Crystal form of revefenacin triphenylacetate and preparation method therefor

Abstract

The present invention belongs to the technical field of pharmaceutical chemistry, and specifically relates to a crystal form of revefenacin triphenylacetate and a preparation method therefor. The crystal form exhibits good chemical stability and crystal form purity, can be readily prepared on a large scale, can be better used in the preparation and large-scale production of pharmaceutical preparations, and has broad application prospects.

Description

A crystalline form of triphenylacetic acid refennaxine and its preparation method Technical Field

[0001] This invention belongs to the field of medicinal chemistry technology, specifically relating to the refennaxine triphenylacetic acid crystal form and its preparation method. This crystal form exhibits good chemical stability and purity, is easy to prepare on a large scale, and is better suited for the preparation of pharmaceutical formulations and large-scale production, thus possessing broad application prospects. Background Technology

[0002] Rafenasine is a long-acting muscarinic antagonist with the structural formula shown in Formula 1. It is primarily administered via inhalation solution to improve lung function, reduce clinical symptoms of COPD, and prevent further deterioration of the disease, serving as maintenance therapy for COPD patients. In 2018, the FDA approved rafenasine for maintenance therapy in patients with chronic obstructive pulmonary disease (COPD). COPD is a progressive, irreversible, persistent airway obstructive lung disease with multiple causes, and is also a general term for chronic bronchitis and emphysema. COPD is estimated to affect approximately 328 million people worldwide and is already the third leading cause of death in the United States, projected to become the third leading cause of death globally by 2020. There is a severe unmet need for COPD treatment.

[0003] The original research company has two patents on crystal forms that have been granted in China: CN101163677B and CN102470130B. CN101163677B discloses three salt forms: diphosphate, monosulfate, and hydrogen oxalate, protecting the diphosphate form. CN102470130B discloses four crystal forms: free base crystal form I, crystal form II, crystal form III, and crystal form IV. The DSC peak temperatures for crystal forms I and II are 102.7℃ and 98.64℃, respectively, while the DSC peak temperatures for crystal forms III and IV are 123.1℃ and 118.8℃, respectively. This patent protects crystal form III, which is anhydrous and has an acceptable level of hygroscopicity and a relatively high melting point. This patent will expire in China on July 13, 2030.

[0004] Salt formation can improve the physicochemical properties of drug molecules, significantly increasing solubility and dissolution rate, and is one of the effective means to enhance their drug-likeness. It can also improve drug compliance, increase drug stability, and reduce adverse reactions. When the stability of free bases or free acids is problematic, salt formation is the preferred option. For ionizable molecules, salt formation can improve the physicochemical properties of compounds, such as solubility, crystallinity, hygroscopicity, melting point, and stability, thereby improving not only bioavailability but also solid-state stability.

[0005] The diphosphate, monosulfate, and hydrogen oxalate salts disclosed in the original patent CN101163677B are obtained through unconventional methods such as freeze-drying, making mass production impossible. Subsequent original patents only protect the free form, and other salt forms are not currently reported in the literature, indicating that refennaxine salt formation is quite difficult. The inventors of this application screened 39 acids, and only triphenylacetic acid showed a tendency to form a salt with refennaxine, but the yield was low. Diphenylacetic acid, with a very similar structure, could not form a salt with refennaxine. Through in-depth optimization of the salt formation conditions of triphenylacetic acid with refennaxine, triphenylacetic acid refennaxine could be stably prepared. The finally prepared triphenylacetic acid refennaxine solid was in good condition, and TG, DSC, PXRD, mass spectrometry, 1H NMR, and 1C NMR all showed that it had formed a salt, and this salt form is currently not reported in any literature or patents.

[0006] Several salt forms of this drug have been disclosed, but there are currently no reports of its triphenylacetic acid crystal form. The inventors of this application obtained the triphenylacetic acid salt form of this drug during the preparation of refennaxine. This crystal form has a high melting point, good chemical stability and crystal purity, is easy to prepare on a large scale, and is better suited for the preparation of pharmaceutical formulations and large-scale production, showing broad application prospects. Summary of the Invention

[0007] This invention belongs to the field of medicinal chemistry technology, specifically relating to the crystalline form of refennaxine triphenylacetic acid and its preparation method. This crystalline form exhibits good chemical stability and purity, is easy to prepare on a large scale, and is better suited for the preparation of pharmaceutical formulations and large-scale production, thus possessing broad application prospects.

[0008] The purpose of this invention is to provide a new salt form (triphenylacetic acid salt form) of a new compound called refinarin and its preparation method. The specific technical solution of this invention is as follows:

[0009] The structural formula of the triphenylacetic acid refennaxine is as follows:

[0010] The X-ray powder diffraction pattern of refennaxine triphenylacetic acid showed characteristic peaks at 2θ diffraction angles of 6.07±0.2°, 12.05±0.2°, 18.58±0.2°, 20.79±0.2°, and 21.46±0.2°.

[0011] Furthermore, the X-ray powder diffraction pattern of the crystal form shows characteristic peaks at 2θ diffraction angles of 3.27±0.2°, 6.94±0.2°, 10.19±0.2°, 13.85±0.2°, 14.87±0.2°, 15.83±0.2°, 22.61±0.2°, 24.24±0.2°, and 31.55±0.2°.

[0012] The aforementioned triphenylacetic acid refennaxine crystal form has an X-ray powder diffraction peak pattern as shown in Figure 1.

[0013] The DSC analysis of the crystal form showed a thermal absorption peak at 172.9±3.0℃, and further, it had the DSC spectrum shown in Figure 2.

[0014] The TG analysis of the crystal form showed thermal weight loss of 7.98±2% and 53.99±2% respectively, and further exhibited the DSC spectrum shown in Figure 3.

[0015] The infrared spectrum of the aforementioned triphenylacetic acid refenapyridine is at 3401±2 cm⁻¹. -1 3290±2cm -1 3149±2cm -1 1717±2cm -1 1673±2cm -1 1624±2cm -1 There is infrared absorption. Furthermore, it exhibits the infrared spectral characteristics shown in the figure.

[0016] The preparation method of the refennaxine triphenylacetic acid crystal form of the present invention includes: (1) adding refennaxine to an acetone solution and heating and stirring until dissolved; (2) adding triphenylacetic acid, cooling to crystallize, separating the crystals, and drying.

[0017] Preferably, the ratio of acetone to revannacin is 1-30 ml: 1 g, more preferably 5-10 ml: 1 g.

[0018] Preferably, the amount of added triphenylacetic acid is 1 to 3 equivalents, more preferably 1.2 to 1.5 equivalents.

[0019] Preferably, the crystallization temperature is 0–30°C, more preferably 20–25°C.

[0020] Advantages of this invention:

[0021] This invention relates to the refennaxine crystalline form of triphenylacetic acid, which exhibits high DSC (Digital Substances Count) and excellent stability under conditions of high temperature, high humidity, and light exposure, demonstrating superior chemical stability and crystal purity. The preparation process is simple, easy to operate, and yields high purity, making it suitable for large-scale industrial production and possessing significant industrial production value. Attached Figure Description

[0022] Figure 1. X-ray powder diffraction pattern of refennaxine triphenylacetic acid.

[0023] Figure 2. Differential scanning calorimetry (DSC) curve of refennaxine triphenylacetic acid.

[0024] Figure 3. Thermogravimetric analysis (TG) curve of refennaxine triphenylacetic acid.

[0025] Figure 4. Infrared spectral image of refennaxine triphenylacetic acid.

[0026] Figure 5. Mass spectrum of refennaxine triphenylacetic acid.

[0027] Figure 6. 1H NMR spectrum of refennaxine triphenylacetic acid.

[0028] Figure 7. Carbon NMR spectrum of refennaxine triphenylacetic acid. Detailed Implementation

[0029] The present invention will now be described in detail with reference to specific embodiments and exemplary examples, but these descriptions should not be construed as limiting the present invention in any way. Those skilled in the art will understand that various equivalent substitutions, modifications, or improvements can be made to the technical solutions and embodiments of the present invention without departing from the spirit and scope of the invention, and all such modifications and improvements fall within the scope of the present invention. The scope of protection of the present invention is defined by the appended claims. Specific embodiments are described below:

[0030] Example 1: Preparation of refennaxine free base (refer to Chinese Patent CN101163677B)

[0031] Add isoperidine carboxamide (59.9 g, 400 mmol), acetic acid (25.7 ml), sodium sulfate (64.4 g), and isopropanol (4000 ml) to a 20 L reactor. Cool to 0–10 °C and slowly add a solution of 1-{2-[(4-formylbenzoyl)methylamino]ethyl}piperidine-4-yl ester (110 g, 227 mmol) in isopropanol (3000 ml). React at room temperature for about 2 h, then cool to 0–10 °C. Add sodium triacetoxyborohydride (151.6 g, 685 mmol) in portions, and react at room temperature for about 16 h. Concentrate the reaction solution to about 500 ml and adjust the pH to about 3 with 1 N HCl (about 2000 ml). Extract with dichloromethane (3 × 2500 ml). Cool to 0–5 °C and adjust the pH to about 10 with 50% NaOH aqueous solution. Extracted with isopropyl acetate (3×3000ml), the organic layer was washed with water (1000ml) and saturated sodium chloride aqueous solution (2×500ml), dried with anhydrous sodium sulfate, filtered, and concentrated to obtain refennaxine free base.

[0032] Example 2: Preparation of revannacin diphosphate (refer to Chinese Patent CN101163677B)

[0033] Dissolve 5.0 g of refennaxine free base in 50 ml of water and 15 ml of 1 M phosphoric acid. Adjust the pH to approximately 5.3 with 2.5 ml of 1 M phosphoric acid. Filter and lyophilize to obtain amorphous diphosphate. Dissolve the amorphous diphosphate in 20 ml of isopropanol:acetonitrile (1:1). Add 1 ml of water, heat to 60 °C, cool to 20–25 °C, and continue stirring for approximately 12 h. Filter and vacuum dry to obtain refennaxine diphosphate.

[0034] Example 3: Preparation of Refennaxine Crystal Form III (refer to Chinese Patent CN102470130B)

[0035] Dissolve 3.07 g of refennaxine free base in 15 ml of acetonitrile and stir at room temperature for about 80 min. Place the mixture in the shaker block for thermal cycling (0 °C to 40 °C in the 1 h zone) for 48 h. Add 15 ml of acetonitrile, then place the mixture back in the shaker block and maintain for 2 h. Filter and vacuum dry to obtain refennaxine crystal form III.

[0036] Example 4: Preparation of refennaxine monosulfate (refer to Chinese Patent CN101163677B)

[0037] Dissolve 44.2 g of refennaxine free base in 500 ml of H2O∶ACN (1∶1), slowly add about 145 ml of 1N sulfuric acid, and adjust the pH to about 3.3. Filter through a 0.2 micron filter and freeze-dry to obtain monosulfate.

[0038] Dissolve 3.0 g of refennax monosulfate in 165 ml of IPA:ACN (10:1), add to a 60 °C water bath and stir for 30 min, then heat to 70 °C and stir for 30 min, then cool to 60 °C and stir for 1 h. Cool to room temperature and let stand at room temperature for about 13 days, then filter and vacuum dry to obtain refennax monosulfate.

[0039] Example 5: Preparation of refennaxine monooxalate (refer to Chinese Patent CN101163677B)

[0040] Dissolve 51.0 g of refennaxine free base in 500 ml of H2O∶ACN (1∶1), slowly add about 170 ml of 1 M aqueous oxalic acid, and adjust the pH to about 3.0. Filter through a 0.2 micron filter and freeze-dry to obtain hydrogen oxalate.

[0041] Dissolve 3.2 g of hydrogen oxalate in 276 ml of 94% IPA / 6% H2O, add to a 60°C water bath and stir for 2.5 h. Cool to room temperature, let stand at room temperature for about 30 days, filter, and vacuum dry to obtain refennaxine monooxalate.

[0042] Example 6: Study on the preparation of refennaxine triphenylacetic acid

[0043] 10.0 g of refennaxine free base was added to a round-bottom flask, followed by different amounts of reaction solvent as shown in Table 1, and then triphenylacetic acid as shown in Table 1. Stirring was started, and the mixture was heated to dissolve. The mixture was then cooled to allow crystals to precipitate, filtered, and dried under vacuum to obtain the refennaxine triphenylacetic acid groups. The experimental results are shown in Table 1.

[0044] Table 1 Preparation of refennaxine triphenylacetic acid

[0045] *: The limit for ignoring this is 0.02%, which has already accounted for the influence of the corresponding acid radicals.

[0046] Refennane triphenylacetic acid PXRD, DSC, TG, IR 1 H NMR, 13 The methods for H NMR and related substance detection are as follows:

[0047] X-ray powder diffraction (PXRD): Empyrean model, step width 0.026°, step time 50s.

[0048] Differential Scanning Thermal Analyzer (DSC): Model DSC214Polyma. Nitrogen purge gas rate: 40 ml / min; protective gas rate: 60 ml / min. Heating rate: 10 K / min. Temperature range: 25–200 °C.

[0049] Thermogravimetric analyzer (TG): Model: TG 209F3. Nitrogen purge gas 20 ml / min, protective gas 20 ml / min. Heating rate 10 K / min. Temperature range 25–400 °C.

[0050] Infrared spectroscopy measurement test: Model: Nicolet iS5 infrared spectrophotometer, and KBr pellet detection was performed.

[0051] High-resolution mass spectrometer (MS): Model: Waters H-Class / G2-XS QTOF liquid chromatography-mass spectrometry system

[0052] Nuclear magnetic resonance spectrometer (NMR spectrometer) 1 H NMR, 13 C NMR: Model: Bruker AVANCEⅢ-600, Solvent: DMSO-d6

[0053] High-performance liquid chromatograph (HPLC): Waters E2695; mobile phase: 0.01 mol / L ammonium dihydrogen phosphate buffer (adjusted to pH 2.9 with phosphoric acid) - acetonitrile (95:5) as mobile phase A, acetonitrile as mobile phase B; column temperature: 40℃; detection wavelength: 220 nm; column: YMC Triart C18 column (4.6 mm × 150 mm, 3 μm).

[0054] The X-ray powder diffraction pattern of refennaxine triphenylacetic acid is shown in Figure 1, the differential scanning calorimetry pattern is shown in Figure 2, the thermogravimetric analysis (TG) curve is shown in Figure 3, the infrared spectrum is shown in Figure 4, the high-resolution mass spectrum is shown in Figure 5, the proton NMR spectrum is shown in Figure 6, and the carbon NMR spectrum is shown in Figure 7.

[0055] Example 7 Study on salt formation of organic / inorganic acids

[0056] In this embodiment, 39 acids were screened, and only triphenylacetic acid could form a salt with refennaxine. The preparation process during screening was the same as that in Example 6, No. 2. The screening results are shown in Table 2.

[0057] Table 2. Screening results for refinasin salt formation

[0058] Example 8: Solubility test of refennaxine triphenylacetic acid of the present invention with other salt forms and crystal forms

[0059] Weigh 100 mg of the test sample and place it in 2 ml of purified water at 25 ± 2 °C. Shake vigorously for 10 seconds every 1 minute and observe the dissolution over 3 minutes. If no solute particles are visible to the naked eye, it is considered completely dissolved; if visible solute particles are present, add 2 ml of water and repeat the above operation until completely dissolved. Record the total amount of water used and the total time. Refennaxine crystal form III was prepared according to Chinese patent document CN102470130A. The results are shown in Table 3.

[0060] Table 3. Solubility of refennaxine triphenylacetic acid with other crystal forms

[0061] As shown in Table 3, the triphenylacetic acid refennaxine of the present invention has better solubility and faster dissolution rate compared with other salt and crystal forms, and can be better used in formulation development.

[0062] Example 9: Stability study of the present invention's triphenylacetic acid refennaxine with other salt forms

[0063] The stability test conditions include:

[0064] 1. Thermal degradation: Take about 200 mg of refennaxine triphenylacetic acid and place it in a drying oven at 60℃;

[0065] 2. Photodegradation: Take about 200 mg of refennaxine triphenylacetic acid and place it in an environment with an illuminance of 4500±500 lx;

[0066] 3. High humidity degradation: Take about 200 mg of triphenylacetic acid refennaxine and place it in a desiccator containing a saturated KNO3 solution, and leave it at room temperature.

[0067] The relevant substance testing methods for refennaxine are as follows:

[0068] Method basis: High performance liquid chromatography (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 0512)

[0069] Chromatographic conditions:

[0070] High-performance liquid chromatograph (HPLC): Waters E2695; mobile phase: 0.01 mol / L ammonium dihydrogen phosphate buffer (adjusted to pH 2.9 with phosphoric acid) - acetonitrile (95:5) as mobile phase A, acetonitrile as mobile phase B; column temperature: 40℃; detection wavelength: 220 nm; column: YMC Triart C18 column (4.6 mm × 150 mm, 3 μm).

[0071] Gradient elution:

[0072] Solution preparation:

[0073] (1) Diluent: Mobile phase A.

[0074] (2) Test solution: Take an appropriate amount of this product, accurately weigh it, add an appropriate amount of acetonitrile to dissolve it, and quantitatively dilute it with mobile phase A to prepare a solution containing about 1 mg per ml.

[0075] (3) Control solution: Accurately measure an appropriate amount of the test solution and quantitatively dilute it with mobile phase A to prepare a solution containing approximately 1 μg per ml.

[0076] The results of the stability study are shown in Table 4.

[0077] Table 4. Stability Study of Refennax Triphenylacetic Acid, Refennax Diphosphate, Refennax Monosulfate, Refennax Monooxalate, and Refennax Crystal Form III

[0078] Note: The ignoring limit is 0.02%, which has already deducted the influence of corresponding acid radicals.

[0079] The DSC peak value of refennax triphenylacetate was 172.9℃, significantly higher than that of diphosphate (154.5℃), monosulfate (approximately 57℃ and 73.2℃), monooxate (69.2℃ and 122.8℃), and crystal form III (123.1℃), theoretically indicating good chemical stability. Table 4 shows that the diphosphate, monosulfate, monooxate, and crystal form III all exhibited a tendency to deteriorate under light, high temperature, and high humidity conditions, especially under high temperature conditions. In contrast, refennax triphenylacetate showed better stability, with no significant increase in impurities and almost no decrease in purity, indicating that refennax triphenylacetate has superior stability compared to other refennax salts and the free base crystal form III.

[0080] Example 10: Preparation of Refennax Triphenylacetate Inhalation Powder

[0081] 1) Weigh 0.087g of magnesium stearate and 2.446g of refennaxine triphenylacetate raw materials, stir and mix for 20 minutes, then transfer to an air jet mill and grind into micro-powder at a pressure of 5 bar to D. 10 : 0.6μm; D 50 : 2.7μm; D 90 5.0 μm, to obtain the refenapyridine complex.

[0082] 2) Weigh 0.313g of the remaining magnesium stearate and 327.154g of the total lactose in the prescription and place them in a TRV high-shear mixer. Mix at a mixing speed of 500.0r / min for 6min, then discharge to obtain the lactose premix.

[0083] 3) Add approximately 1 / 2 of the lactose premix, all of the revanasin complex, and the remaining approximately 1 / 2 of the lactose premix to the TRV mixing device in sequence. Set the mixing speed to 1350 r / min, mix for 3 min, and then discharge to obtain the total revanasin mixture.

[0084] 4) Take the total mixture of refennaxine powder and fill it into capsules at a filling amount of 25mg±1mg to prepare refennaxine inhalation powder capsules that can be delivered to the lungs via a dry powder inhaler.

[0085] 5) The above-mentioned drug-loaded capsules are then packaged in double aluminum foil to obtain the final product.

[0086] Table 5. List of prescriptions for refenapyridine inhalation powder (refenapyridine triphenylacetate)

[0087] Example 11 Preparation of refennax diphosphate, refennax monosulfate, refennax monooxalate, and refennax crystal form III inhalation powder

[0088] Weigh out 0.087g of magnesium stearate and 2.191g of refennane diphosphate raw materials, stir and mix for 20 minutes, then transfer to an air jet mill and grind into micronized powder at a pressure of 5 bar to D. 10 : 0.6μm; D 50 : 2.5μm; D 90 The particle size was 4.9 μm, and a refennaxine complex was obtained. Refennaxine diphosphate inhalation powder was prepared according to the method in Example 10.

[0089] Weigh out 0.087g of magnesium stearate and 1.650g of refennaxine crystal form III raw material, stir and mix for 20 minutes, then transfer to an air jet mill and grind into micronized powder at a pressure of 5 bar to D. 10 : 0.5μm; D 50 : 2.3μm; D90 The particle size was 4.5 μm, and a refennaxine complex was obtained. Refennaxine crystal form III inhalation powder was prepared according to the method in Example 10.

[0090] Weigh out 0.087g of magnesium stearate and 1.921g of refennaxine monosulfate raw materials, stir and mix for 20 minutes, then transfer to an air jet mill and grind into micronized powder at a pressure of 5 bar to D. 10 : 0.7μm; D 50 : 2.7μm; D 90 The refenapyridine complex was obtained at a thickness of 5.5 μm. The refenapyridine monosulfate inhalation powder was prepared according to the method in Example 8.

[0091] Weigh out 0.087g of magnesium stearate and 1.899g of refennaxine monooxalic acid raw materials, stir and mix for 20 minutes, then transfer to an air jet mill and pulverize at a pressure of 5 bar to form D. 10 : 0.6μm; D 50 : 2.4μm; D 90 The particle size was 4.8 μm, and a refennaxine complex was obtained. A refennaxine crystalline monooxalate inhalation powder was prepared according to the method in Example 8.

[0092] Example 12 Stability Study of Different Refnacin Inhalation Powder Formulations

[0093] Different refennaxine inhalation powders prepared in Examples 10 and 11 were placed in an environment of 40℃±2℃ and RH75%±5%, and the changes in the product fine particle dose, fine particle fraction, content and related substances were measured at regular intervals. The results are shown in Table 5.

[0094] The relevant substance testing methods are as follows:

[0095] Method basis: High performance liquid chromatography (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 0512).

[0096] Chromatographic conditions:

[0097] High-performance liquid chromatograph (HPLC): Waters E2695; mobile phase: 0.01 mol / L ammonium dihydrogen phosphate buffer (adjusted to pH 2.9 with phosphoric acid) - acetonitrile (95:5) as mobile phase A, acetonitrile as mobile phase B; column temperature: 40℃; detection wavelength: 220 nm; column: YMC Triart C18 column (4.6 mm × 150 mm, 3 μm).

[0098] Gradient elution:

[0099] Solution preparation:

[0100] (1) Diluent: Mobile phase A.

[0101] (2) Test solution: Take 10 capsules of this product, transfer the contents to a 25ml volumetric flask, wash the capsule shells several times with 10ml of mobile phase A, add the washings to the volumetric flask, shake to dissolve, dilute to the mark with mobile phase A, and shake well to obtain the test solution.

[0102] (3) Control solution: Accurately measure 1 ml of the test solution, place it in a 100 ml volumetric flask, and dilute to the mark with diluent.

[0103] Table 6 Stability Test Conditions

[0104] Table 7. Results of Stability Study

[0105] Table 7 (Continued) shows the results of the stability test.

[0106] The inhaled powder prepared using refennaxine triphenylacetate exhibited significantly higher fine particle dose and fraction than those prepared using refennaxine diphosphate and crystal form III. In stability studies, the fine particle dose and fraction of the refennaxine triphenylacetate inhaled powder remained stable over 3 months of accelerated testing, while those of the refennaxine diphosphate and crystal form III inhaled powders showed a decreasing trend. Related substances (RMS) growth was slow in the refennaxine triphenylacetate inhaled powder over 3 months, while those of the refennaxine diphosphate and crystal form III inhaled powders showed a significant increase. The inhaled powders prepared using refennaxine monosulfate and refennaxine oxalate had the worst fine particle dose and fraction, and both showed the most significant RMS growth in stability studies.

[0107] In summary, the inhaled powder formulation prepared using refennaxine triphenylacetate showed the best performance in the stability study. Example 13: Pharmacokinetics of different refennaxine inhaled powder formulations in rats.

[0108] The rats used in this experiment were male SD rats (SPF grade), aged 9 months, weighing 180–220 g. Throughout the experiment, the rats had free access to food and water. A randomized block design was used to group the experimental SD rats into three groups: a refennaxine triphenylacetate crystal powder inhaler group, a refennaxine diphosphate inhaler group, and a refennaxine crystal form III inhaler group. The drugs were administered via oral and nasal inhalation to the rats at a dose of 375 μg / kg (calculated as refennaxine).

[0109] At 0.1h, 0.2h, 0.5h, 2h, 6h, 12h, 24h, 48h, 60h, 72h, 96h, and 120h after drug administration, 0.5ml of venous blood was collected from the fundus venous plexus and placed in pre-labeled EDTA (4mM) anticoagulant EP tubes. After collection, the blood was placed on ice and then centrifuged at 4℃, 8000rpm, and 5min to collect plasma. The plasma was transferred to 96-well plates and stored at -20℃ for LC-MS / MS analysis. The drug concentration in the plasma of EDTA (4mM) anticoagulated SD rats was determined using LC / MS / MS (Agilent 6460). The relevant pharmacokinetic parameters of the concentrations at each time point after drug administration were calculated using WinNonlin 5.2 software according to statistical distance theory. See Table 8 for details.

[0110] Table 8. Pharmacokinetics of different crystal forms of refennax inhalation powder in rats

[0111] In vivo pharmacokinetic results in rats showed that refennaxine triphenylacetate inhalation powder had the lowest peak plasma concentration (C0.05). max and AUC 0-∞ The results indicate that the refennax triphenylacetate inhalation powder is significantly safer than the refennax diphosphate inhalation powder group and the refennax crystal form III inhalation powder group.

[0112] Example 14: Tissue distribution of different crystal forms of refennaxine inhalation powder in rats.

[0113] Formulation selection: Refennax triphenylacetate inhalation powder, refennax diphosphate inhalation powder, and refennax crystal form III inhalation powder prepared in Examples 10 and 11.

[0114] Dosing frequency: single dose

[0115] Route of administration: Inhalation via mouth and nose

[0116] Experimental animals: SD rats

[0117] Grouping: A total of 108 SD rats were divided into three groups: a 375 μg / kg (calculated as refenapyridine) inhalation powder group, a 375 μg / kg (calculated as refenapyridine) inhalation powder group, and a 375 μg / kg (calculated as refenapyridine) inhalation powder group (refenapyridine crystal form III), with 36 rats in each group. Drug concentrations were collected from bronchoalveolar lavage fluid, lung tissue, main trachea and bronchi, bladder, heart, liver, and kidneys 0.5 h after administration.

[0118] Table 9. Distribution of refennaxine in major organs and tissues of rats 0.5 h after inhalation of the two formulations.

[0119] Rafenacin is a long-acting anticholinergic drug with similar affinity to the M1-M5 subtypes of sarcosine receptors. In the airways, it exerts its pharmacological effects by inhibiting smooth muscle M3 receptors, thus causing bronchodilation. Smooth muscle M3 receptors are mainly located in the trachea and bronchi, which are the primary sites of action for rafenacin. The animal tissue distribution experiments described above showed that, 0.5 hours after inhalation administration, the concentration of refennax triphenylacetate inhaled powder in the lung tissue, main trachea, and bronchi was significantly higher than that of refennax diphosphate inhaled powder and refennax crystalline form III inhaled powder at the sites of efficacy. However, in areas where clinical side effects (such as urinary retention) might occur, such as the bladder and heart, the concentration of refennax triphenylacetate inhaled powder was significantly lower than that of the other two crystalline forms of refennax inhaled powder. This suggests that refennax triphenylacetate inhaled powder may produce a higher bronchodilatory effect than refennax diphosphate inhaled powder and refennax crystalline form III inhaled powder, and may also produce fewer clinical side effects.

Claims

The refennac triphenylacetic acid crystal form has the following structural formula: Its features are: The X-ray powder diffraction pattern of the crystal form has characteristic peaks at 2θ diffraction angles of 6.07±0.2°, 12.05±0.2°, 18.58±0.2°, 20.79±0.2°, and 21.46±0.2°. The refennae crystalline form of triphenylacetic acid according to claim 1 is characterized in that: The X-ray powder diffraction pattern of the crystal form further shows characteristic peaks at 2θ diffraction angles of 3.27±0.2°, 6.94±0.2°, 10.19±0.2°, 13.85±0.2°, 14.87±0.2°, 15.83±0.2°, 22.61±0.2°, 24.24±0.2°, and 31.55±0.2°. The refennaeline crystalline form of triphenylacetic acid according to claim 2 is characterized in that: It has the X-ray powder diffraction peak pattern shown in Figure 1. The refennae saccharide crystal form according to any one of claims 1 to 3 is characterized in that: The DSC analysis of the crystal form showed a thermal absorption peak at 172.9 ± 3.0 °C. The refennaeline crystalline form of triphenylacetic acid according to claim 4 is characterized in that: It has the DSC spectrum shown in Figure 2. The refennae saccharide crystal form according to any one of claims 1 to 3 is characterized in that: The TG analysis of the crystal form showed thermal weight loss of 7.98±2% and 53.99±2% respectively. The refennaeline crystalline form of triphenylacetic acid according to claim 6 is characterized in that: It has the TG spectrum shown in Figure 3. The refennae saccharide crystal form according to any one of claims 1 to 3 is characterized in that: The infrared spectrum of the crystal form is at 3401±2 cm⁻¹. -1 3290±2cm -1 3149±2cm -1 1717±2cm -1 1673±2cm -1 1624±2cm -1 There is infrared absorption at that location. The refennae crystalline form of triphenylacetic acid according to claim 8 is characterized in that... It has the infrared spectral characteristics shown in Figure 6. The method for preparing the refennaxine triphenylacetic acid crystal form according to any one of claims 1 to 9 is characterized in that, Includes the following steps: a) Add refennaxine and acetone to the reactor, and heat and stir until dissolved; b) Add triphenylacetic acid, cool to allow crystals to precipitate, separate the crystals, and dry. The preparation method according to claim 10 is characterized in that: The ratio of acetone to revannacin in step a) is 5-30 ml: 1 g; the equivalent of triphenylacetic acid in step b) is 1-3; and the crystallization temperature in step b) is 0-30 °C.

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