CN114437080A - Yokenafil oxide, preparation method and application thereof - Google Patents

Abstract

The invention discloses a eukenafil oxide shown in a formula I or a pharmaceutically acceptable salt thereof, and a preparation method and application of the compound. The impurity is degradation impurity of eukenafil hydrochloride, and can be used as reference substance for research of related substances, and can be used for hydrochloric acidUkenafil methodology validation and quality control to better control product quality.

Description

Euclaflon oxide and its preparation method and use

This application claims to enjoy the priority rights of the prior invention patent application with the application number 202011205349.0 and the title of "Uconafil oxide and its preparation method and use" filed with the State Intellectual Property Office of China on November 2, 2020. The entirety of this application is incorporated herein by reference.

technical field

The present invention relates to the field of medicinal chemistry, in particular to ucnafil oxide and a preparation method and application thereof.

Background technique

Erectile Dysfunction (ED) refers to the persistent inability to achieve and/or) maintain a sufficient erection for a satisfying sexual life. According to statistics, there are currently many males with different degrees of ED in the world, and the number of patients will double by 2025. There are many treatment options for ED, among which selective phosphodiesterase 5 (PDE5) inhibitors are the most mature ED treatment drugs and the first-line drugs for clinical treatment of ED. There are five such drugs approved for marketing today, namely Sildenafil, Tadalafil, Vardenafil, Udenafil and Mironafil (Mirodenafil).

Patent document CN100374441C discloses a series of pyrrolopyrimidone structural compounds and their preparation methods and uses. Wherein compound 1-HCl, namely 2-[2-ethoxy-5-(4-ethylpiperazine-1-sulfonyl)phenyl]-5-methyl-7-n-propyl-3,7- Dihydropyrrolo[2,3-d]pyrimidin-4-one monohydrochloride has been disclosed in Example 1 of this document, which is ucnafil hydrochloride.

Patent document CN104530054B discloses the polymorphic form of ucnafil hydrochloride and its preparation method, and the crystal form A reported in the patent document has good physical and chemical stability, is easy to industrialized production, and is a pharmaceutically acceptable and stable form. Uxafil hydrochloride crystal form.

However, there are still continuing needs and uncertainties for the quality control methods of euclafil and its salts and/or the improvement of its drug safety and stability, and its research results are very important for the application and development of this drug. significance.

SUMMARY OF THE INVENTION

In order to improve the above-mentioned technical problems, the present invention provides a compound represented by the following formula I or a pharmaceutically acceptable salt thereof:

The present invention also provides a pharmaceutical composition, comprising ucnafil or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable adjuvant.

According to an embodiment of the present invention, the pharmaceutical composition has a light-protected and/or sealed package. Preferably, the pharmaceutical composition has a light-proof and airtight package.

According to an embodiment of the present invention, the packaging material includes an inner packaging material, or includes an inner packaging material and an outer packaging material.

As an example, the inner packaging material may be selected from one, two or three kinds of polyvinyl chloride, polyvinylidene chloride and aluminum foil (eg, medicinal aluminum foil).

As an example, the outer cover may be selected from composite films, such as composite films comprising two or three of polyester, aluminum and polyethylene.

The present invention also provides a medicine box, comprising a box body and a pharmaceutical composition of the present invention located in the box body, for example, comprising a box body and a light-proof and/or sealed package located in the box body of the pharmaceutical composition.

According to an embodiment of the present invention, in the pharmaceutical composition, based on the compound represented by formula I, the weight percentage content of the compound represented by formula I or a pharmaceutically acceptable salt thereof is ≤0.5%.

According to an embodiment of the present invention, in the pharmaceutical composition, based on the compound represented by formula I, the weight percent content of the compound represented by formula I or a pharmaceutically acceptable salt thereof may be ≥0.

According to an embodiment of the present invention, in the pharmaceutical composition, based on the compound represented by formula I, the weight percentage content of the compound represented by formula I or a pharmaceutically acceptable salt thereof may be 0.001%, 0.01%, 0.02% %, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.10%, 0.11%, 0.12%, 0.13%, 0.14%, 0.15%, 0.16%, 0.17%, 0.18%, 0.19%, 0.20%, 0.25%, 0.30%, 0.35%, 0.40%, 0.45% or 0.50%, preferably < 0.2%, more preferably < 0.15%, more preferably < 0.1%.

The present invention also provides a method for preparing the compound represented by formula I or a pharmaceutically acceptable salt thereof, comprising oxidizing euclaflon or a pharmaceutically acceptable salt thereof to obtain the compound represented by formula I or a pharmaceutically acceptable salt thereof. acceptable salt.

According to an embodiment of the present invention, the oxidation is carried out in the presence of an oxidizing agent. For example, the oxidizing agent is hydrogen peroxide, C _1-10 peroxy fatty acid, peroxybenzoic acid, halogenated peroxybenzoic acid, ammonium persulfate, cumene hydrogen peroxide and dicumyl hydrogen peroxide At least one of , preferably hydrogen peroxide.

According to an embodiment of the present invention, the mass ratio of the oxidizing agent to Uxafil or a pharmaceutically acceptable salt thereof is 0.3:1-1:1, preferably 0.4:1-0.6:1, exemplarily 0.3:1 1, 0.4:1, 0.5:1, 0.6:1, 0.7:1, 0.8:1, 0.9:1, 1:1.

According to an embodiment of the present invention, the temperature of the oxidation reaction is 20-100°C, preferably 50-100°C, more preferably 75-85°C, exemplarily 20°C, 30°C, 40°C, 50°C, 60°C, 70°C, 75°C, 80°C, 85°C, 90°C, 100°C.

According to an embodiment of the present invention, the oxidation is carried out in a solvent. For example, the solvent is selected from organic acids that are liquid at the temperature of the oxidation reaction, preferably C _1-10 organic acids, more preferably formic acid, acetic acid, propionic acid, butyric acid, isobutyric acid, n-valeric acid , one, two or more of 1-methylbutanoic acid, 2-methylbutanoic acid, 2,2-dimethylpropionic acid, exemplified by formic acid.

According to an embodiment of the present invention, the solution containing ukenafil or a pharmaceutically acceptable salt thereof may be subjected to a cooling treatment before adding the oxidizing agent. For example, the temperature is lowered to -10 to 10°C, preferably -5 to 5°C. Preferably, the temperature at which the oxidizing agent is added to the solution containing ukenafil or its pharmaceutically acceptable salt is -10-10°C, preferably -5-5°C, more preferably, the oxidizing agent and the solution are the same temperature.

According to an embodiment of the present invention, the mass ratio of the solvent to Uxafil or a pharmaceutically acceptable salt thereof is 3:1-15:1, preferably 7:1-10:1, exemplarily 3:1 1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1.

According to an embodiment of the present invention, the preparation method further includes a post-processing step.

According to an embodiment of the present invention, the post-treatment step includes adding a reducing agent to the oxidation system to quench the reaction.

According to an embodiment of the present invention, the reducing agent is one, two or more of sodium sulfite, sodium bisulfite and ferrous sulfate, preferably sodium sulfite and/or sodium bisulfite.

According to an embodiment of the present invention, the reducing agent is added to the oxidation system in solution. Preferably, the concentration of the reducing agent solution is 0.1mol/L～5.0mol/L, preferably 0.5mol/L～3mol/L, exemplarily 0.5mol/L, 1.0mol/L, 1.5mol/L, 2mol/L L, 3mol/L, 4mol/L, 5mol/L.

According to an embodiment of the present invention, the preparation method further includes a purification step. Preferably, the purification step follows the work-up step.

According to an embodiment of the present invention, the purification step includes the steps of extracting, washing, drying and recrystallizing the reaction product after the above post-treatment is completed.

According to an embodiment of the present invention, after the post-treatment is completed, water and a first solvent are added to the reaction system, and the mixture is stirred and extracted to obtain an organic phase.

According to an embodiment of the present invention, the first solvent is one, two or more of dichloromethane, ethyl acetate and methyl tert-butyl ether, preferably dichloromethane.

According to an embodiment of the present invention, the organic phase is washed. For example, a saturated sodium chloride solution is used for washing.

According to an embodiment of the present invention, the drying is drying the washed organic phase using a drying agent.

According to an embodiment of the present invention, the desiccant may be one, two or more of anhydrous sodium sulfate, anhydrous magnesium sulfate and molecular sieves, preferably anhydrous sodium sulfate and/or anhydrous magnesium sulfate.

According to an embodiment of the present invention, the recrystallization step includes: filtering the dried organic phase, concentrating the obtained filtrate, adding a second solvent to the concentrate, heating up and stirring, and then cooling down for crystallization.

According to an embodiment of the present invention, the temperature of the concentration is 20-80°C, preferably 30-50°C, exemplarily 20°C, 30°C, 40°C, 50°C, 60°C, 70°C, 80°C.

According to an embodiment of the present invention, the second solvent is one, two or more of methanol, ethanol, acetone, ethyl acetate, dichloromethane, diethyl ether, chloroform, toluene, xylene and acetonitrile, preferably to ethanol.

According to an embodiment of the present invention, the mass ratio of the second solvent to Uxafil or a pharmaceutically acceptable salt thereof is 3:1-8:1, preferably 3:1-6:1, exemplified by 3:1, 4:1, 5:1, 6:1, 7:1, 8:1.

According to an embodiment of the present invention, in the recrystallization step, the temperature raised to is 40-80°C, preferably 70-80°C, exemplarily 40°C, 50°C, 60°C, 70°C, 80°C.

According to an embodiment of the present invention, the temperature of the cooling crystallization is -10 to 30°C, preferably -5 to 5°C, exemplarily -10°C, -5°C, 0°C, 5°C, 10°C, 20°C °C, 30 °C.

According to an embodiment of the present invention, the recrystallization step further comprises filtering and drying the product obtained by crystallization. Preferably, the drying temperature is 30-80°C, preferably 40-50°C, exemplarily 30°C, 40°C, 50°C, 60°C, 70°C, 80°C.

According to the exemplary scheme of the present invention, the preparation method of the compound shown in the formula I or a pharmaceutically acceptable salt comprises the following steps:

Adding ucrafil or a pharmaceutically acceptable salt thereof to the solvent, stirring and dissolving, then adding an oxidizing agent therein to carry out an oxidation reaction, and controlling the reaction in TLC until the reaction is completed;

After the reaction is completed, add the reducing agent solution to the reaction system, and stir; then add water and the first solvent to the system, stir and extract; use saturated sodium chloride solution to wash the organic phase obtained by extraction, and dry the organic phase;

The above-mentioned dried organic phase is filtered, the filtrate is concentrated, the second solvent is added to the obtained concentrate, the temperature is raised and stirred, then the temperature is lowered for crystallization, filtered, and the obtained filter cake is dried to obtain the target product.

The present invention also provides a preparation method of the compound represented by formula I or a pharmaceutically acceptable salt thereof, comprising subjecting euclafil or a pharmaceutically acceptable salt thereof to light irradiation to obtain the compound represented by formula I A compound or a pharmaceutically acceptable salt thereof.

According to an embodiment of the present invention, the light may be strong white light and/or ultraviolet light, and the preferred illuminance may be 4500lx±500lx.

The present invention also provides the use of the compound represented by formula I or a pharmaceutically acceptable salt thereof as a standard or reference substance, especially as a pharmaceutical standard or reference substance.

The present invention also provides the use of the compound represented by the above-mentioned formula I or a pharmaceutically acceptable salt thereof as a quality research standard or reference substance of euclafil or a pharmaceutically acceptable salt thereof.

The present invention also provides the use of Uxafil or a pharmaceutically acceptable salt thereof in the manufacture of a pharmaceutical composition, wherein the pharmaceutical composition is as defined above.

The present invention also provides a method of treating and/or preventing erectile dysfunction comprising administering the pharmaceutical composition to a subject in need thereof.

According to embodiments of the present invention, the subject in need includes, but is not limited to, patients with erectile dysfunction or potential patients at risk of erectile dysfunction. Preferably, the patient is a male mammal, especially a male human.

The present invention also provides a method for quality control of euclafil or a pharmaceutically acceptable salt thereof, comprising using the compound represented by formula I or a pharmaceutically acceptable salt thereof as a standard substance or reference substance, especially as a medicine Standard or reference substance is used.

The present invention also provides a method for quality control of euclafil or a pharmaceutically acceptable salt thereof, comprising detecting the compound represented by formula I or a pharmaceutically acceptable salt thereof. Preferably, the detection may be qualitative detection or quantitative detection.

The present invention also provides a method for quality control of ukenafil or a pharmaceutically acceptable salt thereof, comprising adding ukenafil or a pharmaceutically acceptable salt thereof to an unfavorable method for the compound represented by formula I or its pharmaceutically acceptable salt. Store or use under acceptable salt-forming conditions.

The present invention also provides a preparation method of a pharmaceutical composition, which comprises mixing ucnafil or a pharmaceutically acceptable salt thereof with a pharmaceutically acceptable auxiliary material, wherein the preparation method is not conducive to the compound represented by formula I or a pharmaceutically acceptable salt thereof is formed.

The present invention also provides a method for preserving or using Euclafil or a pharmaceutically acceptable salt thereof, wherein the method for preserving or using is not conducive to the formation of the compound represented by Formula I or a pharmaceutically acceptable salt thereof. conditions.

Preferably, the "conditions that are not conducive to the formation of the compound represented by formula I or a pharmaceutically acceptable salt thereof" include, but are not limited to, at least one selected from light-shielding and sealing conditions; more preferably, light-shielding and sealed condition.

According to an optional embodiment of the present invention, the storage or use method of euclafil or a pharmaceutically acceptable salt thereof can be protected from light and sealed, and at a temperature lower than 60°C (preferably lower than 50°C) and / or under the condition of relative humidity lower than 75% RH (preferably lower than 65%).

The present invention also provides a method for detecting a compound represented by formula I or a pharmaceutically acceptable salt, comprising using high performance liquid chromatography to detect the compound represented by formula I or a pharmaceutically acceptable salt in a test solution.

According to an embodiment of the present invention, the test article is a pharmaceutical composition, preferably a preparation, such as a solid preparation or a liquid preparation, containing ucnafil or a pharmaceutically acceptable salt thereof. More preferably, the pharmaceutical composition or preparation comprises a compound represented by formula I or a pharmaceutically acceptable salt.

According to an embodiment of the present invention, the chromatographic column used in the high performance liquid chromatography is a chromatographic column with octadecyl-bonded silica gel as a filler; for example, the chromatographic column is selected from Agilent Eclipse XDB-C18 250×4.6mm, 5μm or a chromatographic column with the same or similar composition and performance.

According to an embodiment of the present invention, the detected wavelength is 220-230 nm, such as 222-228 nm, preferably 226 nm.

According to an embodiment of the present invention, the column temperature of the chromatography column is 20-30°C, such as 22-28°C, exemplarily 25°C.

According to an embodiment of the present invention, the flow rate is 0.5-1.5 mL/min, such as 0.8-1.2 mL/min, exemplarily 1 mL/min.

According to an embodiment of the present invention, the high performance liquid chromatography uses citrate buffer-acetonitrile as the mobile phase. Preferably, the concentration of the citrate buffer in the mobile phase is 0.01-0.04 mol/L, such as 0.02-0.03 mol/L, and exemplarily 0.02 mol/L.

According to an embodiment of the present invention, the pH of the citrate buffer is 5.3-5.5, eg, 5.4.

According to an embodiment of the present invention, the citrate buffer is a citrate-triethylamine buffer.

According to an embodiment of the present invention, the citric acid buffer is prepared by mixing citric acid, triethylamine and water, eg, dissolving citric acid in water, and adjusting the pH to the above range with triethylamine.

According to an embodiment of the present invention, the volume ratio of citrate buffer to acetonitrile in the mobile phase is (45-55):(55-45), eg, 50:50.

According to an embodiment of the present invention, the test solution is prepared from the mobile phase, and in the test solution, the concentration of ucnafil hydrochloride is 0.5-1.5 mg/mL, for example, 1.0 mg/mL. mL.

According to an embodiment of the present invention, the reference substance used in the detection method is the compound represented by formula I or a pharmaceutically acceptable salt.

According to an embodiment of the present invention, in the test article, ucnafil or a pharmaceutically acceptable salt thereof may exist in its amorphous or crystalline form. For example, for the hydrochloride salt of Uxafil (such as Uxafil hydrochloride), it may be in the form of a polymorph, such as the A-type polymorph or B-type polymorph disclosed in the patent document CN104530054B , especially the polymorph of Uxafil hydrochloride type A shown in the XRD spectrum of FIG.

According to embodiments of the present invention, the compound of formula I, or a pharmaceutically acceptable salt thereof, may exist in its amorphous or crystalline form.

According to embodiments of the present invention, euclafil and the compound of formula I may independently exist in the form of various pharmaceutically acceptable acid addition salts. The acid addition salts include, but are not limited to, salts formed with an acid selected from the group consisting of: hydrochloride, hydrofluoride, hydrobromide, hydroiodide, sulfate, pyrosulfate, phosphoric acid Salt, nitrate, mesylate, ethanesulfonate, 2-hydroxyethanesulfonate, benzenesulfonate, tosylate, sulfamate, 2-naphthalenesulfonate, formate, acetyl Acetic acid, pyruvic acid, laurosilicate, cinnamate, benzoate, acetate, glyoxylate, trifluoroacetate, trimethylacetate, propionate, butyrate, Caproate, Enanthate, Undecanoate, Stearate, Ascorbate, Camphorate, Camphorsulfonate, Citrate, Fumarate, Malate, Maleate, Hydroxymarate Acetate, oxalate, salicylate, succinate, gluconate, quinate, pamoate, glycolate, tartrate, lactate, 2-(4-hydroxy Benzoyl)benzoate, cyclopentane propionate, digluconate, 3-hydroxy-2-naphthoate, nicotinate, pamoate, pectinate, 3-benzene propionate, picrate, pivalate, itaconate, triflate, dodecyl sulfate, p-toluenesulfonate, naphthalene disulfonate, malonate, Adipate, alginate, mandelate, glucoheptanoate, glycerophosphate, sulfosalicylate, hemi-sulfuric or thiocyanate, aspartate, etc.

In the context of the present invention the compound of formula I may be referred to as "euclaflox".

Unless otherwise stated, "Uxafil", "Uxafil free base", "Uxafil hydrochloride free base" in the context of the present invention mean the same compound, ie one which does not form an addition salt with an acid The chemical structure of ucnafil is known to those skilled in the art, such as the structure shown in formula II in the examples.

According to a preferred embodiment of the present invention, the pharmaceutically acceptable salt of the compound represented by formula I is selected from its hydrochloride salt. Unless otherwise stated, the hydrochloride salts may be referred to in the context of the present invention as "euxafil oxide hydrochloride", "hydrochloride salt of ucnafil oxide" or "salt of the compound of formula I" acid salt", these terms should be understood to mean the same salt form.

beneficial effect

The present invention confirms the structure of the compound represented by formula I or its pharmaceutically acceptable salt, and its preparation method and detection method through in-depth research on the raw materials and preparations of euclafamide and its pharmaceutically acceptable salts. , to provide standard and reference substances for the qualitative and quantitative analysis of ucnafil hydrochloride, and to provide guidance for the safe use of ucnafil hydrochloride. The inventor unexpectedly found that the compound represented by formula I or a pharmaceutically acceptable salt thereof can affect the safety, effectiveness and/or stability of the euclafil preparation, especially the euclafil as a pharmaceutical active ingredient or the content stability of a pharmaceutically acceptable salt thereof. Therefore, controlling the compound shown in formula I or a pharmaceutically acceptable salt thereof helps to improve the quality of euclafil or a pharmaceutically acceptable salt thereof and its pharmaceutical composition, including its safety, effectiveness and/or or stability. Moreover, the compound represented by formula I or a pharmaceutically acceptable salt thereof can be used as a standard substance or a reference substance for the quality control of the raw material medicines of ukenafil or ukenafil hydrochloride and their preparations.

The present invention also provides a preparation method of the compound represented by formula I or a pharmaceutically acceptable salt thereof, which has the advantages of easily available raw materials, simple and convenient operation, mild reaction conditions, good repeatability, convenient post-processing steps, and concise purification steps.

The present invention also provides a method for detecting the compound shown in formula I or a pharmaceutically acceptable salt thereof, which can detect its content in raw materials and medicines with high precision and accuracy, which is beneficial to the safety and effectiveness of medicines. Significant improvement in performance and/or stability.

Description of drawings

Fig. 1 is the hydrogen nuclear magnetic resonance spectrum of formula I compound;

Fig. 2 is the carbon nuclear magnetic resonance spectrum of the compound of formula I;

Fig. 3 is a partial enlarged view of the carbon nuclear magnetic resonance spectrum of the compound of formula I in Fig. 2;

Fig. 4 is the hydrogen nuclear magnetic resonance spectrum of ucnafil;

Figure 5 is the mass spectrum of the compound of formula I;

Fig. 6 is the HPLC spectrogram of formula I compound;

Fig. 7 is the HPLC spectrogram of Ukenafil hydrochloride finished product;

Figure 8 is an HPLC profile of the 6th month sample in the accelerated stability experiment.

Detailed ways

The technical solutions of the present invention will be described in further detail below with reference to specific embodiments. It should be understood that the following examples are only for illustrating and explaining the present invention, and should not be construed as limiting the protection scope of the present invention. All technologies implemented based on the above content of the present invention are covered within the intended protection scope of the present invention.

Unless otherwise stated, the starting materials and reagents used in the following examples are commercially available or can be prepared by known methods.

Example 1

To 100 mL of formic acid, add 10 g of free base of ucnafil hydrochloride (that is, the compound shown in formula II, also known as ucnafil), stir to dissolve, cool down to -5 ~ 5 ℃, slowly add 4.5 g of 30% concentration After adding the hydrogen peroxide solution, the temperature was slowly raised to 70-80° C., the reaction was incubated for 5 h, and controlled by TLC (developing solvent: volume ratio of ethyl acetate and ethanol=5:1) until the reaction was completed. Add 100mL of 1mol/L sodium bisulfite solution to the reaction solution, stir for 30min, add 40mL of purified water and 200mL of dichloromethane, stir for 30min, and extract by liquid separation. After the obtained organic phase is washed with 100mL of saturated sodium chloride solution, Dry with anhydrous sodium sulfate for 2h. Filtration, the filtrate was concentrated to dryness under reduced pressure at 30～40°C, then 40 g of ethanol was added, the temperature was raised to 70～80°C, the system was dissolved and then cooled to -5～5°C, crystallized, filtered, and the filter cake was heated to 40～50°C It was dried under vacuum to obtain 7.8 g of the compound of formula I with a yield of 75.5% and a purity of 99.1%.

Example 2

To 45mL of formic acid, add 5g of the compound of the structure shown in formula II, stir and dissolve, cool to -5～5°C, slowly add 3.0g hydrogen peroxide solution with a concentration of 30%, be warming up to 70～80°C after adding, and keep the reaction 4h, controlled by TLC (developing solvent: volume ratio of ethyl acetate and ethanol=5:1) to completion of the reaction. 50mL of 1mol/L sodium bisulfite solution was added to the reaction solution, stirred for 30min, added with 25mL of purified water and 100mL of dichloromethane, stirred for 30min, and subjected to liquid separation extraction. The obtained organic phase was washed with 50mL of saturated sodium chloride solution, and then Dry with anhydrous sodium sulfate for 2h. Filtration, the filtrate was concentrated to dryness under reduced pressure at 30-40 °C, 25 g of ethanol was added, the temperature was raised to 70-80 °C, the system was dissolved and then cooled to -5-5 °C, crystallized, filtered, and the filter cake was vacuumed at 40-50 °C After drying, 3.7 g of the compound represented by formula I was obtained, with a yield of 71.6% and a purity of 99.3%.

Example 3

In 45mL of acetic acid, add 5g of the compound of the structure shown in formula II, stir and dissolve, cool down to -5～5°C, slowly add 3.0g hydrogen peroxide solution with a concentration of 30%, be warming up to 70～80°C after adding, and keep the reaction 5h, controlled by TLC (developing solvent: volume ratio of ethyl acetate and ethanol=5:1) to completion of the reaction. 50mL of 1mol/L sodium bisulfite solution was added to the reaction solution, stirred for 30min, added with 25mL of purified water and 100mL of dichloromethane, stirred for 30min, and subjected to liquid separation extraction. The obtained organic phase was washed with 50mL of saturated sodium chloride solution, and then Dry with anhydrous sodium sulfate for 2h. Filtration, the filtrate was concentrated to dryness under reduced pressure at 30-40 °C, 25 g of ethanol was added, the temperature was raised to 70-80 °C, the system was dissolved and then cooled to -5-5 °C, crystallized, filtered, and the filter cake was vacuumed at 40-50 °C After drying, 3.5 g of the compound represented by formula I was obtained, with a yield of 67.8% and a purity of 96.7%.

Example 4

Utilize nuclear magnetic resonance and mass spectrometry to confirm the product structure prepared in Example 1, and the test results are as follows:

Nuclear magnetic resonance spectroscopy (BRUKER AV-500 nuclear magnetic resonance instrument, DMSO-d6)

As shown in Figure 1, ¹ H-NMRδ(ppm): 12.41(H,s), 7.96(1H,s), 7.87(1H,d,J=8.7Hz), 7.42(1H,d,J=8.7Hz) )4.26(2H,q,J＝6.8Hz),3.58(3H,m),2.92(4H,s),2.42(4H,s),2.31(2H,q,J＝7.1Hz),1.64(2H, m), 1.38 (6H, tJ=6.8 Hz), 0.94 (3H, t, J=7.0 Hz), 0.86 (3H, t, J=7.3 Hz).

As shown in Figure 2 and Figure 3, ¹³ C-NMRδ(ppm): 178.23, 161.93, 159.80, 157.33, 156.99, 132.19, 130.01, 126.35, 122.34, 113.47, 102.41, 65.03, 51.12, 50.95, 45.82, 40.23, 38 , 21.03, 14.10, 13.31, 11.71, 10.98.

Mass spectrometry (Agilent 1260-6100LC-MS mass spectrometer)

The mass spectrometry test results are shown in Figure 5, 504.2[M+H] ⁺ , 502.2[MH] ⁺ . The mass spectrometry results are consistent with the structural theoretical molecular weight; the molecular weight is odd, which is consistent with the odd number of Ns in the molecule of the compound of formula I.

It is proved by the above test that the product prepared in Example 1 is the compound represented by formula I, that is, ucnafil oxide.

Example 5

Detection method of ucnafil hydrochloride oxide in finished product of ucnafil hydrochloride:

The compound prepared in Example 1 was used as the reference substance, and the finished product of ucnafil hydrochloride (purity 99.8%, single impurity 0.07%, its hydrogen NMR spectrum as shown in Figure 4) was used as the test sample, and high performance liquid chromatography ( HPLC) method was used to detect the content of ucnafil oxide in the finished product.

Chromatographic conditions:

Chromatographic column: with octadecyl-bonded silica gel as filler (Agilent Eclipse XDB-C18 250×4.6mm, 5μm)

Detection wavelength: 226nm

Column temperature: 25℃

Flow rate: 1mL/min

Mobile phase: 0.02mol/L citrate buffer-acetonitrile=50:50 (volume ratio);

Preparation process of citric acid buffer: take 4.2 g of citric acid, dissolve in water and dilute to 1000 mL, and adjust the pH to 5.4±0.1 with triethylamine.

Test solution: Take an appropriate amount of the finished product of ucnafil hydrochloride, accurately weigh it, add mobile phase to dissolve and dilute to make a solution containing about 1.0 mg of ucnafil hydrochloride per 1 mL, shake well, and use it as the test solution.

Reference substance solution: Take the ucnafil oxide prepared in Example 1, accurately weigh it, add the mobile phase to dissolve and dilute to make a solution containing about 0.01 mg of ucnafil oxide per 1 mL, shake well, and use it as the reference substance solution.

The detection results are shown in Figure 7, compared with the HPLC spectrum of the reference substance (Figure 6), the substance with a relative retention time of about 26.8min in the test solution is the euclaflon oxide with the structure shown in formula I.

The above-mentioned detection method for the related substances in the finished product of ucnafil hydrochloride has been systematically verified by methodology, which fully proves the detection ability of the method for the oxide of ucnafil. The quality of the finished product of ukenafil hydrochloride can be guaranteed by setting the uxenafil oxide into the finished product quality standard of ukenafil hydrochloride and carrying out strict control. Example 6 Strong degradation test of ucnafil hydrochloride preparation

Damaged conditions investigated:

(1) Lighting: Take 10 tablets of this product and place them for 10 days under the mixed lighting conditions of 4500lx±500lx strong white light and ultraviolet light;

(2) High temperature: Take 10 tablets of this product and store it in the dark at 60°C for 10 days;

(3) High temperature + high humidity: Take 10 pieces of this product and store it in the dark for 10 days under the conditions of 60℃±2℃, RH75%±5%;

(4) Oxidation: Take 10 tablets of this product, add 5ml of 3% H ₂ O ₂ , and place at room temperature for 5h in the dark.

Detected according to the method of embodiment 5, the detection result is as follows:

Table 1 Investigation results of strong degradation test

The above results show that under the conditions of high temperature or high temperature and high humidity, the oxides of ucnafil increased to a certain extent; under the conditions of light and oxidation, the oxides of ucnafil increased more significantly. The above results show that the compound of formula I is the degradation product of the ucnafil hydrochloride preparation. In order to ensure the quality of the ucnafil hydrochloride preparation, its content needs to be controlled within the scope of the present invention.

Example 7 Inhibitory effect on hERG potassium channel

Patch-clamp technique was used to detect the concentration-response relationship of blockade of hERG channel by euclafil and euclafloxide, so as to evaluate the risk of inhibition of cardiac hERG potassium channel.

Cell Culture:

The HEK-293 cell line stably expressing hERG potassium channel was used, and the hERG potassium channel cells were purchased from Creacell Company. The HEK293 cell line stably expressing hERG potassium channel was cultured in DMEM medium containing 10% fetal bovine serum and 0.8 mg/mL G418 at a temperature of 37°C and a carbon dioxide concentration of 5%.

cell inheritance:

Remove the old medium and wash once with PBS, then add 1 mL of TrypLE ^™ Express solution and incubate at 37°C for about 0.5 min. When the cells are detached from the bottom of the dish, add about 5 mL of pre-warmed complete medium at 37°C. The cell suspension was gently pipetted to dissociate the aggregated cells. The cell suspension was transferred to a sterile centrifuge tube, and the cells were collected by centrifugation at 1000 rpm for 5 min. For expansion or maintenance culture, cells were seeded in 6 cm cell culture dishes, and the amount of cells seeded in each cell culture dish was 2.5×10 5 cells (final volume: 5 mL). To maintain the electrophysiological activity of the cells, the cell density must not exceed 80%.

Cells were isolated with TrypLE ^™ Express before the experiment, 4×10 3 cells were plated on coverslips, cultured in 24-well plates (final volume: 500 μL), and the assay was performed 18 hours later.

Electrophysiological recording

Extracellular fluid: K-007-1

140 mM NaCl, 3.5 mM _KCl , ₁ mM MgCl2.6H2O, ₂ mM CaCl2.2H2O, 10 mM D-Glucose, ₁₀ mM _HEPES , 1.25 mM _NaH2PO4.2H2O , _NaOH adjusted to pH=7.4.

Intracellular fluid: K-002-2

20 mM KCl, 115 mM K-Aspartic, 1 mM MgCl2.6H2O, 5 mM EGTA, 10 mM HEPES, ₂ mM _Na2 -ATP, KOH to adjust pH=7.2.

The extracellular fluid was stored for 2 weeks. After the intracellular fluid was prepared, it was divided into 1 mL tubes and stored in a -20°C refrigerator. The newly thawed intracellular fluid was used in the test every day. All intracellular fluid was used up within three months. After three months, discard the old intracellular fluid and reconstitute.

Patch Clamp Detection

The voltage stimulation protocol for whole-cell patch-clamp recording of whole-cell hERG potassium currents was as follows: when the whole-cell seal was formed, the cell membrane voltage was clamped at -80mV. The clamp voltage was depolarized from -80mV to -50mV for 0.5s (as leakage current detection), then stepped to 30mV for 2.5s, and then quickly returned to -50mV for 4s to excite the tail current of the hERG channel. Data were collected every 10s to observe the effect of drugs on hERG tail current. A -50mV stimulus for 0.5s was used as leakage current detection. Experimental data were acquired by an EPC-10 amplifier (HEKA) and stored in PatchMaster (HEKA) software.

The capillary glass tube was drawn into the recording electrode with a microelectrode puller. Under the inverted microscope, the microelectrode manipulator was manipulated to contact the recording electrode to the cells, and suction was given to negative pressure to form a GΩ seal. After forming the GΩ seal, perform fast capacitance compensation, and then continue to give negative pressure to break the cell membrane to form a whole-cell recording mode. Compensation for the slow capacitance was then performed and the membrane capacitance and series resistance were recorded. No leakage compensation is given.

When the hERG current recorded in the whole cell is stable, the drug starts to be administered, and each drug concentration is applied for 5 minutes (or the current is stable) and then the next concentration is detected, and each test compound is detected at multiple concentrations. The cell-coated coverslip was placed in the recording bath of the inverted microscope, and the test compound and the compound-free external solution flowed through the recording bath sequentially from low concentration to high concentration by gravity perfusion to act on the cells. Liquid exchange was performed using a vacuum pump during the recording. The current detected by each cell in the compound-free exosome served as its own control. Multiple cells were independently replicated. All electrophysiological experiments were performed at room temperature.

test results

In three independent repeated experiments, the inhibitory effects of ucnafil and ucrafil oxide on hERG channels were detected, and the semi-inhibitory concentration (IC50) of the samples on hERG current was calculated by fitting. The test results are as follows:

The inhibition ratio and IC ₅₀ results of the test product (Ukenafil) on hERG current:

The inhibition ratio and IC ₅₀ results of the test product ucnafil oxide on hERG current:

The patch-clamp technique was used to detect the concentration-effect relationship of the blockade of hERG channels by the test articles (Ukenafil hydrochloride and ucnafil oxide), so as to evaluate the risk of the inhibitory effect of the test articles on the cardiac hERG potassium channel. The test results showed that: within the tested concentration range, the IC50 of the test product ucnafil on hERG current was 3.9μM, and the inhibitory effect of ucnafil oxide on the cardiac hERG potassium channel was significantly stronger than that of ucnafil. The risk of inhibition of cardiac hERG potassium channel by euclaflox is suggested, so the content of the oxide impurities needs to be strictly controlled.

Example 8 Stability test of ucnafil hydrochloride preparation

The formulation used in this example has the same composition as the sample in Example 6.

The packaging materials selected in this embodiment include inner packaging materials and outer packaging materials. Among them, the inner packaging material adopts polyvinyl chloride/polyvinylidene chloride solid pharmaceutical composite hard sheet and pharmaceutical aluminum foil; the outer packaging material adopts polyester/aluminum/polyethylene pharmaceutical composite film bag.

Investigation conditions: The formulation stability test was carried out under accelerated conditions (40°C ± 2°C, 75% ± 5% RH), and samples were taken for testing in 0, January, March, and June respectively. The results are shown in Table 2 below.

Table 2 Accelerated stability retention sample results

According to the method of Example 5, the chromatogram of the 6th month sample detected by HPLC is shown in Figure 8, wherein the peak of ucnafil oxide is at 26.782 min.

The results show that after 6 months of investigation under accelerated conditions (40 ℃ ± 2 ℃, 75% ± 5% RH) in simulated packaging materials, the ucnafil oxide in the related substances increased slowly, and the total impurities increased slightly. There was no significant change in any item, indicating that the oxide was effectively controlled under the above packaging material conditions.

Exemplary embodiments of the present invention have been described above. However, the protection scope of the present invention is not limited to the above-mentioned embodiments. Any modification, equivalent replacement or improvement made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims (10)

1. A compound of formula I or a pharmaceutically acceptable salt thereof:

2. a pharmaceutical composition comprising eukenafil or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient;

preferably, the pharmaceutical composition has a light-tight and/or sealed package.

3. A process for preparing a compound of formula I or a pharmaceutically acceptable salt thereof according to claim 1, comprising oxidizing eukenafil or a pharmaceutically acceptable salt thereof to obtain said compound of formula I or a pharmaceutically acceptable salt thereof;

preferably, the oxidation is carried out in the presence of an oxidizing agent; for example, the oxidizing agent is hydrogen peroxide, C_1-10At least one of peroxyfatty acid, peroxybenzoic acid, haloperoxybenzoic acid, ammonium persulfate, cumene hydroperoxide, and diisopropylbenzene hydroperoxide;

preferably, the mass ratio of the oxidant to the eukenafil or the pharmaceutically acceptable salt thereof is 0.3: 1-1: 1;

preferably, the temperature of the oxidation reaction is 20-100 ℃;

preferably, the oxidation is carried out in a solvent; for example, the solvent is selected from organic acids that are liquid at the temperature of the oxidation reaction, preferably C_1-10The organic acid of (1).

Preferably, the solution containing the eukenafil or the pharmaceutically acceptable salt thereof is subjected to cooling treatment before the oxidizing agent is added;

preferably, the preparation method further comprises a post-treatment step; for example, the post-treatment step comprises adding a reducing agent to the oxidation system and quenching the reaction;

preferably, the preparation method further comprises a purification step;

preferably, the purification step comprises the steps of extracting, washing, drying and recrystallizing the reaction product after the above-mentioned post-treatment is completed.

4. Use of a compound of formula I according to claim 1 or a pharmaceutically acceptable salt thereof as a standard or control, preferably as a pharmaceutical standard or control;

preferably, the compound shown in the formula I or the pharmaceutically acceptable salt thereof is used as a quality research standard or a reference substance of Youcafil or the pharmaceutically acceptable salt thereof.

5. The pharmaceutical composition of claim 2, wherein the content of the compound represented by formula I or the pharmaceutically acceptable salt thereof in the pharmaceutical composition is less than or equal to 0.5% by weight of the compound represented by formula I;

preferably, in the pharmaceutical composition, the content of the compound shown in the formula I or the pharmaceutically acceptable salt thereof in percentage by weight is more than or equal to 0;

preferably, in the pharmaceutical composition, the content of the compound shown in the formula I or the pharmaceutically acceptable salt thereof is less than or equal to 0.2 percent by weight, more preferably less than or equal to 0.15 percent by weight, and even more preferably less than or equal to 0.1 percent by weight of the compound shown in the formula I.

6. Use of eukenafil or a pharmaceutically acceptable salt thereof for the preparation of a pharmaceutical composition, wherein the pharmaceutical composition has the definition as defined in claim 2 or 5.

7. A quality control method of eukenafil or a pharmaceutically acceptable salt thereof, which is characterized in that the quality control method comprises the step of using a compound shown as a formula I or a pharmaceutically acceptable salt thereof as a standard substance or a reference substance, preferably as a drug standard substance or a reference substance;

or, the quality control method comprises detecting a compound represented by formula I or a pharmaceutically acceptable salt thereof; preferably, the detection is a qualitative or quantitative detection;

alternatively, the quality control method comprises storing or using the eukenafil or a pharmaceutically acceptable salt thereof under conditions that are unfavorable for the formation of the compound represented by formula I or a pharmaceutically acceptable salt thereof.

8. A process for preparing a pharmaceutical composition according to claim 2 or 5, comprising mixing eukenafil or a pharmaceutically acceptable salt thereof with a pharmaceutically acceptable excipient, wherein the process is carried out under conditions which do not favour the formation of the compound of formula I or a pharmaceutically acceptable salt thereof.

9. A method of preserving or using eukenafil or a pharmaceutically acceptable salt thereof, wherein the preserving or using method is performed under conditions that are unfavorable for the formation of the compound of formula I or a pharmaceutically acceptable salt thereof;

preferably, the "conditions unfavorable for the formation of the compound represented by formula I or a pharmaceutically acceptable salt thereof" include, but are not limited to, at least one selected from the group consisting of light-shielding and sealing conditions; more preferably selected from the conditions of protection from light and sealing;

preferably, the method of storing or using the eukenafil or a pharmaceutically acceptable salt thereof can be performed under conditions of protection from light and sealing, and at a temperature below 60 ℃ (preferably below 50 ℃) and/or a relative humidity below RH 75% (preferably below 65%).

10. The method for detecting a compound represented by the formula I or a pharmaceutically acceptable salt thereof according to claim 1, which comprises detecting a compound represented by the formula I or a pharmaceutically acceptable salt thereof in a test solution by high performance liquid chromatography;

preferably, the chromatographic column used in the high performance liquid chromatography is a chromatographic column using octadecyl bonded silica gel as a filler;

preferably, the wavelength of the detection is 220-230 nm;

preferably, the temperature of the chromatographic column is 20-30 ℃;

preferably, the flow rate is 0.5-1.5 mL/min;

preferably, the high performance liquid chromatography takes citric acid buffer solution-acetonitrile as a mobile phase;

preferably, the pH value of the citric acid buffer solution is 5.3-5.5;

preferably, the citric acid buffer solution is a citric acid-triethylamine buffer solution;

preferably, the volume ratio of the citric acid buffer solution to the acetonitrile in the mobile phase is (45-55) to (55-45);

preferably, the test solution is prepared from the mobile phase, and the concentration of the test solution in terms of eukenafil hydrochloride is 0.5-1.5 mg/mL;

preferably, the reference substance used in the detection method is a compound shown in formula I or a pharmaceutically acceptable salt thereof, and the test substance is a pharmaceutical composition containing eukenafil or a pharmaceutically acceptable salt thereof.

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