CN119176806A - Aceclidine prodrug, and preparation method and application thereof - Google Patents

Abstract

The present invention discloses an aceclidine prodrug and a preparation method and application thereof. The aceclidine prodrug is a compound shown in formula I or a pharmaceutically acceptable salt or stereoisomer thereof. The aceclidine prodrug provided by the present invention 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, since 1 hour, the drug concentration has been higher than that of the aceclidine group, and is more than ten times that of the aceclidine drug concentration at the 4-hour time point, and is several dozen times that of the aceclidine drug concentration at the 8-hour time point. The drug-time curve is significantly flat and lasting, and is more conducive to oral administration for the treatment of diseases such as dry mouth.

Description

Aceclidine prodrug, and preparation method and application thereof

Technical Field

The invention belongs to the field of medicines, and particularly relates to a aceclidine prodrug, and a preparation method and application thereof.

Background

ACECLIDINE (aceclidine) is a muscarinic drug, a parasympathetic stimulant, for the treatment of open angle glaucoma, in the form of eye drops. It is a muscle type acetylcholine receptor agonist, having weak anticholinesterase activity. Has the following characteristics:

Has potent muscarinic receptor agonism, and can stimulate the contraction of the pupillary and ciliary muscles of the eye, resulting in pupillary constriction and myopia effects. The oral administration or after eye drop has rapid action, usually takes place within a few minutes, and the action time is short. Muscarinic receptor agonists (Muscarinic receptor agonist) are a class of drugs that act on muscarinic receptors and mimic the effects of acetylcholine, stimulating the excitation of muscarinic nerve endings, and thus causing a corresponding physiological response. Muscarinic receptor agonists include natural plant muscarinic, synthetic drugs such as carbachol, procaine choline and the like.

Muscarinic receptor agonists act primarily on muscarinic receptors, which are widely distributed in various tissues and organs within the human body, including the nervous system, cardiovascular system, digestive system, respiratory system, urinary system, eye, etc. Different muscarinic receptor subtypes have different distributions and functions in different tissues, and therefore muscarinic receptor agonists have a number of different roles, such as 1, increasing heart contractility and frequency, and decreasing heart conduction velocity. 2. Promoting peristalsis and secretion of digestive system, increasing gastrointestinal tract movement and secretion, and relieving constipation. 3. Expanding respiratory tract, increasing secretion of respiratory tract, and improving respiratory tract diseases. 4. Stimulating urinary system, increasing bladder contractility and urethral sphincter relaxation, and improving symptoms such as frequent urination and urgent urination. 5. Stimulating the pupil constrictor and ciliary muscle constriction of the eye, resulting in pupil constriction and myopia effects. The muscarinic receptor agonists should be administered at the time of use according to physician recommendations and prescriptions to avoid unnecessary side effects. Some common side effects include headache, dizziness, nausea, vomiting, diarrhea, tachycardia, etc. Can be used for treating eye diseases such as glaucoma and dislocation of crystalline lens, and can also be used for diagnosing eye diseases.

ACECLIDINE is an effective ophthalmic drug, but has not yet been reported for oral treatment of related diseases.

Disclosure of Invention

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

The aceclidine prodrug provided by the invention is a compound shown in a formula I or pharmaceutically acceptable salt and stereoisomer thereof:

in the formula I, R _a is H or methyl.

In the formula I, R _b is unsubstituted or substituted by one or more R, namely (C1-C18) alkane, (C3-C8) carbocyclylalkyl, (C2-C8) alkenyl, (C2-C8) alkynyl, (C6-C20) aryl, (C2-C20) heterocyclic group and (C2-C20) heterocyclyloxy;

In the formula I, each R is 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) carbocyclylalkyl, (C3-C8) carbocyclylalkyloxy, (C2-C8) alkenyl, (C2-C8) alkynyl, (C6-C20) aryl, (C6-C20) aryloxy, (C2-C20) heterocyclyl and (C2-C20) heterocyclyloxy;

Wherein R ₁、R₂, which may be the same or different, are independently selected from H, unsubstituted or optionally substituted with one or more R, a (C1-C18) alkane, (C3-C8) carbocyclylalkyl, (C2-C8) alkenyl, (C2-C8) alkynyl, (C6-C20) aryl, (C2-C20) heterocyclyl;

In the formula I, X can represent anions generated by ionization of inorganic acid or organic acid.

Further, the compound shown in the formula I can be a compound shown in the following formula II:

in the formula II, R _a is H or methyl;

In the formula II, R can be mono-substituted or multi-substituted, each R can be the same or different and is selected from any one of ：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) carbocyclylalkyl, (C3-C8) carbocyclyloxy, (C2-C8) alkenyl, (C2-C8) alkynyl, (C6-C20) aryl, (C6-C20) aryloxy, (C2-C20) heterocyclyl and (C2-C20) heterocyclyloxy, wherein R ₁、R₂ is selected from any one of H, C1-C18 alkane, C3-C8 carbocyclylalkyl, (C2-C8) alkenyl, (C2-C8) alkynyl, (C6-C20) aryl and (C2-C20) heterocyclyl which are unsubstituted or optionally substituted by one or more R;

in the formula II, X can represent anions generated by ionization of inorganic acid or organic acid.

Preferably, R is independently selected from any of hydrogen, F, cl, br, I, methyl, ethyl, propyl, trifluoromethyl.

More preferably, R is independently selected from H or methyl,

Preferably, X is selected from the group consisting of acid anions generated upon ionization of any of 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, malic acid.

Preferably, the pharmaceutically acceptable salt of the compound shown in the formula I can be any salt selected from hydrochloride, mesylate, sulfate, fumarate, citrate, tartrate, nitrate, citrate, phosphate, hydrobromide, hydroiodide, succinate, acetate, lactate, benzoate, cinnamate, salicylate, malonate, glutarate and malate.

Specifically, the compound shown in the formula I can be any one of the following compounds:

In the above formula, X is preferably selected from acid anions generated during ionization of hydrochloric acid, methanesulfonic acid, sulfuric acid, fumaric acid, citric acid and tartaric acid.

Pharmaceutically acceptable salts of the compounds of formula I as described herein are salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like commensurate with a reasonable benefit/risk ratio.

The invention also provides application of the compound shown in the formula I or pharmaceutically acceptable salts and stereoisomers thereof in preparing at least one of the following medicaments:

1) A medicament for preventing and/or treating xerostomia;

2) A medicament for preventing and/or treating dry syndrome;

3) A medicament for preventing and/or treating an ocular tissue disease;

4) A medicament for preventing and/or treating miosis-related diseases.

The invention also provides a medicine or a medicine composition for preventing and/or treating diseases, which comprises the compound shown in the formula I or pharmaceutically acceptable salt, stereoisomer and pharmaceutically acceptable carrier thereof.

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

a) Xerostomia;

b) Drying syndrome;

c) Ocular tissue disease;

d) Miosis-related diseases.

The eye tissue diseases in the invention can comprise glaucoma, presbyopia and xerophthalmia, and the diseases related to miosis can comprise glaucoma and presbyopia.

The medicine can be introduced into body by oral administration, injection, nasal drip, eye drop, permeation, absorption, physical or chemical mediated method, such as muscle, intradermal, subcutaneous, intravenous, mucosal tissue, or mixed or encapsulated with other substances.

Preferably, the dosage form of the medicament or the pharmaceutical composition is an oral solid preparation or a liquid preparation. The medicaments of the various formulations can be prepared according to the conventional method in the pharmaceutical field.

Compared with the prior art, the invention has the following beneficial effects:

Acetoricidine has the effect of promoting salivary secretion, and can be used for treating dry mouth, but after the rats are irrigated with stomach, the drug concentration reaches the highest 2501.17ng/ml after 0.25 hour, the attenuation is 21.28ng/ml after 4 hours, and the drug concentration is more than 100 times of that of 4 hours, which suggests that the peak concentration can be too high to cause side effects in a short time after oral administration, and the peak concentration can be quickly attenuated to 21.28ng/ml after 4 hours, so that the drug concentration is too low to influence the drug effect, because the dry mouth is needed to be taken before sleeping, and after 4 hours, the treatment is influenced because the drug concentration is too low, and the drug concentration too high for 0.25 hour can be regarded as unnecessary and can generate side effects at the same time. Based on the design of screening series of compounds, the compound shown in the formula I can reduce the peak concentration to a lower level in 0.25 hours, reduce side effects caused by the peak concentration, and meanwhile, the drug concentration is always higher than that of aceclidine group from 1 hour, the drug concentration is ten times higher than that of aceclidine at the 4-hour time point, the drug time curve is obviously smooth and durable in tens times higher than that of aceclidine at the 8-hour time point, and the oral administration is more beneficial to treating diseases such as dry mouth and the like.

Detailed Description

The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.

The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

EXAMPLE 1 Synthesis of 3-acetoxyquinuclidine-1-chloroonium salt (Q1)

Quinuclidin-3-ol (10 g,78.7 mmol) was dissolved in methylene chloride (50 mL), acetic anhydride (9.64 g,94.5 mmol) and triethylamine (15.9 g,157 mmol) were added respectively, and stirred at room temperature for 5h. Concentrating the reaction solution, purifying the residual liquid by column chromatography, and salifying the sample in ethyl hydrogen chloride acetate solution to obtain white solid 3-acetoxyquinuclidine-1-chloroonium salt (14.6g,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]⁺.

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

1) 2, 4-Dimethylbenzoic acid chloromethyl ester

2, 4-Dibenzoic acid (10.0 g,66.7 mmol) was dissolved in methylene chloride (100 mL), and water (100 mL), sodium hydrogencarbonate (22.1 g,267 mmol) and tetrabutylammonium bisulfate (2.26 g,6.67 mmol) were added, respectively, and stirred well. The mixture was cooled to 0deg.C and chloromethyl chlorosulfonate (14.3 g,86.7 mmol) was added dropwise and reacted at room temperature for 2h. Standing the reaction solution for layering, concentrating an organic phase, and purifying by column chromatography to obtain transparent oily 2, 4-dimethyl benzyl chloroformate (11.8g,89.5％).¹H 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]⁺.

2) 3-Acetoxy-1- (((2, 4-dimethylbenzoyl) oxy) methyl) quinuclidine-1-chloroonium salt

3-Acetoxyquinuclidine-1-chloroonium salt (2.0 g,9.73 mmol), chloromethyl 2, 4-dimethylbenzoate (1.93 g,9.73 mmol), and sodium iodide (1.46 g,9.73 mmol) were each mixed with anhydrous acetonitrile (20 mL), and reacted under reflux for 8 hours. Concentrating the reaction solution, and performing column chromatography on the residual solution to obtain white solid 3-acetoxyl-1- (((2, 4-dimethylbenzoyl) oxy) methyl) quinuclidine-1-chloroonium salt (2.34g,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⁺].

Example 3 Synthesis of 3-acetoxy-1- (1- ((2, 4-ditolyloxy) ethyl) quinuclidine-1-chloroonium salt (Q3)

1) 2, 4-Dimethylphenyl acid 1-chloroethyl ester

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), and 2, 4-dimethylbenzoyl chloride (5.5 g,32.4 mmol) was added dropwise to the solution in an ice-water bath (5.+ -. 5 ℃ C.) and reacted at constant temperature for 5h. Quenching with water, concentrating the organic phase, and column chromatography to obtain colorless oily 2, 4-dimethylphenyl acid-1-chloroethyl ester (4.16g,60.5％).¹H 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]⁺.

2) 3-Acetyloxy-1- (1- ((2, 4-ditolyl) oxy) ethyl) quinuclidine-1-chloroonium salt

3-Acetoxyquinuclidine-1-chloroonium 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 each mixed with anhydrous acetonitrile (20 mL) and reacted under reflux for 8 hours. Concentrating the reaction solution, and performing column chromatography on the residual solution to obtain white solid 3-acetoxyl-1- (1- ((2, 4-dimethylbenzoyl) oxy) ethyl) quinuclidine-1-chloroonium salt (2.31g,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⁺].

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

Synthesis of examples 1 and 2 by preparing chloromethyl 2-methylbenzoate from 2-methylbenzoic acid and alkylating with 3-acetoxyquinuclidine-1-chloroonium salt to obtain 3-acetoxy-1- (((2-methylbenzoyl) oxy) methyl) quinuclidine-1-chloroonium salt .¹H 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⁺].

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

Synthesis of examples 1 and 3 by preparing 2-methylbenzoyl chloride to give 1-chloroethyl 2-methylbenzoate and alkylating with 3-acetoxyquinuclidine-1-chloroonium salt to give 3-acetoxyquinuclidine-1- (((2-methylbenzoyl) oxy) ethyl) quinuclidine-1-chloroonium salt .¹H 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⁺].

Example 6 Synthesis of 3-acetoxy-1- (((2-methylbenzoyl) oxy) methyl) quinuclidine-1-methanesulfonium salt

3-Acetyloxy-1- (((2-methylbenzoyl) oxy) methyl) quinuclidine-1-chloroonium 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 give pale yellow 3-acetoxy-1- (((2-methylbenzoyl) oxy) methyl) quinuclidine-1-methanesulfonate (0.49g,83.7％).¹H 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⁺].

EXAMPLE 7 Synthesis of 3-acetoxy-1- (((2-methylbenzoyl) oxy) methyl) quinuclidine-1-sulfate

Synthesis of salt-transferring method of example 6 to obtain 3-acetoxy-1- (((2-methylbenzoyl) oxy) methyl) quinuclidine-1-onium sulfate salt .¹H 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⁺].

Example 8 Synthesis of 3-acetoxy-1- (((2-methylbenzoyl) oxy) methyl) quinuclidine-1-fumarate onium salt

Synthesis of salt-transferring method of example 6 to obtain 3-acetoxy-1- (((2-methylbenzoyl) oxy) methyl) quinuclidine-1-fumarate .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⁺].

Example 9 Synthesis of 3-acetoxy-1- (((2-methylbenzoyl) oxy) methyl) quinuclidine-1-citrate onium salt

Synthesis of salt-transferring method of example 6 to obtain 3-acetoxy-1- (((2-methylbenzoyl) oxy) methyl) quinuclidine-1-citrate onium salt .¹H 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⁺].

Example 10 Synthesis of 3-acetoxy-1- (((2-methylbenzoyl) oxy) methyl) quinuclidine-1-phosphate onium salt

Synthesis of salt-transferring method of example 6 to obtain 3-acetoxy-1- (((2-methylbenzoyl) oxy) methyl) quinuclidine-1-phosphate onium salt .¹H 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⁺].

EXAMPLE 11 Synthesis of 3-acetoxy-1- (((2-methylbenzoyl) oxy) methyl) quinuclidine-1-iodonium salt

Synthesis of salt-transferring method of example 6 to obtain 3-acetoxy-1- (((2-methylbenzoyl) oxy) methyl) quinuclidine-1-iodonium salt .¹H 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⁺].

With reference to the above examples, other inorganic and organic salts of the compounds of formula I may be obtained.

SD rat oral administration pharmacokinetic study of Compounds Q1, Q2, Q3, Q4, Q5 example 12

SD rats were given single equimolar gavage of Q2 (hydrochloride), Q3 (hydrochloride), Q4 (hydrochloride), Q5 (hydrochloride) and aceclidine (hydrochloride) Q1, and the generation of active metabolite aceclidine in SD rats of group Q2, group Q3, group Q4, and group Q5 was explored and compared with the pharmacokinetic profile in SD rats of group aceclidine.

SD rats were divided into 5 groups of 3 according to the test sample. The remaining 6 were used for blank plasma collection. Sample collection time points are 0.083h, 0.25h, 0.5h, 1h, 2h, 4h, 6h, 8h, 24h and 48h after administration. Collecting blood from retroorbital venous plexus, collecting about 0.2mL of whole blood, directly connecting into K ₂ EDTA anticoagulation tube, centrifuging at 3600rpm for 10min, and taking upper plasma for freezing at-20deg.C to obtain the final product. Before testing, the plasma samples were removed from the refrigerator (-20 ℃) and left to thaw naturally at room temperature. mu.L to 1.5mL EP tube was taken, 150. Mu.L acetonitrile (containing 1ng/mL diazepam) was added, 5 th vortex was applied for 5min, and the supernatant was centrifuged at 12000rpm for 5min, and 40. Mu.L of the supernatant was analyzed.

1. Experimental materials

1.1 Test article

1.1.1Q1 sample number TN-2261, white powdery solid, molecular weight 205.68, origin of Guangzhou mountain ophthalmology center, lot number 20221117, purity 98.0%, preservation condition of light-proof sealing, cold storage at 2-8 ℃ and effective period of 2023.08.03.

1.1.2Q2 (hydrochloride) sample No. TN-2262, white powdery solid, molecular weight 367.87, source of the product was prepared by the method of example 2, lot No. 20220727, purity 98.37%, and storage conditions were sealed in a dark place, refrigerated at 2-8deg.C, and shelf life up to 2023.08.03.

1.1.3Q3 (hydrochloride) sample No. TN-22109, white powdery solid, molecular weight 381.89, source of self-made product by referring to example 3, lot No. 20221025, purity 99.0%, preservation condition of light-proof sealing, cold storage at 2-8deg.C, and effective period of 2023.11.06.

1.1.4Q4 (hydrochloride) white powdery solid, the molecular weight 353.84, the source of which is self-made by the method of the example 4, the lot number of which is 20221015, the purity of which is 99.0%, and the preservation condition of which is sealed from light, and the refrigerating at 2-8 ℃ and the validity period of which is 2023.10.14.1.1.4Q5 (hydrochloride) white powdery solid, the molecular weight 367.87, the source of which is self-made by the method of the example 5, the lot number 20221225, the purity 99.0%, the preservation condition of which is light-proof sealing, and the refrigerating at 2-8 ℃ and the effective period of which is 2023.12.24. Remarks the sample weighing did not make a conversion of ionic salt and purity.

1.2 Tool medicine and Main reagent

1.2.1 Acetonitrile (chromatographic purity) Merck, germany, lot number K54201330215, expiration date after unsealing to 2023.11.10.1.2.2 methanol (chromatographic purity) Merck, germany, lot number I1154907124, expiration date after unsealing to 2023.11.10.1.2.3 formic acid (chromatographic purity) microphone, lot number C12752526, expiration date after unsealing to 2023.02.05.

1.2.4 Distilled water, guangzhou Chengshi food and beverage Co., ltd., lot number 20210731, and the expiration date after unsealing to 2022.11.11.

1.2.5 100 Mug/mL diazepam acetonitrile solution, which is provided by Hebei Kagaku Kogyo New drug evaluation center, the medical institute of Hebei Kagaku Kogyo, has the effective period of 2020.07.07.

1.3 Experiment System

1.3.1 Animal species SD rats.

Animal grade 1.3.2 SPF grade.

1.3.3 Animal sex and number Male mice, 21 total, 6 were used for plasma collection in blank rats.

1.3.4 Animal weight 190-220 g.

1.3.5 Animal sources purchased from Experimental animal technologies Co., ltd.

1.3.6 Raising conditions, namely raising the strain in Hebei Kagaku Kogyo GmbH of medical institute, wherein the strain is subjected to illumination for 12 hours/day at 20-26 ℃ and the relative humidity is 40-70%.

1.3.7 Quarantine procedure, new animals were adaptively observed for 3 days. Animals were observed for drinking, ingestion, and health status, and for the presence of disease and death symptoms.

1.3.8 Sign, marker pen sign.

2. Test method

2.1 Test design basis

Adopts the technical guidelines of non-clinical pharmacokinetics research of medicines issued by the national drug administration.

2.2 Dose, grouping and sample collection

2.2.1 Grouping 15 rats were taken from the trial and randomly grouped into 5 groups of 3 rats each. The specific groupings and dosing amounts are shown in Table 1.

2.2.2 Doses the test was set to equimolar doses, with doses of 10.0mg/kg for group Q1, 17.9mg/kg for group Q2, 18.6mg/kg for group Q3, 17.2mg/kg for group Q4 and 17.9mg/kg for group Q5.

2.2.3 Sample collection SD rats were divided into 5 groups of 3 according to the test sample. The remaining 6 were used for blank plasma collection. Sample collection time points are 0.083h, 0.25h, 0.5h, 1h, 2h, 4h, 6h, 8h, 24h and 48h after administration. The retroorbital venous plexus is sampled, about 0.2mL of whole blood is collected and directly connected into a K ₂ EDTA anticoagulation tube, the blood is centrifuged at 3600rpm for 10min under the condition of 4 ℃, and the upper plasma is taken out and frozen at-20 ℃ to be tested.

The test animals were fasted for at least 12 hours prior to sample collection, without water. During the sample collection process, the patient can eat and drink water freely after 2 hours of administration.

Table 1 test dose and groupings

Note that the amount of the sample is not converted into purity.

2.3 Methods of administration

The administration route is oral gastric lavage.

Frequency and period of administration, single administration.

The dosage and volume of the medicine are shown in Table 1.

The administration mode is that the injector extracts the corresponding liquid medicine for oral gastric lavage administration.

2.4 Preparation and preservation of samples

Considering that the purity of the compound is equal to/higher than 98.0%, the purity is not converted in the actual preparation process.

And in the group Q1, 15.00mg of Q1 (hydrochloride) is weighed, 7.5mL of distilled water is added, after 5-grade vortex is carried out for 2min, ultrasonic treatment is carried out until the solution is completely dissolved, and the Q2 (hydrochloride) gastric lavage solution with the concentration of 2.00mg/mL is obtained and is prepared for use.

And in the group Q2, 26.85mg of Q2 (hydrochloride) is weighed, 7.5mL of distilled water is added, after 5-grade vortex is carried out for 2min, ultrasonic treatment is carried out until the solution is completely dissolved, and the Q2 (hydrochloride) gastric lavage solution with the concentration of 3.58mg/mL is obtained and is prepared for use.

And in the group Q3, 27.90mg of Q3 (hydrochloride) is weighed, 7.5mL of distilled water is added, 5-grade vortex is carried out for 2min, ultrasonic treatment is carried out until the solution is completely dissolved, and the Q3 (hydrochloride) gastric lavage solution with the concentration of 3.72mg/mL is obtained and is prepared for use.

And in the Q4 group, 25.8mg of Q4 (hydrochloride) is weighed, 7.5mL of distilled water is added, after 5-grade vortex is carried out for 2min, ultrasonic treatment is carried out until the solution is completely dissolved, and the Q4 (hydrochloride) gastric lavage solution with the concentration of 3.44mg/mL is obtained and is prepared for use.

And in the group Q5, 26.85mg of Q5 (hydrochloride) is weighed, 7.5mL of distilled water is added, after 5-grade vortex is carried out for 2min, ultrasonic treatment is carried out until the solution is completely dissolved, and the Q5 (hydrochloride) gastric lavage solution with the concentration of 3.58mg/mL is obtained and is prepared for use.

2.5 Observed metrics, time and content

The plasma sample at each time point is tested for Q1 blood concentration by LC-MS/MS method, and quantitative analysis is performed.

2.6 Pretreatment before sample detection

Before testing, the plasma samples were removed from the refrigerator (-20 ℃) and left to thaw naturally at room temperature. mu.L to 1.5mL EP tube was taken, 150. Mu.L acetonitrile (containing 1ng/mL diazepam) was added, 5 th vortex was applied for 5min, and the supernatant was centrifuged at 12000rpm for 5min, and 40. Mu.L of the supernatant was analyzed.

2.7 Instrument System

LC-MS AB SCIEX Exion LC/Triple Quad 5500 LC-MS was equipped with an electrospray ionization source (ESI source) (AB SCIEX).

Data System-Analyst 1.6.3 (AB SCIEX Co.), multiQuantTM3.0.2 (AB SCIEX Co.).

2.8 Establishment of LC/MS/MS analysis method of Q1 in rat plasma

2.8.1 Chromatographic conditions

Chromatographic column ACQUITYHSS T3 C18,1.8μm,50×2.1mm(Waters)

The sample injection volume was 2. Mu.L, the injector temperature was 15℃and the column temperature was 40 ℃

Mobile phase A is water (0.1% formic acid water solution by volume) and B is acetonitrile

Needle washing liquid, acetonitrile and water (1:1, v/v)

The liquid phase process is shown in Table 2.

TABLE 2 LC liquid phase method

2.8.2 Mass Spectrometry conditions

An ESI ion source was used. And under the positive ion detection mode, selecting an MRM working mode for secondary mass spectrometry analysis. The mass spectrum detection working parameters are shown in table 3.

Table 3 mass spectrometric detection parameters

Note that Q1-1 is a quantitative ion pair and Q1-2 is a qualitative ion pair.

2.9 Data processing and analysis

And calculating by using the peak area ratio of the object to be detected and the internal standard diazepam. Regression calculation was performed by a weighted least square method (w=1/X ²), a linear regression equation was obtained, and the Q1 concentration was calculated by substituting the ratio into the equation. The plasma concentration-time curve was plotted with GRAPHPAD PRISM.8.0.1. The reported results are summarized to provide drug Q1 concentration data detected for all plasma samples. The time-to-drug curve is drawn by adopting average blood concentration and SD value.

3 Results

3.1 Standard Curve

The range of the Q1 standard curve is 1-1000 ng/mL, the lower limit of quantification is 1ng/mL, and the concentration of the quality control sample is 2, 40 and 800ng/mL. In the test process, the standard curve r of each analysis batch is more than 0.99, the accuracy of the quality control samples with more than 2/3 is within +/-15%, and the analysis batch is acceptable.

3.2 Detection results of the concentration of Q1 in plasma of SD rat

The ratio of Q1 to internal standard peak area in the sample was measured by LC/MS/MS analysis of 15 SD rat plasma, and the concentration of Q1 in the plasma was determined from standard curve equation, wherein, below the lower limit point of quantification, the peak was represented by 0 before reaching the peak, the peak was represented by ND after reaching the peak, and the value was not calculated. The plasma drug concentrations of each group of Q1 are shown in tables 4-8.

Conclusion 4

In general, after intragastric administration of Q2 (hydrochloride), Q3 (hydrochloride), Q4 (hydrochloride) and Q5 (hydrochloride) rats, the active metabolite Q1 can be detected in vivo, the half-life of the Q1 in the rats is prolonged to a certain extent, but PK results of clinical tests are required to be further finished, and the effective blood concentration levels of human bodies are summarized, so that further patent drug property evaluation is carried out on the Q2, the Q3, the Q4 and the Q5.

Table 4SD rats were given Q1 by gavage and Q1 plasma drug concentrations (ng/mL)

Table 5SD rats were given Q2 (hydrochloride) by gavage and the metabolite Q1 plasma drug effect concentration (ng/mL)

Table 6SD rats were given plasma drug concentrations (ng/mL) of metabolite Q1 following intragastric administration of Q3 (hydrochloride)

Note that the detection results after dilution of rats No.4 for 0.25h and 0.5h and rats No. 6 for 0.25h and 0.5h for 10 times are greatly different from the first undiluted result, and the first detection result is considered to be in consideration of possible pollution in the dilution process.

TABLE 7 plasma drug effect concentration of metabolite Q1 (ng/mL) following intragastric administration of Q4 (hydrochloride) in SD rats

Table 8SD rats were given Q5 (hydrochloride) by gavage and the metabolite Q1 plasma drug effect concentration (ng/mL)

Claims (10)

1. A compound of formula I or a pharmaceutically acceptable salt, stereoisomer thereof:

In the formula I, R _a is H or methyl;

In the formula I, R _b is unsubstituted or substituted by one or more R, namely (C1-C18) alkane, (C3-C8) carbocyclylalkyl, (C2-C8) alkenyl, (C2-C8) alkynyl, (C6-C20) aryl, (C2-C20) heterocyclic group and (C2-C20) heterocyclyloxy;

In the formula I, each R is 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) carbocyclylalkyl, (C3-C8) carbocyclylalkyloxy, (C2-C8) alkenyl, (C2-C8) alkynyl, (C6-C20) aryl, (C6-C20) aryloxy, (C2-C20) heterocyclyl and (C2-C20) heterocyclyloxy;

Wherein R ₁、R₂, which are identical or different, are independently of one another selected from H, unsubstituted or optionally substituted by one or more R, are (C1-C18) alkanes, (C3-C8) carbocyclylalkyl, (C2-C8) alkenyl, (C2-C8) alkynyl, (C6-C20) aryl, (C2-C20) heterocyclyl;

In the formula I, X represents anions generated by ionization of inorganic acid or organic acid.

2. A compound according to claim 1, or a pharmaceutically acceptable salt, stereoisomer thereof, wherein: the compound shown in the formula I is a compound shown in the following formula II:

in the formula II, R _a is H or methyl;

In the formula II, R is single or multiple substituent groups, each R is the same or different and is selected from any one ：H、F、Cl、Br、I、OH、SH、CN、NO₂、-COOR₁、-CONR₁R₂、-NR₁R₂、-P(O)(OR₁)(OR₂)、-S(O)₂OR₁、(C1-C18) alkyl group, a (C1-C18) alkyloxy group, a (C3-C8) carbocyclylalkyl group, a (C3-C8) carbocyclyloxy group, a (C2-C8) alkenyl group, a (C2-C8) alkynyl group, a (C6-C20) aryl group, a (C6-C20) aryloxy group, a (C2-C20) heterocyclic group and a (C2-C20) heterocyclic oxy group independently of each other;

Wherein R ₁、R₂, which are identical or different, are independently selected from any of H, unsubstituted or optionally substituted by one or more R, are (C1-C18) alkane, (C3-C8) carbocyclylalkyl, (C2-C8) alkenyl, (C2-C8) alkynyl, (C6-C20) aryl, (C2-C20) heterocyclyl;

In the formula II, X represents anions generated by ionization of inorganic acid or organic acid.

3. The compound of claim 2, wherein R is independently selected from any of hydrogen, F, cl, br, I, methyl, ethyl, propyl, trifluoromethyl, preferably, R is independently selected from H or methyl;

Or, the X is selected from acid anions generated when any acid is ionized, such as 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 or pharmaceutically acceptable salt or stereoisomer thereof according to claim 1 or 2, wherein the pharmaceutically acceptable salt of the compound of formula I is any of hydrochloride, mesylate, sulfate, fumarate, citrate, tartrate, nitrate, citrate, phosphate, hydrobromide, hydroiodide, succinate, acetate, lactate, benzoate, cinnamate, salicylate, malonate, glutarate, malate.

5. The compound according to any one of claims 1 to 4, wherein the compound of formula I is selected from any one of the following compounds:

wherein X is selected from acid radical anions generated by ionization of any acid selected from hydrochloric acid, methanesulfonic acid, sulfuric acid, fumaric acid, citric acid and tartaric acid.

6. Use of a compound according to any one of claims 1 to 5, or a pharmaceutically acceptable salt, stereoisomer thereof, for the preparation of at least one of the following:

1) A medicament for preventing and/or treating xerostomia;

2) A medicament for preventing and/or treating dry syndrome;

3) A medicament for preventing and/or treating an ocular tissue disease;

4) A medicament for preventing and/or treating miosis-related diseases.

7. The method according to claim 6, wherein the ocular tissue diseases comprise glaucoma, presbyopia and xerophthalmia, and the diseases related to mydriasis comprise glaucoma and presbyopia.

8. A medicament or pharmaceutical composition for the prevention and/or treatment of a disease comprising a compound according to any one of claims 1 to 5 or a pharmaceutically acceptable salt, stereoisomer thereof, and a pharmaceutically acceptable carrier;

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

a) Xerostomia;

b) Drying syndrome;

c) Ocular tissue disease;

d) Miosis-related diseases.

9. The pharmaceutical composition according to claim 8, wherein the ocular tissue diseases comprise glaucoma, presbyopia and dry eye, and the diseases related to miosis comprise glaucoma and presbyopia.

10. The pharmaceutical composition according to claim 8 or 9, wherein the pharmaceutical composition is in the form of an oral solid or liquid preparation.