PCSK9 inhibitor intermediate as well as preparation process and application thereof

Abstract

The invention provides a key intermediate I of a PCSK9 inhibitor MK-0616 as well as a preparation process and application of the key intermediate I. Compared with the prior art, the key intermediate I is obtained by carrying out reductive amination reaction on an intermediate II and pent-4-olefine aldehyde.Complex protection / deprotection reaction steps are not needed, the intermediate I can be prepared only through one-step reductive amination, and the preparation process is simple and convenient. The reaction route and the production period are greatly shortened, and the production efficiency is improved. The reaction conversion rate and yield are obviously improved, the material loss is reduced, and the production cost is saved. The prepared intermediate I can be used for preparing the PCSK9 inhibitor MK0616 on a large scale.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of synthesis of pharmaceutical intermediates, in particular, to a synthesis method of an intermediate of a PCSK9 inhibitor compound. BACKGROUND

[0002] Proprotein Convertase Subtilisin / kexin Type 9 (PCSK9) is a serine protease mainly secreted by hepatocytes, which has been reported in recent years as an important molecule leading to elevated serum low-density lipoprotein cholesterol (LDL-C), and the elevation of serum LDL-C will further lead to the occurrence of atherosclerosis and cardiovascular and cerebrovascular diseases. Therefore, the development of PCSK9 inhibitors can reduce serum LDL-C levels and further reduce the risk of cardiovascular events.

[0003] Currently marketed PCSK9 inhibitors include antibody drugs Evolocumab and Alirocumab, and RNA interference drug Inclisiran. Although the above drugs are effective to some extent in preventing or treating cardiovascular and cerebrovascular diseases, they still have the following defects:

[0004] ① The administration method is inconvenient, and monoclonal antibodies need to be frequently injected subcutaneously, with low patient compliance; ② The production and treatment cost of monoclonal antibody drugs is high; ③ There is a risk of immunogenicity, and the individual efficacy varies greatly.

[0005] Based on the defects of the above existing macromolecular PCSK9 inhibitors, the development of small molecule PCSK9 inhibitors has great clinical value. MK-0616 is an innovative oral small molecule PCSK9 inhibitor developed by Merck & Co., which is currently in clinical phase III. It prevents the binding of PCSK9 protein to the LDL receptor (LDLR) on the surface of hepatocytes by targeting PCSK9 protein, thereby accelerating the clearance of LDL-C in the blood to treat atherosclerosis and cardiovascular diseases, and the structure is as follows:

[0006]

[0007] The preparation method of compound MK-0616 is disclosed in patent CN112313243A of Merck & Co., and the process route is shown in the following formula: intermediate 77B is prepared by three-step amino protection / deprotection reaction to obtain intermediate 79, intermediate 79 is prepared by substitution reaction to obtain intermediate 80, intermediate 80 is prepared by condensation reaction and deprotection to obtain intermediate 82, and intermediate 82 is prepared by subsequent reaction to obtain MK-0616:

[0008]

[0009] In the above synthetic process for preparing MK-0616, there are the following defects: ① The intermediate 80 is prepared from the intermediate 77B through multiple protection / deprotection steps, which is complicated and time-consuming; ② When the intermediate 80 is synthesized from the intermediate 79 by the original route, a disubstituted impurity (as shown below) is easily produced, and in order to reduce the generation of the impurity, only 0.5 eq (based on 1.0 eq of the intermediate 79) of pent-4-en-1-yl p-toluenesulfonate can be added, which results in large material loss. ③ The actual yield of the intermediate 80 synthesized from the compound 77B by the original patent method is about 19%, which is low in conversion efficiency and is not conducive to industrial production.

[0010]

[0011] In summary, the preparation process for synthesizing the key intermediate 80 from the intermediate 77B of MK-0616 in the prior art has the defects of complex reaction operation, long production cycle, large material loss, low production efficiency, greatly increased cost, and is not suitable for industrial production. It is necessary to improve it and provide a process route with shortened process route, improved production efficiency and saved production cost. BRIEF DESCRIPTION OF DRAWINGS

[0012] Figure 1 The reaction liquid liquid phase detection spectrum of Example 1 (the retention time of the intermediate I is 1.395 min).

[0013] Figure 2 The reaction liquid liquid phase detection spectrum of Comparative Example 2 (the retention time of the intermediate I is 1.391 min, and almost no product is generated). SUMMARY

[0014] In order to solve the defects in the prior art, the present application provides an improved preparation process for a key intermediate of MK-0616, which has a shortened process route, improved production efficiency, reduced production cost and is suitable for industrial production.

[0015] In order to achieve the above-mentioned purpose, the specific scheme is as follows:

[0016] On the one hand, the present application provides a key intermediate I of the PCSK9 inhibitor MK-0616, which has the following structure:

[0017]

[0018] The R is one of 9-fluorenylmethyloxycarbonyl (Fmoc), p-toluenesulfonyl (Tos), benzyl protecting group (Bn) or benzyloxycarbonyl (Cbz) amino protecting group.

[0019] In some embodiments, the R is 9-fluorenylmethyloxycarbonyl (Fmoc).

[0020] In another aspect, the present application provides a process for preparing MK-0616 key intermediate I, which comprises a reductive amination reaction of intermediate II with pent-4- enal to obtain intermediate I, as shown in the following reaction formula:

[0021]

[0022] In some embodiments, the reductive amination reaction comprises the following steps:

[0023] A) intermediate II, pent-4-enal and an organic base are dissolved in an organic solvent and stirred, and the reaction is incubated for 1-12 h;

[0024] B) a reducing agent is added to the reaction system, and the reaction is incubated for 1-12 h;

[0025] C) after the reaction is completed, the reaction is quenched by cooling, extracted, the reaction solution is concentrated, and intermediate I is obtained by purification.

[0026] In some embodiments, the solvent of the reductive amination reaction is selected from any one or more of dichloromethane, tetrahydrofuran, acetonitrile;

[0027] In further embodiments, the solvent is dichloromethane.

[0028] In some embodiments, the reducing agent of the reductive amination is selected from a combination of one or more of sodium borohydride, sodium borohydride acetate, sodium cyanoborohydride, sodium triacetoxyborohydride;

[0029] In further embodiments, the reducing agent is sodium triacetoxyborohydride.

[0030] In some embodiments, the organic base of the reductive amination reaction can be selected from a combination of one or more of, but not limited to, N,N-diisopropylethylamine, triethylamine, N-methylmorpholine;

[0031] In further embodiments, the organic base is N,N-diisopropylethylamine.

[0032] In some embodiments, the molar ratio of intermediate II, pent-4-enal, reducing agent and organic base in the reductive amination is 1:

[0033] (1-5):(1-10):(0.5-2);

[0034] In further embodiments, the molar ratio of intermediate II, pent-4-enal, reducing agent and organic base in the reductive amination is 1:(1-2):(1-5):(1-2).

[0035] In some embodiments, the temperature of the reductive amination reaction is -20°C to 30°C.

[0036] In further embodiments, the temperature of the reductive amination reaction is -10°C to 20°C.

[0037] In some embodiments, the reaction time of the reductive amination is 1 to 24 h.

[0038] In further embodiments, the reaction time is 1 to 6 h.

[0039] In another aspect, the present application provides a use of MK-0616 key intermediate I in the preparation of MK-0616 intermediate IV, comprising the following steps:

[0040] 1) intermediate I is subjected to condensation reaction with monomethyl succinate to obtain intermediate III;

[0041] 2) intermediate III is subjected to deprotection reaction to obtain intermediate IV;

[0042] The reaction formula is as follows:

[0043]

[0044] In some embodiments, the intermediate I is obtained by reductive amination reaction of intermediate II with pent-4-enal.

[0045] In some embodiments, the condensing agent of the condensation reaction of step 1) can be selected from one or more of HATU, HBTU, DCC, EDC, EDCI, or other optional condensing agents.

[0046] In further embodiments, the condensing agent of the condensation reaction of step 1) is HATU.

[0047] In some embodiments, the deprotection reaction of step 2) can be selected from common deprotection methods including but not limited to deprotection under acidic conditions, deprotection under basic conditions, deprotection under hydrogen atmosphere, etc., which can be specifically selected according to the type of R.

[0048] In further embodiments, when R of the intermediate I is 9-fluorenylmethyloxycarbonyl (Fmoc), the deprotection reaction of step 2) is deprotection under basic conditions, and the type of base can be selected from one or more of the combination of piperidine, pyridine, 3-(diethylamino)propylamine, piperazine, triethylamine, N,N-diisopropylethylamine.

[0049] In still another aspect, the present application provides a use of an MK-0616 key intermediate I in the preparation of the PCSK9 inhibitor MK-0616, wherein the intermediate I is obtained by reductive amination of intermediate II with pent-4-enal.

[0050] Compared with the prior art, the technical scheme of the present application has the following beneficial effects:

[0051] 1. Compared with the prior art, the present application does not require complex protection / deprotection reaction steps, and only one step of reductive amination is needed to obtain the intermediate I, greatly shortening the reaction route and production cycle and improving production efficiency.

[0052] 2. The present application uses reductive amination reaction, which significantly improves the reaction conversion rate and yield compared with the prior art, reduces material loss, saves production cost, and is suitable for large-scale production of MK0616. DETAILED DESCRIPTION

[0053] In order to make the technical problems, technical schemes and technical effects solved by the present application clearer, the following will further describe the present application in detail with specific examples. Obviously, the described examples are only part of the examples of the present application, not all examples. Based on the examples in the present application, all other examples obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0054] In the following examples, the experimental methods are described, and if not specifically stated, they are all conventional methods; the materials or reagents are self-made or commercially available, if not specifically stated.

[0055] Liquid chromatography-mass spectrometry detection method:

[0056] Chromatographic conditions:

[0057] A liquid chromatograph (Thermo U3000+ISQ) was used, and octadecyl-bonded silica gel (Welchrom C18 4.6mm x 50mm, 5μm or a chromatographic column with equivalent performance) was used as the filler;

[0058] 0.1% formic acid aqueous solution was used as mobile phase A, and acetonitrile was used as mobile phase B, and linear gradient elution was carried out according to the following table; the column temperature was 30℃; the detection wavelength was 254nm; the injection volume was 5μl; the ion source type was ESI.

[0059]

[0060] Synthesis of intermediate I in Example 1

[0061]

[0062] Into a reaction flask was added p-toluenesulfonic acid salt of intermediate II (9.96 g, 11.7 mmol), N,N-diisopropylethylamine (1.55 g, 12.0 mmol) and 50 mL of dichloromethane (DCM) successively, and stirred until the solid was dissolved. The temperature was lowered to -10 °C, and pent-4-enal (1.41 g, 16.7 mmol) was added, and stirred for 3 h. Sodium triacetoxyborohydride (8.4 g, 39.6 mmol) was added, and stirred for 1 h. The reaction was stopped after TLC detection showed that the reaction was complete. Liquid chromatography showed that the retention time of intermediate I in the reaction solution was 1.395 min, and the content was 81.5%. See Figure 1 .

[0063] Work-up: saturated aqueous NaHCO3solution was added to quench, and stirred until no gas was emitted. The aqueous phase was extracted once more with DCM, and the organic phases were combined and concentrated at 30 °C to obtain the crude product. Column chromatography purification (EA:MeOH = 10:1) and concentration yielded intermediate I in a yield of 55%, ESIm / z: 552.3 [M+H + ].

[0064] Synthesis of intermediate I in Example 2

[0065]

[0066] Into a reaction flask was added p-toluenesulfonic acid salt of intermediate II (1.00 g, 1.20 mmol), N,N-diisopropylethylamine (0.15 g, 1.20 mmol) and 5 mL of tetrahydrofuran (THF) successively, and stirred until the solid was dissolved. The temperature was lowered to -10 °C, and pent-4-enal (0.14 mg, 1.44 mmol) was added, and stirred for 4 h at 20 °C. Sodium triacetoxyborohydride (0.76 g, 3.6 mmol) was added, and stirred for 1 h. The reaction was stopped after TLC detection showed that the reaction was complete. Work-up and purification yielded intermediate I.

[0067] Synthesis of intermediate I in Example 3

[0068]

[0069] Into a reaction flask was added p-toluenesulfonic acid salt of intermediate II (1.00 g, 1.20 mmol), N,N-diisopropylethylamine (0.15 g, 1.20 mmol) and 5 mL of tetrahydrofuran (THF) successively, and stirred until the solid was dissolved. The temperature was lowered to -10 °C, and pent-4-enal (0.14 mg, 1.44 mmol) was added, and stirred for 4 h at 20 °C. Sodium triacetoxyborohydride (0.76 g, 3.6 mmol) was added, and stirred for 1 h. The reaction was stopped after TLC detection showed that the reaction was complete. Work-up and purification yielded intermediate I.

[0070] Synthesis of Intermediate III in Preparation Example 1

[0071]

[0072] Intermediate I (2.00 g, 3.6 mmol) was dissolved in DMF, monomethyl succinate (0.70 g, 5.4 mmol), 1.6 eq of condensing agent HATU (0.82 g, 2.16 mmol) and N,N-diisopropylethylamine (0.79 g, 6.12 mmol) were added sequentially and the reaction was stirred at room temperature for 4 h. Upon completion of the reaction, TLC confirmed the completion of the reaction and water was added and extracted with ethyl acetate twice. The ethyl acetate phases were combined, washed with saturated brine, dried and concentrated and purified by column chromatography to obtain Intermediate III in 75% yield, ESIm / z: 666.3 [M+H + ].

[0073] Synthesis of Intermediate IV in Preparation Example 2

[0074]

[0075] Intermediate III (0.70 g, 1.05 mmol) was dissolved in 8 mL of acetonitrile and piperidine (0.54 g, 6.30 mmol) was added and the reaction was stirred at room temperature for 4 h. Upon completion of the reaction, the reaction was concentrated. The concentrate was dissolved in ethyl acetate and washed with water and saturated brine. Purification by column chromatography yielded 0.4 g of Intermediate IV in 86% yield, ESIm / z: 444.2 [M+H + ].

[0076] Intermediate IV can be further prepared to MK-0616.

[0077] Synthesis of Intermediate I in Comparative Example 1

[0078]

[0079] Reference to Example 1 was made for the feed, with the exception that the reaction solvent was replaced with methanol. TLC detection revealed that there was a large amount of starting material remaining.

[0080] Synthesis of Intermediate I in Comparative Example 2

[0081]

[0082] A three-necked flask was charged with Intermediate II hydrochloride salt (2.5 g, 4.8 mol), penta-4-en-1-yl p-toluenesulfonate (0.58 g, 2.4 mmol), K2CO3(1.33 g, 9.6 mmol) and 12 mL of DMF, stirred and dissolved, replaced with N2, and the temperature was raised to 80 °C for 3 h. Upon completion of the reaction, liquid phase detection revealed that there was almost no product generated in the reaction solution, see Figure 2 .

[0083] Synthesis of intermediate 80 (CN112313243A)

[0084]

[0085] Into a three-necked flask, intermediate 79 (6.6 g, 18.26 mmol), p-toluenesulfonic acid pent-4-en-1-yl ester (2.2 g, 9.13 mmol), K2CO3(5.0 g, 36.52 mmol) and 40 mL of DMF were added successively, stirred to dissolve, replaced by N2, and warmed to 80 °C for reaction. After 3 h of reaction, extraction was performed with ethyl acetate, the organic phase was concentrated, and column chromatography purification was performed to obtain 0.8 g of intermediate 80 with a yield of 10%.

Claims

1. A key intermediate I of the PCSK9 inhibitor MK-0616, characterized in that, has the following structure: The R is one of 9-fluorenylmethyloxycarbonyl (Fmoc), p-toluenesulfonyl (Tos), benzyl protecting group (Bn) or benzyloxycarbonyl (Cbz) amino protecting group, preferably 9-fluorenylmethyloxycarbonyl (Fmoc).

2. A preparation process for the key intermediate I of MK-0616 as described in claim 1, characterized in that, The intermediate II is reacted with pent-4-enal by reductive amination to obtain the intermediate I, and the reaction formula is as follows:

3. The manufacturing process of claim 2, wherein, The reductive amination reaction comprises the following steps: A) The intermediate II, pent-4-enal and an organic base are dissolved in an organic solvent and stirred, and the reaction is kept for 1-12 hours; B) A reducing agent is added to the reaction system, and the reaction is kept for 1-12 hours; C) After the reaction is completed, the reaction is quenched by cooling, extracted, the reaction solution is concentrated, and the intermediate I is obtained by purification.

4. The manufacturing process of claim 3, wherein, The reducing agent is one or a combination of sodium borohydride, sodium borohydride acetate, sodium cyanoborohydride and sodium triacetoxyborohydride, and is preferably sodium triacetoxyborohydride.

5. The manufacturing process of claim 3, wherein, The organic base is one or a combination of N,N-diisopropylethylamine, triethylamine and N-methylmorpholine, and is preferably N,N-diisopropylethylamine.

6. The manufacturing process of claim 3, wherein, The organic solvent is one or a combination of dichloromethane, tetrahydrofuran and acetonitrile, and is preferably dichloromethane.

7. The manufacturing process of claim 3, wherein, The molar ratio of the intermediate II, pent-4-enal, reducing agent and organic base in the reductive amination reaction is 1:(1-5):(1-10):(0.5-2).

8. The manufacturing process of claim 7, wherein, The molar ratio of the intermediate II, pent-4-enal, reducing agent and organic base in the reductive amination reaction is 1:(1-2):(1-5):(1-2).

9. The manufacturing process of claim 3, wherein, The temperature of the reductive amination reaction is -20℃-30℃.

10. Use of the MK-0616 key intermediate I of claim 1 in the preparation of MK-0616 intermediate IV, comprising the following steps: 1) The intermediate I is subjected to condensation reaction with monomethyl succinate to obtain the intermediate III; 2) The intermediate III is subjected to deprotection reaction to obtain the intermediate IV; The reaction formula is as follows:

11. Use of the MK-0616 key intermediate I of claim 1 in the preparation of PCSK9 inhibitor MK-0616.

12. Use according to claim 10 or 11, characterized in that, The intermediate I is obtained by reductive amination of the intermediate II and pent-4-enal.

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