Acecridine prodrug as well as preparation method and application thereof

Abstract

The invention discloses an Acecridine prodrug as well as a preparation method and application thereof. The aceticidine prodrug is a compound as shown in a formula I or pharmaceutically acceptable salts and stereoisomers thereof. The aceticidine prodrug provided by the invention not only can reduce the peak concentration within 0.25 hour to a lower level and reduce side effects caused by the peak concentration, but also has the drug concentration always higher than that of an aceticidine group from 1 hour, the drug concentration of the aceticidine prodrug is dozens of times of that of the aceticidine at the time point of 4 hours and dozens of times of that of the aceticidine at the time point of 8 hours, and the drug concentration of the aceticidine prodrug is higher than that of the aceticidine at the time point of 4 hours. The medicine-time curve is obviously gentle and lasting, and is more beneficial to oral administration for treating diseases such as xerostomia and the like. # imgabs0 #

Description

Technical Field

[0001] The invention belongs to the field of medicine, and specifically relates to an aceclidine prodrug and a preparation method and application thereof. Background Art

[0002] Aceclidine is a muscarinic drug, a parasympathetic stimulant, used in the treatment of open-angle glaucoma in the form of eye drops. It is a muscle acetylcholine receptor agonist with weak anticholinesterase activity. It has the following characteristics:

[0003] It has a strong muscarinic receptor agonist effect, which can stimulate the pupillary contraction muscle and ciliary muscle contraction of the eye, thereby causing pupil constriction and myopia. It works quickly after oral administration or eye drops, usually taking effect within a few minutes, and the action time is short. Muscarinic receptor agonists are a class of drugs that act on muscarinic receptors, which can simulate the effects of acetylcholine, stimulate the excitement of muscarinic nerve endings, and thus cause corresponding physiological reactions. Muscarinic receptor agonists include natural plant muscarines and synthetic drugs such as carbachol and procaine choline.

[0004] Muscarinic receptor agonists mainly act on muscarinic receptors, which are widely distributed in various tissues and organs in the human body, including the nervous system, cardiovascular system, digestive system, respiratory system, urinary system, eyes, etc. Different muscarinic receptor subtypes have different distributions and functions in different tissues, so muscarinic receptor agonists have a variety of different effects, as follows: 1. Increase heart contractility and frequency, and reduce heart conduction velocity. 2. Promote peristalsis and secretion of the digestive system, increase gastrointestinal motility and secretion, and relieve symptoms such as constipation. 3. Dilate the respiratory tract, increase respiratory secretions, and improve respiratory diseases. 4. Stimulate the urinary system, increase bladder contractility and urethral sphincter relaxation, and improve symptoms such as frequent urination and urgency. 5. Stimulate the pupillary contraction muscle and ciliary muscle contraction of the eye, resulting in pupillary contraction and myopic effects. Muscarinic receptor agonists should be used according to the doctor's advice and prescription when used to avoid unnecessary side effects. Some common side effects include headache, dizziness, nausea, vomiting, diarrhea, tachycardia, etc. It can be used to treat eye diseases such as glaucoma and lens dislocation, and can also be used to diagnose ophthalmic diseases.

[0005] Aceclidine is an effective ophthalmic drug, but its oral use in the treatment of related diseases has not been reported. Summary of the invention

[0006] The object of the present invention is to provide an excellent aceclidine prodrug.

[0007] The aceclidine prodrug provided by the present invention is a compound represented by Formula I or a pharmaceutically acceptable salt or stereoisomer thereof:

[0008]

[0009] In the formula I, R a It is H or methyl.

[0010] In the formula I, R b is the following groups which are unsubstituted or optionally substituted by one or more R: (C1-C18)alkane, (C3-C8)carbocycloalkyl, (C2-C8)alkenyl, (C2-C8)alkynyl, (C6-C20)aryl, (C2-C20)heterocyclyl, (C2-C20)heterocyclyloxy;

[0011] In the formula I, each R may be the same or different and is independently selected from H, F, Cl, Br, I, OH, SH, CN, NO2, -COOR1, -CONR1R2, -NR1R2, -P(O)(OR1)(OR2), -S(O)2OR1, (C1-C18)alkyl, (C1-C18)alkyloxy, (C3-C8)carbocyclic alkyl, (C3-C8)carbocyclic alkyloxy, (C2-C8)alkenyl, (C2-C8)alkynyl, (C6-C20)aryl, (C6-C20)aryloxy, (C2-C20)heterocyclyl, (C2-C20)heterocyclyloxy;

[0012] Wherein, R1 and R2 may be the same or different and are independently selected from H, the following groups which are unsubstituted or optionally substituted by one or more R: (C1-C18) alkane, (C3-C8) carbocyclic alkyl, (C2-C8) alkenyl, (C2-C8) alkynyl, (C6-C20) aryl, (C2-C20) heterocyclic group;

[0013] In the formula I, X may represent an anion generated by ionization of an inorganic acid or an organic acid.

[0014] Furthermore, the compound represented by formula I may be a compound represented by the following formula II:

[0015]

[0016] In the formula II, R a is H or methyl;

[0017] In the formula II, R may be monosubstituted or polysubstituted, and each R may be the same or different and independently selected from any one of the following groups: H, F, Cl, Br, I, OH, SH, CN, NO2, -COOR1, -CONR1R2, -NR1R2, -P(O)(OR1)(OR2), -S(O)2OR1, (C1-C18) alkyl, (C1-C18) alkyloxy, (C3-C8) carbocyclic alkyl, (C3-C8) carbocyclic alkyloxy, (C2-C8) alkene wherein R1 and R2 are independently selected from any one of the following groups: H, the following groups which are unsubstituted or optionally substituted with one or more R: (C1-C18) alkane, (C3-C8) carbocyclic alkyl, (C2-C8) alkenyl, (C2-C8) alkynyl, (C6-C20) aryl, (C6-C20) aryloxy, (C2-C20) heterocyclyl, (C2-C20) heterocyclyloxy;

[0018] In the formula II, X may represent an anion generated by ionization of an inorganic acid or an organic acid.

[0019] Preferably, R is independently selected from any of the following groups: hydrogen, F, Cl, Br, I, methyl, ethyl, propyl, trifluoromethyl.

[0020] More preferably, said R is independently selected from H or methyl,

[0021] Preferably, X is selected from the group consisting of acid anions generated when any of the following acids are ionized: hydrochloric acid, methanesulfonic acid, sulfuric acid, fumaric acid, citric acid, tartaric acid, nitric acid, citric acid, phosphoric acid, hydrobromic acid, hydroiodic acid, succinic acid, acetic acid, lactic acid, benzenesulfonic acid, cinnamic acid, salicylic acid, malonic acid, glutaric acid, and malic acid.

[0022] Preferably, the pharmaceutically acceptable salt of the compound represented by Formula I may be any of the following salts: hydrochloride, methanesulfonate, sulfate, fumarate, citrate, tartrate, nitrate, lemon salt, phosphate, hydrobromide, hydroiodide, succinate, acetate, lactate, tosylate, cinnamate, salicylate, malonate, glutarate, and malate.

[0023] Specifically, the compound represented by formula I may be any of the following compounds:

[0024]

[0025] In the above structural formula, X is preferably selected from the acid anions generated when the following acids are ionized: hydrochloric acid, methanesulfonic acid, sulfuric acid, fumaric acid, citric acid, and tartaric acid.

[0026] The pharmaceutically acceptable salt of the compound of formula I described in the present invention refers to a salt that is suitable for contact with the tissues of humans and lower animals without excessive toxicity, irritation, allergic reaction, etc., and is commensurate with a reasonable effect / risk ratio within the scope of reliable medical judgment.

[0027] The present invention also provides the use of the compound represented by the above formula I or its pharmaceutically acceptable salt or stereoisomer in the preparation of at least one of the following drugs:

[0028] 1) Drugs for preventing and / or treating dry mouth;

[0029] 2) Drugs for the prevention and / or treatment of Sjögren's syndrome;

[0030] 3) Drugs related to the prevention and / or treatment of eye tissue diseases;

[0031] 4) Drugs for preventing and / or treating diseases associated with miosis.

[0032] The present invention also provides a medicine or pharmaceutical composition for preventing and / or treating a disease, comprising the compound represented by formula I as described above or a pharmaceutically acceptable salt, stereoisomer thereof, and a pharmaceutically acceptable carrier.

[0033] The disease is selected from at least one of the following:

[0034] a) dry mouth;

[0035] b) Sjögren's syndrome;

[0036] c) diseases of eye tissues;

[0037] d) Diseases associated with miosis.

[0038] The eye tissue diseases described in the present invention may specifically include glaucoma, presbyopia, and dry eye; the diseases associated with miosis may specifically include glaucoma and presbyopia.

[0039] The drug can be introduced into the body such as muscle, intradermal, subcutaneous, intravenous, mucosal tissue by oral administration, injection, spray, nasal drops, eye drops, penetration, absorption, physical or chemical mediation; or introduced into the body after being mixed or wrapped with other substances.

[0040] Preferably, the dosage form of the drug or pharmaceutical composition is an oral solid preparation or liquid preparation. The drugs in the above-mentioned various dosage forms can be prepared according to conventional methods in the pharmaceutical field.

[0041] Compared with the prior art, the present invention has the following beneficial effects:

[0042] Acetaminophen has the function of promoting saliva secretion and can be used to treat dry mouth. However, after oral administration to rats, it was found that the drug concentration reached a maximum of 2501.17 ng / ml at 0.25 hours, and decayed to 21.28 ng / ml after 4 hours. The drug concentration at 0.25 hours was more than 100 times that at 4 hours, indicating that after oral administration, side effects may be caused by excessive peak concentration in a short period of time, and then rapidly decayed to 21.28 ng / ml after 4 hours, so that the drug concentration was too low and affected the efficacy. Because dry mouth needs to be taken before bedtime, after 4 hours, the drug concentration was too low to affect the treatment. Assuming that the drug concentration at 4 hours is the required effective drug concentration, then the excessively high drug concentration at 0.25 hours can be regarded as unnecessary and a drug concentration that produces side effects. Based on this, we designed and screened a series of compounds and found that the compound of formula I can not only reduce the peak concentration at 0.25 hours to a lower level and reduce the side effects caused by the peak concentration, but also the drug concentration has been higher than that of the aceclidine group since 1 hour. At the 4-hour time point, it is more than ten times that of the aceclidine, and at the 8-hour time point, it is several dozen times that of the aceclidine. The drug-time curve is significantly flat and long-lasting, which is more conducive to oral administration for the treatment of diseases such as dry mouth. DETAILED DESCRIPTION

[0043] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0044] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0045] Example 1. Synthesis of 3-acetoxyquinuclidine-1-chloroonium salt (Q1)

[0046]

[0047] Dissolve quinuclidine-3-ol (10 g, 78.7 mmol) in dichloromethane (50 mL), add acetic anhydride (9.64 g, 94.5 mmol) and triethylamine (15.9 g, 157 mmol) respectively, and stir at room temperature for 5 h. Concentrate the reaction solution, purify the residual liquid by column chromatography, and salt the sample in hydrogen chloride ethyl acetate solution to obtain white solid 3-acetoxyquinuclidine-1-chloroium salt (14.6 g, 90.0%). 1H NMR (CDCl3 400MHz) δ10.65(s,1H),4.88(m,1H),3.33-2.81(m,6H),2.14-1.53(m,8H).ESI-MS m / z:170.1[M+H] + .

[0048] Example 2. Synthesis of 3-acetoxy-1-(((2,4-dimethylbenzoyl)oxy)methyl)quinuclidine-1-chloroonium salt (Q2)

[0049]

[0050] 1) 2,4-Dimethylbenzoic acid chloromethyl ester

[0051]

[0052] Dissolve 2,4-dimethylbenzoic acid (10.0 g, 66.7 mmol) in dichloromethane (100 mL), add water (100 mL), sodium bicarbonate (22.1 g, 267 mmol), and tetrabutylammonium hydrogen sulfate (2.26 g, 6.67 mmol) respectively, and stir evenly. The mixed solution is cooled to 0°C, and chloromethyl chlorosulfonate (14.3 g, 86.7 mmol) is added dropwise. After the addition is complete, react at room temperature for 2 h. The reaction solution is allowed to stand for stratification, and the organic phase is concentrated and purified by column chromatography to obtain a transparent oily substance, 2,4-dimethylbenzoic acid chloromethyl ester (11.8 g, 89.5%). 1 H NMR (CDCl3 400MHz) δ7.89(d,1H),7.33(s,1H),7.28(d,1H),5.93(s,2H),2.63(s,3H),2.36(s,3H).ESI-MS m / z:199.6[M+H] + .

[0053] 2) 3-Acetoxy-1-(((2,4-dimethylbenzoyl)oxy)methyl)quinuclidine-1-chloroium salt

[0054]

[0055] 3-Acetoxyquinuclidine-1-chloroium salt (2.0 g, 9.73 mmol), 2,4-dimethylbenzoic acid chloromethyl ester (1.93 g, 9.73 mmol), and sodium iodide (1.46 g, 9.73 mmol) were mixed in anhydrous acetonitrile (20 mL) and refluxed for 8 h. The reaction solution was concentrated, and the residual liquid was subjected to column chromatography to obtain white solid 3-acetoxy-1-(((2,4-dimethylbenzoyl)oxy)methyl)quinuclidine-1-chloroium salt (2.34 g, 65.4%). 1H NMR (CDCl3 400MHz) δ7.95(d,1H),7.12(m,2H),5.92(s,2H),5.19(m,1H),4.33-3.73(m,6H),2.55-2.12(m,14H).ESI-MS m / z:332.2[M + ].

[0056] Example 3. Synthesis of 3-acetoxy-1-(1-((2,4-dimethylbenzene)oxy)ethyl)quinuclidine-1-chloroonium salt (Q3)

[0057]

[0058] 1) 2,4-Dimethylphenyl acid-1-chloroethyl ester

[0059]

[0060] 2,4,6-trimethyl-1,3,5-trioxane (4.28 g, 32.4 mmol) and anhydrous zinc chloride (0.05 g, 0.324 mmol) were dissolved in DCM (40 mL) respectively, and 2,4-dimethylbenzoyl chloride (5.5 g, 32.4 mmol) was added dropwise in an ice-water bath (5±5°C), and the mixture was reacted at constant temperature for 5 h. Water was added to quench the reaction, and the organic phase was concentrated and column chromatography was performed to obtain a colorless oily substance, 2,4-dimethylphenyl acid-1-chloroethyl ester (4.16 g, 60.5%). 1 H NMR (CDCl3 400MHz) δ7.96(d,1H),7.44(s,1H),7.27(d,1H),6.78(m,1H),2.62(s,3H),2.46(s,3H),1.92(d,3H).ESI-MS m / z:213.6[M+H] + .

[0061] 2) 3-Acetoxy-1-(1-((2,4-dimethylbenzene)oxy)ethyl)quinuclidine-1-chloroium salt

[0062]

[0063] 3-Acetoxyquinuclidine-1-chloroium salt (2.0 g, 9.73 mmol), 2,4-dimethylphenyl acid-1-chloroethyl ester (2.07 g, 9.73 mmol), and sodium iodide (1.46 g, 9.73 mmol) were mixed in anhydrous acetonitrile (20 mL) and refluxed for 8 h. The reaction solution was concentrated, and the residual liquid was subjected to column chromatography to obtain a white solid 3-acetoxy-1-(1-((2,4-dimethylbenzene)oxy)ethyl)quinuclidine-1-chloroium salt (2.31 g, 62.5%). 1H NMR (CDCl3 400MHz) δ7.98(d,1H),7.17(m,2H),6.26(m,1H),5.34(m,1H),4.45-3.66(m,6H),2.57-2.04(m,17H).ESI-MS m / z:346.2[M + ].

[0064] Example 4. Synthesis of 3-acetoxy-1-(((2-methylbenzoyl)oxy)methyl)quinuclidine-1-chloroonium salt (Q4)

[0065]

[0066] According to the synthesis methods of Examples 1 and 2, 2-methylbenzoic acid chloromethyl ester was first prepared using 2-methylbenzoic acid, and then alkylated with 3-acetoxyquinuclidine-1-chloroonium salt to obtain 3-acetoxy-1-(((2-methylbenzoyl)oxy)methyl)quinuclidine-1-chloroonium salt. 1 H NMR (CDCl3 400MHz) δ7.87(d,1H),7.15(m,2H),7.03(d,1H),5.79(s,2H),5.15(m,1H),4.38-3.57(m,6H),2.43-2.11(m,11H).ESI-MS m / z:318.2[M + ].

[0067] Example 5. Synthesis of 3-acetoxy-1-(((2-methylbenzoyl)oxy)ethyl)quinuclidine-1-chloroonium salt (Q5)

[0068]

[0069] According to the synthesis methods of Examples 1 and 3, 2-methylbenzoic acid-1-chloroethyl ester was first prepared by 2-methylbenzoyl chloride, and then alkylated with 3-acetoxyquinuclidine-1-chloroonium salt to obtain 3-acetoxy-1-(((2-methylbenzoyl)oxy)ethyl)quinuclidine-1-chloroonium salt. 1 H NMR (CDCl3 400MHz) δ7.86(d,1H),7.18(m,2H),7.04(d,1H),5.98(m,1H),5.22(m,1H),4.52-3.64(m,6H),2.46-2.18(m,14H).ESI-MS m / z:332.2[M + ].

[0070] Example 6. Synthesis of 3-acetoxy-1-(((2-methylbenzoyl)oxy)methyl)quinuclidine-1-methanesulfonate

[0071]

[0072] 3-Acetoxy-1-(((2-methylbenzoyl)oxy)methyl)quinuclidine-1-chloroium salt (0.5 g, 1.41 mmol) was dissolved in water (20 mL), desalted by AG MP-1M anion exchange resin, and the eluate was neutralized with methanesulfonic acid, concentrated and recrystallized to obtain light yellow 3-acetoxy-1-(((2-methylbenzoyl)oxy)methyl)quinuclidine-1-methanesulfonic acid salt (0.49 g, 83.7%). 1 H NMR (CDCl3 400MHz) δ7.88(d,1H),7.16(m,2H),7.05(d,1H),5.80(s,2H),5.25(m,1H),4.38-3.57(m,6H),2.68(S,3H),2.43-2.12(m,11H).ESI-MS m / z:318.2[M + ].

[0073] Example 7. Synthesis of 3-acetoxy-1-(((2-methylbenzoyl)oxy)methyl)quinuclidine-1-sulfate salt

[0074]

[0075] The salt conversion method of Example 6 was combined to prepare 3-acetoxy-1-(((2-methylbenzoyl)oxy)methyl)quinuclidine-1-sulfate salt. 1 H NMR (CDCl3 400MHz) δ12.0(s,1H),7.86(d,1H),7.18(m,2H),7.07(d,1H),5.78(s,2H),5.23(m,1H),4.38-3.57(m,6H),2.43-2.12(m,11H).ESI-MS m / z:318.2[M + ].

[0076] Example 8 Synthesis of 3-acetoxy-1-(((2-methylbenzoyl)oxy)methyl)quinuclidine-1-fumaric acid onium salt

[0077]

[0078] The salt conversion method of Example 6 was combined to prepare 3-acetoxy-1-(((2-methylbenzoyl)oxy)methyl)quinuclidine-1-fumaric acid onium salt. 1H NMR(CDCl3 400MHz)δ11.0(s,1H),7.86(d,1H),7.29-7.11(m,4H),6.45(d,1H),5.78(s,2H),5.23(m,1H),4.38-3.57(m,6H),2.43-2.12(m,11H).ESI-MS m / z:318.2[M + ].

[0079] Example 9. Synthesis of 3-acetoxy-1-(((2-methylbenzoyl)oxy)methyl)quinuclidine-1-citrate

[0080]

[0081] The salt conversion method of Example 6 was combined to prepare 3-acetoxy-1-(((2-methylbenzoyl)oxy)methyl)quinuclidine-1-citrate salt. 1 H NMR (CDCl3 400MHz) δ11.0(s,2H),7.86(d,1H),7.18(m,2H),7.04(d,1H),5.98(m,1H),5.22(m,1H),4.52-3.64(m,7H),2.66-2.18(m,15H).ESI-MS m / z:318.2[M + ].

[0082] Example 10. Synthesis of 3-acetoxy-1-(((2-methylbenzoyl)oxy)methyl)quinuclidine-1-phosphate

[0083]

[0084] The salt conversion method of Example 6 was combined to prepare 3-acetoxy-1-(((2-methylbenzoyl)oxy)methyl)quinuclidine-1-phosphate. 1 H NMR (CDCl3 400MHz) δ12.0(s,2H),7.86(d,1H),7.18(m,2H),7.04(d,1H),5.98(m,1H),5.22(m,1H),4.52-3.64(m,6H),2.46-2.18(m,11H).ESI-MS m / z:318.2[M + ].

[0085] Example 11. Synthesis of 3-acetoxy-1-(((2-methylbenzoyl)oxy)methyl)quinuclidine-1-iodonium salt

[0086]

[0087] The salt conversion method of Example 6 was combined to prepare 3-acetoxy-1-(((2-methylbenzoyl)oxy)methyl)quinuclidine-1-iodonium salt. 1 H NMR (CDCl3 400MHz) δ7.87(d,1H),7.15(m,2H),7.03(d,1H),5.79(s,2H),5.15(m,1H),4.38-3.57(m,6H),2.43-2.11(m,11H).ESI-MS m / z:318.2[M + ].

[0088] By referring to the above examples, other inorganic and organic salts of the compound of formula I can be obtained.

[0089] Example 12. Pharmacokinetic study of compounds Q1, Q2, Q3, Q4 and Q5 in SD rats after oral administration

[0090]

Objective

[0091]

Method

[0092] 1 Experimental Materials

[0093] 1.1 Test Articles

[0094] 1.1.1Q1: Test product number: TN-2261, white powdery solid, molecular weight: 205.68, source: Guangzhou Zhongshan Eye Center, batch number: 20221117, purity: 98.0%, storage conditions: sealed in a dark place, refrigerated at 2-8℃, valid until 2023.08.03.

[0095] 1.1.2Q2 (hydrochloride): Test product number: TN-2262, white powdery solid, molecular weight: 367.87, source: homemade according to the method in Example 2, batch number: 20220727, purity: 98.37%, storage conditions: sealed in a dark place, refrigerated at 2-8°C, valid until 2023.08.03.

[0096] 1.1.3Q3 (hydrochloride): Sample number: TN-22109, white powdery solid, molecular weight: 381.89, source: homemade according to the method in Example 3, batch number: 20221025, purity: 99.0%, storage conditions: sealed in a dark place, refrigerated at 2-8°C, valid until 2023.11.06.

[0097] 1.1.4Q4 (hydrochloride): white powder solid, molecular weight: 353.84, source: homemade with reference to the method of Example 4, batch number: 20221015, purity: 99.0%, storage conditions: sealed in the dark, refrigerated at 2-8°C, valid until 2023.10.14.1.1.4Q5 (hydrochloride): white powder solid, molecular weight: 367.87, source: homemade with reference to the method of Example 5, batch number: 20221225, purity: 99.0%, storage conditions: sealed in the dark, refrigerated at 2-8°C, valid until 2023.12.24. Note: The weighing of the test sample did not convert the ion salt and purity.

[0098] 1.2 Tools and main reagents

[0099] 1.2.1 Acetonitrile (chromatographically pure): Merck, Germany, batch number: K54201330215, valid until 2023.11.10 after opening. 1.2.2 Methanol (chromatographically pure): Merck, Germany, batch number: I1154907124, valid until 2023.11.10 after opening. 1.2.3 Formic acid (chromatographically pure): McLean, batch number: C12752526, valid until 2023.02.05 after opening.

[0100] 1.2.4 Distilled water: Guangzhou Watsons Food and Beverage Co., Ltd., batch number: 20210731, valid until: 2022.11.11 after opening.

[0101] 1.2.5 100 μg / mL diazepam acetonitrile solution: provided by the New Drug Evaluation Center of Hebei Yiling Pharmaceutical Research Institute Co., Ltd., valid until: 2020.07.07.

[0102] 1.3 Experimental system

[0103] 1.3.1 Animal strain: SD rats.

[0104] 1.3.2 Animal grade: SPF grade.

[0105] 1.3.3 Animal sex and number: 21 male rats in total, 6 of which were used for the collection of blank rat plasma.

[0106] 1.3.4 Animal weight: 190-220 g.

[0107] 1.3.5 Animal source: purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd.

[0108] 1.3.6 Rearing conditions: The animals were kept in the New Drug Evaluation Center of Hebei Yiling Pharmaceutical Research Institute Co., Ltd. with 12 hours of light per day, a temperature of 20-26°C, and a relative humidity of 40-70%.

[0109] 1.3.7 Quarantine process: The new animals will be observed for 3 days to adapt to the new environment. The animals’ drinking water, food intake, health status, and signs of disease and death will be observed.

[0110] 1.3.8 Labeling: Marking with a marker pen.

[0111] 2 Test methods

[0112] 2.1 Experimental design basis

[0113] Adoption standards: Technical guidelines for nonclinical pharmacokinetic studies of drugs issued by the State Drug Administration.

[0114] 2.2 Dosage, grouping and sample collection

[0115] 2.2.1 Grouping: In this experiment, 15 rats were randomly divided into 5 groups, with 3 rats in each group. The specific grouping and dosage are shown in Table 1.

[0116] 2.2.2 Dosage: This trial was set up as equimolar dosing, with the dose for group Q1 being 10.0 mg / kg, group Q2 being 17.9 mg / kg, group Q3 being 18.6 mg / kg, group Q4 being 17.2 mg / kg, and group Q5 being 17.9 mg / kg.

[0117] 2.2.3 Sample collection: SD rats were divided into 5 groups according to the test products, with 3 rats in each group. The remaining 6 rats were used for blank plasma collection. Sample collection time points: 0.083h, 0.25h, 0.5h, 1h, 2h, 4h, 6h, 8h, 24h, and 48h after administration. Blood was collected from the retro-orbital venous plexus, and about 0.2mL of whole blood was collected directly into a K2EDTA anticoagulant tube, centrifuged at 3600rpm for 10min at 4℃, and the upper plasma was frozen at -20℃ for testing.

[0118] The test animals were fasted for at least 12 hours before sample collection, but not water. During sample collection, they were free to eat and drink freely 2 hours after administration.

[0119] Table 1 Test doses and groups

[0120]

[0121]

[0122] Note: The purity of the test sample is not converted.

[0123] 2.3 Administration

[0124] Route of administration: oral administration.

[0125] Dosage frequency and cycle: single dose.

[0126] Dosage and volume: see Table 1.

[0127] Administration method: Draw the corresponding drug solution with a syringe and administer it by oral gavage.

[0128] 2.4 Preparation and storage of test samples

[0129] Considering that the purity of the compounds this time is equal to / higher than 98.0%, no purity conversion is performed during the actual preparation process.

[0130] Q1 group: weigh 15.00 mg of Q1 (hydrochloride), add 7.5 mL of distilled water, vortex at gear 5 for 2 min, and sonicate until completely dissolved to obtain a Q2 (hydrochloride) gavage solution with a concentration of 2.00 mg / mL, which was prepared and used immediately.

[0131] Q2 group: weigh 26.85 mg of Q2 (hydrochloride), add 7.5 mL of distilled water, vortex at gear 5 for 2 min, and ultrasonicate until completely dissolved to obtain a Q2 (hydrochloride) gavage solution with a concentration of 3.58 mg / mL, which was prepared and used immediately.

[0132] Q3 group: Weigh 27.90 mg of Q3 (hydrochloride), add 7.5 mL of distilled water, vortex at gear 5 for 2 min, and sonicate until completely dissolved to obtain a Q3 (hydrochloride) gavage solution with a concentration of 3.72 mg / mL, which was prepared and used immediately.

[0133] Q4 group: weigh 25.8 mg of Q4 (hydrochloride), add 7.5 mL of distilled water, vortex at gear 5 for 2 min, and sonicate until completely dissolved to obtain a Q4 (hydrochloride) gavage solution with a concentration of 3.44 mg / mL, which was prepared and used immediately.

[0134] Q5 group: Weigh 26.85 mg of Q5 (hydrochloride), add 7.5 mL of distilled water, vortex at gear 5 for 2 min, and sonicate until completely dissolved to obtain a Q5 (hydrochloride) gavage solution with a concentration of 3.58 mg / mL, which was prepared and used immediately.

[0135] 2.5 Observation indicators, time and content

[0136] The LC-MS / MS method was used to detect the blood concentration of Q1 in plasma samples at each time point for quantitative analysis.

[0137] 2.6 Sample pretreatment before testing

[0138] Before testing, take the plasma sample out of the refrigerator (-20°C) and thaw naturally at room temperature. Take 50 μL to a 1.5 mL EP tube, add 150 μL acetonitrile (containing 1 ng / mL diazepam), vortex at gear 5 for 5 min, centrifuge at 12000 rpm for 5 min, and take 40 μL of the supernatant for injection analysis.

[0139] 2.7 Instrument system

[0140] Liquid spectrometer: AB SCIEX Exion LC / Triple Quad 5500 liquid spectrometer, equipped with an electrospray ionization source (ESI source) (AB SCIEX).

[0141] Data system: Analyst 1.6.3 (AB SCIEX), MultiQuantTM3.0.2 (AB SCIEX).

[0142] 2.8 Establishment of LC / MS / MS analysis method for Q1 in rat plasma

[0143] 2.8.1 Chromatographic conditions

[0144] Column: ACQUITY HSS T3 C18, 1.8μm, 50×2.1mm (Waters)

[0145] Injection volume: 2 μL; Injector temperature: 15°C; Column temperature: 40°C

[0146] Mobile phase: Phase A is water (0.1% formic acid aqueous solution by volume) and phase B is acetonitrile

[0147] Needle washing solution: acetonitrile: water (1:1, v / v)

[0148] The liquid phase method is shown in Table 2.

[0149] Table 2 LC liquid phase method

[0150]

[0151] 2.8.2 Mass spectrometry conditions

[0152] The ESI ion source was used. In the positive ion detection mode, the MRM working mode was selected for secondary mass spectrometry analysis. The mass spectrometry detection working parameters are shown in Table 3.

[0153] Table 3 Mass spectrometry detection parameters

[0154]

[0155]

[0156] Note: Q1-1 is the quantitative ion pair, and Q1-2 is the qualitative ion pair.

[0157] 2.9 Data processing and analysis

[0158] The calculation was based on the peak area ratio of the analyte to the internal standard diazepam. 2 ) The least squares method was used for regression calculation to obtain the linear regression equation, and the ratio was substituted into the equation to calculate the Q1 concentration. The plasma drug concentration-time curve was drawn using GraphPad Prism 8.0.1. The Q1 concentration data of the drug detected in all plasma samples were provided in the summary report results. The average plasma drug concentration and SD value were used to draw the drug-time curve.

[0159] 3 Results

[0160] 3.1 Standard curve

[0161] Q1 standard curve range: 1-1000ng / mL, quantitative lower limit: 1ng / mL, quality control sample concentration: 2, 40, 800ng / mL. During the test, the standard curve of each analytical batch r>0.99; more than 2 / 3 of the quality control samples meet the accuracy within ±15%, and the analytical batch is acceptable.

[0162] 3.2 Results of Q1 concentration test in SD rat plasma

[0163] LC / MS / MS analysis was performed on the plasma of 15 SD rats, and the ratio of the peak area of ​​Q1 in the sample to the internal standard was measured. The concentration of Q1 in the plasma was calculated by the standard curve equation, where the value below the lower limit of quantification, before the peak, was represented by 0, after the peak, it was represented by ND, and - represented that the value could not be calculated. The plasma drug concentration of Q1 in each group is shown in Tables 4 to 8.

[0164] 4 Conclusion

[0165] In general, after oral administration of Q2 (hydrochloride), Q3 (hydrochloride), Q4 (hydrochloride), and Q5 (hydrochloride) to rats, the active metabolite Q1 was detected in the body, and the half-life of Q1 in rats was prolonged to a certain extent; however, it is necessary to further organize the PK results of clinical trials and summarize the effective blood concentration levels in humans, so as to further evaluate the drugability of Q2, Q3, Q4, and Q5.

[0166] Table 4 Q1 plasma drug concentration (ng / mL) after oral administration of Q1 to SD rats

[0167]

[0168] Table 5 Plasma pharmacodynamic concentration of metabolite Q1 (ng / mL) after oral administration of Q2 (hydrochloride) to SD rats

[0169]

[0170]

[0171] Table 6 Plasma drug concentration of metabolite Q1 (ng / mL) after oral administration of Q3 (hydrochloride) to SD rats

[0172]

[0173] Note: The test results of rat No. 4 0.25h, 0.5h and rat No. 6 0.25h, 0.5h after dilution 10 times are quite different from the first undiluted results. Considering the possible contamination during the dilution process, the first test result shall prevail.

[0174] Table 7 Plasma pharmacodynamic concentration of metabolite Q1 (ng / mL) after oral administration of Q4 (hydrochloride) to SD rats

[0175]

[0176] Table 8 Plasma pharmacodynamic concentration of metabolite Q1 (ng / mL) after oral administration of Q5 (hydrochloride) to SD rats

[0177]

[0178]

Claims

1. The compound represented by formula I or its pharmaceutically acceptable salt or stereoisomer: In the formula I, R a is H or methyl; In the formula I, R b is the following groups which are unsubstituted or optionally substituted by one or more R: (C1-C18)alkane, (C3-C8)carbocycloalkyl, (C2-C8)alkenyl, (C2-C8)alkynyl, (C6-C20)aryl, (C2-C20)heterocyclyl, (C2-C20)heterocyclyloxy; In the formula I, each R may be the same or different and is independently selected from H, F, Cl, Br, I, OH, SH, CN, NO2, -COOR1, -CONR1R2, -NR1R2, -P(O)(OR1)(OR2), -S(O)2OR1, (C1-C18)alkyl, (C1-C18)alkyloxy, (C3-C8)carbocyclic alkyl, (C3-C8)carbocyclic alkyloxy, (C2-C8)alkenyl, (C2-C8)alkynyl, (C6-C20)aryl, (C6-C20)aryloxy, (C2-C20)heterocyclyl, (C2-C20)heterocyclyloxy; in, Said R1 and R2 are the same or different and are independently selected from H, the following groups which are unsubstituted or optionally substituted by one or more R: (C1-C18) alkane, (C3-C8) carbocyclic alkyl, (C2-C8) alkenyl, (C2-C8) alkynyl, (C6-C20) aryl, (C2-C20) heterocyclyl; In the formula I, X represents an anion generated by ionization of an inorganic acid or an organic acid.

2. The compound according to claim 1 or its pharmaceutically acceptable salt or stereoisomer, characterized in that: The compound represented by the formula I is a compound represented by the following formula II: In the formula II, R a is H or methyl; In the formula II, R is a single or multiple substituent groups, each R is the same or different, and is independently selected from any one of the following groups: H, F, Cl, Br, I, OH, SH, CN, NO2, -COOR1, -CONR1R2, -NR1R2, -P(O)(OR1)(OR2), -S(O)2OR1, (C1-C18) alkyl, (C1-C18) alkyloxy, (C3-C8) carbocyclic alkyl, (C3-C8) carbocyclic alkyloxy, (C2-C8) alkenyl, (C2-C8) alkynyl, (C6-C20) aryl, (C6-C20) aryloxy, (C2-C20) heterocyclyl, (C2-C20) heterocyclyloxy; Wherein, R1 and R2 are the same or different and are independently selected from any one of the following groups: H, the following groups which are unsubstituted or optionally substituted by one or more R: (C1-C18) alkane, (C3-C8) carbocyclic alkyl, (C2-C8) alkenyl, (C2-C8) alkynyl, (C6-C20) aryl, (C2-C20) heterocyclic group; In the above formula II, X represents an anion generated by ionization of an inorganic acid or an organic acid.

3. The compound according to claim 2 or its pharmaceutically acceptable salt or stereoisomer, characterized in that: The R is independently selected from any of the following groups: hydrogen, F, Cl, Br, I, methyl, ethyl, propyl, trifluoromethyl; preferably, the R is independently selected from H or methyl; Or, X is selected from the group consisting of acid anions generated when any of the following acids are ionized: hydrochloric acid, methanesulfonic acid, sulfuric acid, fumaric acid, citric acid, tartaric acid, nitric acid, citric acid, phosphoric acid, hydrobromic acid, hydroiodic acid, succinic acid, acetic acid, lactic acid, benzenesulfonic acid, cinnamic acid, salicylic acid, malonic acid, glutaric acid, and malic acid.

4. The compound according to claim 1 or 2, or a pharmaceutically acceptable salt or stereoisomer thereof, characterized in that: The pharmaceutically acceptable salt of the compound represented by Formula I is any of the following salts: hydrochloride, methanesulfonate, sulfate, fumarate, citrate, tartrate, nitrate, lemon salt, phosphate, hydrobromide, hydroiodide, succinate, acetate, lactate, tosylate, cinnamate, salicylate, malonate, glutarate, and malate.

5. The compound according to any one of claims 1 to 4 or a pharmaceutically acceptable salt or stereoisomer thereof, characterized in that: The compound represented by formula I is selected from any of the following compounds: In the above structural formula, X is selected from the acid anions generated when any of the following acids are ionized: hydrochloric acid, methanesulfonic acid, sulfuric acid, fumaric acid, citric acid, and tartaric acid.

6. Use of the compound according to any one of claims 1 to 5 or its pharmaceutically acceptable salt or stereoisomer in the preparation of at least one of the following drugs: 1) Drugs for preventing and / or treating dry mouth; 2) Drugs for the prevention and / or treatment of Sjögren's syndrome; 3) Drugs related to the prevention and / or treatment of eye tissue diseases; 4) Drugs for preventing and / or treating diseases associated with miosis.

7. The use according to claim 6, characterized in that: The eye tissue diseases include glaucoma, presbyopia, and dry eye; the diseases associated with miosis include glaucoma and presbyopia.

8. A drug or pharmaceutical composition for preventing and / or treating a disease, comprising the compound according to any one of claims 1 to 5 or a pharmaceutically acceptable salt or stereoisomer thereof, and a pharmaceutically acceptable carrier; The disease is selected from at least one of the following: a) dry mouth; b) Sjögren's syndrome; c) diseases of eye tissues; d) Diseases associated with miosis.

9. The drug or pharmaceutical composition according to claim 8, characterized in that: The eye tissue diseases include glaucoma, presbyopia, and dry eye; the diseases associated with miosis include glaucoma and presbyopia.

10. The drug or pharmaceutical composition according to claim 8 or 9, characterized in that: The dosage form of the medicine or pharmaceutical composition is an oral solid preparation or a liquid preparation.

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