Deuterated compound of benzodiazepine and use thereof

Abstract

Disclosed are a deuterated compound of benzodiazepine, and a use thereof. The deuterated compound of benzodiazepine has the following general formula (I), R1 and R2 being independently selected from ethyl or deuterated ethyl, R3-R18 being independently selected from hydrogen or deuterium, and R1-R18 containing at least one deuterium. The deuterated compound of benzodiazepine provided by the present invention, or a pharmaceutically acceptable salt, hydrate or solvate thereof, has antagonistic activity against muscarinic M2 receptors. Compared with known selective M2 receptor inhibitors, the compound of the present invention has better inhibitory activity and pharmacodynamic properties, and has minimal activity on M3 receptors, thereby avoiding the side effect of pupil dilation (mydriasis). It has promising prospects for clinical applications in treating myopia, preventing myopia, and / or inhibiting the progression of myopia.

Description

Deuterated compounds of benzodiazepines and uses thereof TECHNICAL FIELD

[0001] The present application belongs to the field of chemical medicine technology, and particularly relates to a deuterated compound of benzodiazepines and uses thereof. BACKGROUND

[0002] Myopia is a common refractive abnormality, which is manifested as a decrease in distance vision. After the occurrence of myopia, the balance between accommodation and convergence is disrupted, and symptoms such as double vision, blurred vision, eye swelling, headache, nausea, eye fatigue, dryness, and other symptoms of myopia occur. According to the course of progression and pathological changes, myopia can be divided into two categories: simple myopia and pathological myopia. Simple myopia generally does not have changes in the fundus. Pathological myopia often has symptoms such as eyeball protrusion, deep anterior chamber, large pupil with slow response, and narrow palpebral fissure. Pathological myopia also increases the risk of macular degeneration, retinal detachment, cataract, open-angle glaucoma, and other diseases, and even severely damages vision (Haarman A. et al., Invest Ophthalmol Vis Sci, 2020, 61(4): 49).

[0003] In terms of disease mechanism, myopia is mainly related to external factors (e.g., close-range work, outdoor activities, lighting, reading habits, etc.) and genetic factors. According to the data released by the National Health Commission of China in 2020, the overall incidence of myopia among children and adolescents in China is 53.6%, and the overall incidence among college students is over 90%. With the popularity of computers, mobile phones, tablets, and other video tools, eye use has increased, and these factors have led to a high incidence of myopia at a young age. In 2020, the number of myopia patients aged 5 to 19 reached approximately 130 million. Globally, the World Health Organization reported in the World Vision Report in 2020 that the number of myopia patients has reached 2.6 billion.

[0004] Current methods for controlling myopia include optical correction, refractive surgery, and anticholinergic drug treatment. In drug treatment, muscarinic receptor antagonists represented by atropine are considered to be a more effective means (Walline J.J. et al., Cochrane Database Syst Rev, 2020, 1: D4916). However, it has been reported that after using 0.01%, 0.025%, and 0.05% atropine, adverse reactions such as pupil dilation and changes in accommodation ability have occurred (Joachimsen L. et al., Int Ophthalmol, 2021, 41(6): 2001-2008).

[0005] Therefore, there is an urgent need to find a drug that can significantly reduce or completely prevent side effects caused by mydriasis for preventing myopia and / or inhibiting the development of myopia. SUMMARY

[0006] The present application aims to provide a deuterated benzodiazepine compound which can be used as a muscarinic M2 receptor antagonist for preparing a drug for preventing myopia and / or inhibiting the development of myopia, and can significantly reduce or completely prevent side effects caused by mydriasis.

[0007] To achieve the above-mentioned purpose, the present application provides a deuterated benzodiazepine compound having the following general formula:

[0008] wherein R1, R2 are independently selected from ethyl or deuterated ethyl, R3-R18 are independently selected from H or deuterium, and at least one of R1-R18 contains deuterium.

[0009] Optionally, at least one of R1-R4 contains deuterium.

[0010] Optionally, at least one of R1 and R2 is deuterated ethyl.

[0011] Optionally, the deuterated ethyl contains at least one deuterium, multiple deuteriums or full deuterium.

[0012] Optionally, at least one of R5-R9 contains deuterium.

[0013] Optionally, the deuterated compound comprises any one of the following:

[0014] or a pharmaceutically acceptable salt, hydrate or solvate thereof.

[0015] The present application also provides a pharmaceutical composition comprising the above-mentioned deuterated compound or a pharmaceutically acceptable salt, hydrate or solvate thereof as an active ingredient.

[0016] Optionally, the pharmaceutical composition further comprises a pharmaceutical excipient.

[0017] The present application also provides the use of the above-mentioned deuterated benzodiazepine compound for preparing a drug for preventing myopia and / or inhibiting the development of myopia. The myopia includes at least one of axial myopia, refractive myopia, pseudomyopia, pathological myopia, simple myopia, super-high myopia, severe myopia, high myopia, moderate myopia, low myopia, myopia combined with glaucoma, myopia with risk of glaucoma, or myopia with high intraocular pressure.

[0018] Compared with the prior art, the technical scheme of the present application has at least the following beneficial effects:

[0019] The deuterium-substituted benzodiazepine compound or pharmaceutically acceptable salt, hydrate or solvate thereof provided by the present application has significantly improved muscarinic M2 receptor antagonism by specific selection of the number and position of deuterium substitution. In particular, the Human M2 and M3 receptor inhibition activity of the deuterium-substituted compound of the present application was verified by experiments, and the M3 / M2 IC 50 ratio was significantly increased, and the compound had specific pharmacological properties, which could effectively treat myopia without affecting pupil size. Compared with known selective M2 receptor inhibitors, the compound of the present application maintained M2 receptor inhibition for a longer time than AFDX-116, and had significantly better effects on delaying myopia progression than AFDX-116, better inhibition activity and pharmacokinetic properties, and better development prospects for clinical application in the treatment of myopia, prevention of myopia and / or inhibition of myopia development. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 is a control diagram showing the effect of AFDX-116 and compound 6 on the refractive power of a myopic guinea pig model in Example 17.

[0021] Figure 2 is a control diagram showing the effect of AFDX-116 and compound 6 on the axial length of a myopic guinea pig model in Example 17. DETAILED DESCRIPTION

[0022] The "myopia" described herein includes at least one of axial myopia, refractive myopia, pseudomyopia, pathological myopia, simple myopia, super-high myopia, severe myopia, high myopia, moderate myopia, low myopia, myopia combined with glaucoma, myopia with a risk of glaucoma, or myopia with high intraocular pressure. Among them, super-high myopia, severe myopia, high myopia, moderate myopia and low myopia are categories of myopic eyes classified according to different diopters. In this document, the diopter of super-high myopia is -10.00D or less; the diopter of severe myopia is between -6.00D and -9.99D; the diopter of high myopia is between -4.00D and -5.99D; the diopter of moderate myopia is between -2.00D and -3.99D; and the diopter of low myopia is between -0.50D and -1.99D.

[0023] M2 receptors are mainly distributed in the ciliary muscle and retina of the eye, and are related to the regulation of the focusing and refractive state of the eye. Barathi et al. (Barathi V.A. et al. Dis. Model. Mech. 2013, 6(5): 1146-1158) knocked out the muscarinic M2 receptor gene in mice, and after 4-8 weeks of negative lens induction, the control group of mice showed significant growth in the axial length of the eye, lens, and vitreous cavity depth, showing myopia symptoms; while the M2 gene knockout mice did not have myopia. In M2 receptor gene knockout mice, the increase in scleral collagen type I and the decrease in type V, i.e. the increase in scleral fibrosis, inhibited the elongation of the eye axis. It can be seen that myopia can be treated or prevented by inhibiting the activity of M2 receptors. The document also discloses two examples of selective M2 receptor inhibitor drugs for inhibiting the development of myopia, including AFDX-116. However, the existing technology discloses that AFDX-116 or its optical isomer AFDX0250 has the challenge of mydriatic side effects in treating or preventing myopia.

[0024] M3 receptors, i.e. muscarinic 3 receptors, are mainly distributed in the pupil sphincter muscle of the eye. M3 receptors are related to the contraction of the pupil sphincter muscle, affecting the size of the pupil. When these receptors are activated, they cause the pupil sphincter muscle to contract, thereby causing the pupil to shrink, a process known as miosis. Conversely, when the activity of M3 receptors is reduced or inhibited, the contraction of the pupil sphincter muscle is weakened, and the pupil dilates, i.e. mydriasis occurs.

[0025] Therefore, in order to reduce mydriasis in the treatment or prevention of myopia, the present application considers maximizing the selectivity of the drug to M2 receptors, mainly acting on M2 receptors, and having less effect on other muscarinic receptors (such as M3 receptors), i.e. increasing the IC 50 ratio of M3 / M2.

[0026] The present application designs and prepares deuterated compounds of benzodiazepines, the general structure of which is as follows:

[0027] wherein R1, R2 are independently selected from ethyl or deuterated ethyl, R3-R18 are independently selected from H group or deuterium group, and at least one deuterium group is contained in R1-R18.

[0028] The present application also provides a pharmaceutical composition, the active ingredient of which comprises the above-mentioned deuterated compounds of benzodiazepines, and the pharmaceutical composition further comprises pharmaceutical excipients, such as excipients, etc. The pharmaceutical composition can be in the form of eye drops, eye gels, eye ointments, or injections.

[0029] The pharmaceutical composition is for ocular topical administration. The ocular topical administration includes, but is not limited to, administration by eye drop, administration by eye ointment, conjunctival sac administration, intravitreal administration, subconjunctival administration, subtenon administration, conjunctival sac injection, or eyelid coating, etc. The administration concentration of the pharmaceutical composition can be 0.001% to 5%.

[0030] Compared with the existing selective M2 receptor inhibitor drugs such as AFDX-116, the compound of the present application has better inhibitory activity and pharmacodynamic performance.

[0031] The present application is further illustrated by the following examples, but the scope of the present application is not limited to these examples. The percentages described in the present application are weight percentages unless otherwise specified. The numerical ranges described in the specification, such as measurement units, reaction conditions, physical state of the compound, or percentages, are provided for clear and unambiguous reference. Those skilled in the art can still obtain the expected results when using temperatures, concentrations, amounts, number of carbon atoms, etc. outside the range or different from the individual numerical values in the practice of the present patent.

[0032] The full names of the reagents used in the examples are as follows: HATU 2-(7-oxadiazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate DIPEA diisopropylethylamine DABCO 1,4-diazabicyclo[2.2.2]octane HOBt 1-hydroxybenzotriazole EDCI.HCl 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride DMF N,N-dimethylformamide DIEA diisopropylethylamine THF tetrahydrofuran EA ethyl acetate DCM dichloromethane PE petroleum ether.

[0033] The reagents or raw materials used in the examples are all conventional commercial products.

[0034] Example 1: Preparation of Compound 1

[0035] Synthetic route of Compound 1:

[0036] Step 1: Preparation of intermediate 1a

[0037] 1-(tert-butoxycarbonyl)piperidine-2-carboxylic acid (500 mg, 2.18 mmol), diethylamine (191 mg, 2.62 mmol), N,N-diisopropylethylamine (1.41 g, 10.92 mmol), HATU (1.66 g, 4.37 mmol) were dissolved in dichloromethane (30 mL) and reacted at room temperature for 2 hours under nitrogen.

[0038] The reaction solution was diluted with dichloromethane (100 mL), and the organic layer was washed with water (50 mL), brine (50 mL), dried over anhydrous sodium sulfate, and concentrated in vacuo.

[0039] The resulting residue was purified by normal phase column to give 481 mg of intermediate 1a (yield: 78%) as a colorless oily liquid.

[0040] LCMS: [Ms + H - 100] + = 185.2.

[0041] Step 2: Preparation of intermediate 1b

[0042] Intermediate 1a (481 mg, 1.69 mmol) was dissolved in dichloromethane (10 mL), followed by the addition of trifluoroacetic acid (2 mL), and the resulting system was allowed to react at room temperature for 2 hours under a nitrogen atmosphere.

[0043] The reaction solution was concentrated in vacuo, and the residue was azeotroped with dichloromethane (10 mL) three times to give 311 mg of intermediate 1b as a yellow oily liquid.

[0044] LCMS: [Ms + H] + = 185.2.

[0045] Step 3: Preparation of intermediate 1c

[0046] Intermediate 1b (311 mg, 1.69 mmol) was dissolved in tetrahydrofuran (5 mL), and lithium aluminum hydride (6.77 mL, 6.77 mmol, 1 mol per liter of tetrahydrofuran solution) was added at 0°C, and the mixture was allowed to react at 70°C for 1.5 hours under a nitrogen atmosphere.

[0047] Methanol (2 mL) was added to quench the remaining lithium aluminum hydride, and the mixture was dried over anhydrous sodium sulfate and concentrated in vacuo. This resulted in 1.56 g of intermediate 1c as a white solid.

[0048] LCMS: [Ms + H] + = 171.2.

[0049] Step 4: Preparation of intermediate 1d

[0050] 2-Chloropyridin-3-amine (973 mg, 7.6 mmol) and triethylamine (1.54 g, 15.21 mmol) were dissolved in dichloromethane (40 mL), followed by the dropwise addition of 5-bromo-2-nitrobenzoyl chloride (2 g, 7.6 mmol), and the resulting system was allowed to react at room temperature for 18 hours under a nitrogen atmosphere.

[0051] The reaction solution was concentrated in vacuo. The resulting residue was purified by normal phase column to give 2.38 g of intermediate 1d (yield: 88%) as a colorless oily liquid.

[0052] LCMS: [Ms+H] = 356.0.

[0053] Step 5: Preparation of intermediate 1e

[0054] Intermediate 1d (1.6 g, 5.63 mmol), ammonium chloride (1.52 g, 28.17 mmol) were dissolved in methanol (25 mL) and water (25 mL), and iron powder (1.58 g, 28.17 mmol) was added. The resulting mixture was stirred at 70 °C for 1.5 h under nitrogen.

[0055] The reaction solution was filtered through celite, and the filtrate was concentrated in vacuo. The resulting residue was purified by normal phase column to give 1.07 g of intermediate 1e (yield: 59%) as a white solid.

[0056] LCMS: [Ms+H] = 326.0.

[0057] Step 6: Preparation of intermediate 1f

[0058] Intermediate 1e (600 mg, 1.85 mmol) was dissolved in n-butanol (15 mL), and sulfuric acid (1.2 mL) was added dropwise to the reaction solution at 0 °C. The system was stirred at 120 °C for 1.5 h.

[0059] The reaction solution was diluted with ethyl acetate (100 mL), and the organic layer was washed successively with water (50 mL) and brine (50 mL), dried over anhydrous sodium sulfate, and concentrated in vacuo. The resulting residue was purified by normal phase column to give 485 mg of intermediate 1f (yield: 91%) as a yellow solid.

[0060] LCMS: [Ms+H] = 290.0.

[0061] Step 7: Preparation of intermediate 1g

[0062] Palladium on carbon (50 mg) and deuterium water (1.5 mL) were added to a 50 mL three-necked flask, followed by hydrogen gas, and then the reaction was continued at room temperature for 24 h in a closed environment. Intermediate 1f (50 mg, 0.17 mmol) was dissolved in deuterium methanol (0.6 mL) and deuterium dimethyl sulfoxide (0.2 mL), and added to the reaction solution, and the reaction was continued at room temperature for 5 h in a closed environment.

[0063] The reaction solution was filtered through celite, and the filtrate was concentrated in vacuo. The resulting residue was purified by normal phase column to give 1.07 g of intermediate 1e (yield: 59%) as a white solid.

[0064] LCMS: [Ms+H] = 213.2.

[0065] Step 8: Preparation of intermediate 1h

[0066] Intermediate 1g (37 mg, 0.17 mmol) was dissolved in dioxane (5 mL) and warmed to 100 °C under nitrogen for 15 min, then cooled to room temperature. A solution of triethylamine (69 mg, 0.68 mmol) and chloroacetyl chloride (39 mg, 0.34 mmol) in dioxane (3 mL) was added and the reaction was stirred at 100 °C under nitrogen for 19.5 h.

[0067] The reaction was diluted with ethyl acetate (50 mL) and the organic layer was washed sequentially with saturated sodium bicarbonate solution (20 mL), brine (20 mL), dried over anhydrous sodium sulfate and concentrated in vacuo. This gave 50 mg of intermediate 1h as a yellow oil.

[0068] LCMS: [Ms+H]+= 289.0.

[0069] Step: Preparation of compound 1

[0070] Intermediate 1c (89 mg, 0.52 mmol), potassium iodide (58 mg, 0.35 mmol) and N,N- diisopropylethylamine (112 mg, 0.87 mmol) were dissolved in dioxane (8 mL) and intermediate 1h (50 mg, 0.17 mmol) was added. The reaction was stirred at 80 °C under nitrogen for 3.5 h.

[0071] The reaction was concentrated in vacuo. The resulting residue was purified by reverse phase preparative chromatography to give 54 mg of compound 1 (yield: 75%).

[0072] LCMS: [Ms+H]+= 423.5.

[0073] 1 H NMR (400 MHz, CDC13) δ 8.73 (d, J = 7.1 Hz, 1H), 8.50 (d, J = 7.0 Hz, 1H), 8.47 - 8.41 (m, 1H), 7.95 - 7.84 (m, 2H), 7.02 (t, J = 7.3 Hz, 1H), 4.30 (dd, J = 34.5, 16.3 Hz, 2H), 3.93 (s, 1H), 3.80 (d, J = 9.7 Hz, 1H), 3.59 - 3.15 (m, 7H), 2.12 (d, J = 13.7 Hz, 1H), 2.03 - 1.80 (m, 4H), 1.76 (s, 1H), 1.37 (t, J = 6.5 Hz, 6H).

[0074] Example 2: Preparation of compound 2

[0075] Synthetic route of compound 2:

[0076] Step 1: Preparation of intermediate 2b

[0077] 2-amino-4-pentenoic acid (10 g, 87 mmol), di-tert-butyl dicarbonate (24.7 g, 113.1 mmol) and triethylamine (14.1 g, 139.2 mmol) were dissolved in super dry dichloromethane (100 mL) and stirred at room temperature for 16 hours under nitrogen protection. The reaction was judged to be completed by TLC. Concentration under reduced pressure gave yellow oil 2a. It was used directly for the next step without further purification.

[0078] 2a (19 g, 87 mmol), iodomethane (24.7 g, 174 mmol) and potassium carbonate (24.2 g, 174 mmol) were added to 200 mL of super dry N,N-dimethylformamide and stirred at 60 °C for 4 hours under refluxing and nitrogen protection.

[0079] Ethyl acetate (300 mL) was added to the reaction solution, which was washed twice with saturated aqueous sodium bicarbonate solution and once with saturated brine, dried over anhydrous sodium sulfate, filtered and rotary evaporated.

[0080] The obtained residue was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 4 / 1) to give 2.78 g of intermediate 2b as yellow oily liquid, with a yield of 15%.

[0081] 1 H NMR (400 MHz, CDC13) δ 5.75-5.63 (m, 1H), 5.17-5.08 (m, 2H), 5.02 (s, 1H), 4.42-4.32 (m, 1H), 3.74 (s, 3H), 2.60-2.40 (m, 2H), 1.44 (s, 9H).

[0082] Step 2: Preparation of intermediate 2c

[0083] Intermediate 2b (2.78 g, 12.1 mmol) was dissolved in N,N-dimethylformamide (50 mL) at 0 °C, 3-bromoprop-1-ene (1.6 g, 13.3 mmol) and sodium hydride (320 mg, 13.3 mmol) were added, and the reaction was stirred at 0 °C for 1.5 hours and at room temperature for 0.5 hours under nitrogen protection.

[0084] The reaction was quenched by adding 50 mL of saturated aqueous ammonium chloride solution to the reaction solution, which was extracted with ethyl acetate (30 mL x 3), the organic phases were combined and washed twice with saturated brine, dried over anhydrous sodium sulfate, filtered and rotary evaporated.

[0085] The obtained residue was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 10 / 1) to give 1.52 g of intermediate 2c as yellow oily liquid 2c, with a yield of 46%.

[0086] 1 H NMR (400 MHz, CDC13) δ 5.92 - 5.69 (m, 2H), 5.24 - 5.03 (m, 4H), 4.69 - 4.41 (m, 1H), 4.09 - 3.74 (m, 2H), 3.70 (s, 3H), 2.65 (d, J = 62.9 Hz, 2H), 1.44 (s, 9H).

[0087] Step 3: Preparation of intermediate 2d

[0088] 2c (1.52 g, 5.65 mmol) was dissolved in dichloromethane (20 mL), and Grubbs second generation catalyst (480 mg, 0.56 mmol) was added. The reaction was carried out at room temperature for 2 hours under nitrogen protection.

[0089] The obtained residue was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 10 / 1) to obtain 1 g of intermediate 2d in the form of yellow oil with a yield of 73%.

[0090] 1 H NMR (400 MHz, CDC13) δ 5.66 (dd, J = 19.3, 16.8 Hz, 2H), 4.97 (dd, J = 7.65, 6.0 Hz, 1H), 4.18 - 3.74 (m, 2H), 3.71 (d, J = 5.4 Hz, 3H), 2.71 - 2.44 (m, 2H), 1.48 (d, J = 12.5 Hz, 9H).

[0091] Step 4: Preparation of intermediate 2e

[0092] Palladium-carbon (14 mg) was dissolved in heavy water (5 mL), and after the reaction was carried out at room temperature for 24 hours under hydrogen protection, a solution of 2d (50 mg, 0.2 mmol) in deuterated methanol (2 mL) was added, and the reaction was carried out at room temperature for 16 hours.

[0093] The reaction solution was filtered through diatomite, and the filtrate was collected, then 20 mL of water was added, extracted with ethyl acetate (10 mL x 3), the organic phases were combined and washed with NaCl aqueous solution for 2 times, dried over anhydrous sodium sulfate, filtered, and rotary evaporated.

[0094] The obtained residue was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 10 / 1) to obtain 39 mg of intermediate 2e in the form of dark green oil with a yield of 77%.

[0095] LCMS: [Ms + H-Boc] = 146.2. +

[0096] Step 5: Preparation of intermediate 2f ​

[0097] Intermediate 2e (118 mg, 0.48 mmol) was dissolved in a mixture of methanol (5 mL) and water (1 mL), sodium hydroxide (96 mg, 2.4 mmol) was added, and the reaction was allowed to proceed at room temperature for 2 hours.

[0098] The methanol was removed by rotary evaporation, 20 mL of water was then added, and the pH was adjusted to precipitate a white solid by dropwise addition of 1 M aqueous hydrochloric acid. The solid was extracted with ethyl acetate (10 mL x 3), the organic phases were combined and washed twice with aqueous NaCl, dried over anhydrous sodium sulfate, filtered, and rotary evaporated. This resulted in 88 mg of intermediate 2f as a yellow oil in 79% yield.

[0099] LCMS: [Ms + H - Boc] = 132.2. +

[0100] Step 6: Preparation of intermediate 2g

[0101] Intermediate 2f (88 mg, 0.38 mmol) was dissolved in dichloromethane (6 mL), HATU (288 mg, 0.76 mmol), N,N-diisopropylethylamine (243 mg, 1.9 mmol), and diethylamine (41 mg, 0.51 mmol) were added, and the reaction was allowed to proceed at room temperature for 4 hours under nitrogen.

[0102] The reaction was diluted with 20 mL of dichloromethane, washed with water (10 mL x 3), washed once with aqueous NaCl, dried over anhydrous sodium sulfate, filtered, and rotary evaporated. The residue was purified by column chromatography on silica gel (eluent: petroleum ether / ethyl acetate = 10 / 1) to give 63 mg of intermediate 2g as a yellow oil in 58% yield.

[0103] LCMS: [Ms + H - Boc] = 187.2. +

[0104] Step 7: Preparation of intermediate 2h

[0105] Intermediate 2g (63 mg, 0.22 mmol) was dissolved in tetrahydrofuran (6 mL), and borane tetrahydrofuran complex (1.1 mL, 1.1 mmol) was added dropwise at 0°C. The reaction was allowed to proceed at 70°C for 12 hours under nitrogen.

[0106] The reaction was cooled to room temperature, quenched by the addition of 20 mL of methanol, then warmed to 70°C for 1 hour, then cooled to room temperature and rotary evaporated. The residue was azeotroped with methanol (10 mL) three times.

[0107] This resulted in 63 mg of crude intermediate 2h. This was used directly in the next step without purification.

[0108] LCMS: [Ms + H]​​+ = 273.3.

[0109] Step 8: Preparation of Intermediate 2i

[0110] Intermediate 2h (63 mg, 0.23 mmol) was dissolved in dichloromethane (2 mL) and trifluoroacetic acid (0.5 mL) was added dropwise at 0 °C. The reaction was stirred at room temperature for 2 h.

[0111] The reaction was rotary evaporated and the residue was azeotroped with dichloromethane (10 mL) for 3 times. The crude 63 mg of intermediate 2i was obtained as a yellow oil. It was used in the next step without purification.

[0112] LCMS: [Ms + H] + = 173.3.

[0113] Step 9: Preparation of Compound 2

[0114] Intermediate 2i (63 mg, 0.36 mmol) was dissolved in 1,4-dioxane (5 mL) and N,N- diisopropylethylamine (236 mg, 0.72 mmol), 11-(2-chloroacetyl)-5,11-dihydro-6H- benzo[3,2-b][1,4]diazepin-6-one (103 mg, 0.36 mmol) and potassium iodide (120 mg, 0.72 mmol) were added successively. The reaction was stirred at 80 °C for 2 h under nitrogen.

[0115] The reaction was rotary evaporated and the residue was purified by Pre-HPLC to give 25 mg of Compound 2 with a yield of 16%.

[0116] LCMS: [Ms + H - Boc] + = 424.3.

[0117] 1 H NMR (400 MHz, DMSO) δ 10.86 (s, 1H), 8.22 (s, 1H), 7.77 (d, J = 7.7 Hz, 1H), 7.71 - 7.55 (m, 2H), 7.53 - 7.21 (m, 3H), 4.15 (d, J = 15.5 Hz, 0.5H), 3.62 (d, J = 15.5 Hz, 0.5H), 3.00 - 2.73 (m, 0.5H), 2.64 - 2.50 (m, 0.5H), 2.40 - 2.19 (m, 5H), 2.16 - 1.74 (m, 2.5H), 1.68 - 1.40 (m, 1.5H), 1.27 (dd, J = 16.6, 9.8 Hz, 2H), 1.17 - 0.97 (m, 1.5H), 0.86 (d, J = 7.2 Hz, 6H), 0.76 - 0.54 (m, 0.5H).

[0118] Example 3: Preparation of compound 3

[0119] Synthetic route of compound 3:

[0120] Step 1: Preparation of intermediate 3a

[0121] tert-Butyl 2-(aminomethyl)piperidine-1-carboxylate (107 mg, 0.5 mmol) and bromoethane-D5 (230 mg, 2 mmol) were dissolved in super dry DMF (8 mL), DIEA (323 mg, 2.5 mmol) and potassium iodide (332 mg, 2 mmol) were added, and the mixture was stirred at 100 °C for 4 h under nitrogen protection.

[0122] Ethyl acetate (30 mL) was added to the reaction solution, which was washed with water (10 mL) for 3 times and saturated brine (15 mL) for 1 time, dried over anhydrous sodium sulfate, filtered and concentrated. The obtained residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 20 / 1) to obtain 115 mg of intermediate 3a as a yellow oily liquid, with a yield of 82.1%.

[0123] LCMS: 281.3 [M+1] + .

[0124] Step 2: Preparation of intermediate 3b

[0125] Trifluoroacetic acid (1 mL) was added dropwise to a solution of intermediate 3a (115 mg, 0.4 mmol) in dichloromethane (4 mL) at 0 °C, and the mixture was stirred at room temperature for 2 h.

[0126] The reaction solution was concentrated to obtain 115 mg of crude 3b. Without further purification, the crude product was directly used in the next reaction.

[0127] LCMS: 181.3 [M+1] + .

[0128] Step 3: Preparation of compound 3

[0129] Intermediate 3b (115 mg, 0.6 mmol) was dissolved in 1,4-dioxane (6 mL), and N,N-diisopropylethylamine (387 mg, 3 mmol), 11-(2-chloroacetyl)-5,11-dihydro-6H-benzo[3,2-b][1,4]diazepin-6-one (258 mg, 0.9 mmol) and potassium iodide (199 mg, 1.2 mmol) were added successively, and the mixture was stirred at 80 °C for 4 h under nitrogen protection.

[0130] The reaction solution was rotary evaporated to dryness, and the residue was purified by Pre-HPLC to give 58 mg of compound 3 in a yield of 22.4%.

[0131] LCMS: 432.4 [M+1] + .

[0132] 1 H NMR (400 MHz, DMSO) δ 10.86 (s, 1H), 8.27 (d, J = 2.8 Hz, 1H), 7.79 (d, J = 7.3 Hz, 1H), 7.68 (d, J = 8.0 Hz, 2H), 7.47 (d, J = 4.5 Hz, 3H), 4.00 - 3.70 (m, 1H), 3.57 - 3.45 (m, 2H), 2.96 - 2.62 (m, 2H), 2.43 - 2.06 (m, 2H), 1.58 - 0.86 (m, 6H).

[0133] Example 4: Preparation of compound 4

[0134] Synthetic route of compound 4:

[0135] Step 1: Preparation of intermediate 4a

[0136] 1-Boc-2-aminomethylpiperidine (600 mg, 2.8 mmol) and triethylamine (1.7 g, 16.82 mmol) were dissolved in dichloromethane (10 mL), and a solution of acetyl chloride (660 mg, 8.41 mmol) in dichloromethane (5 mL) was added dropwise at 0 °C, followed by stirring at room temperature for 2 hours under nitrogen.

[0137] Diluted with dichloromethane (50 mL), then washed successively with saturated sodium bicarbonate solution (30 mL), water (30 mL), brine (30 mL), dried over anhydrous sodium sulfate, and rotary evaporated to dryness. Without further purification, 580 mg of intermediate 4a (yield: 81%) was obtained as a yellow oily liquid, which was directly used in the next reaction.

[0138] LCMS: [Ms+H-100] = 157.2.

[0139] Step 2: Preparation of intermediate 4b

[0140] 4a (580 mg, 2.27 mmol) was dissolved in dichloromethane (10 mL), followed by the addition of trifluoroacetic acid (3 mL) and stirring at room temperature for 2 hours.

[0141] The reaction solution was evaporated to dryness and the residue was co-evaporated with dichloromethane (10 mL) three times. Without further purification, 300 mg of 4b was obtained as a yellow oily liquid which was used directly in the next step.

[0142] LCMS: [Ms+H] = 157.1.

[0143] Step 3: Preparation of intermediate 4c

[0144] Intermediate 4b (270 mg, 1.73 mmol) and N,N-diisopropylethylamine (1.12 g, 8.65 mmol) were dissolved in acetonitrile (20 mL), followed by the addition of benzyl bromide (326 mg, 1.90 mmol) and reaction at 70 °C for 1.5 h under nitrogen.

[0145] The reaction solution was evaporated to dryness. The residue was purified by normal phase column to give 405 mg of intermediate 4c as a white solid in 95% yield.

[0146] LCMS: [Ms+H] = 247.2.

[0147] Step 4: Preparation of intermediate 4d

[0148] 4c (405 mg, 1.65 mmol) was dissolved in tetrahydrofuran (20 mL), followed by the addition of lithium aluminum deuteride (287 mg, 6.83 mmol) at 0 °C and reaction at 70 °C for 3 h under nitrogen.

[0149] The remaining lithium aluminum deuteride was quenched by the addition of water (2 mL) to the reaction solution, followed by dilution with dichloromethane (100 mL). The organic layer was washed successively with water (30 mL), brine (30 mL), dried over anhydrous sodium sulfate and evaporated to dryness. The residue was purified by normal phase column to give 276 mg of intermediate 4d as a colorless oily liquid in 67% yield.

[0150] LCMS: [Ms+H] = 235.2.

[0151] Step 5: Preparation of intermediate 4e

[0152] 4d (253 mg, 1.08 mmol) and triethylamine (656 mg, 6.49 mmol) were dissolved in dichloromethane (15 mL), followed by the dropwise addition of a solution of acetyl chloride (255 mg, 3.24 mmol) in dichloromethane (5 mL) at 0 °C and stirring at room temperature for 2 h under nitrogen.

[0153] The reaction was diluted with dichloromethane (50 mL) and washed sequentially with saturated sodium bicarbonate solution (20 mL), water (20 mL), brine (20 mL), dried over anhydrous sodium sulfate and concentrated in vacuo. The residue was purified by normal phase column to give 220 mg of intermediate 4e as a colorless oily liquid in 74% yield.

[0154] LCMS: [Ms + H]+ = 277.2.

[0155] Step 6: Preparation of intermediate 4f

[0156] Intermediate 4e (186 mg, 0.67 mmol) was dissolved in tetrahydrofuran (10 mL) and lithium aluminium deuteride (113 mg, 2.7 mmol) was added at 0 °C. The resulting system was allowed to react at 70 °C for 2 hours under nitrogen atmosphere.

[0157] The reaction was diluted with dichloromethane (50 mL) and washed sequentially with saturated sodium bicarbonate solution (20 mL), water (20 mL), brine (20 mL), dried over anhydrous sodium sulfate and concentrated in vacuo. The residue was purified by normal phase column to give 220 mg of intermediate 4e as a colorless oily liquid in 74% yield.

[0158] LCMS: [Ms + H]+ = 277.2.

[0159] Step 7: Preparation of intermediate 4g

[0160] Intermediate 4f (126 mg, 0.47 mmol) was dissolved in methanol (8 mL) and dichloromethane (2 mL) and palladium on carbon catalyst (38 mg, 10% palladium content) was added. The resulting system was allowed to react at room temperature for 2 hours under hydrogen atmosphere.

[0161] The filtrate was filtered over celite and concentrated in vacuo. Without further purification, 80 mg of 4g was obtained as a colorless oily liquid which was used directly in the next step.

[0162] LCMS: [Ms + H]+ = 277.2.

[0163] Step 8: Preparation of compound 4

[0164] Compound 4 was prepared from intermediate 4g (63 mg, 0.36 mmol) and 5,11-dihydro-11-chloroacetyl-6H-pyrido[2.3-b][1,4]benzodiazepin-6-one (114 mg, 0.40 mmol) according to the method of Example 3, step 3 in 46% yield.

[0165] LCMS: [Ms + H] + = 277.2.

[0166] LCMS: [Ms + H]1 H NMR (400 MHz, DMSO) δ 10.86 (s, 1H), 8.22 (s, 1H), 7.78 (d, J = 7.7 Hz, 1H), 7.64 (ddd, J = 7.9, 2.8, 1.7 Hz, 2H), 7.55 - 7.18 (m, 3H), 4.12 (d, J = 16.0 Hz, 0.5H), 3.61 (d, J = 15.3 Hz, 0.5H), 3.30 (d, J = 15.5 Hz, 0.5H), 2.89 (d, J = 16.2 Hz, 0.5H), 2.58 (d, J = 10.6 Hz, 0.5H), 2.42 - 2.21 (m, 2H), 2.21 - 1.95 (m, 2H), 1.94 - 1.84 (m, 0.5H), 1.62 (dd, J = 9.2, 5.3 Hz, 0.5H), 1.48 (brs, 1H), 1.32 (brs, 2H), 1.19 - 0.99 (m, 2H), 0.86 (d, J = 6.8 Hz, 6H), 0.81 - 0.64 (m, 0.5H).

[0167] Example 5: Preparation of compound 5

[0168] Synthetic route of compound 5:

[0169] Step 1: Preparation of intermediate 5a

[0170] Borane tetrahydrofuran solution (70.11 ml, 70.11 mmol, 1 mole per liter) was added to the reaction at 0 °C. The mixture was reacted at 70 °C for 2 hours under nitrogen atmosphere.

[0171] The reaction was quenched by dropwise addition of methanol (20 ml) at 0 °C, then the reaction was rotary evaporated. The obtained residue was azeotroped with methanol (20 ml) for three times. The obtained residue was purified by column chromatography to obtain 700 mg of intermediate 5a as colorless oily liquid, with a yield of 41%.

[0172] LCMS: [Ms+H] = 243.2.

[0173] Step 2: Preparation of intermediate 5b

[0174] 5a (300 mg, 1.24 mmol) was dissolved in dichloromethane (10 ml) with triethylamine (753 mg, 7.44 mmol), acetyl chloride (292 mg, 3.72 mmol) was added dropwise to the reaction. The mixture was reacted at room temperature for 18 hours under nitrogen atmosphere.

[0175] The reaction mixture was washed with saturated sodium bicarbonate solution (30 mL), water (30 mL), brine (30 mL), dried over anhydrous sodium sulfate and concentrated in vacuo. The resulting residue was purified by column chromatography to give 220 mg of intermediate 5b as a yellow oily liquid in 77% yield.

[0176] LCMS: [Ms + H - 100] = 185.2.

[0177] Step 3: Preparation of intermediate 5c

[0178] 5b (240 mg, 0.85 mmol) was dissolved in dichloromethane (10 mL) and trifluoroacetic acid (4 mL) was added dropwise to the mixture at 0 °C. The mixture was allowed to react at room temperature for 1 hour.

[0179] The reaction mixture was concentrated in vacuo. The resulting residue was azeotroped with dichloromethane (10 mL) for three times. Concentration in vacuo gave 170 mg of intermediate 5c as a yellow oily liquid, which was used directly in the next step.

[0180] LCMS: [Ms + H] = 185.2.

[0181] Step 4: Preparation of intermediate 5d

[0182] 5c (145 mg, 0.79 mmol) was dissolved in acetonitrile (15 mL) with N,N- diisopropylethylamine (303 mg, 0.87 mmol) and benzyl bromide (148 mg, 0.87 mmol) was added to the reaction mixture. The mixture was allowed to react at 70 °C for 1.5 hours under nitrogen.

[0183] The reaction mixture was concentrated in vacuo. The resulting residue was purified by column chromatography to give 220 mg of intermediate 5d as a colorless oily liquid in 98% yield.

[0184] LCMS: [Ms + H] = 275.2.

[0185] Step 5: Preparation of intermediate 5e

[0186] 5d (220 mg, 0.8 mmol) was dissolved in tetrahydrofuran (15 mL) and lithium aluminum deuteride (135 mg, 3.21 mmol) was added to the mixture at 0 °C. The mixture was allowed to react at 70 °C for 2 hours under nitrogen.

[0187] The reaction was quenched by adding water (1 mL) and then filtered through celite. The resulting filtrate was dried over anhydrous sodium sulfate and concentrated in vacuo. The resulting residue was purified by column chromatography to give 187 mg of intermediate 5e as a white solid in 89% yield.

[0188] LCMS: [Ms + H] + = 263.3.

[0189] Step 6: Preparation of Intermediate 5f

[0190] To a solution of 5e (160 mg, 0.61 mmol) in methanol (8 mL) and dichloromethane (2 mL) was added palladium on carbon catalyst (53 mg, 10% palladium content). The mixture was stirred at room temperature under hydrogen atmosphere for 2 hours.

[0191] The mixture was filtered through celite and the filtrate was concentrated. Concentration gave 117 mg of intermediate 5f as a colorless oily liquid which was used directly in the next step.

[0192] LCMS: [Ms + H] = 173.3. +

[0193] Step 7: Preparation of Compound 5

[0194] Compound 5 was prepared from intermediate 5f (117 mg, 0.68 mmol) and 5,11- dihydro-11-chloroacetyl-6H-pyrido[2.3-b][l,4]benzodiazepin-6-one (195 mg, 0.68 mmol) following the procedure of Example 3, Step 3 in 35% yield.

[0195] LCMS: [Ms + H] = 424.2. +

[0196] 1 H NMR (400 MHz, DMSO) δ 10.87 (s, 1H), 8.28 (d, J = 2.7 Hz, 1H), 7.79 (d, J = 7.5 Hz, 1H), 7.72 - 7.57 (m, 2H), 7.46 (dd, J = 7.7, 4.4 Hz, 3H), 3.87 (d, J = 18.1 Hz, 0.5H), 3.59 - 3.41 (m, 2H), 2.93 - 2.61 (m, 3.5H), 2.59 - 2.50 (m, 0.5H), 2.44 - 2.28 (m, 1.5H), 2.13 (s, 1H), 1.64 - 1.34 (m, 2H), 1.34 - 1.06 (m, 4H), 1.08 - 0.91 (m, 6H).

[0197] Example 6: Preparation of Compound 6

[0198] Synthetic route of Compound 6:

[0199] Step 1: Preparation of Intermediate 6a

[0200] ​​To a solution of 1-Boc-2-aminomethylpiperidine (530 mg, 2.47 mmol), 2-(7- azabenzotriazol-1 -yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (1.13 g, 2.97 mmol) and N,N-diisopropylethylamine (959 mg, 7.41 mmol) in N,N- dimethylformamide (7.5 mL) was added deuterated acetic acid-D4 (159 mg, 2.47 mmol). The mixture was stirred at room temperature under nitrogen for 2 hours.

[0201] Ethyl acetate (50 mL) and saturated aqueous ammonium chloride solution (50 mL) were added, followed by washing the aqueous layer with ethyl acetate (30 mL) three times, combining the organic layers and drying over anhydrous sodium sulfate, filtering and evaporating.

[0202] This gave 1.1 g of intermediate 6a as a yellow oil which was used directly in the next step.

[0203] LCMS: [Ms + H - 100] = 160.2.

[0204] Step 2: Preparation of intermediate 6b

[0205] Intermediate 6b was prepared from intermediate 6a (914 mg, 3.53 mmol) following the procedure of Example 5, Step 1 as a colourless oil in 31% yield.

[0206] LCMS: [Ms + H] = 246.2.

[0207] Step 3: Preparation of intermediate 6c

[0208] Intermediate 6c was prepared from intermediate 6b (225 mg, 0.92 mmol) and deuterated acetic acid-D4 (59 mg, 0.92 mmol) following the procedure of Example 6, Step 1 as a colourless oil in 99% yield.

[0209] LCMS: [Ms + H] = 291.2 [Ms + H - 100] = 191.2.

[0210] Step 4: Preparation of intermediate 6d

[0211] Intermediate 6d was prepared from intermediate 6c (330 mg, 1.14 mmol) following the procedure of Example 5, Step 1 as a colourless oil in 58% yield.

[0212] LCMS: [Ms + H] + = 277.4.

[0213] Step 5: Preparation of intermediate 6e

[0214] Intermediate 6e (110 mg) was prepared from intermediate 6d (183 mg, 0.66 mmol) following the procedure of Example 5, step 3 as a yellow oily liquid.

[0215] LCMS: [Ms + H] = 288.3. + = 177.3.

[0216] Step 6: Preparation of compound 6

[0217] Compound 6 was prepared from intermediate 6e (116 mg, 0.66 mmol) and 5,11- dihydro-11-chloroacetyl-6H-pyrido[2.3-b][1,4]benzodiazepin-6-one (228 mg, 0.79 mmol) following the procedure of Example 3, step 3 in 13% yield.

[0218] LCMS: [Ms + H] = 288.3. + = 428.4.

[0219] 1 H NMR (400 MHz, DMSO) δ 10.86 (s, 1H), 8.25 (s, 1H), 7.79 (d, J = 7.1 Hz, 1H), 7.67 (d, J = 7.9 Hz, 2H), 7.46 (s, 3H), 3.52 (dd, J = 22.9, 15.4 Hz, 2H), 3.22 - 2.95 (m, 1H), 2.61 (s, 5H), 2.33 (s, 2H), 2.04 (d, J = 24.1 Hz, 1H), 1.65 - 1.08 (m, 5H), 0.91 (d, J = 45.9 Hz, 1H).

[0220] Example 7: Preparation of compound 7

[0221] Synthetic route for compound 7:

[0222] Step 1 : Preparation of intermediate 7a

[0223] Intermediate 7a was prepared from intermediate 6b (461 mg, 1.88 mmol) and acetyl chloride (443 mg, 5.64 mmol) following the procedure of Example 5, step 2 as a colorless oily liquid in 36% yield.

[0224] LCMS: [Ms + H] = 288.3.

[0225] Step 2: Preparation of intermediate 7b

[0226] Intermediate 7b was prepared from intermediate 7a (196 mg, 0.68 mmol) following the procedure of Example 5, step 1 as a colorless oily liquid in 55% yield.

[0227] LCMS: [Ms+H] = 274.3.

[0228] Step 3: Preparation of intermediate 7c

[0229] Intermediate 7c (60 mg) was prepared from intermediate 7b (102 mg, 0.37 mmol) following the procedure of Example 5, step 3 as a yellow oily liquid.

[0230] LCMS: [Ms+H] + = 174.2.

[0231] Step 4: Preparation of compound 7

[0232] Compound 7 was prepared from intermediate 7c (60 mg, 0.34 mmol) and 5,11- dihydro-11-chloroacetyl-6H-pyrido[2.3-b][1,4]benzodiazepin-6-one (147 mg, 0.51 mmol) following the procedure of Example 3, step 3 in 13% yield.

[0233] LCMS: [Ms+Na] + = 425.3.

[0234] 1 H NMR (400 MHz, DMSO) δ 10.88 (s, 1H), 8.28 (d, J = 4.1 Hz, 1H), 7.80 (d, J = 6.9 Hz, 1H), 7.69 (t, J = 7.8 Hz, 2H), 7.48 (s, 3H), 3.70 (dd, J = 79.2, 14.3 Hz, 1.5H), 3.48 (d, J = 16.1 Hz, 1H), 2.95 - 2.55 (m, 7H), 2.43 - 2.09 (m, 1.5H), 1.67 - 1.11 (m, 6H), 1.06 (s, 3H).

[0235] Example 8: Preparation of compound 8

[0236] Synthetic route for compound 8:

[0237] Step 1: Preparation of intermediate 8a

[0238] tert-Butyl 2-(aminomethyl)piperidine-1 -carboxylate (100 mg, 0.47 mmol) and deuterated acetaldehyde (112 mg, 2.34 mmol) were dissolved in methanol (10 mL) and acetic acid (28 mg, 0.47 mmol) and NaBH3CN (147 mg, 2.34 mmol) were added to the mixture which was allowed to react at 40 °C for 48 h.

[0239] The reaction was concentrated to dryness. The resulting residue was purified by reverse phase preparative chromatography to give 80 mg of intermediate 8a as a white solid in 61% yield.

[0240] LCMS: [Ms + H] = 279.3. +

[0241] Step 2: Preparation of intermediate 8b

[0242] Intermediate 8b (50 mg) was prepared from intermediate 8a (80 mg, 0.29 mmol) following the procedure of Example 5, step 3 as a yellow oil liquid.

[0243] LCMS: [Ms + H] = 179.2. +

[0244] Step 3: Preparation of compound 8

[0245] Compound 8 was prepared from intermediate 8b (50 mg, 0.28 mmol) and 5,11- dihydro-11-chloroacetyl-6H-pyrido[2.3-b][1,4]benzodiazepin-6-one (80 mg, 0.28 mmol) following the procedure of Example 3, step 3 in 13% yield.

[0246] LCMS: [Ms + Na] = 430.3. +

[0247] 1 H NMR (400 MHz, DMSO) δ 10.85 (s, 1H), 8.22 (s, 1H), 7.78 (d, J = 7.7 Hz, 1H), 7.70 - 7.56 (m, 2H), 7.56 - 7.25 (m, 3H), 4.10 (d, J = 13.8 Hz, 1H), 3.32 (d, J = 15.7 Hz, 1H), 2.92 (d, J = 13.9 Hz, 0.5H), 2.58 (d, J = 11.4 Hz, 0.5H), 2.44 - 1.80 (m, 6H), 1.66 (brs, 0.5H), 1.29 (dd, J = 87.4, 53.4 Hz, 5H), 0.85 (dd, J = 27.3, 19.7 Hz, 0.5H).

[0248] ​​​Example 9: Preparation of compound 9

[0249] Synthetic route of compound 9:

[0250] Step 1: Preparation of intermediate 9a

[0251] Dissolve 5a (150 mg, 0.62 mmol) in dichloromethane (4 mL), add acetaldehyde (136 mg, 3 mmol), sodium cyanoborohydride (81 mg, 1.24 mmol) and acetic acid (74 mg, 1.24 mmol), and react at room temperature for 2 hours under nitrogen protection.

[0252] Quench the reaction with water (50 mL), extract with dichloromethane (25 mL x 3), combine the organic phases, wash with NaCl aqueous solution, dry over anhydrous sodium sulfate, filter, and rotary evaporate. Purify the obtained residue by silica gel column chromatography (eluent: dichloromethane / petroleum ether = 20 / 1) to obtain 69 mg of intermediate 9a in the form of a yellow oil with a yield of 41%.

[0253] LCMS: [Ms + H - Boc] = 146.2. +

[0254] Step 2: Preparation of intermediate 9b

[0255] Intermediate 9b (69 mg) is prepared from intermediate 9a (69 mg, 0.25 mmol) according to the method of Example 5, Step 3, in the form of a yellow oily liquid.

[0256] LCMS: [Ms + H] = 172.2. +

[0257] Step 3: Preparation of compound 9

[0258] Compound 9 is prepared from intermediate 9b (69 mg, 0.4 mmol) and 5,11-dihydro-11- chloroacetyl-6H-pyrido[2.3-b][1,4]benzodiazepin-6-one (115 mg, 0.4 mmol) according to the method of Example 3, Step 3, with a yield of 13%.

[0259] LCMS: [Ms + H] = 423.4. +

[0260] 1 ​​​H NMR (400 MHz, DMSO) δ 10.89 (s, 1H), 8.30 (d, J = 4.3 Hz, 1H), 7.80 (d, J = 6.7 Hz, 1H), 7.70 (t, J = 8.5 Hz, 2H), 7.49 (s, 3H), 3.80 - 3.57 (m, 2H), 2.92 (br s, 4H), 2.83 - 2.55 (m, 2.5H), 2.41 - 2.19 (m, 1.5H), 1.65 - 1.14 (m, 6H), 1.08 (d, J = 4.5 Hz, 6H).

[0261] Example 10: Preparation of compound 10

[0262] Synthetic route of compound 10:

[0263] Step 1: Preparation of intermediate 10a

[0264] Intermediate 10a was prepared from 1-Boc-2-aminomethylpiperidine (6.18 g, 28.88 mmol) and iodoacetic acid (5.37 g, 28.88 mmol) following the procedure of Example 2, step 6, in 63% yield.

[0265] LCMS: [Ms + H - 100] = 283.0.

[0266] Step 2: Preparation of intermediate 10b

[0267] 10a (6.90 g, 18.70 mmol) was dissolved in tetrahydrofuran (100 mL) and then lithium aluminum deuteride (987 mg, 23.5 mmol) was added at 0 °C. The resulting system was allowed to react at 0 °C for 2 hours under nitrogen atmosphere, and then the temperature was raised to room temperature and allowed to react for 30 minutes.

[0268] Water (10 mL) was added to quench the remaining lithium aluminum deuteride, and then dichloromethane (300 mL) was added to dilute the reaction mixture. The organic layer was washed with water (200 mL) and brine (200 mL) successively, dried over anhydrous sodium sulfate, and concentrated in vacuo. The resulting residue was purified by normal phase column to give 1.72 g of intermediate 10b as a yellow oily liquid in 36% yield.

[0269] LCMS: [Ms + H - 100] = 283.0.

[0270] Step 3: Preparation of intermediate 10c

[0271] Intermediate 10c (950 mg) was prepared from intermediate 10b (1.72 g, 6.69 mmol) following the procedure of Example 5, step 3, as a yellow oily liquid.

[0272] LCMS: [Ms + H] = 158.2.

[0273] Step 4: Preparation of Intermediate 10d

[0274] Intermediate 10d (1.15 g) was prepared from Intermediate 10c (950 mg, 6.05 mmol) and benzyl bromide (1.24 g, 7.26 mmol) following the procedure of Example 5, Step 4 as a white solid.

[0275] LCMS: [Ms + H] = 248.2.

[0276] Step 5: Preparation of Intermediate 10e

[0277] Intermediate 10e (484 mg) was prepared from Intermediate 10d (1.15 g, 4.65 mmol) and lithium aluminum deuteride (650 mg, 15.48 mmol) following the procedure of Example 4, Step 4 as a colorless oily liquid.

[0278] LCMS: [Ms + H]+= 236.2.

[0279] Step 6: Preparation of Intermediate 10f

[0280] Intermediate 10f (yield: 97%) was prepared from Intermediate 10e (484 mg, 2.06 mmol) and deuterated acetic acid (145 mg, 2.27 mmol) following the procedure of Example 2, Step 6 as a yellow oily liquid.

[0281] LCMS: [Ms + H - 100] = 281.2.

[0282] Step 7: Preparation of Intermediate 10g

[0283] Intermediate 10g (500 mg) was prepared from Intermediate 10f (570 mg, 2.04 mmol) and lithium aluminum deuteride (341 mg, 8.14 mmol) following the procedure of Example 4, Step 4 as a colorless oily liquid.

[0284] LCMS: [Ms + H] + = 269.4.

[0285] Step 8: Preparation of Intermediate 10h

[0286] Intermediate 10h (200 mg) was prepared from Intermediate 10g (500 mg, 1.87 mmol) following the procedure of Example 4, Step 7 as a colorless oily liquid.

[0287] Step 9: Preparation of Compound 10

[0288] Compound 10 was prepared from intermediate 10h (200 mg, 1.12 mmol) and 5,11- dihydro-11-chloroacetyl-6H-pyrido[2.3-b][l,4]benzodiazepin-6-one (384 mg, 1.35 mmol) following the procedure of Example 3, step 3 in 20% yield.

[0289] LCMS: [Ms+H] = 430.3. + = 430.3.

[0290] 1 H NMR (400 MHz, CDC13) δ 8.30 (s, 1H), 7.90 (t, J = 7.4 Hz, 1H), 7.76 (dd, J = 17.9, 7.5 Hz, 1H), 7.61 (t, J = 7.5 Hz, 1H), 7.52 (t, J = 8.2 Hz, 1H), 7.45 (t, J = 7.3 Hz, 1H), 7.41 - 7.35 (m, 1H), 4.27 (d, J = 16.7 Hz, 1H), 3.99 - 3.51 (m, 3H), 3.32 (d, J = 14.8 Hz, 1H), 3.22 - 2.77 (m, 2H), 2.12 - 1.86 (m, 1H), 1.59 (s, 4H), 1.24 (d, J = 4.1 Hz, 3H).

[0291] Example 11: Preparation of Compound 11

[0292] Synthetic route for compound 11:

[0293] Step 1: Preparation of intermediate 11a

[0294] Intermediate 11a (45 mg) was prepared from intermediate 4d (120 mg, 0.51 mmol) and bromoethane-D5 (58 mg, 0.51 mmol) following the procedure of Example 3, step 1 as a colorless oily liquid.

[0295] LCMS: [Ms+H] = 268.3.

[0296] Step 2: Preparation of intermediate 11b

[0297] Intermediate 11b (45 mg) was prepared from intermediate 11a (45 mg, 0.17 mmol) following the procedure of Example 4, step 7 as a colorless oily liquid.

[0298] LCMS: [Ms+H] = 178.2. + = 178.2.

[0299] Step 3: Preparation of compound 11

[0300] Compound 11 was prepared from intermediate 11b (116 mg, 0.66 mmol) and 5,11- dihydro-11-chloroacetyl-6H-pyrido[2.3-b][1,4]benzodiazepin-6-one (228 mg, 0.79 mmol) following the procedure of Example 3, step 3 in 12% yield.

[0301] LCMS: [Ms + Na] = 429.4. +

[0302] 1 H NMR (400 MHz, DMSO) δ 10.86 (s, 1H), 8.24 (s, 1H), 7.78 (d, J = 7.5 Hz, 1H), 7.66 (d, J = 7.7 Hz, 2H), 7.57 - 7.30 (m, 3H), 4.01 (d, J = 14.5 Hz, 0.5H), 3.56 (d, J = 14.8 Hz, 0.5H), 3.41 (d, J = 15.4 Hz, 0.5H), 3.03 (d, J = 14.0 Hz, 0.5H), 2.70 - 2.52 (m, 1H), 2.44 - 2.10 (m, 3H), 2.08 - 1.77 (m, 1H), 1.49 (t, J = 13.4 Hz, 1H), 1.42 - 1.15 (m, 3.5H), 1.10 (s, 1H), 0.92 (s, 3H), 0.89 - 0.74 (m, 0.5H).

[0303] Example 12: Preparation of Compound 12

[0304] Synthetic route of compound 12:

[0305] Step 1: Preparation of intermediate 12a

[0306] Intermediate 12a was prepared from intermediate 10e (158 mg, 0.67 mmol) and acetyl chloride (159 mg, 2.02 mmol) following the procedure of Example 5, step 2 in 69% yield as a colorless oily liquid.

[0307] LCMS: [Ms + H] = 278.2. +

[0308] Step 2: Preparation of intermediate 12b

[0309] Intermediate 12b was prepared from intermediate 12a (100 mg, 0.36 mmol) and lithium aluminum deuteride (46 mg, 1.08 mmol) following the procedure of Example 4, step 4 in 92% yield as a colorless oily liquid.​​

[0310] LCMS: [Ms + H] = 266.4. +

[0311] Step 3: Preparation of intermediate 12c

[0312] Intermediate 12c (55 mg) was prepared from intermediate 12b (75 mg, 0.28 mmol) following the procedure of Example 4, step 7 as a colorless oily liquid.

[0313] LCMS: [Ms + H] = 176.2. +

[0314] Step 4: Preparation of compound 12

[0315] Compound 12 was prepared from intermediate 12c (55 mg, 0.31 mmol) and 5,11- dihydro-11-chloroacetyl-6H-pyrido[2.3-b][1,4]benzodiazepin-6-one (98 mg, 0.31 mmol) following the procedure of Example 3, step 3 in 15% yield.

[0316] LCMS: [Ms + H] = 427.3. +

[0317] 1 H NMR (400 MHz, DMSO) δ 10.85 (s, 1H), 8.22 (s, 1H), 7.77 (d, J = 7.4 Hz, 1H), 7.71 - 7.51 (m, 2H), 7.51 - 7.11 (m, 3H), 4.13 (d, J = 15.3 Hz, 0.5H), 3.61 (d, J = 16.3 Hz, 0.5H), 2.88 (d, J = 11.0 Hz, 0.5H), 2.44 - 2.20 (m, 2.5H), 2.20 - 1.95 (m, 3H), 1.92 - 1.75 (m, 0.5H), 1.71 - 1.54 (m, 0.5H), 1.54 - 1.41 (m, 1H), 1.31 (br s, 2H), 1.16 - 0.97 (m, 2H), 0.85 (d, J = 7.1 Hz, 5H).

[0318] Example 13: Preparation of compound 13

[0319] Synthetic route for compound 13:

[0320] Step 1: Preparation of intermediate 13a

[0321] ​​​Intermediate 13a (55 mg) was prepared from intermediate 5a (100 mg, 0.41 mmol) and bromoethane-D5 (93 mg, 0.83 mmol) following the procedure of Example 3, step 1 as a colorless oily liquid.

[0322] LCMS: [Ms + H] = 276.4. + LCMS: [Ms + H] = 276.4.

[0323] Step 2: Preparation of intermediate 13b

[0324] Intermediate 13b (35 mg) was prepared from intermediate 13a (55 mg, 0.2 mmol) following the procedure of Example 5, step 3 as a yellow oily liquid.

[0325] LCMS: [Ms + H] = 176.4. + LCMS: [Ms + H] = 176.4.

[0326] Step 3: Preparation of compound 13

[0327] Compound 13 was prepared from intermediate 13b (35 mg, 0.2 mmol) and 5,11- dihydro-11-chloroacetyl-6H-pyrido[2.3-b][1,4]benzodiazepin-6-one (115 mg, 0.4 mmol) following the procedure of Example 3, step 3 in 23% yield.

[0328] LCMS: [Ms + H] = 427.4. + LCMS: [Ms + H] = 427.4.

[0329] 1 H NMR (400 MHz, DMSO) δ 10.88 (s, 1H), 8.30 (d, J = 3.7 Hz, 1H), 7.80 (d, J = 7.4 Hz, 1H), 7.68 (dd, J = 16.4, 8.1 Hz, 2H), 7.49 (d, J = 6.2 Hz, 3H), 3.96 - 3.44 (m, 2H), 3.23 - 2.57 (m, 6H), 2.46 - 2.19 (m, 1H), 1.71 - 0.85 (m, 10H).

[0330] Example 14: Preparation of compound 14

[0331] Synthetic route of compound 14:

[0332] Step 1: Preparation of intermediate 14a

[0333] Intermediate 14a (830 mg) was prepared from intermediate lb (920 mg, 5 mmol) and benzyl bromide (940 mg, 5.5 mmol) following the procedure of Example 5, step 4 as a yellow solid.

[0334] LCMS: [Ms+H] = 275.2.

[0335] Step 2: Preparation of intermediate 14b

[0336] Intermediate 14b (yield: 40%) was prepared from intermediate 14a (400 mg, 1.5 mmol) and lithium aluminum deuteride (252 mg, 6 mmol) following the procedure of Example 4, step 4 as a yellow solid.

[0337] LCMS: [Ms+H] + = 263.2.

[0338] Step 3: Preparation of intermediate 14c

[0339] Intermediate 14c (100 mg) was prepared from intermediate 14b (160 mg, 0.61 mmol) following the procedure of Example 4, step 7 as a yellow solid.

[0340] LCMS: [Ms+H] + = 173.3.

[0341] Step 4: Preparation of compound 14

[0342] Compound 14 was prepared from intermediate 14c (100 mg, 0.58 mmol) and 5,11- dihydro-11-chloroacetyl-6H-pyrido[2.3-b][l,4]benzodiazepin-6-one (184 mg, 0.64 mmol) following the procedure of Example 3, step 3 in 27% yield.

[0343] LCMS: [Ms+H] + = 424.2.

[0344] 1 H NMR (400 MHz, DMSO) δ 10.92 (s, 1H), 8.33 (d, J = 4.4 Hz, 1H), 7.83 (d, J = 7.5 Hz, 1H), 7.80 - 7.62 (m, 2H), 7.61 - 7.41 (m, 3H), 4.41 - 3.49 (m, 2H), 3.29 - 3.05 (m, 5H), 3.07 - 2.85 (m, 1H), 2.82 - 2.61 (m, 1H), 1.81 - 1.33 (m, 6H), 1.17 (q, J = 6.7 Hz, 6H).

[0345] Example 15: Detection of compound inhibition activity on Human M2, M3 receptor

[0346] Principle of experiment

[0347] The CHO cell lines stably expressing Human M2 and Human M3 respectively were used to determine the effect of the compound on Human M2 and Human M3 receptor activity by FLIPR Calcium 6 Assay Kit. The ability of the test compound to affect Human M2 and Human M3 was studied according to the change in cell signal intensity, and the corresponding concentration-effect curve was calculated.

[0348] The experimental reagents, consumables and instruments are shown in Table 1 and Table 2.

[0349] Table 1: Source information of reagents and consumables

[0350] Table 2: Instrument information

[0351] Experimental procedure

[0352] 1. Cell plating: The Human M2-CHO and Human M3-CHO cells were collected by digestion, counted after resuspension, and inoculated into 384-well cell plates at a seeding density of 1.2 x 10 4 cells / 25μL / well. Then the cell plates were incubated in a 37℃, 5% CO2 incubator for about 16-20h;

[0353] 2. The next day: Prepare the Assay Buffer according to the FLIPR Calcium 6 Assay Kit instructions, thaw 20x Component A (dilute the component A dry powder from the kit 20 times with the Assay Buffer) to room temperature, dilute it to 1x loading buffer with Assay buffer, and place it at room temperature for standby;

[0354] 3. Remove the culture medium from the cell plates, quickly add 35μL of the above 1x loading buffer to each well, centrifuge the cell plates, and then incubate them at 37℃ in the dark for 120min;

[0355] 4. Prepare the positive compound and the test compound working solution respectively, and transfer 5μL to the corresponding cell wells, and incubate them at 37℃ in the dark for 30min;

[0356] 5. Prepare agonist working solution and transfer 20 μΐ / νβΙΙ to 384-well compound plates (Corning, 264573);

[0357] 6. Place cell plates, inhibitor compound plates (Corning, 264573) and tips into the FLIPR instrument, add 10 μΐ of agonist diluted in step 5 to each well using the FLIPR Tetra and collect data at 515-575 nm wavelength;

[0358] 7. Curve fitting and IC50 calculation by non-linear regression method using GraphPad Prism software by plotting signal value vs. compound concentration.

[0359] Data analysis

[0360] 1) Z' factor = 1 - 3 * (SD Max + SD Min ) / (AVG Max - AVG Min )

[0361] 2) CV Max = (SD Max / AVG Max )* 100%

[0362] 3) CV Min = (SD Min / AVG Min )* 100%

[0363] 4) Signal to background ratio S / B = AVG Max / AVG Min

[0364] 5) Calculate compound IC 50 using GraphPad non-linear fitting equation:

[0365] Y = Bottom + (Top - Bottom) / (1 + 10^((LogIC 50 - X) * Hill Slope))

[0366] 6) % Inhibition equation:

[0367] Average of positive control.

[0368] Average of negative control (0.1% DMSO).

[0369] Experimental results

[0370] According to the above experimental method, the Human M2, M3 receptor inhibitory activity of the compound is detected by taking AFDX-116 as a positive control compound, and the data summary is shown in the following table (Table 3).

[0371] Table 3: Inhibition selectivity of compounds 1-14 prepared in Examples 1-14 on Human M2, M3

[0372] As can be seen from Table 3, the inhibition activity of most of the compounds in Examples 1-14 on Human M2 receptor is better than that of AFDX-116, and the inhibition selectivity of M3 / M2 of the other compounds except compound 1 is significantly better than that of AFDX-116.

[0373] Example 16: Study on in vivo pharmacokinetic properties

[0374] The efficacy of a drug is positively correlated with the concentration and residence time of the drug in the target tissue. If the concentration of the drug in the target tissue rises and falls quickly, the inhibition effect on the target tissue receptor will be maintained for a short time. The compounds provided in the present application have a longer maintenance time of the inhibition effect on M2 receptor than AFDX-116, and are expected to have better efficacy. Among them, the drug concentration of compound 6 at 6h is the highest at each time point. The following is an example of comparison experiment between compound 6 and AFDX-116.

[0375] Purpose of the experiment: determination of drug concentration in the iris-ciliary body tissue of the New Zealand white rabbit after eye drop administration.

[0376] Experimental materials: New Zealand white rabbits, male and female, 1.7-2.3 kg, 2-2.5 months old, from Qingdao Kangda Aibo.

[0377] Experimental operation: the candidate compound is configured into a clear solution. The eye drops are configured according to a certain proportion of sodium citrate, tromethamine, glycerol, hydrochloric acid, sodium hydroxide and sodium chloride. The drug concentration of the eye drops is 5 mg / mL. The New Zealand white rabbits are given eye drop administration. The iris-ciliary body is collected for homogenization at 0.25h, 1h, 2h and 6h, respectively. The concentration of the candidate compound in the iris-ciliary body of the New Zealand white rabbit after eye drop administration at each time point is detected by LC-MS / MS method.

[0378] Experimental results: as shown in Table 4.

[0379] Table 4: Comparison of pharmacokinetic test results of compound 6 and AFDX-116

[0380] From the pharmacokinetic experiment, it can be seen that the iris-ciliary body tissue absorption of AFDX-116 rapidly decreased after 2 h, while the absorption of compound 6 steadily increased from 0.25 h to 6 h, and the absorption of compound 6 was twice that of AFDX-116 at the 6 h time point.

[0381] Example 17: In vivo efficacy evaluation

[0382] Purpose of the experiment: To evaluate the inhibitory effect of AFDX-116 and compound 6 on the development of myopia (ocular axial length elongation and refractive error) in a guinea pig myopia model. Ocular axial length increase refers to the increase in the length of the eyeball from front to back. Ocular axial length increase is one of the key biological characteristics of myopia development. In myopic eyes, due to excessive ocular axial length, light is focused before reaching the retina, resulting in the formation of an image in front of the retina, causing blurred vision of distant objects. In myopia treatment, the smaller the decrease in diopter, the better. The decrease in diopter refers to the decrease in myopia degree, which usually means that the refractive state of the eye is improving, i.e., from myopia to emmetropia or hyperopia. However, the goal of treatment is to achieve a balance point that is suitable for the visual needs of the patient and the health of the eye, i.e., the diopter should be kept stable.

[0383] Sample preparation: 0.1% AFDX-116 eye drops and 0.1% compound 6 eye drops were prepared by mixing sodium citrate, tromethamine, glycerol, hydrochloric acid, sodium hydroxide, sodium chloride, and water in a certain proportion.

[0384] Grouping: According to the right eye refraction value, the animals were evenly divided into 3 groups: model control group, AFDX-116 group, and compound 6 group.

[0385] Modeling: A myopia model was established by right eye eyelid suture method, and the left eye was used as a control eye. The modeling procedure was as follows: after removing the hair from the right eye of all animals, the right eye was sutured with a U-shaped suture. After the first suture, the animals were raised for 28 days.

[0386] Drug administration: Starting from the day of suture modeling, the animals in each group were administered with eye drops according to the set (the drug samples are shown in Table 5), 20 μL / eye, once a day, for 28 consecutive days.

[0387] Table 5: Drug administration and dosage design for each group

[0388] Detection: On the 15th and 29th days of modeling, the ocular axial length was measured using an A-mode ultrasonic measuring instrument (MD-1000A, Tianjin Meda Medical Technology Co., Ltd.), and the diopter was measured using an infrared optometry instrument (ST-PR.01, Striatech).

[0389] The results are shown in Figures 1 and 2, in which the axial length growth = the axial length at the time of detection - the axial length at the time of grouping (before administration). As can be seen from the figures, in the experimental conditions, the refractive value of the animals in the compound 6 group remained stable on the 15th day of administration and on the 29th day of administration; on the 29th day of administration, the degree of slowing down of the progression of refractive reduction in the compound 6 group was equivalent to that of AFDX-116, and the effect of slowing down the growth of the axial length was superior to that of AFDX-116. Based on the stability of the progression of refractive and the slowing down of the growth of the axial length, the effect of the compound 6 group on delaying the progression of myopia was obviously superior to that of AFDX-116.

[0390] In summary, the deuterated compound provided by the present application is used as the active component of a pharmaceutical composition, which can significantly improve the selectivity for M2 receptors, significantly reduce the inhibitory effect on M3 receptors, significantly increase the IC 50 ratio of M3 / M2, has specific pharmacological properties, and can effectively treat myopia without affecting pupil size. This selective action mechanism helps to improve the targeting of treatment and the comfort of patients.

[0391] Although the content of the present application has been described in detail through the above preferred embodiments, it should be recognized that the above description should not be considered as limiting the present application. After reading the above content, various modifications and alternatives of the present application will be apparent to those skilled in the art. Therefore, the scope of protection of the present application should be defined by the appended claims.

Claims

1. A deuterated compound of a benzodiazepine, characterized in that: It has the following general formula: Wherein, R1 and R2 are independently selected from ethyl or deuterated ethyl, R3-R18 are independently selected from H group or deuterium group, and R1-R18 contain at least one deuterium group.

2. The deuterated compound of benzodiazepine according to claim 1, wherein R1-R4 contain at least one deuterium group.

3. The deuterated compound of benzodiazepine according to claim 1, wherein At least one of R1 and R2 is a deuterated ethyl group.

4. The deuterated compound of benzodiazepine according to claim 3, wherein The deuterated ethyl group includes at least one deuteration, multiple deuterations or full deuteration.

5. The deuterated compound of benzodiazepine according to claim 1, wherein R5-R9 contain at least one deuterium group.

6. The deuterated compound of benzodiazepine according to claim 1, wherein The deuterated compound comprises: Any one of them or a pharmaceutically acceptable salt, hydrate or solvate thereof.

7. A pharmaceutical composition, characterized in that Comprising: the deuterated compound according to any one of claims 1 to 6 or a pharmaceutically acceptable salt, hydrate or solvate thereof as an active ingredient.

8. The pharmaceutical composition according to claim 7, wherein Also contains: pharmaceutical excipients.

9. Use of a deuterated benzodiazepine compound according to claims 1 to 6, characterized in that: The invention is used for preparing medicine for preventing myopia and / or inhibiting the development of myopia.

10. The use according to claim 9, characterized in that The myopia includes at least one of axial myopia, refractive myopia, pseudomyopia, pathological myopia, simple myopia, extreme myopia, severe myopia, high myopia, moderate myopia, low myopia, myopia combined with glaucoma, myopia with risk of glaucoma, or myopia accompanied by high intraocular pressure.

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