A lactone compound, its preparation method and application

Abstract

The present invention discloses a lactone compound and a preparation method and application thereof. The structural formula of the lactone compound is shown in Formula I. The compound has suitable solubility and is relatively stable under storage conditions. Pharmacokinetic experiments show that pilocarpine can be detected in ocular tissues, and the drug concentration in the ciliary body target tissue is significantly higher than that in other tissues, avoiding the side effects caused by the burst effect of pilocarpine eye drops. After 4 hours, the drug concentration in the ciliary body is higher than that in the pilocarpine eye drops group, and the action time is prolonged, which is significantly better than pilocarpine. The compound can be prepared into liquid preparations, such as eye drops and injections, for the treatment of ocular tissue diseases. #imgabs0#

Description

Technical Field

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

[0002] Pilocarpine, also known as pilocarpine, is an M cholinergic receptor agonist that mainly stimulates M receptors to produce M-like effects. In 1875, Gutinha of Brazil extracted the alkaloids from the leaves of the American Pilocarpa plant. It is a tertiary amine compound with a stable aqueous solution and can now be artificially synthesized.

[0003] Pilocarpine can directly act on the M cholinergic receptors of the effector organs innervated by the postganglionic fibers of the parasympathetic nerves (including the sympathetic nerves innervating the sweat glands), especially on the eyes and glands. This drug can excite the M cholinergic receptors of the pupillary sphincter, which manifests as pupil constriction. Lowering intraocular pressure: Pilocarpine can pull the iris toward the center through the pupil constriction effect, thinning the root of the iris, thereby widening the anterior chamber angle gap around the iris, making it easier for aqueous humor to enter the scleral venous sinus through the filter curtain, and reducing intraocular pressure. Pilocarpine (10-15 mg subcutaneous injection) can significantly increase the secretion of sweat glands and salivary glands. In addition, the secretion of other glands such as lacrimal glands, gastric glands, pancreas, small intestinal glands and respiratory glands also increases.

[0004] In addition to stimulating the pupillary sphincter and ciliary muscle in the eye, pilocarpine can also stimulate the intestinal smooth muscle, bronchial smooth muscle, uterus, bladder and bile duct smooth muscle.

[0005] Clinical Application:

[0006] 1. Glaucoma: Low concentrations of pilocarpine eye drops can be used to treat angle-closure glaucoma. After use, the patient's pupils will shrink, the anterior chamber angle will expand, and the intraocular pressure will drop. However, high concentrations may aggravate the patient's symptoms, so it is not suitable for use. This product also has a certain effect on the early stage of open-angle glaucoma, but the mechanism is unclear.

[0007] 2. Alternate use of mydriatic drugs for iritis to prevent adhesion of the iris and lens.

[0008] 3. Others: This drug can increase saliva secretion and can be taken orally for dry mouth after neck radiation, but sweat secretion is also significantly increased. It can also be used as a rescue for poisoning by the anticholinergic drug atropine.

[0009] In our experiment, we found that after administering pilocarpine eye drops to New Zealand rabbits, the highest drug concentration detected in the aqueous humor reached 25580.00 ng / ml in 0.5 hours, and then rapidly decayed to 3113.60 ng / ml in 1 hour and 50.73 ng / ml in 4 hours. This phenomenon of rapid release to a high drug concentration in a short period of time is called the burst effect. The burst effect not only causes side effects due to excessively high drug concentrations in the initial period, but also due to rapid decay, the drug maintains its efficacy for a very short time, a double negative effect. At the same time, we It was found that the drug concentration of pilocarpine (P1 group) in the aqueous humor and cornea was much higher than that in the ciliary body, while the drug concentration in the ciliary body of the P2 and P3 groups was significantly higher than that in the aqueous humor and cornea. The ciliary body is the target organ tissue for the treatment of aging. The drug concentrations in various ocular tissues of the P2 and P3 groups tended to be flat at different time periods, avoiding the burst release effect of pilocarpine. After 4 hours, the ciliary body drug concentrations of the P2 and P3 groups began to be higher than that of the P1 group until 12 hours. This shows that P2 and P3 are more beneficial for the treatment of presbyopia and other diseases related to miosis. Summary of the invention

[0010] The purpose of the present invention is to provide an excellent pilocarpine prodrug.

[0011] The pilocarpine prodrug provided by the present invention is a compound represented by Formula I or a pharmaceutically acceptable salt thereof:

[0012]

[0013] In the formula I, R m A mono-substituted or poly-substituted R fragment compound of benzoic acid, wherein R can be the same or different substituents when poly-substituted, and the number of substituents m=0-5;

[0014] In the formula I, R is independently selected from hydrogen, halogen (Cl, Br, F, I), (C1-C5) alkane or (C1-C5) alkane containing a substituent, (C3-C8) carbocyclyl alkyl, (C2-C8) alkenyl or (C2-C8) alkenyl containing a substituent, (C2-C8) alkynyl or (C2-C8) alkynyl containing a substituent, (C6-C20) aryl or (C6-C20) aryl containing a substituent, (C2-C20) heterocyclyl or (C2-C20) heterocyclyl containing a substituent, -OR a , -NR b R c ,-SO2(OR d ).

[0015] The R a , R b , R c , Rd Independently selected from hydrogen, (C1-C5)alkane or (C1-C5)alkane containing a substituent, (C3-C8)carbocyclylalkyl, (C2-C8)alkenyl or (C2-C8)alkenyl containing a substituent, (C2-C8)alkynyl or (C2-C8)alkynyl containing a substituent, (C6-C20)aryl or (C6-C20)aryl containing a substituent, (C2-C20)heterocyclyl or (C2-C20)heterocyclyl containing a substituent.

[0016] Preferably, the R is independently selected from hydrogen, halogen, methyl, ethyl, propyl, trifluoromethyl, and the structural formula of the mono- or poly-substituted benzoic acid is as follows:

[0017]

[0018]

[0019] Further, in the formula I, the The acid anions are selected from the group consisting of hydrochloric acid, methanesulfonic acid, sulfuric acid, fumaric acid, citric acid, tartaric acid, nitric acid, citric acid, phosphoric acid, HBr, HI, succinic acid, acetic acid, hydrogen sulfate, lactic acid, benzenesulfonic acid, cinnamic acid, salicylic acid, malonic acid, glutaric acid, and malic acid.

[0020] Specifically, the compound represented by formula I may be any of the following compounds:

[0021]

[0022] In the above structural formula Preferred are acid anions produced when the following acids are ionized: hydrochloric acid, benzenesulfonic acid, sulfuric acid, fumaric acid, citric acid, tartaric acid.

[0023] 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.

[0024] Preferably, the pharmaceutically acceptable salt of the compound represented by Formula I is: hydrochloride, methanesulfonate, sulfate, fumarate, citrate, tartrate, nitrate, citrate, phosphate, hydrobromide, hydroiodide, succinate, acetate, bisulfate, lactate, toluenesulfonate.

[0025] The present invention also provides the use of the compound represented by the above formula I or a pharmaceutically acceptable salt thereof in the preparation of a drug for preventing and / or treating eye tissue diseases.

[0026] The eye tissue diseases are presbyopia, glaucoma, and adhesion of the iris and lens.

[0027] The present invention also provides the use of the compound represented by the above formula I or a pharmaceutically acceptable salt thereof in the preparation of a drug for preventing and / or treating diseases associated with miosis.

[0028] The present invention also provides a medicine or pharmaceutical composition for preventing and / or treating eye tissue diseases, comprising the compound represented by formula I as described above or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0029] The drug can be introduced into the body such as muscle, intradermal, subcutaneous, intravenous, mucosal tissue by injection, spraying, nasal drops, eye drops, penetration, absorption, physical or chemical mediation; or introduced into the body after being mixed or wrapped with other substances.

[0030] Preferably, the eye tissue disease is a disease related to pupil dilation such as presbyopia; or the eye tissue disease is an eye tissue disease with the ciliary body as the target tissue organ.

[0031] Preferably, the drug or pharmaceutical composition is in the form of a liquid preparation; preferably, the liquid preparation is an eye drop or an injection. The drugs in the above-mentioned various dosage forms can be prepared according to conventional methods in the pharmaceutical field.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] The compound represented by formula I of the present invention has suitable solubility and is an excellent pilocarpine prodrug. The compound represented by formula I of the present invention can avoid the burst release effect of pilocarpine eye drops, and the drug concentration is significantly gentle. Moreover, after 4 hours, the drug concentration of the compound represented by formula I in the ciliary body is higher than that of pilocarpine eye drops, and the drug concentration in the ciliary body targeted tissue organs is significantly higher than that of other tissues such as the cornea and aqueous humor, which is more conducive to the treatment of eye tissue diseases such as presbyopia. DETAILED DESCRIPTION

[0034] 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.

[0035] 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.

[0036] Example 1. Synthesis of 5-(((3R,4S)-4-ethyl-5-oxotetrahydrofuran-3-yl)methyl)-1-methyl-3-(((2-methylbenzoyl)oxy)methyl)-1-hydrogen-imidazole-3-chloroium salt (P-2)

[0037]

[0038] 1) Chloromethyl 2-methylbenzoate

[0039]

[0040] Dissolve 2-methylbenzoic acid (20.0 g, 147 mmol) in dichloromethane (200 mL), add water (200 mL), sodium bicarbonate (49.0 g, 588 mmol), and tetrabutylammonium hydrogen sulfate (5.0 g, 14.7 mmol) respectively, and stir evenly. The mixed solution is cooled to 0°C, and chloromethyl chlorosulfonate (29.0 g, 176 mmol) is added dropwise. After the addition is complete, react at room temperature for 2 hours. 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, chloromethyl 2-methylbenzoate (27.0 g, 99.9%). 1 HNMR(DMSO-d6 400MHz)δ7.97(d,1H),7.44(m,1H),7.26(m,2H),5.93(s,2H),2.63(s,3H).ESI-MS m / z:185.6[M+H] + .

[0041] 2) 5-(((3R,4S)-4-ethyl-5-oxotetrahydrofuran-3-yl)methyl)-1-methyl-3-(((2-methylbenzoyl)oxy)methyl)-1-hydrogen-imidazol-3-yl chloride

[0042]

[0043] Acetonitrile (100 mL), pilocarpine nitrate (20.0 g, 73.8 mmol), anhydrous sodium iodide (11.1 g, 73.8 mmol), and chloromethyl 2-methylbenzoate (16.3 g, 88.6 mmol) were added to a three-necked round-bottom flask and stirred at 60°C for 20 h. The reaction solution was filtered, concentrated, and purified by column chromatography to obtain a foamy solid 5-(((3R, 4S)-4-ethyl-5-oxotetrahydrofuran-3-yl)methyl)-1-methyl-3-(((2-methylbenzoyl)oxy)methyl)-1-hydrogen-imidazole-3-chloroium salt (14.5 g, 50.0%). 1H NMR(DMSO-d6 400MHz) δ9.89(s,1H),8.03(d,1H),7.72(s,1H),7.47(m,1H),7.28(m,2H),6.44(s,2H),4 .38-4.15(m,2H),4.01(s,3H),2.94-2.61(m,7H),1.17-1.50(m,2H),1.01(s,3H).ESI-MS m / z:357.1[M] + .

[0044] Example 2: Synthesis of 5-(((3R,4S)-4-ethyl-5-oxotetrahydrofuran-3-yl)methyl)-1-methyl-3-(((2-methylbenzoyl)oxy)methyl)-1-hydrogen-imidazole-3-methanesulfonate

[0045]

[0046] 5-(((3R,4S)-4-ethyl-5-oxotetrahydrofuran-3-yl)methyl)-1-methyl-3-(((2-methylbenzoyl)oxy)methyl)-1 hydrogen-imidazole-3-chloroium salt (1.0 g, 2.54 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 light yellow 5-(((3R,4S)-4-ethyl-5-oxotetrahydrofuran-3-yl)methyl)-1-methyl-3-(((2-methylbenzoyl)oxy)methyl)-1 hydrogen-imidazole-3-methanesulfonic acid onium salt (0.98 g, 85.0%).

[0047] 1 H NMR(DMSO-d6 400MHz) δ9.89(s,1H),8.03(d,1H),7.72(s,1H),7.47(m,1H),7.28(m,2H),6.44(s,2H),4 .38-4.15(m,2H),4.01(s,3H),2.94-2.61(m,7H),1.17-1.50(m,2H),1.01(s,3H).ESI-MS m / z:357.1[M] + .

[0048] Example 3. Synthesis of 5-(((3R,4S)-4-ethyl-5-oxotetrahydrofuran-3-yl)methyl)-1-methyl-3-(((2-methylbenzoyl)oxy)methyl)-1-hydrogen-imidazole-3-sulfate salt

[0049]

[0050] According to the salt conversion method of Example 2, 5-(((3R,4S)-4-ethyl-5-oxotetrahydrofuran-3-yl)methyl)-1-methyl-3-(((2-methylbenzoyl)oxy)methyl)-1-hydrogen-imidazole-3-sulfate salt can be obtained. 1 H NMR(DMSO-d6400MHz)δ9.89(s,1H),8.03(d,1H),7.72(s,1H),7.47(m,1H),7.28(m,2H),6.44(s,2H),4 .38-4.15(m,2H),4.01(s,3H),2.94-2.61(m,7H),1.17-1.50(m,2H),1.01(s,3H).ESI-MSm / z:357.1[M] + .

[0051] Example 4. Synthesis of 5-(((3R,4S)-4-ethyl-5-oxotetrahydrofuran-3-yl)methyl)-1-methyl-3-(((2-methylbenzoyl)oxy)methyl)-1-hydrogen-imidazole-3-fumaric acid onium salt

[0052]

[0053] According to the salt conversion method of Example 2, 5-(((3R,4S)-4-ethyl-5-oxotetrahydrofuran-3-yl)methyl)-1-methyl-3-(((2-methylbenzoyl)oxy)methyl)-1-hydrogen-imidazole-3-fumaric acid onium salt can be obtained. 1 H NMR(DMSO-d6400MHz)δ12.0(s,1H),9.89(s,1H),8.03(d,1H),7.72(s,1H),7.47(m,1H),7.28(m,2H),6.44(s ,2H),6.25(s,2H),4.38-4.15(m,2H),4.01(s,3H),2.94-2.61(m,7H),1.17-1.50(m,2H),1.01(s,3H).ESI-MS m / z:357.1[M] + .

[0054] Example 5. Synthesis of 5-(((3R,4S)-4-ethyl-5-oxotetrahydrofuran-3-yl)methyl)-1-methyl-3-(((2-methylbenzoyl)oxy)methyl)-1-hydrogen-imidazole-3-citrate

[0055]

[0056] According to the salt conversion method of Example 2, 5-(((3R,4S)-4-ethyl-5-oxotetrahydrofuran-3-yl)methyl)-1-methyl-3-(((2-methylbenzoyl)oxy)methyl)-1-hydrogen-imidazole-3-citrate salt can be obtained. 1 H NMR(DMSO-d6400MHz)δ12.0(s,2H),9.89(s,1H),8.03(d,1H),7.72(s,1H),7.47(m,1H),7.28(m,2H),6.44(s ,2H),6.25(s,2H),4.38-4.15(m,2H),4.01(s,3H),2.94-2.61(m,7H),1.17-1.50(m,6H),1.01(s,3H).ESI-MS m / z:357.1[M] + .

[0057] Example 6. Synthesis of 5-(((3R,4S)-4-ethyl-5-oxotetrahydrofuran-3-yl)methyl)-1-methyl-3-(((2-methylbenzoyl)oxy)methyl)-1-hydrogen-imidazolium-3-tartrate

[0058]

[0059] According to the salt conversion method of Example 2, 5-(((3R,4S)-4-ethyl-5-oxotetrahydrofuran-3-yl)methyl)-1-methyl-3-(((2-methylbenzoyl)oxy)methyl)-1-hydrogen-imidazole-3-tartrate salt can be obtained. 1 H NMR(DMSO-d6400MHz)δ12.0(s,1H),9.89(s,1H),8.03(d,1H),7.72(s,1H),7.47(m,1H),7.28(m,2H),6.44(s ,2H),5.05(s,2H),4.38-4.15(m,2H),4.01(s,3H),2.94-2.61(m,7H),1.17-1.50(m,2H),1.01(s,3H).ESI-MS m / z:357.1[M] + .

[0060] Example 7. Synthesis of 3-((Benzoyl)methyl)-5-(((3R,4S)-4-ethyl-5-oxotetrahydrofuran-3-yl)methyl)-1-methyl-1-hydrogen-imidazole-3-chloroium salt (P-3)

[0061]

[0062] According to the synthesis method of Example 1, chloromethyl benzoate was first prepared from benzoic acid, and then alkylated with pilocarpine nitrate to obtain a foamy solid 3-((benzoyl)methyl)-5-(((3R,4S)-4-ethyl-5-oxotetrahydrofuran-3-yl)methyl)-1-methyl-1-hydrogen-imidazole-3-chloroium salt. 1 H NMR(DMSO-d6 400MHz) δ9.89(s,1H),8.03(d,1H),7.72(s,1H),7.47(m,1H),7.28(m,2H),6.44(s,2H),4 .38-4.15(m,2H),4.01(s,3H),2.94-2.61(m,4H),1.17-1.50(m,2H),1.01(s,3H).ESI-MS m / z:343.1[M] + .

[0063] Example 8, 5-(((3R,4S)-4-ethyl-5-oxotetrahydrofuran-3-yl)methyl)-1-methyl-3-(((2,4-dimethylbenzoyl)oxy)methyl)-1-hydrogen-imidazole-3-chloroium salt (P-4)

[0064]

[0065] According to the synthesis method of Example 1, 2,4-dimethylbenzoic acid was used as a starting material to synthesize chloromethyl 2,4-dimethylbenzoate, which was then alkylated with pilocarpine nitrate to obtain a foamy solid 5-(((3R,4S)-4-ethyl-5-oxotetrahydrofuran-3-yl)methyl)-1-methyl-3-(((2,4-dimethylbenzoyl)oxy)methyl)-1-hydrogen-imidazole-3-chloroium salt. 1 HNMR(DMSO-d6 400MHz) δ9.89(s,1H),8.03(d,1H),7.72(s,1H),7.47(m,1H),7.28(m,2H),6.44(s,2H),4 .38-4.15(m,2H),4.01(s,3H),2.94-2.61(m,10H),1.17-1.50(m,2H),1.01(s,3H).ESI-MS m / z:371.1[M] + .

[0066] Other organic and inorganic salts can be obtained by using the synthesis methods of other salts in the above examples.

[0067] Similarly, other salts of the compound represented by Formula 1 can also be obtained.

[0068] Example 9: Exploration of pharmacokinetic characteristics of P-1 (pilocarpine), P-2, P-3, and P-4 in New Zealand rabbits after ocular administration

[0069]

Method

[0070] [Results] After 3.7 μmol / rabbit P-1, P-2, P-3, and P-4 eye drops were administered into the conjunctival sac of New Zealand rabbits, the main pharmacokinetics of pilocarpine in the iris-ciliary body were shown in the table below.

[0071]

[0072]

in conclusion

[0073] 1) After 3.7 μmol / rabbit of P-1, P-2, P-3, and P-4 eye drops were administered to the conjunctival sac of New Zealand rabbits, the iris-ciliary body pilocarpine reached its peak rapidly around 1 hour, and the exposure level (AUC (0-t) , Cmax) average values: P-1>P-4>P-2>P-3; however, the average drug concentration after 4 hours in P-4 group was significantly higher than that in P-1, P-2 and P-3 groups in the same time period.

[0074] 2) The concentration of pilocarpine in the iris-ciliary body of New Zealand rabbits in groups P-2, P-3, and P-4 1 / 2 , MRT (0-t) Compared with the P-1 group, it was significantly prolonged.

[0075] The detailed experimental methods and results are as follows:

[0076] Purpose of the test

[0077] The contents of the metabolite pilocarpine in the aqueous humor, cornea, conjunctiva, iris-ciliary body and other tissues were investigated 0.5, 1, 2, 4, 8 and 12 h after equimolar administration of P-1, P-2, P-3 and P-4 eye drops into the conjunctival sac of New Zealand rabbits, and the pharmacokinetic characteristics of P-1, P-2, P-3 and P-4 in the eyes of New Zealand rabbits were explored.

[0078] 1 Experimental Materials

[0079] 1.1 Test Articles

[0080] 1.1.1P-1 (pilocarpine): white powdery solid, molecular weight: 271, batch number: 20220422, purity: 99.0%, storage conditions: sealed in a dark place, refrigerated at 2-8°C, valid until 2023.04.21.

[0081] 1.1.2P-2: white powdery solid, molecular weight: 393, batch number: 20220422, purity: 99.0%, storage conditions: sealed and protected from light, refrigerated at 2-8°C, valid until 2023.04.21.

[0082] 1.1.3P-3: white powdery solid, molecular weight: 343.1, batch number: 20220422, purity: 99.0%, storage conditions: sealed and protected from light, refrigerated at 2-8°C, valid until 2023.04.21.

[0083] 1.1.4P-4: white powdery solid, molecular weight 371, batch number: 20220422, purity: 99.0%, storage conditions: sealed in a dark place, refrigerated at 2-8℃, valid until 2023.04.21.

[0084] 1.2 Tools and main reagents

[0085] 1.2.1 Acetonitrile (chromatographically pure): Merck, Germany, batch number: JB107430, valid until 2023.02.27 after opening. 1.2.2 Methanol (chromatographically pure): Merck, Germany, batch number: I1156207217, valid until 2023.06.14. 1.2.3 Formic acid (chromatographically pure): McLean, batch number: C12752526, valid until 2023.02.05 after opening.

[0086] 1.2.4 Distilled water: Guangzhou Watsons Food and Beverage Co., Ltd., batch number: 20210731, valid until: 2022.07.23 after opening.

[0087] 1.2.5 100 μg / mL diazepam acetonitrile solution, valid until: 2020.07.07 (expired, but can still be used).

[0088] 1.2.6 Ulitose: Yuanye Biotechnology, batch number: S16GS161476, valid until 2023.05.17 after opening.

[0089] 1.2.7 Sodium hydroxide: Comeau Reagent, batch number: 20140105, valid until 2023.01.02 after opening. 1.2.8 0.9% Sodium Chloride Injection: Shijiazhuang Siyao Co., Ltd., batch number: 2102283202, valid until 2022.07.20 after opening.

[0090] 1.3 Experimental system

[0091] 1.3.1 Animal strain: New Zealand rabbit.

[0092] 1.3.2 Animal level: ordinary level.

[0093] 1.3.3 Animal sex and number: 75 male New Zealand rabbits were purchased, 72 of which were selected for this experiment, and the remaining 3 were used to take blank tissue samples.

[0094] 1.3.4 Animal age at the start of drug administration: The protocol required 3 to 5 months of age, and the actual age was 3 to 5 months.

[0095] 1.3.5 Animal weight at the start of dosing: The protocol required that the animals weigh 1.5-2.0 kg at the time of purchase, and the actual animal weight was 1.523-1.803 kg. In this experiment, dosing was performed on a per-animal basis, so the actual body weight was not measured before dosing.

[0096] 1.3.6 Animal license issuing unit: Beijing Jinmuyang Experimental Animal Breeding Co., Ltd., license number: SCXK (Beijing) 2020-0002, acceptance date: July 11, 2022, certificate number: No.110333220100075037. 1.3.7 Adaptive feeding: Newly received animals are adaptively fed for 3 days. During this period, observe the animals' drinking water, food intake, health status, and whether there are signs of disease and death.

[0097] 1.3.8 Labeling: Ears are marked with pen numbers.

[0098] 2 Test methods

[0099] 2.1 Experimental design basis

[0100] 2.1.1 Adoption standards: Technical guidelines for non-clinical pharmacokinetic studies of drugs issued by the State Food and Drug Administration.

[0101] 2.2 Dosage and grouping

[0102] 2.2.1 Grouping: 72 male New Zealand rabbits were randomly divided into 4 groups, with 18 rabbits in each group.

[0103] 2.2.2 Dosage: The concentration of commercially available pilocarpine eye drops is usually 1%, i.e. 37 μmol / mL. Considering that the maximum volume of a single eye of a New Zealand rabbit is 50 μL (i.e. 100 μL / New Zealand rabbit), this experiment is designed to be an equimolar dose, 3.7 μmol / New Zealand rabbit: the dose of the P-1 group is 1.00 mg / New Zealand rabbit, the P-2 group is 1.45 mg / New Zealand rabbit, and the P-3 group is 1.27 mg / New Zealand rabbit. The P-4 group is 1.37 mg / New Zealand rabbit. See Table 1 for specific grouping and dosage.

[0104] Table 1 Dosage and grouping

[0105]

[0106] 2.3 Administration

[0107] The administration method consistent with clinical medication was adopted, that is, ocular administration, and the left and right eyes of the rabbit were administered the drug simultaneously.

[0108] 2.4 Preparation and storage of test samples

[0109] Considering that the purity of the compounds this time is 99%, no purity conversion is performed during the actual preparation process.

[0110] 1.00% P-1 eye drops: weigh 36.1 mg of pilocarpine (nitrate), add 3.61 mL of 0.9% sodium chloride injection, vortex at gear 5 for 2 min to dissolve, add 16 μL of 1 mol / L sodium hydroxide solution to adjust the pH to 6.02, and use immediately after preparation.

[0111] 1.45% P-2 eye drops: weigh 35.6 mg of P-2, add 2.46 mL of 0.9% sodium chloride injection, vortex at gear 5 for 2 min and ultrasonicate for 5 min, vortex at gear 5 for 2 min to dissolve, add 2 μL of 1 mol / L sodium hydroxide solution to adjust the pH to 6.28, and use immediately after preparation.

[0112] 1.27% P-3 eye drops: weigh 38.1 mg of P-3, add 3.00 mL of 0.9% sodium chloride injection, vortex at gear 5 for 2 min and ultrasonicate for 5 min, vortex at gear 5 for 2 min to dissolve, add 3 μL of 1 mol / L sodium hydroxide solution to adjust the pH to 6.41, and use immediately after preparation.

[0113] 1.37% P-4 eye drops: weigh 41.1 mg of P-4, add 3.00 mL of 0.9% sodium chloride injection, vortex at gear 5 for 2 min and ultrasonicate for 5 min, vortex at gear 5 for 2 min to dissolve, add 2 μL of 1 mol / L sodium hydroxide solution to adjust the pH to 6.11, and use immediately after preparation.

[0114] 2.5 Administration of test articles

[0115] The drug was administered to the left and right eyes of the rabbits at the same time, with a drug volume of 100 μL / New Zealand rabbit, 50 μL in each eye. The drug was accurately administered using a pipette: the rabbit eyelids were gently pulled down into a cup shape, and 50 μL of eye drops were accurately drawn with a pipette and dropped into the eyelids, and the rabbit eyes were passively closed for 10 seconds.

[0116] 2.6 Observation indicators, time and content

[0117] 2.6.1 Sample collection

[0118] The aqueous humor, cornea, conjunctiva, and iris-ciliary body were collected at 0.5, 1, 2, 4, 8, and 12 hours after administration. Three New Zealand rabbits were selected at each time point.

[0119] Collection of aqueous humor: For sacrificed animals, use a 1 mL syringe to insert the needle at the junction of the pupil and iris to extract about 200 μL of aqueous humor (note that the cut surface of the syringe needle should face downward when extracting aqueous humor to prevent aqueous humor from spraying out).

[0120] Collection of eyeballs: Use forceps to grab the eyeball, cut the tissue around it (be careful not to cut off the conjunctiva on the eye surface), take out the eyeball, and store it in the refrigerator.

[0121] Collection of conjunctiva: Remove the conjunctiva around the eyeball, wash it with saline, and then absorb the surface liquid with filter paper.

[0122] Collection of cornea and iris-ciliary body: Fix the eyeball with forceps, cut the cornea and iris, cut the cornea along the junction of the two, and then remove the iris-ciliary body. Wash the removed cornea and iris-ciliary body with physiological saline, and then use filter paper to absorb the surface liquid.

[0123] 2.6.2 Tissue sample processing

[0124] Preparation of corneal, conjunctival, and iris-ciliary body homogenates: First, use small scissors to cut the cornea / iris-ciliary body of the left and right eyes into small pieces, accurately weigh them, add 50% methanol aqueous solution (cornea, conjunctival mass volume ratio is 1:10, iris-ciliary body mass volume ratio is 1:20), use a grinder to grind (the program is 4 cycles of grinding, each cycle is 6500rpm for 30s and wait for 20s), use a low-speed centrifuge to centrifuge at 4000rpm for 10min, aspirate the supernatant, and freeze it for testing.

[0125] 2.7 Sample pretreatment before testing

[0126] Aqueous humor sample: Take 50 μL of aqueous humor, add 200 μL of internal standard working solution (1 ng / mL diazepam acetonitrile solution), vortex mix for 5 min at gear 5, centrifuge at 12000 rpm for 10 min, take the supernatant and put it into the automatic sampler vial for testing.

[0127] Cornea, conjunctiva, iris-ciliary body samples: Take 50 μL of the homogenate supernatant, add 200 μL of the internal standard working solution (1 ng / mL diazepam acetonitrile solution), vortex mix for 5 min at gear 5, centrifuge at 12000 rpm for 10 min, take the supernatant and put it into the automatic sampler vial for testing.

[0128] 2.8 Instrument system

[0129] Liquid spectrometer: AB SCIEX Exion LC / Triple Quad 5500 liquid spectrometer, equipped with an electrospray ionization source (ESI source) (AB SCIEX).

[0130] Data system: Analyst 1.6.3 (AB SCIEX), MultiQuantTM3.0.2 (AB SCIEX).

[0131] 2.9 Establishment of LC / MS / MS analysis method for pilocarpine in aqueous humor, corneal homogenate, conjunctival homogenate, and iris-ciliary body homogenate of New Zealand rabbits

[0132] 2.9.1 Chromatographic conditions

[0133] Chromatographic column: HSS T3C18, 1.8μm, 50×2.1mm (Waters)

[0134] Injection volume: 2 μL; Injector temperature: 15°C; Column temperature: 40°C

[0135] Mobile phase: Phase A is water (0.1% formic acid) and phase B is acetonitrile

[0136] Needle washing solution: acetonitrile: water (1:1, v / v)

[0137] The liquid phase method is shown in Table 2.

[0138] Table 2 LC liquid phase method

[0139]

[0140] 2.9.2 Mass spectrometry conditions

[0141] 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.

[0142] Table 3 Mass spectrometry detection working conditions

[0143]

[0144] Note: Pilocarpine-1 is the quantitative ion pair of pilocarpine, and DXP is the quantitative ion pair of internal standard diazepam.

[0145] 2.10 Data Processing

[0146] The calculation is based on the peak area ratio of the sample to the internal standard. 2 ) The least square method was used for regression calculation to obtain the linear regression equation, and the ratio was substituted into the equation to calculate the drug concentration of pilocarpine. PhoenixWinNonlin 8.3.5.340 software was used to calculate the pharmacokinetic parameters.

[0147] 3 Results

[0148] 3.1 Standard curve

[0149] During the test, the standard curve of each analytical batch had r>0.99; the quality control samples all met the accuracy within ±15%, and the analytical batch was acceptable.

[0150] 3.2 Results of pilocarpine concentration test in aqueous humor, cornea, conjunctiva, and iris-ciliary body of New Zealand rabbits

[0151] LC / MS / MS analysis was performed on aqueous humor, corneal homogenate, conjunctival homogenate, and iris-ciliary body homogenate of 72 New Zealand rabbits. The ratio of the peak area of ​​pilocarpine to the internal standard in the sample was measured, and the concentration of pilocarpine in different tissues was calculated by the standard curve equation. The concentration of the test substance in the cornea and conjunctiva was 11 times the concentration of the test substance in the tissue homogenate, and the concentration of the test substance in the iris-ciliary body was 21 times the concentration of the test substance in the tissue homogenate. The test results are shown in Tables 4 to 7.

[0152] Table 4 Drug concentration of pilocarpine in aqueous humor after administration of 3.7 μmol / rabbit P-1, P-2, P-3, P-4 eye drops to New Zealand rabbits (ng / mL)

[0153]

[0154]

[0155] Table 5 Drug concentration of pilocarpine in the cornea of ​​New Zealand rabbits after administration of 3.7 μmol / rabbit P-1, P-2, P-3, P-4 eye drops (ng / g)

[0156]

[0157] Table 6 Drug concentration of pilocarpine in the conjunctiva of New Zealand rabbits after administration of 3.7 μmol / rabbit P-1, P-2, P-3, P-4 eye drops (ng / g)

[0158]

[0159]

[0160] Table 7 Drug concentration of pilocarpine in the iris-ciliary body of New Zealand rabbits after administration of 3.7 μmol / rabbit P-1, P-2, P-3, P-4 eye drops (ng / g)

[0161]

[0162] 3.3 Calculation results of pharmacokinetic parameters of active metabolite pilocarpine in New Zealand rabbits

[0163] The pharmacokinetic software Phoenix WinNonlin 8.3.5.340 was used to calculate the main pharmacokinetic parameters of the active metabolite pilocarpine after administration of 3.7 μmol / rabbit P-1, P-2, P-3, and P-4 eye drops into the conjunctival sac of New Zealand rabbits. The specific results are shown in Tables 8 to 11.

[0164] Table 8 Pharmacokinetic parameters in aqueous humor of New Zealand rabbits after administration of 3.7 μmol / rabbit P-1, P-2, P-3, P-4 eye drops

[0165]

[0166] Table 9 Pharmacokinetic parameters in the cornea of ​​New Zealand rabbits after administration of 3.7 μmol / rabbit P-1, P-2, P-3, P-4 eye drops

[0167]

[0168]

[0169] Table 10 Pharmacokinetic parameters in the conjunctiva of New Zealand rabbits after administration of 3.7 μmol / rabbit P-1, P-2, P-3, P-4 eye drops

[0170]

[0171]

[0172] Table 11 Pharmacokinetic parameters in the iris-ciliary body of New Zealand rabbits after administration of 3.7 μmol / rabbit P-1, P-2, P-3, P-4 eye drops into the conjunctival sac

[0173]

[0174]

[0175] 4 Conclusion

[0176] After 3.7 μmol / rabbit of P-1, P-2, P-3, and P-4 eye drops were administered into the conjunctival sac of New Zealand rabbits, pilocarpine in the iris-ciliary body quickly reached a peak around 1 hour. The drug concentrations in the target organ iris and ciliary body of the P-2, P-3, and P-4 groups were significantly higher than those in other tissues. There was no excessive drug concentration within 0.5 hour, avoiding the side effects caused by the drug burst effect of the P-1 group. The drug was gradually released, the drug concentration was significantly flat, and after 4 hours the drug concentration was higher than that of the P-1 group. The half-life was significantly prolonged, which is more conducive to the treatment of eye tissue diseases, such as presbyopia.

[0177] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.

Claims

1. A compound represented by formula I or a pharmaceutically acceptable salt thereof: In the formula I, Represents anion.

2. The compound of formula I or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: In the formula I, The acid anions are selected from the group consisting of hydrochloric acid, methanesulfonic acid, sulfuric acid, fumaric acid, citric acid, tartaric acid, nitric acid, citric acid, phosphoric acid, HBr, HI, succinic acid, acetic acid, hydrogen sulfate, lactic acid, benzenesulfonic acid, cinnamic acid, salicylic acid, malonic acid, glutaric acid, and malic acid.

3. Use of the compound according to claim 1 or 2 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for preventing and / or treating eye tissue diseases; The eye tissue diseases are presbyopia, glaucoma, and adhesion of the iris and lens.

4. Use of the compound according to claim 1 or 2 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for preventing and / or treating a disease associated with miosis.

5. A pharmaceutical composition for preventing and / or treating eye tissue diseases, comprising the compound according to claim 1 or 2 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

6. The pharmaceutical composition according to claim 5, characterized in that: The eye tissue disease is a disease associated with pupil dilation.

7. The pharmaceutical composition according to claim 5 or 6, characterized in that: The dosage form of the pharmaceutical composition is a liquid preparation.

8. The pharmaceutical composition according to claim 7, characterized in that: The liquid preparation is eye drops or injection.

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