Crystalline form, preparation method and application of cucurbituril derivative

Abstract

The invention relates to a crystal form, a preparation method and application of a cucurbituril derivative. Specifically, the invention provides a crystal form B and a crystal form I of a compound shown as a formula 1, and the crystal form B and the crystal form I have good stability and can be better used for clinical treatment.

Description

Technical Field

[0001] This disclosure belongs to the field of pharmaceutical technology and relates to the crystalline form of a cucurbituril derivative, its preparation method and uses. Background Technology

[0002] Muscle relaxation is one of the three essential elements of general anesthesia. While muscle relaxants meet the needs of endotracheal intubation and surgery, they also bring safety risks—residual muscle relaxation. This can lead to subjective discomfort in patients and a series of pulmonary complications such as hypoxemia, regurgitation, and aspiration. To reduce the incidence of residual muscle relaxation, measures such as the use of intermediate- and short-acting muscle relaxants, optimized intraoperative muscle relaxation management, postoperative antagonism of muscle relaxant effects, and perioperative objective muscle relaxation monitoring have made continuous progress in addressing this clinical challenge.

[0003] Postoperative muscle relaxant antagonism refers to the use of muscle relaxant antagonists to reverse the residual effects of non-depolarizing muscle relaxants. Currently, commonly used muscle relaxant antagonists can be broadly classified into two categories: competitive muscle relaxant antagonists, including neostigmine as an acetylcholine inhibitor; and selective muscle relaxant antagonists, including sugambogia sodium as a steroid muscle relaxant antagonist and cysteine ​​as a benzylisoquinoline muscle relaxant antagonist.

[0004] PCT / CN2023 / 131007 discloses a series of non-closed ring CB[n] type molecular containers with cucurbituril structures, which can efficiently bind to benzylisoquinoline and steroid muscle relaxants. By covering the quaternary ammonium sites of benzylisoquinoline and steroid muscle relaxants, the binding of muscle relaxants to neuromuscular cholinergic receptors is prevented, thereby rapidly reversing muscle relaxation.

[0005] The crystal form of a pharmaceutical active ingredient often affects its chemical stability. Different crystallization and storage conditions can lead to changes in the crystal structure of the compound, sometimes even resulting in other crystal forms. Generally, amorphous drug products lack regular crystal structures and often have other defects, such as poor product stability, fine crystals, difficulty in filtration, easy agglomeration, and poor flowability. Polymorphism of drugs places different requirements on product storage, production, and scale-up. Therefore, in-depth research on the crystal forms of the aforementioned compounds and the improvement of their various properties is essential. Summary of the Invention

[0006] This disclosure provides a novel crystal form of the compound shown in Formula 1, which has good stability and can be better applied in clinical practice.

[0007]

[0008] The crystal form A of the compound of Formula 1 provided in this disclosure has characteristic peaks at 4.869, 11.989, 13.582, and 14.425 in its X-ray powder diffraction pattern expressed as a diffraction angle 2θ.

[0009] In some embodiments, the X-ray powder diffraction pattern of crystal form A of the compound shown in Formula 1, expressed in terms of diffraction angle 2θ, is as follows: Figure 2 As shown.

[0010] This disclosure also provides a method for preparing crystal form A of the compound shown in Formula 1, the method comprising the steps of adding the compound of Formula 1 to solvent I and stirring; wherein solvent I is selected from one of acetone, isopropyl acetate, tetrahydrofuran, 2-methyltetrahydrofuran, cyclohexane, and tert-butanol.

[0011] The crystal form B of the compound shown in Formula 1 provided in this disclosure has characteristic peaks at 5.620, 8.213, 11.362, 14.698, 17.815, and 21.598 in its X-ray powder diffraction pattern expressed as a diffraction angle of 2θ.

[0012] In some embodiments, the X-ray powder diffraction pattern of crystal form B of the compound shown in Formula 1, expressed as a diffraction angle 2θ, has characteristic peaks at 5.620, 6.162, 6.468, 8.188, 8.213, 11.362, 13.125, 13.821, 14.698, 15.546, 17.815, and 21.598.

[0013] In some embodiments, the X-ray powder diffraction pattern of crystal form B of the compound shown in Formula 1, expressed as a diffraction angle 2θ, has characteristic peaks at 5.620, 6.162, 6.468, 8.188, 8.213, 11.362, 13.125, 13.821, 14.698, 15.546, 17.815, 19.510, 20.297, 21.598, 23.232, 24.745, and 26.258.

[0014] In some embodiments, the X-ray powder diffraction pattern of crystal form B of the compound shown in Formula 1, expressed in terms of diffraction angle 2θ, is as follows: Figure 3 As shown.

[0015] This disclosure also provides a method for preparing crystal form B of the compound shown in Formula 1, the method comprising dissolving the compound of Formula 1 in a 90% methanol / water solution, adding solvent II, and stirring; wherein solvent II is selected from tert-butanol, methyl tert-butyl ether, and isopropyl ether.

[0016] The crystal form C of the compound shown in Formula 1 provided in this disclosure has characteristic peaks at 8.275, 9.766, 12.368, 14.006, 16.725, and 19.006 in its X-ray powder diffraction pattern expressed as a diffraction angle 2θ.

[0017] In some embodiments, the X-ray powder diffraction pattern of crystal form C of the compound shown in Formula 1, expressed as a diffraction angle 2θ, has characteristic peaks at 8.275, 8.801, 9.766, 11.316, 12.368, 14.006, 14.737, 16.725, and 19.006.

[0018] In some embodiments, the X-ray powder diffraction pattern of crystal form C of the compound shown in Formula 1, expressed as a diffraction angle 2θ, has characteristic peaks at 8.275, 8.801, 9.766, 11.316, 12.368, 14.006, 14.737, 16.725, 19.006, 20.790, and 22.398.

[0019] In some embodiments, the X-ray powder diffraction pattern of crystal form C of the compound shown in Formula 1, expressed in terms of diffraction angle 2θ, is as follows: Figure 1 As shown.

[0020] This disclosure also provides a method for preparing the crystal form C of the compound shown in Formula 1, the method comprising dissolving the compound of Formula 1 in a 90% methanol / water solution, adding isopropyl acetate, and stirring.

[0021] The crystal form D of the compound of Formula 1 provided in this disclosure has characteristic peaks at 6.560, 9.806, 10.780, 15.654, 18.051, 19.863, and 24.544 in its X-ray powder diffraction pattern expressed as a diffraction angle of 2θ.

[0022] In some embodiments, the crystal form D of the compound shown in Formula 1, expressed as a diffraction angle 2θ, has characteristic peaks at 6.560, 9.080, 9.806, 10.780, 11.849, 12.289, 15.654, 18.051, 19.863, 20.963, 22.691, 24.544, 30.108, and 32.874.

[0023] In some embodiments, the X-ray powder diffraction pattern of crystal form D of the compound shown in Formula 1, expressed as a diffraction angle 2θ, has characteristic peaks at 6.560, 9.080, 9.806, 10.780, 11.849, 12.289, 14.740, 15.654, 16.689, 17.412, 18.051, 19.863, 20.963, 22.691, 24.544, 25.594, 27.783, 30.108, and 32.874.

[0024] In some embodiments, the X-ray powder diffraction pattern of the crystal form D of the compound shown in Formula 1, expressed in terms of the diffraction angle 2θ, is as follows: Figure 5 As shown.

[0025] This disclosure also provides a method for preparing the crystal form D of the compound shown in Formula 1, the method comprising any of the following methods:

[0026] Method 1: Dissolve the compound of formula 1 in dimethyl sulfoxide and stir; then add isopropanol and stir.

[0027] Method 2: Dissolve the compound of Formula 1 in dimethyl sulfoxide, add solvent III, stir, and then add isopropanol and stir; the solvent III is selected from one of ethanol, isopropanol, ethyl acetate, acetone, acetonitrile, isopropyl acetate, dichloromethane, and 2-methyltetrahydrofuran.

[0028] The crystal form E of the compound of Formula 1 provided in this disclosure has characteristic peaks at 7.127, 10.107, 12.052, 13.087, 14.404, and 21.618 in its X-ray powder diffraction pattern expressed as a diffraction angle 2θ.

[0029] In some embodiments, the X-ray powder diffraction pattern of the crystal form E of the compound shown in Formula 1, expressed in terms of the diffraction angle 2θ, is as follows: Figure 6 As shown.

[0030] This disclosure also provides a method for preparing the crystal form E of the compound shown in Formula 1, the method comprising any of the following methods:

[0031] Method 1: Dissolve the compound of formula 1 in water, add isopropanol, and stir;

[0032] Method 2: Dissolve the compound of formula 1 in 30% methanol / water, add isopropanol, and stir.

[0033] The crystal form F of the compound of Formula 1 provided in this disclosure has characteristic peaks at 5.950, 7.764, 10.758, 15.624, 16.747, and 26.479 in its X-ray powder diffraction pattern expressed as a diffraction angle 2θ.

[0034] In some embodiments, the X-ray powder diffraction pattern of the crystal form F of the compound shown in Formula 1, expressed as a diffraction angle 2θ, has characteristic peaks at 4.626, 5.950, 7.764, 10.758, 13.004, 15.624, 16.747, 19.828, and 26.479.

[0035] In some embodiments, the X-ray powder diffraction pattern of the crystal form F of the compound shown in Formula 1, expressed in terms of the diffraction angle 2θ, is as follows: Figure 7 As shown.

[0036] This disclosure also provides a method for preparing the crystal form F of the compound shown in Formula 1, the method comprising the steps of adding the compound of Formula 1 to N,N-dimethylformamide and stirring.

[0037] The crystal form G of the compound shown in Formula 1 provided in this disclosure has characteristic peaks at 9.800, 14.102, 19.125, and 21.088 in its X-ray powder diffraction pattern expressed as a diffraction angle 2θ.

[0038] In some embodiments, the X-ray powder diffraction pattern of the crystal form G of the compound shown in Formula 1, expressed as a diffraction angle 2θ, is as follows: Figure 8 As shown.

[0039] This disclosure also provides a method for preparing the crystal form G of the compound shown in Formula 1, the method comprising adding the compound of Formula 1 to a 90% methanol / water solution, then adding 2-methyltetrahydrofuran, and stirring.

[0040] In some embodiments, the method for preparing the crystal form G of the compound of Formula 1 further includes dissolving the compound of Formula 1 in a 50% ethanol / water solution or a 30% methanol / water solution, adding ethanol, and stirring.

[0041] The X-ray powder diffraction pattern of the compound of Formula 1 H provided in this disclosure, expressed as a diffraction angle 2θ, has characteristic peaks at 10.913, 19.269, 20.707, 22.324, 24.421, and 26.817.

[0042] In some embodiments, the X-ray powder diffraction pattern of the crystal form H of the compound shown in Formula 1, expressed in terms of the diffraction angle 2θ, is as follows: Figure 9 As shown.

[0043] This disclosure also provides a method for preparing the crystal form H of the compound shown in Formula 1, the method comprising the steps of dissolving the compound of Formula 1 in water, adding N,N-dimethylformamide, and stirring.

[0044] The X-ray powder diffraction pattern of the compound of Formula 1 provided in this disclosure, expressed in terms of diffraction angle 2θ, has characteristic peaks at 5.792, 11.909, 13.741, 19.465, and 21.297.

[0045] In some embodiments, the X-ray powder diffraction pattern of crystal form I of the compound shown in Formula 1, expressed as a diffraction angle 2θ, has characteristic peaks at 5.792, 9.423, 10.535, 11.909, 13.741, 15.802, 19.465, 21.297, 21.984, and 24.470.

[0046] In some embodiments, the X-ray powder diffraction pattern of crystal form I of the compound shown in Formula 1, expressed as a diffraction angle 2θ, has characteristic peaks at 5.792, 9.423, 10.535, 11.909, 13.741, 14.624, 15.279, 15.802, 17.339, 19.465, 21.297, 21.984, 23.293, and 24.470.

[0047] In some embodiments, the X-ray powder diffraction pattern of crystal form I of the compound shown in Formula 1, expressed in terms of diffraction angle 2θ, is as follows: Figure 10 As shown.

[0048] This disclosure also provides a method for preparing crystal form I of the compound shown in Formula 1, the method comprising dissolving the compound of Formula 1 in a 70% methanol / water solution and adding tert-butanol while stirring.

[0049] In some embodiments, the preparation method described in this disclosure further includes any one of the steps of crystallization, centrifugation (filtration), washing, or drying.

[0050] The crystallization methods disclosed herein include, but are not limited to, stirred crystallization, static crystallization, or evaporative crystallization. In some embodiments, the crystallization is stirred crystallization. In some embodiments, the crystallization is static crystallization.

[0051] This disclosure also provides a pharmaceutical composition comprising the aforementioned crystal form A, crystal form B, crystal form C, crystal form D, crystal form E, crystal form F, crystal form G, crystal form H or crystal form I, and a pharmaceutical excipient optionally selected from pharmaceutically acceptable excipients.

[0052] This disclosure also provides a pharmaceutical composition prepared from the aforementioned crystal form A, crystal form B, crystal form C, crystal form D, crystal form E, crystal form F, crystal form G, crystal form H or crystal form I, and optionally a pharmaceutically acceptable excipient.

[0053] This disclosure also provides a method for preparing a pharmaceutical composition, comprising the step of mixing the aforementioned crystal form A, crystal form B, crystal form C, crystal form D, crystal form E, crystal form F, crystal form G, crystal form H or crystal form I with a pharmaceutically acceptable excipient.

[0054] This disclosure also provides the use of the aforementioned crystal form A, crystal form B, crystal form C, crystal form D, crystal form E, crystal form F, crystal form G, crystal form H or crystal form I, or the aforementioned composition, in the preparation of a medicament for reversing drug-induced neuromuscular blockade and / or anesthesia.

[0055] The "2θ or 2θ angle" mentioned in this disclosure refers to the diffraction angle, where θ is the Bragg angle, and the unit is ° or degree; the error range of 2θ for each characteristic peak is ±0.20 (including the case where the number has more than one decimal place after rounding), specifically -0.20, -0.19, -0.18, -0.17, -0.16, -0.15, -0.14, -0.13, -0.12, -0.11, -0.10, -0.09, -0.08, -0.07, -0.06, -0.05, -0.04, -0.03, -0.02, -0.01, 0.00, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20.

[0056] The numerical values ​​in this disclosure, such as those relating to the content of certain substances, are calculated data and inevitably contain a certain degree of error. Generally, ±10% is within the reasonable error range. The error may vary to some extent depending on the context in which it is used, but this variation shall not exceed ±10%, and may be ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1%, preferably ±5%.

[0057] The starting material used in the crystal form preparation method disclosed herein can be any form of compound, including but not limited to: amorphous, arbitrary crystal form, hydrate, solvate, etc.

[0058] The drying temperature described in this disclosure is generally 25℃-100℃, preferably 40℃-70℃, and can be dried under normal pressure or reduced pressure.

[0059] The crystallization methods described in this disclosure include room temperature crystallization, cooling crystallization, solvent evaporation crystallization, and seed crystallization induction. The cooling temperature is selected from below 65°C, preferably from -10°C to 60°C. Stirring can also be performed during the crystallization process.

[0060] The “differential scanning calorimetry or DSC” described in this disclosure refers to measuring the temperature difference and heat flow difference between the sample and the reference material during the sample heating or isothermal process, in order to characterize all physical and chemical changes related to thermal effects and obtain phase transition information of the sample.

[0061] According to the description of hygroscopic characteristics and the definition of hygroscopic weight gain in the "Guiding Principles on Hygroscopicity of Drugs" in Part IV of the 2015 edition of the Chinese Pharmacopoeia,

[0062] Deliquescence: Absorbs sufficient moisture to form a liquid;

[0063] Extremely hygroscopic: the weight gain due to hygroscopic absorption is not less than 15%;

[0064] It has hygroscopic properties: the weight gain due to hygroscopic absorption is less than 15% but not less than 2%;

[0065] Slightly hygroscopic: the weight gain due to moisture absorption is less than 2% but not less than 0.2%;

[0066] It has little or no hygroscopicity: the weight gain due to moisture absorption is less than 0.2%.

[0067] The “excipients” described in this disclosure include, but are not limited to, any adjuvants, carriers, flow aids, sweeteners, diluents, preservatives, dyes / colorants, flavoring agents, surfactants, wetting agents, dispersants, suspending agents, stabilizers, isotonic agents, or emulsifiers that have been approved by the U.S. Food and Drug Administration for use in humans or livestock. Attached Figure Description

[0068] Figure 1 The TOF 0.9 time for cisatracurium administration of muscle relaxant antagonists was as follows: *p<0.05, **p<0.01, ***p<0.001.

[0069] Figure 2 XRPD spectrum of crystal form A of compound of formula 1.

[0070] Figure 3 XRPD spectrum of crystal form B of compound of formula 1.

[0071] Figure 4 XRPD spectrum of crystal form C of compound 1.

[0072] Figure 5 XRPD spectrum of compound D of Formula 1.

[0073] Figure 6 XRPD spectrum of crystal form E of compound 1.

[0074] Figure 7 XRPD spectrum of compound F (Formula 1)

[0075] Figure 8 XRPD spectrum of compound G (Formula 1)

[0076] Figure 9 XRPD spectrum of compound H (Formula 1)

[0077] Figure 10 XRPD spectrum of compound I of Formula 1.

[0078] Figure 11 XRPD spectrum of the amorphous compound of Formula 1. Detailed Implementation

[0079] The present disclosure will be explained in more detail below with reference to embodiments or experimental examples. The embodiments or experimental examples in the present disclosure are only used to illustrate the technical solutions in the present disclosure and are not intended to limit the substance and scope of the present disclosure.

[0080] Test conditions of the instruments used in the experiment:

[0081] The structure of the compound was determined by nuclear magnetic resonance (NMR) and / or mass spectrometry (MS). NMR shifts (δ) were expressed in 10⁻¹⁰ ohms. -6 The unit (ppm) is given. NMR measurements were performed using a Bruker AVANCE NEO 500M NMR spectrometer. The solvents used were deuterated dimethyl sulfoxide (DMSO-d6), deuterated chloroform (CDCl3), and deuterated methanol (CD3OD), with tetramethylsilane (TMS) as the internal standard.

[0082] MS measurements were performed using an Agilent 1200 / 1290DAD-6110 / 6120 Quadrupole MS LC-MS system (manufacturer: Agilent, MS model: 6110 / 6120 Quadrupole MS), a Waters ACQuity UPLC-QD / SQD system (manufacturer: Waters, MS model: Waters ACQuity Qda Detector / Waters SQ Detector), or a THERMO Ultimate3000-Q Exactive system (manufacturer: THERMO, MS model: THERMO Q Exactive).

[0083] High-performance liquid chromatography (HPLC) analysis was performed using an Agilent HPLC 1200DAD, an Agilent HPLC 1200VWD, and a Waters HPLCe2695-2489 HPLC system.

[0084] High performance liquid chromatography (HPLC) was performed using Waters 2767, Waters 2767-SQ Detecor2, Shimadzu LC-20AP, and Gilson-281 preparative chromatographs.

[0085] Silica gel column chromatography generally uses Yantai Huanghai silica gel with a mesh size of 200-300 as the carrier.

[0086] XRPD (X-ray Powder Diffraction) was used for analysis: measurements were performed using a BRUKER D8 X-ray diffractometer. Specific data collected included: Cu anode (40 kV, 40 mA), Cu-Kα1 rays. Kα2 rays Kβ rays Scanning mode: θ / 2θ, scanning range (2θ range): 5°~45°.

[0087] DSC stands for Differential Scanning Calorimetry: Measurements were performed using a METTLER TOLEDO DSC 3+ differential scanning calorimeter with a heating rate of 10℃ / min. The specific temperature range was referenced from the corresponding spectra (mostly 25-300℃), and the nitrogen purging rate was 50mL / min.

[0088] TGA is thermogravimetric analysis: the test was performed using a METTLER TOLEDO TGA 2 thermogravimetric analyzer, with a heating rate of 10℃ / min, and the specific temperature range was referenced from the corresponding spectrum (mostly 30-350℃). The nitrogen purging rate was 50mL / min.

[0089] DVS stands for Dynamic Moisture Adsorption: The detection method is SMSDVS Advantage. At 25℃, the humidity changes from 50% to 95% to 0% to 95% to 50%, with a step of 10% (the last step is 5%). (The specific humidity range is subject to the corresponding spectrum. The methods listed here are the most commonly used methods.) The judgment standard is that dm / dt is not greater than 0.002%.

[0090] The known starting materials disclosed herein can be synthesized using or in accordance with methods known in the art, or can be purchased from companies such as ABCR GmbH & Co. KG, Acros Organics, Aldrich Chemical Company, AccelaChemBio Inc, and Darui Chemicals.

[0091] Unless otherwise specified in the examples, all reactions can be carried out under an argon or nitrogen atmosphere.

[0092] Argon or nitrogen atmosphere refers to a reaction flask connected to an argon or nitrogen gas balloon with a volume of approximately 1L.

[0093] The reaction process in the examples was monitored using thin-layer chromatography (TLC). The developing solvent used in the reaction, the eluent system used for column chromatography to purify the compounds, and the developing solvent system for TLC included: A: n-hexane / ethyl acetate system, B: dichloromethane / methanol system. The volume ratio of the solvent was adjusted according to the polarity of the compounds, and small amounts of basic or acidic reagents such as triethylamine and acetic acid could also be added for adjustment.

[0094] Example 1

[0095]

[0096] Step 1: Urea (38.89 g, 647.46 mmol), 0.3 M dilute hydrochloric acid (80 mL), and 1,2-cyclohexanedione 1a (22.0 g, 196.20 mmol) were added to a 500 mL three-necked flask. The mixture was heated to 50 °C and stirred for 16 hours. The reaction mixture was cooled to room temperature, filtered, and the filter cake was washed with 100 mL of water and 100 mL of anhydrous ethanol. After drying, cyclohexylglycerol, compound 1b (pale yellow body, 27.4 g, yield: 71%) was obtained.

[0097] MSm / z(ESI):197.1[M+1]+.

[0098] 1H NMR (400MHz, DMSO-d6): δ7.02 (s, 4H), 1.72-1.68 (m, 4H), 1.42-1.35 (m, 4H).

[0099] Step 2: Add compound 1b (27.4 g, 139.65 mmol), 140 mL of 9 M hydrochloric acid, and paraformaldehyde (20.9 g, 698.23 mmol) to a 1 L three-necked flask. Stir the reaction mixture at room temperature for 24 hours. Add 500 mL of water to the reaction system and continue stirring at room temperature for 16 hours. Filter the reaction mixture, wash, and dry to obtain cyclohexylglycerol diether and compound 1c (white solid, 20.2 g, yield: 52%).

[0100] MS(ESI):281.1[M+1]+.

[0101] 1H NMR (400MHz, DMSO-d6): δ5.20(d,4H,J=11.6), 4.91(d,4H,J=11.2), 2.25-2.18(m,4H), 1.56-1.50(m,4H).

[0102] Step 3: Compound 1c (2.73 g, 9.73 mmol) was weighed into a dry three-necked flask, purged with argon, dissolved in methanesulfonic acid (10 mL), and then glycourea dimer 1d (1 g, 3.24 mmol, prepared using the known method "WO2012051407A2") was added. The mixture was stirred at room temperature for 24 hours. The reaction solution was slowly added to 100 mL of water (cooled in an ice-water bath), and then brought to room temperature. The mixture was filtered. 1.77 g of crude product was obtained by drying. The crude product was heated and dissolved in TFA (4 mL), then 16 mL of water was added, stirred, filtered, and dried under vacuum to obtain compound 1e (1.21 g, yield: 44.9%).

[0103] MS(ESI):837.3[M+1]+.

[0104] 1H NMR (400MHz, CDCl3): δ5.72~5.37(m,10H),5.15(d,4H),4.75(d,4H),4.15~4.11(m,6H),2.28(br,4H),2.05(br,4H),1.45(br,8H).

[0105] Step 4: Weigh compound 1e (1.06 g, 1.27 mmol) into a dry three-necked flask, replace with argon gas, add TFA (10 mL) to dissolve, then add sodium 3,3'-(naphthalene-1,4-dioxy)bis(propane-1-sulfonic acid) and compound 1f (1.42 g, 3.17 mmol, prepared by the known method "WO2012051407A2"). After the addition is complete, heat the reaction to 60 °C and stir for 3 hours. Evaporate the TFA under reduced pressure, add 20 mL of ethanol to the resulting solid, heat under reflux for 2 hours, cool to room temperature, and filter. The filter cake was washed with ethanol, dried, and the resulting solid was dissolved in 10 mL of water by heating. Then, 30 mL of ethanol was added, and the mixture was filtered. The filter cake was then purified by high-performance liquid chromatography (HPLC) (column: SharpSil-T, 30*150 mm, 5 μm; mobile phase: aqueous phase and acetonitrile, gradient ratio: aqueous phase 25%-42%) to obtain compound 1 (0.27 g, yield: 12.6%). X-ray powder diffraction analysis showed that the product was in a free, amorphous state. The XRPD spectrum is shown below. Figure 11 .

[0106] MSm / z (negative ion ESI): 801.0 [M / 2-1] -

[0107] 1H NMR (400MHz, D2O): δ7.85-7.61(m,4H),7.37-6.99(m,4H),5.58-5.42(m,6H),5.32-5.10(m,8H), 4.42-4.25(m,4H),4.17-3.81(m,14H),3.20-3.05(m,8H),2.28-1.92(m,16H),1.56-1.19(m,8H).

[0108] Test Example 1. Test of the in vitro binding activity of the disclosed compounds against muscle relaxants.

[0109] I. Experimental Objective:

[0110] The in vitro binding activity of the disclosed compound with the muscle relaxant was tested by isothermal titration calorimetry (ITC), and the Kd value of the binding was evaluated.

[0111] II. Experimental Materials:

[0112] Isothermal titration calorimeter (including computer host and supporting software), CB2, compound 1, cisatracurium ammonium, deionized water.

[0113] III. Experimental Methods and Procedures:

[0114] Using a preset washing program, wash the tubing, sample cell, and titration needle of the isothermal titration calorimetry system with deionized water. Prepare an aqueous solution of cisatracurium ammonium and an aqueous solution of the analyte, with a cisatracurium ammonium:analyte ratio of 10:1 to 20:1. Fill the sample cell with CB2 and the analyte using the dispensing needle, and then pipette cisatracurium ammonium into the titration needle using the dispensing program. Place the titration needle into the sample cell, start stirring, and allow the system to equilibrate for 5–10 minutes. Set the titration parameters: 2.5 μL of sample per drop, 20 drops in total, and a titration interval of 150 s. Begin the titration and record the thermal curve.

[0115] After titration, the Kd value of the combination of the test compound and cisatracurium is calculated by thermal curve. The tubing, sample cell and titration needle are then washed using a washing program before testing the next compound.

[0116] CB2 (prepared using the known method "WO2012051407A2") has the following structure:

[0117]

[0118] IV. Test Results:

[0119] The binding capacity of CB2 and compound 1 to cisatracurium in vitro was tested using the ITC method. The dissociation constant Kd values ​​are shown in Table 1. The results show that compound 1 has a stronger ability to bind cisatracurium in vitro than CB2.

[0120] Table 1: In vitro binding affinity of the test compounds to cisatracurium

[0121] Test compound <![CDATA[K d (M)]]> CB2 2.506E-5 Compound 1 7.475E-6

[0122] Test Example 2. Test on the antagonistic effect of the disclosed compound on muscle relaxants.

[0123] I. Experimental Objective

[0124] The effects of the compounds disclosed herein on the reversal of muscle relaxant effects in the gastrocnemius muscle were tested in a rat neuromuscular model. Onset time, TOF and other indicators were evaluated and compared with CB2 and neostigmine.

[0125] II. Experimental Materials

[0126] SPF grade male SD rats, BL-420A biological function experimental system (main unit, stimulator, tension transducer), ventilator, electronic scale, surgical instruments, syringe, clippers, electronic scale, iron stand, foam board, urethane, sodium chloride, CB2, compound 1, cisatracurium, succinylcholine, neostigmine, sterile water, 95% alcohol.

[0127] III. Experimental Methods

[0128] SPF-grade male SD rats that have passed quarantine were used and housed in an environment with a room temperature of 22±0.5℃, an air exchange rate of 20-50 / h, an airflow velocity of 0.05-0.18m / s, and a 12 / 12-hour day / night light / dark cycle. The animals were allowed to acclimatize in the facility for at least 3 days, with 6 rats per cage, until their weight reached 220g-250g before the start of the experiment.

[0129] The test sample was reconstituted using 0.9% sodium chloride. After calculating the content, the required amount of test sample was weighed, dissolved in 0.9% sodium chloride injection (within 30 minutes), and then mixed thoroughly using a vortex mixer. CB2 was reconstituted with purified water.

[0130] Actual drug weight (mg) = Dosage concentration A (mg / mL) × Solvent volume (mL) / Content (%)

[0131] Rats were randomly divided into CB2, compound 1, and neostigmine groups, with 5 rats in each group (based on the actual number of rats in each group). The drug administration volume was 2 mL / kg for each group.

[0132] CB2 (prepared using the known method "WO2012051407A2") has the following structure:

[0133]

[0134] After anesthetizing rats, the sciatic nerve and gastrocnemius muscle were isolated. The sciatic nerve was stimulated, and muscle tone signals were recorded using a tension transducer. The rats were then intubated and mechanically ventilated using a small animal ventilator. After a period of stable muscle tone recording, the drug was administered: the initial dose was twice the ED. 90 Administer a dose (0.8 mg / kg) of muscle relaxant (cistracurium). The muscle tone curve should decrease at this point. Inject the antagonist (test sample and neostigmine) 30-60 seconds after administration. Once the muscle tone curve recovers to above 95%, administer the ED. 90 When administering a muscle relaxant (succinylcholine) at a dose of 0.9 mg / kg, a decrease in muscle tone should be observed. The experiment can be stopped once muscle tone naturally recovers to above 95%. Muscle tone signals should be continuously recorded during the experiment, and indicators such as onset time and clinical efficacy should be statistically analyzed. By comparing muscle tone signals after administration, the reversal effect of the antagonist can be determined and compared.

[0135] IV. Experimental Procedure

[0136] 4.1 Weighing and anesthetizing rats

[0137] The rats were weighed and, once they were in a stable mood, 25% urethane was prepared from ethyl carbamate and anesthetized by intraperitoneal injection at a rate of 1 mL / 100 g. After the pain reflex disappeared, the rats were fixed in a prone position on a foam board, and hair was removed from the buttocks and the outer side of the right thigh.

[0138] 4.2 Separation of the sciatic nerve

[0139] Behind the hip joint, make an incision along the outer edge of the femur in the middle of the thigh, lift the skin and superficial fascia, bluntly dissect the muscles to expose the sciatic nerve. Be careful to use a glass dissecting needle during dissection to prevent damage to the nerve with metal instruments.

[0140] 4.3 Separation of the gastrocnemius muscle

[0141] Cut open the skin of the lower leg at the ankle joint, cut the ligaments at the front of the ankle joint, separate the gastrocnemius muscle, tie a suture at the gastrocnemius tendon at the ankle, and cut the tendon at the distal end of the suture.

[0142] 4.4 Signal Collection

[0143] The gastrocnemius muscle ligation suture was connected to the tension transducer, and the stimulator was connected to the sciatic nerve. The input signal was set to tension, and the parameters were set as square wave, fine voltage, series stimulation, delay 0.05ms, pulse width 0.2ms, frequency 2Hz, intensity 0.225±0.025V, intensity increment 0, series length 4, main period 12s, and number of stops 30000. The muscle contraction curve was recorded. During the measurement, the muscle nerve was kept moist with physiological saline every 3-5 minutes.

[0144] 4.5 Connect to ventilator and administer muscle relaxants and antagonists.

[0145] Wipe the ventilator tubing with alcohol, make an incision in the neck skin to locate the jugular vein and trachea, cut the trachea, connect the ventilator, and set the parameters as follows: tidal volume 6 mL, respiratory rate to exhalation ratio 5:4, and respiratory rate 80 breaths / min. After stabilizing for approximately 5 minutes, administer the drug via the jugular vein. Based on the reference settings, administer 2 times the initial dose. 90 After administering the appropriate dose of muscle relaxant (cistracurium), the muscle tone curve should decrease. Inject the antagonist (test sample or neostigmine) 30-60 seconds later. Once the muscle tone curve recovers to over 95%, administer the ED. 90 After administering the appropriate dose of muscle relaxant (succinylcholine), wait until the muscle tone curve recovers to above 95% before stopping the experiment. Continue recording the muscle tone curve throughout the experiment.

[0146] Table 2

[0147]

[0148] 4.6 Statistics on Timeliness Indicators

[0149] The onset time and secondary muscle relaxation onset time were statistically analyzed using a biological function experimental system, and the statistical standards are as follows.

[0150] 1) Time to 90% recovery of TOF (TOF 0.9): The time for the T4 / T1 value of TOF string stimulation to recover to approximately 90% - the time of antagonist administration.

[0151] 2) Duration of muscle relaxation by succinylcholine: Time to reduce muscle tone to its lowest value - Time to administer succinylcholine

[0152] V. Experimental Conclusions

[0153] Depend on Figure 1 It can be seen that compound 1 can achieve the efficacy level of neostigmine at 20 mg / kg, which is superior to CB2.

[0154] Example 2 Preparation of free crystal form A

[0155] Add 5 mg of the compound shown in Formula 1 to 2 ml of acetone, stir overnight at 40 °C, centrifuge, and vacuum dry to obtain a solid. X-ray powder diffraction analysis identified the product as free crystalline form A, and the XRPD spectrum is shown below. Figure 2 The positions of its characteristic peaks are shown in Table 3. The DSC spectrum shows that the endothermic peaks are at 64.13℃ and 280.57℃. The TGA spectrum shows a weight loss of 3.84% between 30℃ and 100℃.

[0156] Table 3

[0157]

[0158]

[0159] Example 3 Preparation of free crystal form A

[0160] Add 5 mg of the compound shown in Formula 1 to 2 ml of solvent (as shown in Table 4), stir overnight at 40°C, centrifuge, and vacuum dry to obtain a solid. X-ray powder diffraction analysis showed that the product is in free crystalline form A.

[0161] Table 4

[0162] solvent result Isopropyl acetate Crystal form A Tetrahydrofuran Crystal form A 2-Methyltetrahydrofuran Crystal form A Cyclohexane Crystal form A tert-Butanol Crystal form A

[0163] Example 4: Preparation of free crystal form B

[0164] 5 mg of the compound shown in Formula 1 was added to 1 ml of 80% methanol / water solution and dissolved at 60 °C. Then, 1.0 ml of tert-butanol was added, and the mixture was stirred at room temperature to induce crystallization. After centrifugation and vacuum drying, a solid was obtained. X-ray powder diffraction analysis identified the product as free crystalline form B. The XRPD spectrum is shown below. Figure 3 The positions of its characteristic peaks are shown in Table 5. The DSC spectrum shows endothermic peaks at 59.81℃, 233.8℃, and 277.27℃. The TGA spectrum shows a weight loss of 6.20% between 30℃ and 110℃, and a weight loss of 18.61% between 150℃ and 310℃.

[0165] Table 5

[0166]

[0167]

[0168] Example 5: Preparation of free crystal form B

[0169] 5 mg of the compound shown in Formula 1 was added to 1 ml of 90% methanol / water solution and dissolved at 60 °C. Then, 0.8 ml of the solvent from Table 6 was added, and the mixture was stirred at room temperature to induce crystallization. After centrifugation and vacuum drying, a solid was obtained. X-ray powder diffraction analysis showed that the product was in a free crystalline state, form B.

[0170] Table 6

[0171] solvent result tert-Butanol Crystal form B Methyl tert-butyl ether Crystal form B Isopropyl ether Crystal form B

[0172] Example 6: Preparation of Free Crystal Form C

[0173] 5 mg of the compound shown in Formula 1 was added to 1 ml of 90% methanol / water solution and dissolved at 60 °C. Then, 0.8 ml of isopropyl acetate was added, and the mixture was stirred at room temperature to induce crystallization. The crystals were then centrifuged and dried under vacuum to obtain a solid. X-ray powder diffraction analysis identified the product as free crystalline form C. The XRPD spectrum is shown below. Figure 4The positions of its characteristic peaks are shown in Table 7. The DSC spectrum shows endothermic peaks at 60.13℃, 236.78℃, and 280.74℃. The TGA spectrum shows a weight loss of 4.39% between 30℃ and 110℃, and a weight loss of 19.47% between 170℃ and 320℃.

[0174] Table 7

[0175]

[0176] Example 7 Preparation of free crystal form D

[0177] 80 mg of the compound shown in Formula 1 was added to 0.8 mL of dimethyl sulfoxide and dissolved at 60 °C. Crystallization was carried out by stirring at room temperature, followed by centrifugation. The solid was then added to 0.8 mL of isopropanol, stirred overnight at room temperature, centrifuged, and dried under vacuum to obtain the solid. X-ray powder diffraction analysis identified the product as free crystal form D. The XRPD spectrum is shown below. Figure 5 The positions of its characteristic peaks are shown in Table 8. The DSC spectrum shows endothermic peaks at 51.15℃, 160.14℃, and 278.11℃. The TGA spectrum shows a weight loss of 2.37% between 30℃ and 80℃, and a weight loss of 17.83% between 150℃ and 310℃.

[0178] Table 8

[0179]

[0180]

[0181] Example 8: Preparation of free crystal form D

[0182] 10 mg of the compound shown in Formula 1 was added to 0.7 mL of dimethyl sulfoxide and dissolved at 60 °C. Then, 1 mL of solvent from Table 9 was added, and the mixture was stirred at room temperature to crystallize. After centrifugation, the solid was added to 0.8 mL of isopropanol, stirred overnight at room temperature, centrifuged, and dried under vacuum to obtain the solid. X-ray powder diffraction analysis showed that the product was in a free crystalline form, D.

[0183] Table 9

[0184] solvent result ethanol Crystal form D Isopropanol Crystal form D Ethyl acetate Crystal form D acetone Crystal form D Acetonitrile Crystal form D Isopropyl acetate Crystal form D dichloromethane Crystal form D 2-Methyltetrahydrofuran Crystal form D

[0185] Example 9 Preparation of free crystal form E

[0186] 5 mg of the compound shown in Formula 1 was dissolved in 0.065 ml of water, and 0.6 ml of isopropanol was added. The mixture was stirred at room temperature to induce crystallization, centrifuged, and dried under vacuum to obtain a solid. X-ray powder diffraction analysis identified the product as free crystalline form E. The XRPD spectrum is shown below. Figure 6The positions of its characteristic peaks are shown in Table 10. The DSC spectrum shows endothermic peaks at 58.63℃ and 281.39℃. The TGA spectrum shows a weight loss of 3.62% between 30℃ and 90℃.

[0187] Table 10

[0188]

[0189] Example 10 Preparation of free crystal form E

[0190] 10 mg of the compound shown in Formula 1 was added to 0.1 ml of 30% methanol / water solution and dissolved at 60 °C. Then, 0.5 ml of isopropanol was added, and the mixture was stirred at room temperature to induce crystallization. After centrifugation and vacuum drying, a solid was obtained. X-ray powder diffraction analysis showed that the product was in the free crystalline form E.

[0191] Example 11 Preparation of free crystal form F

[0192] Add 5 mg of the compound shown in Formula 1 to 0.5 mL of N,N-dimethylformamide, stir at 40 °C to crystallize, centrifuge, and vacuum dry to obtain a solid. X-ray powder diffraction analysis identified the product as free crystalline form F, and the XRPD spectrum is shown below. Figure 7 The positions of its characteristic peaks are shown in Table 11. The DSC spectrum shows endothermic peaks at 67.46℃ and 281.89℃. The TGA spectrum shows a weight loss of 6.79% between 30℃ and 120℃.

[0193] Table 11

[0194]

[0195] Example 12 Preparation of free crystal form G

[0196] 5 mg of the compound shown in Formula 1 was dissolved in 1 ml of 90% methanol / water solution, and 0.8 ml of 2-methyltetrahydrofuran was added. The mixture was stirred at room temperature to induce crystallization, centrifuged, and dried under vacuum to obtain a solid. X-ray powder diffraction analysis identified the product as free-state crystalline form G. The XRPD spectrum is shown below. Figure 8 The positions of its characteristic peaks are shown in Table 12. The DSC spectrum shows endothermic peaks at 69.45℃ and 284.71℃. The TGA spectrum shows a weight loss of 4.95% between 30℃ and 120℃.

[0197] Table 12

[0198]

[0199]

[0200] Example 13 Preparation of free crystal form G

[0201] 10 mg of the compound shown in Formula 1 was dissolved in 0.1 ml of solvent from Table 13, dissolved completely at 60 °C, and 0.5 ml of ethanol was added. The mixture was stirred at room temperature to induce crystallization, centrifuged, and dried under vacuum to obtain a solid. X-ray powder diffraction analysis showed that the product was in a free crystalline state, G.

[0202] Table 13

[0203] solvent result 50% ethanol / aqueous solution Crystal form G 30% methanol / water solution Crystal form G

[0204] Example 14 Preparation of free crystalline form H

[0205] 5 mg of the compound shown in Formula 1 was dissolved in 0.065 ml of water, and 0.6 ml of N,N-dimethylformamide was added. The mixture was stirred at room temperature to induce crystallization, centrifuged, and dried under vacuum to obtain a solid. X-ray powder diffraction analysis identified the product as free crystalline form H. The XRPD spectrum is shown below. Figure 9 The positions of its characteristic peaks are shown in Table 14. The DSC spectrum shows endothermic peaks at 70.94℃ and 282.7℃. The TGA spectrum shows a weight loss of 4.06% between 30℃ and 100℃, and a weight loss of 19.43% between 175℃ and 315℃.

[0206] Table 14

[0207]

[0208] Example 15 Preparation of Free Crystal Form I

[0209] 10 mg of the compound shown in Formula 1 was added to 0.6 ml of 70% methanol / water solution and dissolved at 60 °C. Then, 1 ml of tert-butanol was added, and the mixture was stirred at room temperature to induce crystallization. The crystals were then centrifuged and dried under vacuum to obtain a solid. X-ray powder diffraction analysis identified this product as free-state crystal form I. The XRPD spectrum is shown below. Figure 10 The positions of its characteristic peaks are shown in Table 15. The DSC spectrum shows endothermic peaks at 73.97℃ and 283.44℃. The TGA spectrum shows a weight loss of 7.43% between 30℃ and 110℃.

[0210] Table 15

[0211]

[0212]

[0213] Example 16 Preparation of free amorphous state

[0214] 5 mg of the compound shown in Formula 1 was dissolved in solvent 1 (Table 16), solvent 2 was added, and the mixture was stirred at room temperature to crystallize. After centrifugation and solid-state vacuum drying, the title product was obtained. X-ray powder diffraction analysis showed that the product was in a free, amorphous state, as shown in the XRPD spectrum. Figure 11 .

[0215] Table 16

[0216] Solvent 1 Solvent 2 result 0.065ml water 0.6ml acetone amorphous 1 ml 90% methanol / water solution 0.8ml acetone amorphous

[0217] Example 17 Preparation of free amorphous state

[0218] Add 5 mg of the compound shown in Formula 1 to 2 ml of Table 17 solvent, stir overnight at 40°C, centrifuge, and dry the solid under vacuum to obtain the title product.

[0219] Table 17

[0220]

[0221]

Claims

1. A crystal form B of the compound shown in Formula 1, characterized in that, The X-ray powder diffraction pattern, expressed as a diffraction angle 2θ, shows characteristic peaks at 5.620, 8.213, 11.362, 14.698, 17.815, and 21.598, with preferred peaks at 5.620, 6.162, 6.468, 8.188, 8.213, 11.362, 13.125, 13.821, 14.698, 15.546, and 17. Characteristic peaks are found at 815 and 21.598, and more preferably at 5.620, 6.162, 6.468, 8.188, 8.213, 11.362, 13.125, 13.821, 14.698, 15.546, 17.815, 19.510, 20.297, 21.598, 23.232, 24.745, and 26.

258.

2. The crystal form B according to claim 1, characterized in that, The X-ray powder diffraction pattern expressed in terms of the diffraction angle 2θ is shown in Figure 3.

3. A method for preparing crystal form B as described in claim 1 or 2, the method comprising the steps of dissolving the compound of formula 1 in methanol / water solution, adding solvent II, and stirring; wherein solvent II is selected from tert-butanol, methyl tert-butyl ether, and isopropyl ether.

4. A crystal form I of the compound shown in Formula 1, characterized in that, The X-ray powder diffraction pattern, expressed as a diffraction angle 2θ, has characteristic peaks at 5.792, 11.909, 13.741, 19.465, and 21.297, preferably at 5.792, 9.423, 10.535, 11.909, 13.741, 15.802, 19.465, 21.297, 21.984, and 24.470, and more preferably at 5.792, 9.423, 10.535, 11.909, 13.741, 14.624, 15.279, 15.802, 17.339, 19.465, 21.297, 21.984, 23.293, and 24.

470.

5. The crystal form I according to claim 4, characterized in that, The X-ray powder diffraction pattern expressed in terms of the diffraction angle 2θ is shown in Figure 10.

6. A method for preparing crystal form I as described in claim 4 or 5, the method comprising the steps of dissolving the compound of formula I in 70% methanol / water solution and adding tert-butanol while stirring.

7. The crystal form according to any one of claims 1-2 and 4-5, wherein the 2θ angle error range is ±0.

20.

8. A pharmaceutical composition comprising the crystal form according to any one of claims 1-2, 4-5 and optionally a pharmaceutically acceptable excipient.

9. A method for preparing a pharmaceutical composition, comprising the step of mixing the crystal form according to any one of claims 1-2, 4-5 and a pharmaceutically acceptable excipient.

10. Use of the crystal form according to any one of claims 1-2, 4-5, or the pharmaceutical composition according to claim 8 in the preparation of a medicament for reversing drug-induced neuromuscular blockade and / or anesthesia.

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