Comefon hydrochloride and crystal form, preparation method and application thereof

Abstract

The invention provides comeflufen hydrochloride as well as a crystal form, a preparation method and application thereof. The comeflufen hydrochloride disclosed by the invention is 3-(3-ethyl-1-methyl-1H-hexahydroazepine-3-yl) and 2-(2-fluoro-4-biphenyl)-phenyl propionate hydrochloride, has good solubility and stability, is quick in drug effect, and can avoid the first-pass effect of the liver, improve the bioavailability and achieve the effects of reducing toxicity and enhancing efficiency; the preparation method of the comeflufen hydrochloride is simple, and the product yield is high.

Description

Comefloxacin hydrochloride, its crystal form, preparation method and application Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to a clomiphene hydrochloride, its crystal form, preparation method, and application. Background Technology

[0002] Flurbiprofen (chemical formula C) 15 H 13 Flurbiprofen (FO2) is a widely used nonsteroidal anti-inflammatory drug (NSAID) with propionic acid. It possesses good anti-inflammatory, analgesic, and antipyretic effects, and is primarily used clinically for rheumatoid arthritis, osteoarthritis, and ankylosing spondylitis. It can also be used for the symptomatic treatment of soft tissue injuries (such as sprains and strains) and mild to moderate pain (such as dysmenorrhea, postoperative pain, and toothache). However, studies have shown that oral flurbiprofen has adverse reactions, including gastrointestinal reactions such as indigestion, diarrhea, bloating, constipation, and gastrointestinal bleeding. Occasionally, it can cause central nervous system reactions such as headache, drowsiness, and dizziness. Furthermore, due to its poor water solubility and the irritating nature of its sodium carboxyl salt, it is difficult to formulate into injectable preparations.

[0003] Mepitafen is a potent analgesic with a structure similar to morphine. It acts as both an agonist and antagonist of opioid μ receptors. Clinically, it is used for short-term treatment of moderate to severe pain, such as rheumatoid arthritis, traumatic pain, musculoskeletal pain, postoperative pain, dysmenorrhea, obstetric pain, and renal colic. However, studies have shown that mepitafen, like many narcotic analgesics containing phenolic hydroxyl groups, exhibits a severe first-pass effect in the liver, with an oral bioavailability of only 8.69%, indicating low bioavailability. Oral administration also causes significant gastrointestinal irritation and a high incidence of adverse clinical reactions. Summary of the Invention

[0004] To address one of the aforementioned technical problems in the prior art, this invention provides a clomiflufen hydrochloride, its preparation method, and its application. Compared to flurbiprofen and meptaprol, the clomiflufen hydrochloride of this invention has significant advantages in terms of good water solubility, good stability, good efficacy, high safety, and low incidence of adverse reactions; it retains the original efficacy advantages of flurbiprofen and meptaprol while overcoming the disadvantage of numerous adverse reactions associated with single-drug use.

[0005] On one hand, the present invention provides a compound, which is 3-(3-ethyl-1-methyl-1H-hexahydroazaphen-3-yl),2-(2-fluoro-4-biphenyl)-propionic acid phenyl ester hydrochloride (i.e., clomiflufen hydrochloride).

[0006] According to some embodiments of the present invention, the compounds of the present invention have the following structures:

[0007] Another aspect of the invention provides a crystal form of the compound described in the first aspect.

[0008] According to some embodiments of the present invention, the compound is crystal form I, and its X-ray powder diffraction pattern, expressed in 2θ angles, has diffraction peaks at 11.4°±0.2°, 14.5°±0.2°, and 21.4°±0.2° when irradiated with Cu-Kα.

[0009] According to some embodiments of the present invention, the X-ray powder diffraction pattern of crystal form I, expressed in 2θ angles, also has diffraction peaks at one or more of the following 2θ angles: 16.5°±0.2°, 17.9°±0.2°, and 18.3°±0.2°.

[0010] According to some embodiments of the present invention, the X-ray powder diffraction pattern of crystal form I, expressed in 2θ angles, has diffraction peaks at 11.4°±0.2°, 14.5°±0.2°, 16.5°±0.2°, 17.9°±0.2°, 18.3°±0.2°, and 21.4°±0.2°.

[0011] According to some embodiments of the present invention, the X-ray powder diffraction pattern of crystal form I, expressed in 2θ angles, also has diffraction peaks at one or more of the following 2θ angles: 9.1°±0.2°, 16.9°±0.2°, 17.5°±0.2°, 19.9°±0.2°, 23.0°±0.2°, and 25.2°±0.2°.

[0012] According to some embodiments of the present invention, the X-ray powder diffraction pattern of crystal form I, expressed in 2θ angles, has diffraction peaks at 9.1°±0.2°, 11.4°±0.2°, 14.5°±0.2°, 16.5°±0.2°, 16.9°±0.2°, 17.5°±0.2°, 17.9°±0.2°, 18.3°±0.2°, 19.9°±0.2°, 21.4°±0.2°, 23.0°±0.2°, and 25.2°±0.2°.

[0013] According to some embodiments of the present invention, the X-ray powder diffraction pattern of crystal form I, expressed in 2θ angles, also has diffraction peaks at one or more of the following 2θ angles: 7.9°±0.2°, 13.6°±0.2°, 15.6°±0.2°, and 27.3°±0.2°.

[0014] According to some embodiments of the present invention, the X-ray powder diffraction pattern of crystal form I, expressed in 2θ angles, has diffraction peaks at 7.9°±0.2°, 9.1°±0.2°, 11.4°±0.2°, 13.6°±0.2°, 14.5°±0.2°, 15.6°±0.2°, 16.5°±0.2°, 16.9°±0.2°, 17.5°±0.2°, 17.9°±0.2°, 18.3°±0.2°, 19.9°±0.2°, 21.4°±0.2°, 23.0°±0.2°, 25.2°±0.2°, and 27.3°±0.2°.

[0015] According to some embodiments of the present invention, the X-ray powder diffraction pattern of crystal form I, expressed in 2θ angle, is substantially the same as that in Figure 9.

[0016] According to some embodiments of the present invention, the compound is crystal form II. Using Cu-Kα radiation, the X-ray powder diffraction pattern of crystal form II, expressed in 2θ angles, has diffraction peaks at 13.6°±0.2°, 16.2°±0.2°, 18.4°±0.2°, and 21.4°±0.2°.

[0017] According to some embodiments of the present invention, using Cu-Kα radiation, the X-ray powder diffraction pattern of the crystal form II, expressed in 2θ angles, also has diffraction peaks at one or more of the following 2θ angles: 8.0°±0.2°, 11.4°±0.2°, 14.5°±0.2°, 19.1°±0.2°, and 20.0°±0.2°.

[0018] According to some embodiments of the present invention, using Cu-Kα radiation, the X-ray powder diffraction pattern of crystal form II, expressed in 2θ angles, has diffraction peaks at 8.0°±0.2°, 11.4°±0.2°, 13.6°±0.2°, 14.5°±0.2°, 16.2°±0.2°, 18.4°±0.2°, 19.1°±0.2°, 20.0°±0.2°, and 21.4°±0.2°.

[0019] According to some embodiments of the present invention, using Cu-Kα radiation, the X-ray powder diffraction pattern of the crystal form II, expressed in 2θ angles, also has diffraction peaks at one or more of the following 2θ angles: 9.1°±0.2°, 11.9°±0.2°, 13.9°±0.2°, 22.9°±0.2°, 24.0°±0.2°, and 27.3°±0.2°.

[0020] According to some embodiments of the present invention, using Cu-Kα radiation, the X-ray powder diffraction pattern of crystal form II, expressed in 2θ angles, has diffraction peaks at 8.0°±0.2°, 9.1°±0.2°, 11.4°±0.2°, 11.9°±0.2°, 13.6°±0.2°, 13.9°±0.2°, 14.5°±0.2°, 16.2°±0.2°, 18.4°±0.2°, 19.1°±0.2°, 20.0°±0.2°, 21.4°±0.2°, 22.9°±0.2°, 24.0°±0.2°, and 27.3°±0.2°.

[0021] According to some embodiments of the present invention, using Cu-Kα radiation, the X-ray powder diffraction pattern of crystal form II expressed at a 2θ angle is substantially the same as that in Figure 8.

[0022] According to some embodiments of the present invention, using Cu-Kα radiation, the X-ray powder diffraction pattern of the crystal form II, expressed at a 2θ angle, is substantially the same as that in Figure 10.

[0023] Another aspect of the present invention provides a pharmaceutical composition comprising the compound described above or the crystal form described above.

[0024] According to some embodiments of the present invention, the pharmaceutical composition further includes pharmaceutically acceptable excipients.

[0025] The pharmaceutical dosage forms of the pharmaceutical compositions of the present invention include solutions and suspensions, (micro)emulsions, injections, chewable or suckable solid dosage forms, sprays, aerosols, powder sprays, lotions, liniments, ointments, gels, gel ointments, patches, tablets, pills, soft-shell or hard-shell capsules, granules, powders, suppositories, powders, lyophilized preparations, effervescent tablets, drops, drop pills, syrups, and elixirs, etc.

[0026] The pharmaceutical compositions of the present invention are suitable for administration via any suitable route, such as oral (including oral or sublingual), rectal, nasal, local (including oral, sublingual, or percutaneous), vaginal, or parenteral (including subcutaneous, intradermal, intramuscular, intra-articular, intrasynovial, intrasternal, intrathecal, intralesional, intravenous, intravenous, or subdermal injection or infusion). Such formulations can be prepared by any method known in the field of pharmaceutical science, for example by mixing the active ingredient with a carrier or excipient. The type of excipient added to the pharmaceutical composition depends on various factors, such as the physical and chemical properties of the drug, the route of administration, and the preparation steps. Pharmaceutical excipients exist in this field and include those listed in various pharmacopoeias.

[0027] In addition, suitable binders, lubricants, disintegrants, and colorants may be incorporated into the mixture when needed or necessary. Suitable binders include starch, gelatin, natural sugars (e.g., glucose or β-lactose), corn sweeteners, natural and synthetic gums (e.g., gum arabic, tragacanth, or sodium alginate), carboxymethyl cellulose, polyethylene glycol, etc. Lubricants used in these dosage forms include sodium oleate, sodium chloride, etc. Disintegrants include, but are not limited to, starch, methyl cellulose, agar, bentonite, xanthan gum, etc. Tablets are prepared, for example, by forming a powder mixture, granulating or pre-compressing, adding lubricants and disintegrants, and compressing into tablets. Powder mixtures are prepared by mixing appropriately pulverized compounds with diluents or bases as described above, optionally with binders (e.g., carboxymethyl cellulose, alginate, gelatin, or polyvinylpyrrolidone), dissolution inhibitors (e.g., paraffin), absorption accelerators (quaternary salts), and / or absorbents (e.g., bentonite, kaolin, or dicalcium phosphate). The powdered mixture can be granulated by wetting it with a binder (e.g., syrup, starch paste, gum arabic, or solutions of cellulose or polymeric materials) and then pressing and sieving it. An alternative method of granulation is to pass the powdered mixture through a tableting machine, resulting in the further breaking down of poorly formed clumps into granules. The granules can be lubricated by adding stearic acid, stearates, talc, or mineral oil to prevent them from sticking to the tableting machine's die. The lubricated mixture is then compressed into tablets. Free-flowing inert carriers can also be added to the pharmaceutical compositions of the present invention, allowing for tableting without granulation or pre-compression steps. Transparent or opaque protective coating materials consisting of shellac sealing, sugar coating, or polymeric material coating and a polished wax coating are available. Dyes can be added to these coating materials to differentiate different unit doses.

[0028] Oral liquid formulations, such as solutions, syrups, and elixirs, can be prepared in dosage units, thus containing a predetermined amount of a compound in a given quantity. Syrups can be prepared by dissolving the compound in an appropriately flavored aqueous solution, while elixirs can be prepared using a non-toxic solvent. Solubilizers and emulsifiers (e.g., ethoxylated isostearyl alcohol and polyoxyethylene sorbitol ether), preservatives, flavoring additives (e.g., peppermint oil or natural sweeteners or saccharin or other artificial sweeteners) may also be added.

[0029] According to some embodiments of the present invention, the pharmaceutical composition is an oral formulation or an injection.

[0030] According to some embodiments of the present invention, the pharmaceutical composition is an injection.

[0031] Another aspect of the present invention provides the use of the compounds or crystal forms described above, or the pharmaceutical compositions described above, in the preparation of pharmaceuticals for anti-inflammatory, analgesic, or antipyretic purposes.

[0032] According to some embodiments of the application described in this invention, the drug is used to treat moderate or severe pain.

[0033] According to some embodiments of the application described in this invention, the drug is used to treat rheumatoid arthritis, ankylosing spondylitis, degenerative arthritis, soft tissue sprains, toothache, trauma, musculoskeletal pain, postoperative pain, gynecological dysmenorrhea, obstetric pain, acute inflammatory visceral pain such as renal colic, central nervous system pain, peripheral nerve pain, or cancer pain, etc.

[0034] Another aspect of the present invention provides a method for preparing the above-described compound or crystal form of the present invention, comprising: mixing an ethyl acetate solution of the compound of formula I with an ethyl acetate solution of HCl and reacting them to generate the compound;

[0035] (Comeflufenicol)

[0036] According to some embodiments of the present invention, the concentration of HCl in the ethyl acetate solution of HCl is 1 to 6 M, for example, 1 M, 2 M, 3 M, 3.5 M, 4 M, 4.5 M, 5 M, 6 M or any value between them, preferably 3 to 5 M, more preferably 3.5 to 4.5 M. In some embodiments, the concentration of HCl in the ethyl acetate solution of HCl is 4 M.

[0037] According to some embodiments of the present invention, the temperature of the reaction is less than or equal to 0°C. In some embodiments, the temperature of the reaction is -5 to 0°C, for example, 0°C, -1°C, -3°C, or -5°C.

[0038] According to some embodiments of the present invention, the amount of ethyl acetate solution of HCl added is sufficient to adjust the pH of the mixed solution to 1-5; for example, 1, 2, 3, 4, 5, preferably 2-3.

[0039] According to some embodiments of the present invention, the reaction time is 1-4 hours, preferably 1.5-2.5 hours.

[0040] According to some embodiments of the present invention, the preparation method further includes the following step: recrystallizing the product after reaction using a mixed solvent of acetone and n-hexane.

[0041] In the above method of the present invention, the product after the reaction is crystal form I of the compound; crystal form I is obtained by recrystallizing crystal form I using a mixed solvent of acetone and n-hexane.

[0042] In some embodiments, the volume ratio of acetone to n-hexane is 1:(1 to 3), for example 1:1, 1:1.2, 1:1.5, 1:1.8, 1:2, 1:2.2, 1:2.5, 1:2.8, 1:3 or any value between them, preferably 1:(1.5 to 2.5), more preferably 1:(1.8 to 2.2).

[0043] According to some embodiments of the present invention, the compound represented by Formula I is prepared by a method comprising the following steps:

[0044] Flurbiprofen (Formula I-1) and oxalyl chloride are subjected to an acyl chloride reaction to produce flurbiprofen acyl chloride (Formula I-2).

[0045] Flurbiprofen acyl chloride (Formula I-2) and meptaptaphenol (Formula I-3) are esterified in the presence of an alkaline substance and a catalyst to produce the compound shown in Formula I.

[0046] In some embodiments, the acyl chloride reaction is carried out in halogenated hydrocarbon and amide organic solvents, wherein the halogenated hydrocarbon organic solvents of the present invention include, but are not limited to, dichloromethane; and the amide organic solvents of the present invention include, but are not limited to, DMF.

[0047] In some embodiments, the mass ratio of flurbiprofen to oxalyl chloride is (1.5–2):1.

[0048] In some embodiments, the temperature of the acyl chloride reaction is 20-25°C.

[0049] In some embodiments, the alkaline substance includes organic amines, such as triethylamine.

[0050] In some embodiments, the catalyst comprises 4-dimethylaminopyridine.

[0051] In some embodiments, the mass ratio of meprobamate to flurbiprofen acyl chloride is 1:(1 to 1.2).

[0052] In some embodiments, the esterification reaction is carried out at a temperature of 20-25°C.

[0053] According to some embodiments of the present invention, the compound represented by Formula I is prepared by a method comprising the following steps:

[0054] Flurbiprofen (Formula I-1) and meptaphen (Formula I-3) were condensed to produce the compound shown in Formula I.

[0055] In some embodiments, the condensation reaction is carried out in the presence of ethyldimethylaminopropylcarbodiimide and 4-dimethylaminopyridine.

[0056] In some embodiments, the temperature of the condensation reaction is 20-25°C.

[0057] In some embodiments, the mass ratio of flurbiprofen to meptaprol is (0.8–1.2):1, preferably (0.9–1):1.

[0058] Another aspect of the present invention provides a method for preventing, alleviating, or treating diseases related to inflammation, pain, and / or fever, the method comprising: contacting cells with the above-described compound or crystal form or pharmaceutical composition of the present invention; or comprising: administering an effective dose of the above-described compound or crystal form or pharmaceutical composition of the present invention to a subject.

[0059] According to some embodiments of the present invention, the inflammation, pain, or fever-related diseases include, but are not limited to: rheumatoid arthritis, ankylosing spondylitis, degenerative arthritis, soft tissue sprains, toothache, trauma, musculoskeletal pain, postoperative pain, gynecological dysmenorrhea, obstetric pain, acute inflammatory visceral pain such as renal colic, central nervous system pain, peripheral nerve pain, or cancer pain, etc.

[0060] Another aspect of the present invention provides the use of the compounds or crystal forms or pharmaceutical compositions described above in the treatment of painful or inflammatory diseases.

[0061] In some embodiments, the painful or inflammatory diseases include, but are not limited to: rheumatoid arthritis, ankylosing spondylitis, degenerative arthritis, soft tissue sprains, toothache, trauma, musculoskeletal pain, postoperative pain, gynecological dysmenorrhea, obstetric pain, acute inflammatory visceral pain such as renal colic, central nervous system pain, peripheral nerve pain, or cancer pain.

[0062] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0063] (1) The compound of the present invention (cometofen hydrochloride) reduces the irritation of the carboxyl group in flurbiprofen and the instability of the hydroxyl group in meprofen, while having better solubility and stability compared with that before salt formation.

[0064] (2) In this invention, different crystal forms can be obtained by recrystallizing clomiphene hydrochloride in acetone / n-hexane.

[0065] (3) The compound of the present invention (cometofen hydrochloride) can be made into an injection solution, which has a faster effect and can avoid the first-pass effect of the liver, improve bioavailability, and achieve the effect of reducing toxicity and increasing efficacy.

[0066] (4) The preparation method of the compound (cometofen hydrochloride) of the present invention is simple, and the resulting product has high purity and high yield. Attached Figure Description

[0067] Figure 1 is a single crystal diagram of the morphology I of clofenac hydrochloride prepared in Example 1 of the present invention.

[0068] Figure 2 is the 1H NMR spectrum of the methylflufenicol hydrochloride crystal form I prepared in Example 1 of this invention.

[0069] Figure 3 is the mass spectrum of the crystalline form I of clofenac hydrochloride prepared in Example 1 of the present invention.

[0070] Figure 4 shows the results of the high-temperature stability study of the cyclophosphamide hydrochloride crystal form I prepared in Example 1 of the present invention and cyclophosphamide.

[0071] Figure 5 shows the crystal form I of clomiflufen hydrochloride prepared in Example 1 of the present invention and the effect of temperature on impurities in clomiflufen.

[0072] Figure 6 shows the results of the photostress study of the crystal form I of clomiflufen hydrochloride prepared in Example 1 of the present invention and clomiflufen.

[0073] Figure 7 shows the crystal form I of clomiflufen hydrochloride prepared in Example 1 of the present invention and the effect of light on impurities in clomiflufen.

[0074] Figure 8 shows the XRD pattern of the morphology II of clomiphene hydrochloride prepared in Example 1 of the present invention.

[0075] Figure 9 shows the XRD pattern of the morphology I of clofenac hydrochloride prepared in Example 2 of the present invention.

[0076] Figure 10 shows the XRD pattern of the morphology II of clomiphene hydrochloride prepared in Example 2 of the present invention. Detailed Implementation

[0077] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments and accompanying drawings. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the scope of the invention in any way.

[0078] This invention addresses the technical problems of existing anti-inflammatory and analgesic drugs, such as the poor water solubility of flurbiprofen and the irritant nature of its sodium carboxyl salt, making it difficult to formulate into injectable preparations; and the severe first-pass effect of meptaphen in the liver, resulting in low oral bioavailability and difficulty in achieving rapid analgesia. However, even esterification of flurbiprofen and meptaphen still presents problems with poor water solubility, high-temperature stability, and photostability. This invention creatively discovers that by esterifying flurbiprofen and meptaphen and then preparing them into hydrochloride forms, the water solubility and stability of the drugs can be improved without altering their pharmacodynamic structural sites, providing multiple possibilities for the development of dosage forms for flurbiprofen and meptaphen.

[0079] In particular, through extensive experiments, this invention unexpectedly discovered that the esterification product of flurbiprofen and meptaphen (colmeflufen) can only form salts with hydrochloric acid and crystallize. It does not form salts with other monobasic acids (such as hydrochloric acid, nitric acid, acetic acid, hydrobromic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, etc.), dibasic acids (such as sulfuric acid, sulfurous acid, oxalic acid, succinic acid, etc.), or polybasic acids (such as citric acid, phosphoric acid, etc.). This invention also found that the crystallization conditions after colmeflufen forms a salt with hydrochloric acid are quite demanding. Only when ethyl acetate is used as the salt-forming solvent can colmeflufen crystallize after forming a salt with hydrochloric acid. Using other solvents, such as methanol, ethanol, isopropanol, tetrahydrofuran, acetone, acetonitrile, isopropyl acetate, dichloromethane, n-hexane / ethyl acetate (1:1), cyclohexane / ethyl acetate (1:1), methyl tert-butyl ether, etc., will not result in salt formation and crystallization.

[0080] Furthermore, the present invention unexpectedly discovered that recrystallizing clomiphene hydrochloride in a mixed solvent of acetone and n-hexane resulted in a change in its crystal structure, yielding a new crystal form.

[0081] Unless otherwise specified, all reagents used in the following experiments of this invention are commercially available products or reagents prepared according to conventional methods. Unless otherwise specified, all methods used in the experiments are conventional experimental methods. Unless otherwise specified, all instruments used in the experiments are commercially available.

[0082] The HCl-ethyl acetate solution used in the embodiments of the present invention was obtained commercially and was a 4M HCl-ethyl acetate solution.

[0083] Example 1

[0084] The synthetic route for clomiphene hydrochloride is as follows:

[0085] The specific operating steps are as follows:

[0086] Step 1, Preparation of flurbiprofen (FM): Add 1.93g flurbiprofen and 20mL dichloromethane to the reaction flask, stir to dissolve, then add 1.50g 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCI) and 105mg 4-dimethylaminopyridine (DMAP), stir and cool to 0-5℃ under nitrogen protection, and react for 0.5h.

[0087] Add 2.0 g of meptaptaphenol to the above reaction solution. After the addition is complete, slowly heat to 20-25℃ and stir for 2-3 hours. Monitor the reaction progress by TLC. After the reaction is complete, slowly add 20 mL of purified water to the reaction solution to quench the reaction. After stirring for 5 minutes, allow to stand and separate the organic layer. Wash the organic layer once with 20 mL of water, separate the organic layer again, add 2.00 g of anhydrous magnesium sulfate to the organic layer, stir and dry, filter, and concentrate the filtrate to dryness under reduced pressure at 45℃ to obtain 3.90 g of FM, with a yield of 99.0% and a purity of 94.7%.

[0088] Step 2, Preparation of FMY (comyfluphene hydrochloride): Add 3.9 g of FM to a reaction flask, then add 39 mL of anhydrous ethyl acetate and stir to dissolve. After dissolution, stir and cool to -5℃ to 0℃. Slowly add 2.2 mL of 4M HCl-ethyl acetate solution to adjust the pH of the reaction solution to 2-3. After the addition is complete, continue to maintain the temperature at -5℃ to 0℃ and stir to induce crystallization for 2 hours. Filter under reduced pressure, and dry the resulting solid at 45℃ to obtain 2.5 g of white solid (comyfluphene hydrochloride crystal form I), yield 59.5%, purity 99.5%.

[0089] Figure 1 shows the single crystal diagram of the morphology I of clomiphene hydrochloride prepared in Example 1. As can be seen from Figure 1, the structural formula of clomiphene hydrochloride matches this single crystal structure.

[0090] The single crystal was tested using a single-crystal X-ray diffractometer (SC-XRD) (D8 VENTURE).

[0091] Detection results: The basic structural information of this compound is: molecular formula C 30 H 35 ClFNO2, crystal system Monoclinic, space group P21 / C The unit cell parameters are α=γ=90°, β=91.837(5)°, volume

[0092] The 1H NMR spectrum of the methylflufenicol hydrochloride crystal form I prepared in Example 1 is shown in Figure 2. Its 1H NMR spectrum is as follows: 1¹H NMR (400 MHz, CDCl₃) 7.59 (m, 2H), 7.48 (td, 3H), 7.41 (m, 1H), 7.29 (m, 3H), 7.20 (d, 1H), 7.01 (q, 1H), 6.89 (m, 1H), 4.03 (q, 1H), 2.86 (d, 1H), 2.54 (ddt, 3H), 2.40 (d, 3H), 2.11 (m, 1H), 1.69 (m, 9H), 1.55 (m, 1H), 0.62 (t, 3H). Its ¹H NMR assignments are consistent with the structure of clofenac hydrochloride (FMY).

[0093] The mass spectrum of the crystalline form I of clomiphene hydrochloride prepared in Example 1 is shown in Figure 3. Its mass spectrometry value is 460.4, which is consistent with the theoretical molecular formula of the free base of clomiphene hydrochloride (FMY) (C1). 30 H 34 The FNO2+H combination is consistent, indicating the rationality of the FMY structure.

[0094] The elemental analysis results of the methylflufenicol hydrochloride crystal form I prepared in Example 1 are shown in Table 1.

[0095] Testing basis: General Rules for Elemental Analyzer Analysis Methods (JY / T0580-2020);

[0096] Testing instrument: German elementar vario EL cube elemental analyzer.

[0097] Table 1

[0098] As can be seen from Table 1, the measured values ​​of the samples are basically consistent with the theoretical calculation values. The errors of C, H, and N are all within three per thousand, which is consistent with the molecular formula of FMY. 30 H 35 The FClNO2 formula matches the theoretical value, indicating that the molecular formula of the sample is consistent with the theoretical value.

[0099] Preparation of clomiphene hydrochloride crystal form II:

[0100] The obtained methylflufenicol hydrochloride crystal form I was recrystallized in a mixed solvent of acetone / n-hexane (volume ratio 1:2) to obtain 2.3 g of methylflufenicol hydrochloride (FMY) crystal form II, with a recrystallization yield of 92.0% and a purity of 99.6%. The XRD characterization results of methylflufenicol hydrochloride crystal form II are shown in Figure 8 and Table 2.

[0101] Table 2

[0102] Example 2

[0103] The synthetic route for clofenac hydrochloride (FMY) is as follows:

[0104] The specific operating steps are as follows:

[0105] Step 1, Preparation of Flurbiprofen Acyl Chloride (FMA): 25.0 g of flurbiprofen was added to a reaction flask, followed by 250 mL of dichloromethane. The mixture was stirred and cooled to 0-5 °C. Under nitrogen protection, 14.3 g of oxaloyl chloride was slowly added dropwise to the reaction solution. After the addition was complete, 3 drops of DMF were added dropwise. The mixture was then heated to 20-25 °C and stirred. The reaction progress was monitored by TLC. After the reaction was complete, the reaction solution was concentrated to dryness under reduced pressure at 45 °C. Dichloromethane was added to dissolve the solution, and concentration under reduced pressure was continued to remove excess oxaloyl chloride. The solution was then concentrated to dryness again to obtain 28.0 g of a pale yellow oily substance, flurbiprofen acyl chloride (FMA), exceeding the theoretical yield.

[0106] Step 2, Preparation of clomiflufen (FM): Add 23.9 g of metoprolol to a reaction flask, add 239 mL of dichloromethane and stir to dissolve. Add 15.5 g of triethylamine and 626 mg of DMAP (4-dimethylaminopyridine). Under nitrogen protection, stir and cool to 0-5℃. Dissolve 28.0 g of FMA in dichloromethane and slowly add it dropwise to the reaction flask. After the addition is complete, slowly raise the temperature to 20-25℃ and stir for 2-3 hours. After the reaction is complete, slowly add 120 mL of purified water to the reaction solution to quench the reaction. After the addition is complete, continue stirring for 5 minutes, allow to stand to separate the organic layer, wash once with 120 mL of purified water, add 23.9 g of anhydrous magnesium sulfate to the organic layer and stir to dry. Filter, concentrate the filtrate to dryness under reduced pressure at 45℃ to obtain 39.5 g of pale yellow oily clomiflufen (FM), yield 83.9%, purity 94.3%.

[0107] Step 3, Preparation of FMY (comfrey hydrochloride): 39.5 g of FM was added to a reaction flask, followed by 395 mL of anhydrous ethyl acetate. The mixture was stirred until dissolved, and then cooled to -5°C to 0°C. 22 mL of 4M HCl-ethyl acetate solution was slowly added dropwise to adjust the pH of the reaction solution to 2-3. After the addition was complete, the mixture was kept at -5°C to 0°C and stirred to induce crystallization for 2 hours. The mixture was filtered under reduced pressure, and the resulting solid was dried at 45°C with a forced-air drying process to obtain 23.0 g of a white solid (comfrey hydrochloride crystal form I), with a yield of 54.0% and a purity of 99.3%. The XRD characterization results of FMY (comfrey hydrochloride crystal form I) are shown in Figure 9 and Table 3.

[0108] Table 3

[0109] Preparation of Clemaflufenicol Hydrochloride Crystal Form II

[0110] The methylflufenicol hydrochloride crystal form I prepared in Example 2 was recrystallized again with a mixed solvent of acetone / n-hexane (volume ratio 1:2) to obtain 21.2 g of methylflufenicol hydrochloride (FMY) crystal form II, with a recrystallization yield of 91.2% and a purity of 99.6%. The XRD characterization results of methylflufenicol hydrochloride crystal form II prepared in Example 2 are shown in Figure 10 and Table 4.

[0111] Table 4

[0112] Example 3

[0113] In the FMY salt formation process, various acids were screened for salt formation. A systematic screening was conducted based on the type of acid.

[0114] Monobasic acids: hydrochloric acid, nitric acid, acetic acid, hydrobromic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid;

[0115] Dicarboxylic acids: sulfuric acid, sulfurous acid, oxalic acid, succinic acid;

[0116] Tribasic acids: citric acid, phosphoric acid.

[0117] 1. FM reacts with monobasic acids to form salts.

[0118] Procedure: Weigh FM and add it to the reaction flask. Add ethyl acetate and stir to dissolve. Cool the mixture to 0-5℃. Weigh different monocarboxylic acids, dissolve and dilute them separately with ethyl acetate, and slowly add them dropwise to the reaction solution to carry out the salt formation reaction. After the addition is complete, keep the mixture at 0-5℃ and stir to induce crystallization. The material ratios with different monocarboxylic acids are shown in Table 5.

[0119] Table 5 Material Proportions Note: " / " indicates that the field should not be filled in.

[0120] After multiple reaction attempts, only the 4M HCl-ethyl acetate solution group and the methanesulfonic acid group produced solid precipitation. The 4M HCl-ethyl acetate solution group produced more solid precipitation, while the methanesulfonic acid group produced only trace amounts of solid precipitation.

[0121] During the reaction experiments, it was found that the hydrobromic acid group, nitric acid group, methanesulfonic acid group, and ethylsulfonic acid group all experienced severe hydrolysis of FM during the salt formation process. The analysis suggests that FM may be unstable and unable to crystallize when exposed to a strong acid environment during salt formation.

[0122] The remaining groups concentrated the reaction solution to dryness and attempted to dissolve and crystallize it using different solvents. The solvents used included methanol, ethanol, isopropanol, tetrahydrofuran, acetone, acetonitrile, isopropyl acetate, dichloromethane, n-hexane / ethyl acetate (1:1), cyclohexane / ethyl acetate (1:1), and methyl tert-butyl ether. The solutions were then transferred to vials, and crystals were slowly evaporated using capillary tubes. After long-term observation, no solid precipitated.

[0123] With other operating conditions unchanged, attempts were made to replace the salt solvent (i.e., to replace the salt solvent ethyl acetate with the following solvents) such as methanol, ethanol, isopropanol, tetrahydrofuran, acetone, acetonitrile, isopropyl acetate, dichloromethane, n-hexane / ethyl acetate (1:1), cyclohexane / ethyl acetate (1:1), methyl tert-butyl ether, etc., but no solid precipitated.

[0124] 2. Salt formation reaction of FM with dibasic acids

[0125] Procedure: Weigh FM and add it to the reaction flask. Add ethyl acetate and stir to dissolve. Cool the mixture to 0-5℃. Weigh different acids, dissolve and dilute them separately with ethyl acetate, and slowly add them dropwise to the reaction solution to carry out the salt formation reaction. After the addition is complete, keep the mixture at 0-5℃ and stir to induce crystallization. The material ratios with different dicarboxylic acids are shown in Table 6.

[0126] Table 6 Material Proportions Note: " / " indicates that the field should not be filled in.

[0127] After multiple reaction attempts, no solid was precipitated in any of the groups.

[0128] During the reaction experiment, it was found that FM hydrolysis was severe in both sulfuric acid and sulfurous acid groups during salt formation. Analysis suggests that the solvent environment was highly acidic during salt formation, which accelerated FM hydrolysis, resulting in poor product purity and inability to crystallize.

[0129] The reaction solutions of the oxalic acid group and the succinic acid group were concentrated to dryness, and each was dissolved in an insoluble solvent. The solvents used included methanol, ethanol, isopropanol, tetrahydrofuran, acetone, acetonitrile, isopropyl acetate, dichloromethane, n-hexane / ethyl acetate (1:1), cyclohexane / ethyl acetate (1:1), and methyl tert-butyl ether. The solutions were then transferred to vials, and the solvent was slowly evaporated using a capillary tube to induce crystallization. After long-term observation, no solid precipitated.

[0130] With other operating conditions unchanged, attempts were made to replace the salt solvent (i.e., to replace the salt solvent ethyl acetate with the following solvents) such as methanol, ethanol, isopropanol, tetrahydrofuran, acetone, acetonitrile, isopropyl acetate, dichloromethane, n-hexane / ethyl acetate (1:1), cyclohexane / ethyl acetate (1:1), methyl tert-butyl ether, etc., but no solid precipitated.

[0131] 3. Salt formation reaction of FM with tribasic acids

[0132] Procedure: Weigh FM and add it to the reaction flask. Add ethyl acetate and stir to dissolve. Cool the mixture to 0-5℃. Weigh different tribasic acids, dissolve and dilute them separately with ethyl acetate, and slowly add them dropwise to the reaction solution to carry out the salt formation reaction. After the addition is complete, keep the mixture at 0-5℃ and stir to induce crystallization. The material ratios with different tribasic acids are shown in Table 7.

[0133] Table 7 Material Proportions Note: " / " indicates that the field should not be filled in.

[0134] After multiple reaction attempts, no solid was precipitated in any of the groups.

[0135] Both reaction solutions were concentrated to dryness and dissolved in insoluble solvents, including methanol, ethanol, isopropanol, tetrahydrofuran, acetone, acetonitrile, isopropyl acetate, dichloromethane, n-hexane / ethyl acetate (1:1), cyclohexane / ethyl acetate (1:1), and methyl tert-butyl ether. The solutions were then transferred to vials, and the solvent was slowly evaporated using a capillary tube to induce crystallization. No solid precipitated during long-term observation.

[0136] With other operating conditions unchanged, attempts were made to replace the salt solvent (i.e., to replace the salt solvent ethyl acetate with the following solvents) such as methanol, ethanol, isopropanol, tetrahydrofuran, acetone, acetonitrile, isopropyl acetate, dichloromethane, n-hexane / ethyl acetate (1:1), cyclohexane / ethyl acetate (1:1), methyl tert-butyl ether, etc., but no solid precipitated.

[0137] Summarize:

[0138] FM has a unique structure, being an ester compound with a tertiary amine group as its salt-forming group. It is inherently weakly basic and requires a strong acid to form a salt. The ester structure in FM is unstable under acidic conditions and readily decomposes, especially in strongly acidic solvents with high water content.

[0139] During FM salt formation, it is necessary to minimize product decomposition caused by the salt formation environment, making the salt formation conditions extremely stringent. The FM salt-formed product exhibits better solubility and greater stability. However, the crystallization conditions after FM salt formation are even more demanding, making crystallization difficult. Through systematic optimization and screening of various solvents and conditions, only a stable hydrochloride structure was ultimately obtained; other salt forms did not yield stable products.

[0140] Example 4: Performance Comparison of FMY (colofen hydrochloride) Crystal Form I and FM (colofen)

[0141] 1. Solubility data:

[0142] FMY crystal form I is extremely soluble in water, while FM is insoluble in water. Therefore, FMY has better water solubility than FM.

[0143] 2. Product purity:

[0144] The FMY product purified by the crystallization method of this invention has a high purity, greater than 99%. Existing technologies directly obtain FM with poor purity, below 95%, and the purification methods are complex; even column purification cannot achieve a purity of over 99%.

[0145] It is evident that the FMY prepared by the method of the present invention has better quality and stability, and the purification process is simpler.

[0146] 3. FMY stability data:

[0147] (1) High temperature stability:

[0148] Stability conditions: Placed at 80°C for a maximum of 30 hours.

[0149] Test method: The sample is spread in a petri dish to a thickness of about 1-2 mm. The sample is placed in a forced-air drying oven and heated to 80°C for continuous placement. Samples are taken periodically and the purity of the product is determined by high performance liquid chromatography.

[0150] The results are shown in Figures 4 and 5. It can be seen that at the same time point, FMY crystal form I has less degradation of the main peak and fewer impurities compared to FM. The degradation rate of the main peak is also slower. It can still maintain a high purity (above 99%) after being placed at 80°C for 30 hours. This indicates that FMY has better high-temperature stability than FM.

[0151] (2) Light stability:

[0152] Stability conditions: Irradiation with 5000 Lux light for up to 72 hours.

[0153] Test method: The sample is spread in a petri dish with a thickness of about 1-2 mm. The sample is placed in a light box and continuously irradiated with a light intensity of 5000 Lux. The purity of the product is determined by high performance liquid chromatography at regular intervals.

[0154] The results are shown in Figures 6 and 7. It can be seen that at the same time point, FMY crystal form I has less degradation of the main peak and fewer impurities compared to FM. The degradation rate of the main peak is also slower. After 72 hours of irradiation with 5000 Lux strong light, it still has a high purity (above 99%). This indicates that FMY has higher light stability than FM.

[0155] It is evident that FMY is more resistant to high temperatures and strong light than FM, and has better stability.

[0156] Example 5: Evaluation of the analgesic and anti-inflammatory activity of clomiphene hydrochloride crystal form I

[0157] 1. Materials

[0158] 1.1 Test Drug

[0159] Mepitafen and flurbiprofen were both supplied by Hefei Keda Biotechnology Co., Ltd.

[0160] Comfrefen hydrochloride is crystalline form I of comfrefen hydrochloride.

[0161] Before the experiment, the drugs were prepared into corresponding concentration suspensions with 0.2% CMC-Na, and the administration volume was 20 mL / kg for all.

[0162] 1.2 Animals and feeding

[0163] ICR mice, male and female, 18 - 22 g, SPF grade, production license number SCXK(Zhe)2019 - 0002, purchased from Hangzhou Medical College.

[0164] The mice were housed in the Animal Center of China Pharmaceutical University (animal use license number: SYXK(Su)2021 - 0011). The temperature of the housing laboratory was 24 ± 2 °C; the relative humidity was 40% - 70%; the number of air exchanges per hour was 10 - 15 times / hour; the light cycle was 12 (day) / 12 (night) hours, and no more than 5 mice were kept in each cage.

[0165] Feed: Complete pellet feed for mice, purchased from Jiangsu Xietong Pharmaceutical Biotechnology Co., Ltd., and its quality conforms to the General Quality Standard for Experimental Animal Compound Feed GB14924.1 - 2010.

[0166] Drinking water: Purified water.

[0167] 1.3 Main reagents

[0168] Xylene, acetic acid, analytical pure, all purchased from Nanjing Chemical Reagent Co., Ltd.

[0169] 1.4 Main instruments

[0170] FEJ - 200 electronic balance (0.1 - 200 g), Fuzhou Furihangzhibao Electronics Co., Ltd.;

[0171] BS210S precision electronic balance (0.1 mg - 10 g), Sartorius, Germany;

[0172] Digital display constant temperature circulating water bath, Changzhou Guohua Electric Appliance Co., Ltd.

[0173] 2. Methods

[0174] 2.1 Dosage setting basis

[0175] In this experiment, meptazinol and flurbiprofen are both marketed drugs, and the mouse test doses are converted according to the clinical human doses. The dose of kemeifufen hydrochloride is converted according to the meptazinol mass it contains (Table 8).

[0176] Table 8. Basis for setting the doses of test drugs a Clinical maximum dose conversion multiple; b Clinical maximum dose conversion multiple converted according to the meptazinol contained

[0177] 2.2 Effects on acetic acid-induced writhing response in mice (analgesic effect)

[0178] Male ICR mice, weighing 18–22 g, were purchased and acclimatized before being randomly divided into 9 groups of 8 mice each, based on their weight: model group, metoprolol group (60 mg / kg, 30 mg / kg, and 15 mg / kg), flurbiprofen group (27 mg / kg and 13.5 mg / kg), and clomiflufenicol hydrochloride group (55 mg / kg, 22.5 mg / kg, and 11.25 mg / kg).

[0179] Animals in each group were fasted for 12 hours before the experiment, but water intake was unrestricted. Sixty minutes after gavage administration of the corresponding drug, 0.6% acetic acid (0.2 mL / mouse) was injected intraperitoneally. The number of writhing episodes in mice within 15 minutes of injection was recorded. A positive writhing response was defined as repeated contraction of the lumbar muscles, arching of the back, twisting of the hips, and extension of the hind limbs. The percentage of writhing inhibition by the drug was calculated using the following formula:

[0180] Inhibition rate (%) = (Number of writhing movements in the control group - Number of writhing movements in the treatment group) / Number of writhing movements in the control group × 100%

[0181] 2.3 Effect on pain threshold of hot plate in mice (analgesic effect)

[0182] Female ICR mice, weighing 18–22 g, were purchased and acclimatized before being randomly divided into 7 groups of 8 mice each, based on their weight: metoprolol group (60 mg / kg, 30 mg / kg, and 15 mg / kg), flurbiprofen group (27 mg / kg), and clomiflufen hydrochloride group (110 mg / kg, 55 mg / kg, and 22.5 mg / kg).

[0183] Animals in each group were fasted for 12 hours before the experiment, but water intake was unrestricted. Mice were placed on a constant-temperature hot plate (55±0.5℃), and the time required for them to lick their paws was recorded as the pain threshold. Qualified mice (pain response time less than 60 seconds) were selected and randomly assigned to groups. The pain threshold was measured twice before drug administration, and the average value was taken as the baseline pain threshold. After gavage administration of the corresponding drug, the pain threshold of each mouse was measured at 0.5 and 1 hour post-administration (if there was no pain response within 60 seconds, it was calculated as 60 seconds). Using the baseline pain threshold as a reference, the increase rate of pain threshold in each mouse at 0.5 and 1 hour post-administration was calculated.

[0184] 2.4 Effects of xylene on mouse ear swelling (anti-inflammatory effect)

[0185] ICR mice, male, weighing 18-22g, were purchased and acclimatized. They were then randomly divided into 5 groups of 8 mice each, based on their weight: model group, meptaphen group (30mg / kg), flurbiprofen group (27mg / kg), and clomiflufen hydrochloride group (55mg / kg and 22.5mg / kg).

[0186] Animals in each group were fasted for 12 hours before the experiment, but water intake was unrestricted. Sixty minutes after gavage administration of the corresponding drug, 50 μl of xylene was applied to both sides of the right ear of each mouse to induce inflammation; the left ear was left untreated as a non-inflammatory ear. Two hours later, the animals were sacrificed, and both ears were removed. Circular ear pieces were punched from the same location using an 8 mm diameter punch and weighed using an electronic balance. The difference between the weight of the inflammatory ear piece and the weight of the non-inflammatory ear piece was used as the degree of swelling. The percentage of swelling inhibition by the drug was calculated using the following formula:

[0187] Swelling inhibition rate (%) = (Swelling degree in model group - Swelling degree in treatment group) / Swelling degree in model group × 100%

[0188] 3. Results

[0189] 3.1 Effects on acetic acid-induced writhing response in mice (analgesic effect)

[0190] In mice, the 30 mg / kg dose of meptaprol significantly inhibited the writhing response induced by intraperitoneal injection of acetic acid compared to the model group, with an inhibition rate of 37.9% (P<0.05). However, no improvement was observed in the 60 mg / kg and 15 mg / kg dose groups. Flurbiprofen (27 mg / kg and 13.5 mg / kg) and clomiflufen hydrochloride (55 mg / kg, 22.5 mg / kg and 11.25 mg / kg) significantly inhibited the writhing response in mice (P<0.05, P<0.01), with inhibition rates >90% in the high-dose groups. Clomiflufen hydrochloride showed greater analgesic efficacy than meptaprol and flurbiprofen (Table 9).

[0191] Table 9. Effect of acetic acid on the number of writhing movements in mice (M±SD, N=8) *P<0.05, **P<0.01, compared with the model group.

[0192] 3.2 Effect on pain threshold of hot plate in mice (analgesic effect)

[0193] Mepitafen (60 mg / kg, 30 mg / kg, and 15 mg / kg) at various doses increased the hot plate pain threshold in mice to varying degrees, with the 30 mg / kg dose showing a significant effect (P<0.01). Flurbiprofen (27 mg / kg) also increased the hot plate pain threshold in mice (P<0.05, P<0.01). Clemflufen hydrochloride (110 mg / kg, 55 mg / kg, and 22.5 mg / kg) also increased the hot plate pain threshold in mice to varying degrees, with the medium-dose group showing a statistically significant difference (P<0.01) (Table 10).

[0194] Table 10. Effect on pain threshold of hot plate in mice (M±SD, N=8)

[0195] *P<0.05, **P<0.01, compared with the baseline pain threshold of each group.

[0196] 3.3 Effects of xylene on mouse ear swelling (anti-inflammatory effect)

[0197] Mepitafen (30 mg / kg) had no significant effect on xylene-induced ear swelling in mice; flurbiprofen (27 mg / kg) significantly inhibited xylene-induced ear swelling in mice (P<0.05); clomiflufen hydrochloride (55 mg / kg and 22.5 mg / kg) also significantly inhibited ear swelling in mice, with the high-dose group showing a statistically significant difference (P<0.05) (Table 11).

[0198] Table 11. Effects of xylene on ear swelling in mice (M±SD, N=8)

[0199] *P<0.05, compared with the model group.

[0200] 4. Discussion

[0201] Meptazinol has a chemical structure similar to morphine, a natural opioid analgesic, and is a potent centrally acting analgesic; flurbiprofen is a nonsteroidal anti-inflammatory drug (NSAID). This experiment investigated the analgesic and anti-inflammatory effects of meptazinol, flurbiprofen, and clomiflufen hydrochloride using three mouse models.

[0202] Intraperitoneal injection of acetic acid in mice caused visceral inflammation and pain due to the irritant effect of acetic acid on the peritoneum, manifested as writhing behavior. In this model, meptafol at 30 mg / kg showed some analgesic effect, but 60 mg / kg and 15 mg / kg did not. Given the unclear dose-response relationship, this suggests that meptafol's analgesic effect on this type of acute inflammatory visceral pain is not significant. Flurbiprofen showed a significant dose-dependent inhibitory effect on the writhing response in mice, indicating a significant analgesic effect on this type of acute inflammatory visceral pain. Using meptafol at 30 mg / kg as an equivalent dose (inhibition rate of writhing count 37.9%), clomiflufen hydrochloride (55 mg / kg) inhibited the writhing count in mice by 100%, indicating that the analgesic effect of clomiflufen hydrochloride was significantly stronger than that of meptafol alone, and slightly stronger than that of flurbiprofen alone. In other words, when meptapone is combined with flurbiprofen, it can significantly increase the inhibitory effect of meptapone on acute inflammatory pain, without weakening the anti-inflammatory and analgesic effect of flurbiprofen.

[0203] The hot plate test, which uses temperature stimulation to induce pain responses in mice, is a method for evaluating the analgesic mechanisms of the central and peripheral nervous systems. In this model, meptaprol at 30 mg / kg and 15 mg / kg showed significant analgesic effects as a central nervous system analgesic, while the analgesic effect decreased somewhat at 60 mg / kg. Flurbiprofen (27 mg / kg) at clinically equivalent doses also had some analgesic effect, but its potency was weaker than meptaprol. Using meptaprol 30 mg / kg (which increased pain threshold by 70.5% and 86.1% at 0.5h and 1h, respectively) as an equivalent dose, clomiflufenicol hydrochloride (55 mg / kg) increased the pain threshold in mice by 47.5% and 78.8% at 0.5h and 1h, respectively. Although weaker than meptaprol monomer, this was comparable to flurbiprofen.

[0204] When xylene comes into contact with the skin, it can induce a local inflammatory response, manifested as tissue exudation and swelling. In a xylene-induced mouse ear swelling model, metoprolol (30 mg / kg) had no significant anti-inflammatory effect, while flurbiprofen (27 mg / kg) had a significant anti-inflammatory effect; based on an equivalent amount of metoprolol 30 mg / kg, clomiphene hydrochloride (55 mg / kg) had a significant anti-inflammatory effect.

[0205] In summary, meptaprol significantly inhibits central neural transmission of pain and significantly increases the hot plate pain threshold in mice, but it has no anti-inflammatory effect and its effect on improving inflammatory visceral pain is relatively weak. Flurbiprofen has a significant anti-inflammatory effect and can also significantly reduce inflammatory visceral pain response, while also having a certain inhibitory effect on neural perception of pain. Compared with meptaprol, clomiflufenicol hydrochloride has a weaker inhibitory effect on neural transmission of pain, but it significantly enhances the anti-inflammatory effect and the analgesic effect on inflammatory pain.

[0206] Example 6 Acute Toxicity Evaluation Report of Comiflophen Hydrochloride

[0207] 1. Materials and Methods

[0208] 1.1 Tested Drugs

[0209] Meptazinol, provided by Hefei University of Science and Technology Biotechnology Co., Ltd.;

[0210] Comiflophen Hydrochloride, which is Comiflophen Hydrochloride Polymorph I.

[0211] Before the experiment, the drugs were prepared into corresponding concentration suspensions with 0.2% CMC-Na, and the administration volume was 20 mL / kg.

[0212] 1.2 Animals and Feeding

[0213] ICR mice, male and female, 18 - 22 g, SPF grade, production license number SCXK(Zhe)2019 - 0002, purchased from Hangzhou Medical College.

[0214] The mice were raised in the Animal Center of China Pharmaceutical University (animal use license number: SYXK(Su)2021 - 0011). The temperature of the breeding laboratory was 24 ± 2°C; the relative humidity was 40% - 70%; the number of air exchanges per hour was 10 - 15 times / hour; the light cycle was 12 (day) / 12 (night) hours, and no more than 5 mice were placed in each cage.

[0215] Feed: Complete mouse pellet feed, purchased from Jiangsu Xietong Pharmaceutical Bioengineering Co., Ltd., and its quality meets the General Quality Standard for Experimental Animal Compound Feed GB14924.1 - 2010.

[0216] Drinking water: Purified water.

[0217] 1.3 Dosage and Grouping

[0218] 1.3.1 Dosage Setting

[0219] According to the pre - test results, a total of 2 tested groups were set, namely meptazinol and comiflophen hydrochloride, and each tested group was set with 5 dosage groups as follows:

[0220] (1) Meptazinol: 500 mg / kg, 400 mg / kg, 320 mg / kg, 256 mg / kg, 204.8 mg / kg;

[0221] (2) Comiflophen Hydrochloride: 625 mg / kg, 500 mg / kg, 400 mg / kg, 320 mg / kg, 256 mg / kg.

[0222] 1.3.2 Interval between agents: 1:0.8

[0223] 1.3.3 Dosage volume: 0.4 mL / 20 g

[0224] 1.4 Route of administration

[0225] Administered via gavage (ig)

[0226] 1.5 Test Methods

[0227] (1) Laboratory environment: room temperature 24±2℃, relative humidity 60~70%.

[0228] (2) Observation indicators: The test drugs were prepared into drug suspensions of corresponding concentrations according to the above dosage and the volume of administration by the proportional dilution method. The suspensions were administered by g once, and various poisoning symptoms and death of mice were recorded. Autopsies were performed on the dead animals.

[0229] (3) Observation period: 14 days.

[0230] 3. Results

[0231] 3.1 General Observation

[0232] Following administration of the test drug to mice via intragastric gavage, all mice exhibited a significant decrease in spontaneous activity, weight loss, abdominal lying posture, drooping eyelids, and sluggish response to external stimuli, eventually dying quietly. All deaths occurred within 1 hour of drug administration; no further deaths were observed thereafter. Twenty-four hours after administration, animal activity began to increase and gradually returned to normal.

[0233] 3.2 Autopsy Results

[0234] Autopsy of deceased mice revealed gastric distension and watery or white contents in the gastrointestinal tract. Gross examination of the dead mice showed no obvious organ lesions. The mice likely died from systemic failure due to the drug's effects on the gastrointestinal tract and central nervous system.

[0235] 3.3 Mouse mortality

[0236] The doses at which all mice died after administration of meptaphen and clomiphene hydrochloride were 500 mg / kg and >625 mg / kg, respectively, while the doses at which no mortality was caused were 204.8 mg / kg and 256 mg / kg, respectively. LD50 was calculated using GraphPad software. 50 The values ​​for meprofen and clofenac hydrochloride were 272.4 mg / kg and 516.9 mg / kg, respectively (Table 12).

[0237] Table 12. Mortality of mice in the acute toxicity test after a single gavage administration of the test substance

[0238] a Number of dead mice / Number of mice administered drugs

[0239] 4. Conclusion

[0240] When mice were administered drugs by gavage, the dose of hydrochloride of kemeflofen that caused mouse death was higher than that of meptazinol, and the LD 50 value was about twice that of meptazinol, indicating that the acute toxicity of hydrochloride of kemeflofen was weaker than that of meptazinol.

[0241] Report on Comparative Evaluation of Analgesic Activity of Crystal Form I (FMY) of Hydrochloride of Kemeflofen and Kemeflofen (FM) in Example 7

[0242] 1. Materials

[0243] 1.1 Tested drugs

[0244] Crystal Form I (FMY) of hydrochloride of kemeflofen and kemeflofen (FM) were both provided by Hefei University of Science and Technology Biotechnology Co., Ltd.

[0245] Before the experiment, FM was prepared into a solution with corresponding concentration using vegetable oil, and FMY was prepared into a solution with corresponding concentration using distilled water. The administration volume was 20 ml / kg for both.

[0246] 1.2 Animals and feeding

[0247] ICR mice, male and female, 18 - 22 g, SPF grade, were purchased from Jiangsu Qinglongshan Biotechnology Co., Ltd., and the production license number: SCXK(Shanghai)2024 - 0001.

[0248] The mice were raised in the Animal Center of China Pharmaceutical University (Animal Use License Number: SYXK(Jiangsu)2021 - 0011). The temperature of the feeding laboratory was 24 ± 2°C; the relative humidity was 40% - 70%; the number of air exchanges per hour was 10 - 15 times / hour; the light cycle was 12 (day) / 12 (night) hours, and no more than 5 mice were kept in each cage.

[0249] Feed: Complete pellet feed for mice, purchased from Jiangsu Xietong Pharmaceutical Bio - engineering Co., Ltd., and its quality complied with the General Quality Standard for Experimental Animal Compound Feed GB14924.1 - 2010.

[0250] Drinking water: Purified water.

[0251] 1.3 Main reagents

[0252] Golden Arowana edible soybean oil, commercially available.

[0253] 1.4 Main instruments

[0254] FEJ - 200 electronic balance (0.1 - 200 g), Fuzhou Furhengzhibao Electronic Co., Ltd.;

[0255] BS210S Precision Electronic Balance (0.1mg~10g), Sartorius, Germany;

[0256] Digital display constant temperature circulating water bath, Changzhou Guohua Electric Appliance Co., Ltd.

[0257] 2. Methods

[0258] 2.1 Basis for Dosage Setting

[0259] The recommended clinical dose of meptaprol is 200 mg orally per dose. In this study, the doses of FM and FMY were converted based on the mass of meptaprol they contained and set at 55 mg / kg, which is approximately twice the clinical dose.

[0260] 2.2 Effects on acetic acid-induced writhing response in mice (analgesic effect)

[0261] ICR mice, male, weighing 18-22g, were purchased and acclimatized. They were then randomly divided into three groups of 10 mice each, based on their weight: model group (vegetable oil), FM 55mg / kg, and FMY 55mg / kg.

[0262] Animals in each group were fasted for 12 hours before the experiment, but water intake was unrestricted. Sixty minutes after gavage administration of the corresponding drug, 0.6% acetic acid (0.2 ml / mouse) was injected intraperitoneally. The number of writhing episodes in mice within 15 minutes of injection was recorded. A positive writhing response was defined as repeated contraction of the lumbar muscles, arching of the back, twisting of the hips, and extension of the hind limbs. The percentage of writhing inhibition by the drug was calculated using the following formula:

[0263] Inhibition rate (%) = (Number of writhing movements in the control group - Number of writhing movements in the treatment group) / Number of writhing movements in the control group × 100%

[0264] 2.3 Effect on pain threshold of hot plate in mice (analgesic effect)

[0265] Female ICR mice, weighing 18–22g, were purchased and acclimatized before being randomly divided into two groups of 10 mice each, with FM 55mg / kg and FMY 55mg / kg respectively.

[0266] Animals in each group were fasted for 12 hours before the experiment, but water intake was unrestricted. Mice were placed on a constant-temperature hot plate (55±0.5℃), and the time required for them to lick their paws was recorded as the pain threshold. Qualified mice (pain response time less than 60 seconds) were selected and randomly assigned to groups. The pain threshold was measured twice before drug administration, and the average value was taken as the baseline pain threshold. After gavage administration of the corresponding drug, the pain threshold of each mouse was measured at 0.5 and 1 hour post-administration (if there was no pain response within 60 seconds, it was calculated as 60 seconds). Using the baseline pain threshold as a reference, the increase rate of pain threshold in each mouse at 0.5 and 1 hour post-administration was calculated.

[0267] 3. Results

[0268] 3.1 Effects on acetic acid-induced writhing response in mice (analgesic effect)

[0269] Compared with the model group, FM 55 mg / kg significantly inhibited the writhing response induced by intraperitoneal injection of acetic acid in mice, with an inhibition rate of 54.8% (P<0.01); FMY 55 mg / kg significantly inhibited the writhing response in mice (P<0.01), with an inhibition rate of 83.1%. The analgesic effect of FMY 55 mg / kg was significantly stronger than that of the same dose of FM (P<0.01) (Table 13).

[0270] Table 13. Effect of acetic acid on the number of writhing movements in mice (M±SD, N=10)

[0271] **P<0.01, compared with the model group; ## P<0.01, compared with the FM group.

[0272] 3.2 Effect on pain threshold of hot plate in mice (analgesic effect)

[0273] FM 55 mg / kg increased the hot plate pain threshold in mice, with a significant effect observed 0.5 h after administration (P<0.05); FMY 55 mg / kg significantly increased the hot plate pain threshold in mice at both 0.5 h and 1 h after administration (P<0.0, P<0.05). The analgesic effect of FMY 55 mg / kg was significantly stronger than that of FM at the same dose (Table 14).

[0274] Table 14. Effects on the pain threshold of a mouse hot plate (M±SD, N=10)

[0275] *P<0.05, **P<0.01, compared with the baseline pain threshold of each group; # P<0.05, compared with the FM group.

[0276] 4. Conclusion

[0277] The results of this experiment show that FM and FMY both have analgesic effects on mouse acetic acid writhing and hot plate models, and the analgesic effect of FMY is stronger than that of FM at the same dose.

[0278] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.

Claims

1. A compound, which is 3-(3-ethyl-1-methyl-1H-hexahydroazaphen-3-yl),2-(2-fluoro-4-biphenyl)-propionic acid phenyl ester hydrochloride.

2. The compound according to claim 1, characterized in that, The compound has the structure shown in the following formula:

3. The crystal form of the compound as described in claim 1 or 2.

4. The crystal form according to claim 3, characterized in that, The crystal form is crystal form I. Using Cu-Kα radiation, its X-ray powder diffraction pattern, expressed in 2θ angles, shows diffraction peaks at 11.4°±0.2°, 14.5°±0.2°, and 21.4°±0.2°. Preferably, its X-ray powder diffraction pattern, expressed in 2θ angles, also has diffraction peaks at one or more of the following 2θ angles: 16.5°±0.2°, 17.9°±0.2°, 18.3°±0.2°; Preferably, its X-ray powder diffraction pattern, expressed in 2θ angles, also has diffraction peaks at one or more of the following 2θ angles: 9.1°±0.2°, 16.9°±0.2°, 17.5°±0.2°, 19.9°±0.2°, 23.0°±0.2°, 25.2°±0.2°; Preferably, its X-ray powder diffraction pattern, expressed in 2θ angles, also has diffraction peaks at one or more of the following 2θ angles: 7.9°±0.2°, 13.6°±0.2°, 15.6°±0.2°, 27.3°±0.2°; Preferably, its X-ray powder diffraction pattern expressed in 2θ angle is substantially the same as that in Figure 9.

5. The crystal form according to claim 3, characterized in that, The crystal form is crystal form II. Using Cu-Kα radiation, its X-ray powder diffraction pattern, expressed in 2θ angles, has diffraction peaks at 13.6°±0.2°, 16.2°±0.2°, 18.4°±0.2°, and 21.4°±0.2°. Preferably, its X-ray powder diffraction pattern, expressed in 2θ angles, also has diffraction peaks at one or more of the following 2θ angles: 8.0°±0.2°, 11.4°±0.2°, 14.5°±0.2°, 19.1°±0.2°, 20.0°±0.2°; Preferably, its X-ray powder diffraction pattern, expressed in 2θ angles, also has diffraction peaks at one or more of the following 2θ angles: 9.1°±0.2°, 11.9°±0.2°, 13.9°±0.2°, 22.9°±0.2°, 24.0°±0.2°, 27.3°±0.2°; Preferably, its X-ray powder diffraction pattern expressed in 2θ angle is substantially the same as that in Figure 8; Preferably, its X-ray powder diffraction pattern expressed in 2θ angle is substantially the same as that in Figure 10.

6. A pharmaceutical composition comprising the compound of claim 1 or 2, or comprising the crystal form of any one of claims 3 to 5; Preferably, the pharmaceutical composition further includes pharmaceutically acceptable excipients; Preferably, the pharmaceutical composition is an oral formulation or an injection.

7. The use of the compound of claim 1 or 2, or the crystal form of any one of claims 3 to 5, or the pharmaceutical composition of claim 6 in the preparation of a medicament for anti-inflammatory, analgesic, or antipyretic purposes; Preferably, the drug is used to treat moderate or severe pain, such as rheumatoid arthritis, ankylosing spondylitis, degenerative arthritis, soft tissue sprains, toothache, trauma, musculoskeletal pain, postoperative pain, gynecological dysmenorrhea, obstetric pain, acute inflammatory visceral pain such as renal colic, central nervous system pain, peripheral neuropathy, or cancer pain.

8. A method for preparing the compound according to claim 1 or 2, comprising the following steps: An ethyl acetate solution of the compound of formula I is mixed with an ethyl acetate solution of HCl and reacted to generate the compound; 9. The preparation method according to claim 8, characterized in that, In the ethyl acetate solution of HCl, the concentration of HCl is 1–6 M, preferably 3–5 M, more preferably 3.5–4.5 M; and / or, The reaction temperature is below 0°C, preferably -5°C to 0°C; and / or, The amount of ethyl acetate solution of HCl added is sufficient to adjust the pH of the mixed solution to 1-5, preferably 2-3.

10. The preparation method according to claim 8, characterized in that, It also includes the following steps: The product after the reaction was recrystallized using a mixed solvent of acetone and n-hexane. Preferably, the volume ratio of acetone to n-hexane is 1:(1-3), more preferably 1:(1.5-2.5).

11. The preparation method according to any one of claims 8 to 10, characterized in that, The compound shown in Formula I was prepared by a method comprising the following steps: Flurbiprofen (Formula I-1) and oxalyl chloride are subjected to an acyl chloride reaction to produce flurbiprofen acyl chloride (Formula I-2). Flurbiprofen acyl chloride (Formula I-2) and meptaptaphenol (Formula I-3) are esterified in the presence of an alkaline substance and a catalyst to produce the compound shown in Formula I. Preferably, the acyl chloride reaction is carried out in a halogenated hydrocarbon and an amide organic solvent; the halogenated hydrocarbon organic solvent preferably includes dichloromethane, and the amide organic solvent preferably includes DMF; Preferably, the mass ratio of flurbiprofen to oxalyl chloride is (1.5–2):1; Preferably, the temperature of the acyl chloride reaction is 20–25°C; Preferably, the alkaline substance includes an organic amine, more preferably triethylamine; Preferably, the catalyst comprises 4-dimethylaminopyridine; Preferably, the mass ratio of meprofen to flurbiprofen acyl chloride is 1:(1-1.2); Preferably, the temperature of the esterification reaction is 20–25°C.

12. The preparation method according to any one of claims 8 to 10, characterized in that, The compound shown in Formula I was prepared by a method comprising the following steps: Flurbiprofen (Formula I-1) and meptaphen (Formula I-3) are condensed together to produce the compound shown in Formula I. Preferably, the condensation reaction is carried out in the presence of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and 4-dimethylaminopyridine; Preferably, the temperature of the condensation reaction is 20–25°C; Preferably, the mass ratio of flurbiprofen to meptaprol is (0.8-1.2):1, more preferably (0.9-1.0):1.

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