Enadostat impurity and preparation method thereof

Abstract

The invention discloses an enadostat impurity and a preparation method thereof, and belongs to the technical field of pharmaceutical chemicals. The preparation method comprises the following steps: firstly, carrying out nucleophilic substitution reaction on a compound 1 and a compound 2 under the action of an acid-binding agent to generate a compound 4; hydrolyzing the compound 4 in an alkaline solution, and carrying out a decarboxylation reaction under an acidic condition to generate a compound 5; then carrying out amide condensation reaction on the compound 5 and the compound 3 under the action of a condensing agent, a solvent and alkali to generate a compound 6; and finally, carrying out hydrolysis reaction on the compound 6 in an alkaline solution and an organic solvent to generate a compound 7. The enrodostat impurity synthesized by the invention can be used for qualitative and quantitative analysis of impurities in production of enrodostat, and is convenient for effective monitoring and timely reduction of impurity content by adopting necessary means, so that the quality standard of the enrodostat intermediate is improved.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical and chemical technology, specifically relating to an ennadustat impurity and its preparation method. Background Technology

[0002] Ennadustat, chemical name: N-[7-hydroxy-5-(2-phenylethyl)[1,2,4]triazolo[1,5-a]pyridine-8-carbonyl]glycine, structural formula as follows:

[0003]

[0004] Ennadulstat is an oral prescription drug used to treat anemia in non-dialysis adult patients with chronic kidney disease (CKD). Brand name: Ennarol, it is manufactured by Shenzhen Sinopharm Pharmaceutical Co., Ltd., and is a Class B drug covered by medical insurance. Ennadulstat stabilizes hypoxia-inducible factor by inhibiting prolyl hydroxylase activity, promotes the body's production of endogenous erythropoietin, downregulates hepcidin levels, improves iron metabolism, and has a certain anti-inflammatory effect, thereby improving anemia in CKD patients. Ennadulstat is a drug for treating renal anemia. With the increasing prevalence and younger age of onset of kidney disease, growing international attention to renal anemia, and driven by significant patient demand, the market prospects for ennadulstat are very broad.

[0005] Chinese patent application CN110214139B discloses a method for producing triazolopyridine compounds, which details the specific preparation steps of ennadustat. Step 8 (CN110214139B,

[00421] -

[00423] ) discloses the following synthetic route:

[0006]

[0007] The specific procedure was as follows: 29.3 kg (78.6 mol) of methyl 2-({[7-chloro-5-(2-phenylethyl)-[1,2,4]triazolo[1,5-a]pyridin-8-yl]carbonyl}amino)acetate (compound (3)) in a suspension of 2-ethoxyethanol (150 L) was added to a 5 mol / L sodium hydroxide aqueous solution (88 L, 440 mol), and the mixture was stirred at 87 °C for 9.5 hours. After the reaction was complete, a mixed solution of ethanol (146.5 L) and water (14.5 L) was added dropwise to the reaction mixture at 70 °C. The mixture was cooled to room temperature and stirred at the same temperature for 9 hours. The precipitated crystals were collected by filtration and washed with a mixed solution of ethanol (78.3 L) and water (11.7 L). The obtained wet crystals were dried under reduced pressure to obtain 2-({[7-hydroxy-5-(2-phenylethyl)-[1,2,4]triazolo[1,5-a]pyridin-8-yl]carbonyl}amino) disodium acetate (compound (2), a salt of compound (1)) (32.9 kg, 85.6 mol, yield 109%).

[0008] When reproducing the above synthesis steps, the applicant discovered the following impurity compounds:

[0009]

[0010] This impurity compound is a byproduct of the reaction process and significantly affects the activity and toxic side effects of the finished enaladustrol product. Establishing scientific detection and monitoring methods, as well as qualitative and quantitative analysis of the impurity, is crucial for improving the quality of enaladustrol. However, there are no existing records or studies of this compound in the current technology. Therefore, its preparation, identification, and research are urgently needed, which is of great significance for improving the quality of enaladustrol. Summary of the Invention

[0011] The technical problem to be solved by the present invention is to provide an ennadustat impurity, which can be used for qualitative and quantitative analysis of impurities in the production of ennadustat, so as to facilitate effective monitoring and timely use of necessary means to reduce the impurity content, thereby improving the quality standard of ennadustat intermediates; another technical problem to be solved by the present invention is to provide a method for preparing ennadustat impurities, which is simple to operate, has mild reaction conditions, and high atom economy.

[0012] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0013] An endostat impurity has the following structural formula:

[0014] .

[0015] Furthermore, the method for preparing the ennadustat impurity includes the following steps:

[0016] 1) Compound 1 and compound 2 undergo a nucleophilic substitution reaction under the action of an acid-binding agent to generate compound 4;

[0017] 2) After hydrolyzing compound 4 in an alkaline solution, it undergoes a decarboxylation reaction under acidic conditions to generate compound 5;

[0018] 3) Compound 5 and compound 3 undergo an amide condensation reaction under the action of a condensing agent, solvent, and alkali to generate compound 6;

[0019] 4) Compound 6 is hydrolyzed in an alkaline solution and an organic solvent to produce compound 7;

[0020] The reaction formula is as follows:

[0021] .

[0022] Further, in step 1), the acid-binding agent is selected from one or more of sodium hydroxide, cesium carbonate, potassium hydroxide, and sodium ethoxide.

[0023] Further, in step 1), the molar ratio of compound 1, compound 2 and acid-binding agent is 1:1.05~1.35:1.2~1.8; the temperature of the nucleophilic substitution reaction is 30~40℃ and the time is 5~6h.

[0024] Further, in step 2), the alkaline solution is selected from one or more of sodium hydroxide solution, potassium hydroxide solution, and sodium ethoxide; the mass concentration of the alkaline solution is 10-30%; the hydrolysis reaction temperature is 40-60℃ and the time is 5-10h.

[0025] Further, in step 2), the acidic solution is a hydrochloric acid solution, and the molar ratio of compound 4 to the acidic solution is 1:4~8; the decarboxylation reaction temperature is 50~80℃, and the time is 10~12h.

[0026] Further, in step 3), the solvent is acetonitrile / water, and the volume ratio of acetonitrile to water is 1:0.5~1; the alkali is selected from one or more of sodium hydroxide, triethylamine, potassium tert-butoxide, and sodium carbonate.

[0027] Further, in step 3), the condensing agent is one or more of N,N'-dicyclohexylcarbodiimide / 1-hydroxybenzotriazole, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride / 1-hydroxybenzotriazole, and N,N'-diisopropylcarbodiimide / 1-hydroxybenzotriazole; the reaction temperature is 20~40℃ and the time is 3~4h.

[0028] Further, in step 4), the alkaline solution is selected from one or more of sodium hydroxide and potassium hydroxide, and the mass concentration of the alkaline solution is 20-30%; the organic solvent is selected from one or more of ethanol, methanol, and 2-ethoxyethanol.

[0029] Furthermore, in step 4), the reaction temperature is 70~80℃ and the time is 9~10h.

[0030] Compared with the prior art, the present invention has the following advantages:

[0031] The ennadustat impurities synthesized in this invention can be used for qualitative and quantitative analysis of impurities in ennadustat production, facilitating effective monitoring and timely implementation of necessary measures to reduce impurity content, thereby improving the quality standards of ennadustat intermediates. The process is simple, the reaction conditions are mild, and it boasts high atom economy. Attached Figure Description

[0032] Figure 1 The ennadustat impurity (compound 6) prepared in Example 1 of this application 1 H-NMR spectrum;

[0033] Figure 2 The ennadustat impurity (compound 6) prepared in Example 1 of this application 13 C-NMR spectrum;

[0034] Figure 3 The ennadustat impurity (compound 7) prepared in Example 1 of this application 1 H-NMR spectrum;

[0035] Figure 4 The ennadustat impurity (compound 7) prepared in Example 1 of this application 13 C-NMR spectrum;

[0036] Figure 5 MS spectrum of ennadustat impurity (compound 7) prepared in Example 1 of this application;

[0037] Figure 6 HPLC chromatogram of ennadustat impurity (compound 7) prepared in Example 1 of this application;

[0038] Figure 7 HPLC chromatogram of impurity (intermediate 4) prepared in Example 4 of this application;

[0039] Figure 8 The detection spectrum of impurity (intermediate 4) prepared in Example 4 of this application. Detailed Implementation

[0040] The present invention will be further illustrated below with reference to specific embodiments. These embodiments are implemented based on the technical solutions of the present invention, and it should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0041] The raw materials used in the following examples are shown in Table 1.

[0042] Table 1 Raw Material Information Table

[0043]

[0044] In the following examples, DCC is N,N'-dicyclohexylcarbodiimide, HOBT is 1-hydroxybenzotriazole, EDCI is 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and DIC is N,N'-diisopropylcarbodiimide.

[0045] Example 1

[0046] A method for preparing an endostat impurity includes the following steps:

[0047] (1) The synthesis of compound 4 is shown in the following reaction formula:

[0048]

[0049] Weigh 50.00g of compound 1 and add it to a 500mL three-necked flask. Then add 250mL of DMSO, 66.12g of compound 2 and 99.32g of cesium carbonate in sequence. Start stirring and heat to 35℃. Keep the temperature at 35±5℃ for 6h.

[0050] Sampling and TLC monitoring continued until the reaction was complete; after the reaction was complete, 250 mL of ethyl acetate and 250 mL of water were added to the reaction vessel, stirring was started, the mixture was allowed to stand and separated, and the upper organic phase was collected.

[0051] Add 250 mL of saturated sodium chloride aqueous solution to the organic phase for washing, allow to stand and separate, collect the upper organic phase; concentrate under reduced pressure to remove ethyl acetate, and obtain compound 4, which can be used directly in the next reaction.

[0052] (2) The synthesis of compound 5 is shown in the following reaction formula:

[0053]

[0054] Compound 4 obtained in step (1) was added to a 500 mL three-necked flask, 250 mL of ethanol was added, stirring was started, and 142.25 g of 20 wt% sodium hydroxide solution was added dropwise at a controlled temperature of 25 ± 5 °C. After the addition was completed, the mixture was kept at 55 ± 5 °C for 6 h.

[0055] Sampling was performed for TLC monitoring until the reaction endpoint; after the reaction was complete, 250 mL of water was added to the system, stirring was started, and the mixture was allowed to stand and separate to obtain intermediate solution 2; 100 mL of ethanol was added to a 1000 mL flask, stirring was started, 103.01 g of concentrated hydrochloric acid was added, and the temperature was controlled at 55±5℃. Intermediate solution 2 was added dropwise; after the addition was complete, the system was heated to 55±5℃ and the reaction was maintained at this temperature for 12 h.

[0056] Samples were taken and analyzed by HPLC to the endpoint; after passing the intermediate control, the reaction solution was cooled to 5±5℃ and kept at 5±5℃ for stirring to precipitate crystals for 4 hours; the mixture was filtered, and the filter cake was washed with a mixed solvent of 100mL ethanol and 100mL drinking water, and dried under vacuum to obtain wet product of compound 5; the filter cake was transferred to 60~70℃ and vacuum dried for 12 hours, and 49.05g of compound 5 was obtained, with a molar yield of 80%.

[0057] (3) The synthesis of compound 6 is shown in the following reaction formula:

[0058]

[0059] Weigh 45.00g of compound 5 and add 225mL of acetonitrile, 45mL of water, 15.38g of DCC, 20.15g of HOBT, 24.34g of compound 3 and 27.34g of potassium tert-butoxide. Start stirring, heat to 35±5℃, and keep at 35±5℃ for 4h.

[0060] Sampling was performed and monitored by HPLC until the reaction endpoint was reached. After the reaction was completed, 225 mL of water and 225 mL of ethanol were added to the reaction solution, and the mixture was kept at 25 ± 5 °C and stirred to crystallize for 2 h. The mixture was filtered, and the filter cake was washed with 45 mL of ethanol and dried under vacuum to obtain a wet product. The product was transferred to 65 °C for vacuum drying, and 51.23 g of compound 6 was obtained, with a molar yield of 92%. 1 H-NMR spectrum as follows Figure 1 As shown, 13 C-NMR spectra as follows Figure 2 As shown.

[0061] (4) The synthesis of compound 7 is shown in the following reaction formula:

[0062]

[0063] Weigh 50.00g of compound 6 and add it to a 500mL three-necked flask. Then add 250mL of ethanol and 82.00g of 25wt% sodium hydroxide solution. Start stirring and heat to 75±5℃ and keep the temperature for 10h.

[0064] Sampling was performed for HPLC monitoring until the reaction endpoint; 250 mL of water was added to the reaction solution, and after the addition was complete, the system was cooled to 5±5℃ and maintained at 5±5℃ for stirring to induce crystallization for 2 hours; the mixture was filtered, the filter cake was washed with 50 mL of ethanol, dried under vacuum to obtain a wet product, and then transferred to 55℃ for vacuum drying. 43.00 g of compound 7 was obtained, with a molar yield of 91.3%. The prepared ennadustat impurity (compound 7)... 1 H-NMR spectrum as follows Figure 3 As shown, 13 C-NMR spectra as follows Figure 4 As shown, the MS spectrum is as follows Figure 5 As shown, the HPLC chromatogram is as follows: Figure 6 As shown.

[0065] Example 2

[0066] A method for preparing an endostat impurity includes the following steps:

[0067] (1) Weigh 50.00g of compound 1 and add it to a 500mL three-necked flask. Add 250mL of DMSO, 66.12g of compound 2 and 12.19g of sodium hydroxide in sequence. Turn on the stirring, heat to 35℃, and keep the temperature at 35±5℃ for 6h.

[0068] Sampling and TLC monitoring continued until the reaction was complete; after the reaction was complete, 250 mL of ethyl acetate and 250 mL of water were added to the reaction vessel, stirring was started, the mixture was allowed to stand and separated, and the upper organic phase was collected.

[0069] Add 250 mL of saturated sodium chloride aqueous solution to the organic phase for washing, allow to stand and separate, collect the upper organic phase; concentrate under reduced pressure to remove ethyl acetate, and obtain compound 4, which can be used directly in the next reaction.

[0070] (2) The steps are the same as in Example 1.

[0071] (3) The steps were the same as in Example 1, but the mass of compound 6 was 49.1 g, and the molar yield was 88.2%.

[0072] (4) The steps are the same as in Example 1, the mass of compound 7 is 30.5g, and the molar yield is 64.8%.

[0073] Example 3

[0074] A method for preparing an endostat impurity includes the following steps:

[0075] (1) The steps are the same as in Example 1.

[0076] (2) The steps are the same as in Example 1.

[0077] (3) Weigh 45.00g of compound 5 and add 225mL of acetonitrile, 45mL of water, 14.30g of EDCI, 20.15g of HOBT, 24.34g of compound 3 and 27.34g of potassium tert-butoxide. Start stirring, heat to 35±5℃, and keep warm at 35±5℃ for 4h.

[0078] Sampling was performed and monitored by HPLC until the reaction endpoint was reached. After the reaction was completed, 225 mL of water and 225 mL of ethanol were added to the reaction solution, and the mixture was kept at 25 ± 5 °C and stirred to crystallize for 2 h. The mixture was filtered, and the filter cake was washed with 45 mL of ethanol and dried under vacuum to obtain a wet product. The product was transferred to 65 °C for vacuum drying, and 50.2 g of compound 6 was obtained, with a molar yield of 90.2 g.

[0079] (4) The steps are the same as in Example 1, the mass of compound 7 is 26.8 g, and the molar yield is 56.9%.

[0080] Example 4

[0081] The reaction formula is:

[0082]

[0083] 90 mL of 2-ethoxyethanol, 18.00 g of intermediate 3, and 46.30 g of 5N sodium hydroxide solution were added sequentially. Heating was initiated and the temperature was raised to 70±5℃, then maintained at 70±5℃ for 8 hours. During the reaction, samples were taken for monitoring. After the reaction was complete, 71.00 g of ethanol was added dropwise while maintaining the temperature at 70±5℃. After the addition was complete, the temperature was lowered to 20-30℃, and the mixture was stirred and maintained at 20-30℃ for 12 hours to induce crystallization. The mixture was filtered, and the filter cake was transferred to 80℃ for forced-air drying. The product was collected to obtain 18.02 g of intermediate 4, with a molar yield of 98.1%. The HPLC chromatogram is shown below. Figure 7 As shown.

[0084] Chromatographic conditions: Octadecylsilane-bonded silica gel was used as the stationary phase (Agilent ZORBAX SB-C18, 100×4.6mm 3.5µm or equivalent column); 0.1% trifluoroacetic acid aqueous solution was used as mobile phase A, and 0.1% trifluoroacetic acid acetonitrile solution was used as mobile phase B, with gradient elution according to Table 2; the detection wavelength was 220nm; the flow rate was 1.0mL per minute; the column temperature was 35℃; the injection volume was 10μL; the sample tray temperature was controlled at 4℃; and the solvent was acetonitrile-water (500:300).

[0085] Table 2 Gradient Elution Table

[0086]

[0087] Preparation of test solution: Take an appropriate amount of this product, accurately weigh it, dissolve it in solvent and dilute it quantitatively to prepare a solution containing about 1 mg per 1 mL.

[0088] Preparation of control solution: Accurately measure an appropriate amount of the test solution and dilute it with solvent to prepare a solution containing approximately 5 μg per 1 mL.

[0089] Inject the test solution and control solution according to the chromatographic conditions described in the method, and record the chromatograms. Figure 8 The test results show that intermediate 4 impurity was indeed generated during the preparation of ennadustat, along with the formation of the target product.

[0090] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An ennadustat impurity, characterized in that, The structural formula is as follows: 。 2. The method for preparing ennadustat impurities according to claim 1, characterized in that: Includes the following steps: 1) Compound 1 and compound 2 undergo a nucleophilic substitution reaction under the action of an acid-binding agent to generate compound 4; 2) After hydrolyzing compound 4 in an alkaline solution, it undergoes a decarboxylation reaction under acidic conditions to generate compound 5; 3) Compound 5 and compound 3 undergo an amide condensation reaction under the action of a condensing agent, solvent, and alkali to generate compound 6; 4) Compound 6 is hydrolyzed in an alkaline solution and an organic solvent to produce compound 7; The reaction formula is as follows: 。 3. The method for preparing ennadustat impurities according to claim 1, characterized in that: In step 1), the acid-binding agent is selected from one or more of sodium hydroxide, cesium carbonate, potassium hydroxide, and sodium ethoxide.

4. The method for preparing ennadustat impurities according to claim 1, characterized in that: In step 1), the molar ratio of compound 1, compound 2 and acid-binding agent is 1:1.05~1.35:1.2~1.8; the nucleophilic substitution reaction is carried out at a temperature of 30~40℃ for 5~6 hours.

5. The method for preparing ennadustat impurities according to claim 1, characterized in that: In step 2), the alkaline solution is selected from one or more of sodium hydroxide solution, potassium hydroxide solution, and sodium ethoxide; the mass concentration of the alkaline solution is 10-30%; the hydrolysis reaction temperature is 40-60℃ and the time is 5-10h.

6. The method for preparing ennadustat impurities according to claim 1, characterized in that: In step 2), the acidic solution is hydrochloric acid solution, and the molar ratio of compound 4 to the acidic solution is 1:4~8; the decarboxylation reaction temperature is 50~80℃, and the time is 10~12h.

7. The method for preparing ennadustat impurities according to claim 1, characterized in that: In step 3), the solvent is acetonitrile / water, and the volume ratio of acetonitrile to water is 1:0.5~1; the base is selected from one or more of sodium hydroxide, triethylamine, potassium tert-butoxide, and sodium carbonate.

8. The method for preparing ennadustat impurities according to claim 1, characterized in that: In step 3), the condensing agent is one or more of N,N'-dicyclohexylcarbodiimide / 1-hydroxybenzotriazole, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride / 1-hydroxybenzotriazole, and N,N'-diisopropylcarbodiimide / 1-hydroxybenzotriazole; the reaction temperature is 20~40℃ and the reaction time is 3~4h.

9. The method for preparing ennadustat impurities according to claim 1, characterized in that: In step 4), the alkaline solution is selected from one or more of sodium hydroxide and potassium hydroxide, and the mass concentration of the alkaline solution is 20-30%; the organic solvent is selected from one or more of ethanol, methanol, and 2-ethoxyethanol.

10. The method for preparing ennadustat impurities according to claim 1, characterized in that: In step 4), the reaction temperature is 70~80℃ and the time is 9~10h.

Images