulized inhalation formulation.

[0393] Table 6: Results of Stability Testing of Formulations

[0394]

[0395]

Claims

1. A compound of formula (I) or a stereoisomer, tautomer, isotopic derivative, nitride, hydrate, solvate, metabolite, pharmaceutically acceptable salt, or prodrug thereof, in, M is independently selected from O, NH and S; Ring A and ring B are independently selected from five- or six-membered aromatic heterocycles, benzene rings, and C3-C8 aliphatic rings containing one or more heteroatoms selected from O, N, and S; R1 and R2 are independently selected from hydrogen, deuterium, halogens, and C. 1～6 Alkyl, C 3～6 cycloalkyl, cyano, C 1～6 Alkoxy, C 3～6 Cycloalkoxy, halogenated C 1～6 Alkyl and Halogenated C 3～6 cycloalkyl; R3 and R4 are independently selected from C 1～6 Alkyl, C 3～6 cycloalkyl, halogenated C 1～6 Alkyl and Halogenated C 3～6 Cycloalkyl; or R3 and R4 together with the N atom attached to them to form a five- or six-membered ring; Z is selected from single bonds, -O-, -S-, and -(CH2). 1～5 -、-S(=O)-、-SO2-、-NR5-;R5 is selected from H、C 1～6 Alkyl, C 3～6 cycloalkyl, halogenated C 1～6 Alkyl and Halogenated C 3～6 cycloalkyl; X - The anion having a negative charge is selected from fluoride, chloride, bromide, iodide, formate, hydrogen sulfate, sulfate, phosphate, trifluoromethanesulfonate, methanesulfonate, nitrate, maleate, acetate, citrate, fumarate, tartrate, oxalate, succinate, benzoate, and p-toluenesulfonate. Y is selected from:

2. The compound or its stereoisomers, tautomers, isotope derivatives, nitrogen oxides, hydrates, solvates, metabolites, pharmaceutically acceptable salts or prodrugs according to claim 1, wherein, The compound represented by formula (I) can be a compound represented by formulas (II), (III), (IV), (V), (VI), (Va), or (VI-a):

3. The compound or its stereoisomers, tautomers, isotope derivatives, nitrogen oxides, hydrates, solvates, metabolites, pharmaceutically acceptable salts or prodrugs according to claim 1 or 2, wherein, Ring A and ring B are selected independently from: More preferably, ring A and ring B are independently selected from: More preferably, ring A is selected from: Ring B is selected from: More preferably, ring A is selected from: Ring B is selected from:

4. The compound or its stereoisomers, tautomers, isotopic derivatives, nitrogen oxides, hydrates, solvates, metabolites, pharmaceutically acceptable salts or prodrugs according to any one of claims 1 to 3, wherein, R1 and R2 are independently selected from hydrogen, deuterium, halogens, and C. 1～4 Alkyl, C 3～5 cycloalkyl, cyano, C 1～4 Alkoxy, C 3～5 Cycloalkoxy, halogenated C 1～4 Alkyl and Halogenated C 3～5 cycloalkyl; Preferably, R1 and R2 are independently selected from hydrogen, deuterium, halogens (e.g., F, Cl, Br), and C. 1～4 Alkyl (e.g., methyl, ethyl), halogenated C 1～4 Alkyl groups (e.g., trifluoromethyl); Preferably, R1 and R2 are independently selected from hydrogen, deuterium, F, Cl, methyl, ethyl, and trifluoromethyl; Preferably, R3 and R4 are independently selected from C 1～4 Alkyl, C 3～5 cycloalkyl, halogenated C 1～4 Alkyl and Halogenated C 3～5 Cycloalkyl; or R3 and R4 together with the N atom attached to them to form a five- or six-membered ring; Preferably, R3 and R4 are independently selected from methyl, ethyl, propyl, and isopropyl; or R3 and R4 together with the N atom they are attached to form a five- or six-membered ring.

5. The compound or its stereoisomers, tautomers, isotopic derivatives, nitrogen oxides, hydrates, solvates, metabolites, pharmaceutically acceptable salts or prodrugs according to any one of claims 1 to 4, wherein, Z is selected from single bonds, -O-, -S-, and -(CH2). 1～2 -; Preferably, Z is a single bond; Preferably, X - Selected from chloride ions and bromide ions; Preferably, Y is selected from Preferably, Y is 6. The compound or its stereoisomers, tautomers, isotopic derivatives, nitrogen oxides, hydrates, solvates, metabolites, pharmaceutically acceptable salts or prodrugs according to any one of claims 1 to 5, wherein, In equations (I), (II), (III), (IV), (V), (Va), and (VI-a), if any, M is independently selected from O, NH, and S, and ring A and ring B are independently selected from: R1 and R2 are independently selected from hydrogen, deuterium, halogens, and C. 1～4 Alkyl, C 3～5 cycloalkyl, cyano, C 1～4 Alkoxy, C 3～5 Cycloalkoxy, halogenated C 1～4 Alkyl and Halogenated C 3～5 cycloalkyl; R3 and R4 are independently selected from C 1～4 Alkyl, C 3～5 cycloalkyl, halogenated C 1～4 Alkyl and Halogenated C 3～5 Cycloalkyl; or R3 and R4 together with the N atom attached to them to form a five- or six-membered ring; Z is selected from single bonds, -O-, -S-, and -(CH2). 1～2 -; X - Selected from chloride ions and bromide ions; Y is selected from Preferably, in formulas (I), (II), (III), (IV), (V), (Va), (VI-a), if any, M is independently selected from O, NH, and S, and ring A and ring B are independently selected from: R1 and R2 are independently selected from hydrogen, deuterium, halogens (e.g., F, Cl, Br), and C. 1～4 Alkyl (e.g., methyl, ethyl), halogenated C 1～4 Alkyl groups (e.g., trifluoromethyl); R3 and R4 are independently selected from methyl, ethyl, propyl, and isopropyl; or R3 and R4 together with the N atom they are attached to form a five- or six-membered ring; Z is selected from single bonds, -O-, -S-, and -(CH2). 1～2 -; X - Selected from chloride ions and bromide ions; Y is selected from Preferably, in formulas (I), (II), (III), (IV), (V), (Va), (VI-a), if present, M is independently selected from O, NH, and S, and ring A is selected from: Ring B is selected from: R1 and R2 are independently selected from hydrogen, deuterium, F, Cl, methyl, ethyl, and trifluoromethyl; R3 and R4 are independently selected from methyl, ethyl, propyl, and isopropyl; or R3 and R4 together with the N atom they are attached to form a five- or six-membered ring; Z represents a single bond; X - Selected from chloride ions and bromide ions; Y is Preferably, in formulas (I), (II), (III), (IV), (V), (Va), (VI-a), if present, M is independently selected from O, NH, and S, and ring A is selected from: Ring B is selected from: R1 and R2 are independently selected from hydrogen, deuterium, F, Cl, methyl, ethyl, and trifluoromethyl; R3 and R4 are independently selected from methyl, ethyl, propyl, and isopropyl; or R3 and R4 together with the N atom they are attached to form a five- or six-membered ring; Z represents a single bond; X - Selected from chloride ions and bromide ions; Y is Preferably, the compound is selected from the following preparation example compounds:

7. A method for preparing the compound (Va) of formula (I) according to any one of claims 1 to 6, the method comprising the following steps: (1) The substituted 2-thiophene boronic acid V-7 (or boronic ester) and the substituted 2-bromobenzoic acid ester V-8 undergo a coupling reaction under the first basic condition to generate intermediate V-1. (2) Intermediate V-1 is hydrolyzed under the second alkaline condition and then acidified to generate intermediate V-2; (3) Intermediate V-2 was treated with reagents such as SOCl2, oxalyl chloride or phosphorus oxychloride to obtain intermediate acyl chloride V-3. The crude product was directly used in the next step of the reaction. (4) Intermediate V-3 undergoes a ring-closing reaction under Lewis acid conditions (aluminum trichloride, tin tetrachloride, etc.) to generate intermediate V-4; (5) Intermediate V-4 reacts with trimethylsilyl cyanide in the presence of zinc iodide to generate intermediate V-5. The crude product is directly used in the next step of the reaction. (6) Intermediate V-5 in alcohol (ROH(wherein, R is C 1-6 The reaction is carried out by passing hydrogen chloride gas into a solution of alkyl groups (such as methanol, ethanol, etc.) or in a commercially available solution of hydrogen chloride alcohol to generate V-6. (7) Intermediates V-6 and Va-0 undergo transesterification under the third alkaline conditions to generate intermediate Va-1; (8) Intermediate Va-1 undergoes a quaternization reaction with R4X (such as bromomethane) to obtain Va; Wherein, R1, R2, R3, R4, X, and Y are as described in any one of claims 1 to 8, and R is C. 1-6 Alkyl groups; Preferably, in step (1), the base used in the first alkaline condition is an inorganic base or an organic base, such as sodium carbonate; Preferably, in step (2), the base used in the second alkaline condition is an inorganic base or an organic base, such as sodium hydroxide; Preferably, in step (2), the acid used for acidification is an inorganic acid or an organic acid, such as hydrochloric acid; Preferably, in step (7), the base used in the third alkaline condition is an inorganic base or an organic base, such as 60% NaH, potassium carbonate, etc. Preferably, the method for preparing the compound (VI-a) of formula (I) according to any one of claims 1 to 6 comprises the following steps: (1) The substituted 3-thiophene boronic acid VI-7 (or boronic ester) and the substituted 2-bromobenzoic acid ester VI-8 undergo a coupling reaction under the first basic condition to generate intermediate VI-1. (2) Intermediate VI-1 is hydrolyzed under the second alkaline condition and then acidified to generate intermediate VI-2; (3) Intermediate VI-2 was treated with reagents such as SOCl2, oxalyl chloride or phosphorus oxychloride to prepare intermediate acyl chloride VI-3. The crude product was directly used in the next step of the reaction. (4) Intermediate VI-3 undergoes a ring-closing reaction under Lewis acid conditions (aluminum trichloride, tin tetrachloride, etc.) to generate intermediate VI-4; (5) Intermediate VI-4 reacts with trimethylsilyl cyanide in the presence of zinc iodide to generate intermediate VI-5. The crude product is directly used in the next step of the reaction. (6) Intermediate VI-5 in alcohol (ROH(where R is C 1-6 Hydrogen chloride gas is passed into a solution of alkyl groups (such as methanol, ethanol, etc.), or a commercially available solution of hydrogen chloride alcohol is reacted to generate VI-6. (7) Intermediates VI-6 and VI-a-0 undergo transesterification under the third alkaline conditions to generate intermediate VI-a-1; (8) Intermediate VI-a-1 undergoes a quaternization reaction with R4X (such as bromomethane) to obtain VI-a; Wherein, R1, R2, R3, R4, X, and Y are as described in any one of claims 1 to 6, and R is C. 1-6 Alkyl groups; Preferably, in step (1), the base used in the first alkaline condition is an inorganic base or an organic base, such as sodium carbonate; Preferably, in step (2), the base used in the second alkaline condition is an inorganic base or an organic base, such as sodium hydroxide; Preferably, in step (2), the acid used for acidification is an inorganic acid or an organic acid, such as hydrochloric acid; Preferably, in step (7), the base used in the third alkaline condition is an inorganic base or an organic base, such as potassium carbonate.

8. A pharmaceutical composition comprising the compound of any one of claims 1 to 6 or its stereoisomers, tautomers, isotope derivatives, nitrides, hydrates, solvates, metabolites, pharmaceutically acceptable salts or prodrugs thereof, and a pharmaceutically acceptable carrier.

9. Use of the compound of any one of claims 1 to 6 or its stereoisomers, tautomers, isotope derivatives, nitrogen oxides, hydrates, solvates, metabolites, pharmaceutically acceptable salts or prodrugs thereof, or the pharmaceutical composition of claim 8, in the preparation of a medicament for the prevention and / or treatment of the following diseases: lung diseases including asthma, chronic obstructive pulmonary disease (COPD), bronchopulmonary dysplasia (BPD); urinary incontinence; and schizophrenia.

10. A combination drug comprising the compound of any one of claims 1 to 6 or its stereoisomers, tautomers, isotope derivatives, nitrides, hydrates, solvates, metabolites, pharmaceutically acceptable salts or prodrugs thereof, or the pharmaceutical composition of claim 8, and other therapeutic agents.

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