Synthesis method of Fmoc-L-(4, 4-difluorocyclohexyl) glycine

Abstract

The invention relates to the technical field of non-natural amino acid synthesis, and particularly discloses a synthesis method of Fmoc-L-(4, 4-difluorocyclohexyl) glycine. The invention relates to a synthesis method of Fmoc-L-(4, 4-difluorocyclohexyl) glycine, which comprises the following steps: reacting 4, 4-difluorocyclohexanone with Z-alpha-phosphonoglycine trimethyl ester and DBU to generate a compound 1; performing palladium-carbon catalysis and normal-pressure hydrogenation on the compound 1 to generate a compound 2; carrying out acetylation reaction on the compound 2 and acetic anhydride, pulping and purifying to obtain a compound 3; performing alkaline hydrolysis reaction on the compound 3 to obtain a compound 4; hydrolyzing the compound 4 through acetylase to obtain a compound 5; reacting the compound 5 with Fmoc-Osu, pulping and purifying to obtain a target product. The synthesis scheme provided by the invention has the advantages of cheap and easily available raw materials, mild reaction conditions and easy purification, and is suitable for large-scale production in laboratories and factories.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of non-natural amino acid synthesis, more particularly, it relates to a synthesis method of Fmoc-L-(4,4-difluorocyclohexyl) glycine (CAS: 2248184-59-8). BACKGROUND

[0002] As a key building block for chemical modification of polypeptide drugs, non-natural amino acids play an irreplaceable role in further improving the effectiveness, biological activity and metabolic stability of peptides, reducing protein hydrolysis, and improving bioavailability, and have become an important research direction in the field of new drug research. Among them, fluorine-containing amino acids have shown significant potential in the treatment of inflammation, cancer and autoimmune diseases by regulating protein folding, lipophilicity and metabolic stability.

[0003] Patent US2020247785A1 discloses IL-17 ligands and their use as inhibitors developed by DICEALPHAINC company, which discloses Fmoc-L-(4,4-difluorocyclohexyl) glycine (CAS: 2248184-59-8) as a core structural unit for structural optimization of IL-17 inhibitors. Such compounds can be used, for example, to treat and / or prevent inflammation, cancer or autoimmune diseases, and have clear clinical value.

[0004] In the prior art, patent WO2020146194 discloses a similar substance Cbz-L-(4,4-difluorocyclohexyl) glycine related to the present application, which depends on a selective hydrogenation route for synthesis. This method has harsh reaction conditions, high requirements for equipment and low safety, and needs to use expensive and difficult-to-obtain chiral catalysts (such as chiral phosphine ligand modified transition metal catalysts). The product purification relies on complex chromatography technology, which is difficult to meet the needs of industrial scale-up.

[0005] Therefore, developing a Fmoc-L-(4,4-difluorocyclohexyl) glycine synthesis method with cheap and readily available raw materials, mild reaction conditions, simple purification process and suitable for scale-up production has important practical significance for promoting its application in the medical field. SUMMARY

[0006] In order to improve the production efficiency of Fmoc-L-(4,4-difluorocyclohexyl) glycine and adapt to the scale-up production scale, the present application provides a synthesis method of Fmoc-L-(4,4-difluorocyclohexyl) glycine.

[0007] In a first aspect, the present application provides a synthesis method of Fmoc-L-(4,4-difluorocyclohexyl) glycine, which adopts the following technical solution: A synthesis method of Fmoc-L-(4,4-difluorocyclohexyl) glycine comprises the following steps: S1: 4,4-difluorocyclohexanone is reacted with Z-alpha-phosphoryl glycine trimethyl ester and DBU to generate compound 1, and the product does not need to be purified; S2: Compound 1 is catalyzed by a palladium-carbon catalyst to generate compound 2 through normal-pressure hydrogenation reaction, and the product does not need to be purified; S3: Compound 2 is acetylated with acetic anhydride, and is purified by beating to obtain compound 3; S4: Compound 3 is hydrolyzed by an alkali to obtain compound 4, and the product does not need to be purified; S5: Compound 4 is hydrolyzed by an acetylation enzyme to obtain compound 5, and the product does not need to be purified; S6: Compound 5 is reacted with Fmoc-Osu, and the obtained crude product is purified by beating to obtain the target product Fmoc-L-(4,4-difluorocyclohexyl) glycine; the synthesis route is as follows:

[0008] By adopting the technical scheme, the whole synthesis process comprises 6 reaction steps, wherein compound 1, 2, 4, 5 and intermediate products do not need to be purified and can be directly subjected to the next step reaction, only compound 3 and the final product need to be purified by beating, compared with the complex purification means such as column chromatography and recrystallization that may be needed in the prior art, the beating operation has the characteristics of less solvent consumption, simple operation and scalable operation, greatly reduces the process conversion difficulty from laboratory small-dose preparation to factory mass production, and is suitable for industrialized scale-up production.

[0009] In step S1-2, DBU is used to promote the occurrence of condensation reaction, and then hydrogenation reduction is performed under normal-pressure hydrogen, the intermediate does not need to be purified and is directly used in the next step reaction, the high-pressure hydrogenation and twice column chromatography purification in the traditional process are omitted, continuous operation is realized, the yield loss in purification is reduced, the yield is higher, and the safety is higher.

[0010] In step S5, selective hydrolysis is realized by acetylation enzyme, chiral resolution is realized, and the L-form compound 5 is specifically obtained, compared with the limitation of the expensive and scarce noble metal chiral catalyst in the prior art, the enzyme method is more economical, the conditions are mild, the selectivity is higher, the optical purity is higher, the harsh requirements of peptide drug synthesis on chiral amino acids can be met, and the method is suitable for scale-up production.

[0011] Optionally, the amount of the palladium-carbon catalyst is 8-12% of the compound 1.

[0012] Optionally, the molar ratio of 4,4-difluorocyclohexanone, Z-alpha-phosphoryl glycine trimethyl ester and DBU in step S1 is 1:(1-1.05):(1-1.05).

[0013] Optionally, the mass ratio of compound 4 to acetylation enzyme in step S5 is (235-283):30.

[0014] Optionally, the solvent used for beating and purification in steps S3 and S6 is a mixed solvent comprising petroleum ether and ethyl acetate.

[0015] By using the above technical solution, the end product is subjected to beating and purification with a mixed solvent of petroleum ether and ethyl acetate to obtain a drug-grade purity product in a green and low-carbon manner. According to HPLC test, the chemical purity is > 99.3%, which can completely replace column chromatography change, reduces waste liquid discharge, realizes efficient separation, and meets the requirements of large-scale production.

[0016] Optionally, the reaction temperature in step S1 is 20-50℃, and the reaction time is 4-12h.

[0017] Optionally, the reaction temperature in step S1 is 30℃, and the reaction time is 8h.

[0018] By using the above technical solution, step S1 is a condensation reaction of 4,4-difluorocyclohexanone and Z-a-phosphoryl glycine trimethyl ester. When the temperature is lower than 20℃, the reaction rate decreases significantly, and when the temperature is higher than 50℃, the amount of defluorination byproduct increases, which will affect the yield and purity of compound 1. When the reaction temperature is 30℃ and the reaction time is 8h, the yield and purity of compound 1 are the highest.

[0019] Optionally, the reaction temperature in step S2 is 20-50℃, and the reaction time is 3-15h.

[0020] Optionally, the reaction temperature in step S2 is 30℃, and the reaction time is 10h.

[0021] By using the above technical solution, step S2 is an atmospheric hydrogenation reaction. When the temperature is lower than 20℃, the hydrogenation rate is too slow, and when the temperature is higher than 50℃, it will lead to over-reduction, affecting the yield and purity of compound 2. When the reaction temperature is 30℃ and the reaction time is 10h, the yield and purity of compound 2 are the highest.

[0022] Optionally, the reaction temperature in step S4 is 20-60℃, and the reaction time is 2-8h.

[0023] Optionally, the reaction temperature in step S4 is 30℃, and the reaction time is 4h.

[0024] By using the above technical solution, step S4 is an ester hydrolysis reaction. When the reaction is carried out at 20-60℃ for 2-8h, it can ensure the ester hydrolysis rate and the integrity of compound chirality. When the reaction is carried out at 30℃ for 4h, the efficiency is maximized.

[0025] Optionally, the reaction temperature of step S6 is 0-40℃, and the reaction time is 4-20h.

[0026] Optionally, the reaction temperature of step S6 is 20℃, and the reaction time is 8h.

[0027] By adopting the above technical scheme, the condensation reaction of compound 5 and Fmoc-Osu occurs in step S6, and the degradation of the Fmoc group can be precisely prevented at 0-40℃ for 4-20h, the generation of impurities is reduced, the reaction conversion rate is improved, the production cycle is shortened, and the efficiency is maximized at 20℃ for 8h.

[0028] In summary, the present application has the following beneficial effects: 1. The synthesis process of the present application has 6 steps, among which 5 steps of compounds 1, 2, 4, 5 and intermediate products do not need to be purified and can be directly used in the next step reaction, only compound 3 and the final product need to be purified by beating, the solvent consumption is low, the operation steps are simple, the scale operation can be carried out, the production efficiency is significantly improved, the process conversion difficulty from small-dose production in the laboratory to industrial production is greatly reduced, and the production is suitable for scaling up.

[0029] 2. The present application uses acetylation enzyme selective hydrolysis to realize the resolution of chiral compounds, and specifically obtains L-form compound 5, without relying on expensive and scarce chiral catalysts, the complete separation of L-isomer and D-isomer is realized, the optical purity is significantly improved, and the harsh requirements of peptide drug synthesis on chiral amino acids are met.

[0030] 3. The present application provides a synthesis method of Fmoc-L-(4,4-difluorocyclohexyl) glycine, which has the advantages of cheap and easily available raw materials, outstanding economy, low equipment requirement, mild reaction condition, high safety, high yield and high purity of the product, high process stability, and is suitable for scaling up production in the laboratory and factory. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1 is the liquid chromatogram of the target product of Example 1 of the present application; Figure 2 is the chiral liquid chromatogram of the target product of Example 1 of the present application; Figure 3 is the mass spectrum of the target product of Example 1 of the present application; Figure 4 is the nuclear magnetic resonance spectrum of the target product of Example 1 of the present application. DETAILED DESCRIPTION

[0032] The present application will be further described in detail below in combination with examples and comparative examples. EMBODIMENT

[0033] Example 1 A method for synthesizing Fmoc-L-(4,4-difluorocyclohexyl) glycine, comprising the following steps, wherein the palladium-carbon catalyst is selected from Shanxi Ruikexin Material, D10H5A, the acetylation enzyme is derived from Escherichia coli, and the R009566 is selected from the Ron reagent: S1: 1.49 mol of 4,4-difluorocyclohexanone and 1.49 mol of Z-alpha-phosphoryl glycine trimethyl ester were dissolved in 2 L of DMF, 1.5 mol of DBU was added, and then stirred at 30°C for 8 h to obtain reaction liquid 1. The reaction liquid 1 was poured into 10 L of water, stirred, filtered, and dried to obtain compound 1 with a yield of 97%, MS (ESI) M / Z: 340.2 [M+H]+; S2: 490 g, 1.44 mol of compound 1 was dissolved in 3.5 L of methanol, 10%, 49 g of palladium-carbon catalyst was added to obtain reaction liquid 2, hydrogen was introduced into the reaction liquid 2, and stirred at 30°C for 10 h. Filtration and concentration of the filtrate obtained compound 2, MS (ESI) M / Z: 208.3 [M+H]+; S3: 300 g, 1.44 mol of compound 2 and 218.7 g, 2.16 mol of triethylamine were dissolved in 3 L of dichloromethane to obtain reaction liquid 3. 176.6 g, 1.73 mol of acetic anhydride was added dropwise to the reaction liquid 3 under ice water bath. After the addition was completed, the reaction liquid 3 was stirred at room temperature for 3 h. Then 2 L of 1M dilute hydrochloric acid was added, stirred, separated, and then the organic phase was washed with saturated brine. After drying over anhydrous sodium sulfate, filtering, and drying under reduced pressure, a crude compound 3 was obtained. The crude compound 3 was purified by slurry with 1 L of petroleum ether / ethyl acetate mixed solvent with a volume ratio of 2:1. After filtration and drying, compound 3 was obtained, MS (ESI) M / Z: 250.1 [M+H]+, and the yield of steps S1 and S2 was 75%; S4: 270 g, 1.08 mol of compound 3 was dissolved in 2 L of tetrahydrofuran, and 2 mL of 1N sodium hydroxide aqueous solution was added to obtain reaction liquid 4. The reaction liquid 4 was stirred at 30°C for 4 h. After the reaction liquid 4 was acidified with concentrated hydrochloric acid, it was extracted with ethyl acetate, and the organic phase was washed with saturated brine. After drying over anhydrous sodium sulfate, filtering, and drying under reduced pressure, compound 4 was obtained, MS (ESI) M / Z: 236.1 [M+H]+; S5: 255 g, 1.08 mol of compound 4 was added into 1 L, 1 N sodium hydroxide aqueous solution, after stirring and dissolving, 2 M hydrochloric acid was used to adjust the pH to (8±0.2), to obtain reaction liquid 5, reaction liquid 5 was heated to 38℃, then 30 g of acetylation enzyme was added, and stirred at this temperature overnight, then reaction liquid 5 was acidified with concentrated hydrochloric acid, and extracted twice with 1 L of ethyl acetate each time, the aqueous phase was adjusted to neutral with sodium hydroxide aqueous solution, to obtain an aqueous solution of compound 5, MS (ESI) M / Z: 194.1 [M+H]+; S6: 136.1 g, 1.62 mol of sodium bicarbonate and 1 L of tetrahydrofuran were added into 0.54 mol of aqueous solution of compound 5 to obtain reaction liquid 6, which was cooled in an ice water bath, then 168.6 g, 0.5 mol of Fmoc-Osu was added into reaction liquid 6, which was stirred at 20℃ for 8 h, then reaction liquid 6 was acidified with concentrated hydrochloric acid, and extracted twice with 1 L of ethyl acetate each time, the combined organic phase was washed with water and then saturated brine, then dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and dried to obtain a crude product, which was purified by slurry with 800 mL of a mixture of petroleum ether / ethyl acetate with a volume ratio of 4:1, filtered and dried to obtain the target product, MS (ESI) M / Z: 382.2 [M+H]+, the yield was 69%; The synthesis route is as follows:

[0034] Example 2 A method for synthesizing Fmoc-L-(4,4-difluorocyclohexyl) glycine, which is different from example 1 in that in step S1, 4,4-difluorocyclohexanone is 1.49 mol, Z-α-phosphoryl glycine trimethyl ester is 1.49 mol, and DBU is 1.49 mol, and other steps are the same as example 1.

[0035] Example 3 A method for synthesizing Fmoc-L-(4,4-difluorocyclohexyl) glycine, which is different from example 1 in that in step S1, 4,4-difluorocyclohexanone is 1.49 mol, Z-α-phosphoryl glycine trimethyl ester is 1.56 mol, and DBU is 1.56 mol, and other steps are the same as example 1.

[0036] Example 4 A method for synthesizing Fmoc-L-(4,4-difluorocyclohexyl) glycine, which is different from example 1 in that in step S2, the amount of palladium-carbon catalyst is 8%, 39.2 g, and other steps are the same as example 1.

[0037] Example 5 A synthesis method of Fmoc-L-(4,4-difluorocyclohexyl) glycine, which is different from example 1 in that the amount of palladium-carbon catalyst in step S2 is 12%, 58.8g, and other steps are the same as example 1.

[0038] Example 6 A synthesis method of Fmoc-L-(4,4-difluorocyclohexyl) glycine, which is different from example 1 in that the amount of compound 4 in step S5 is 235g, 1mol, and other steps are the same as example 1.

[0039] Example 7 A synthesis method of Fmoc-L-(4,4-difluorocyclohexyl) glycine, which is different from example 1 in that the amount of compound 4 in step S5 is 283g, 1.2mol, and other steps are the same as example 1.

[0040] Example 8 A synthesis method of Fmoc-L-(4,4-difluorocyclohexyl) glycine, which is different from example 1 in that the reaction temperature of step S1 is 20℃, the reaction time is 12h, the reaction temperature of step S2 is 50℃, the reaction time is 3h, the reaction temperature of step S4 is 20℃, the reaction time is 8h, the reaction temperature of step S6 is 40℃, the reaction time is 4h, and other steps are the same as example 1.

[0041] Example 9 A synthesis method of Fmoc-L-(4,4-difluorocyclohexyl) glycine, which is different from example 1 in that the reaction temperature of step S1 is 50℃, the reaction time is 4h, the reaction temperature of step S2 is 20℃, the reaction time is 15h, the reaction temperature of step S4 is 60℃, the reaction time is 2h, the reaction temperature of step S6 is 0℃, the reaction time is 20h, and other steps are the same as example 1.

[0042] Performance detection test Test example 1 The target product synthesized by the synthesis method of Fmoc-L-(4,4-difluorocyclohexyl) glycine in example 1 was detected by HPLC, Chiral HPLC, MS and NMR respectively, and the detection results are shown in the following table. Figures 1-4 .

[0043] Figure 1 The liquid chromatogram of the target product of example 1 shows a single main peak, and the area ratio of impurity peak is very low, and the chemical purity of the target product is ≥99.3%. It can be seen that after the beating purification with petroleum ether / ethyl acetate mixed solvent, there is almost no residue of unreacted raw material, byproduct or solvent in the target product, and the chemical purity meets the requirements of drug research and development and industrial production, verifying the effectiveness of the synthesis and purification steps of the present application.

[0044] Figure 2 The chiral liquid chromatogram of the target product of Example 1 shows only a single peak corresponding to the L-form, without a peak of the D-form, and the enantiomeric excess value reaches 100%, which indicates that the enzymatic resolution using acetylation enzyme in step S5 of the synthesis process of the present application has extremely high selectivity, can only retain the L-form product, and ensures that the optical purity of the target product meets the strict requirements of chiral drug research and development, significantly improving the problems of expensive chiral control raw materials, difficult process, and low purity in the prior art.

[0045] Figure 3 The mass spectrum of the target product of Example 1 shows no significant interference peaks or abnormal fragment peaks, indicating that the molecule has not been degraded or modified, and the mass-to-charge ratio verifies that the measured value is highly consistent with the theoretical molecular weight 381.3 g / mol of the target product Fmoc-L-(4,4-difluorocyclohexyl) glycine, plus H + and Cl, which is 382.3 g / mol.

[0046] Figure 4 The nuclear magnetic resonance spectrum of the target product of Example 1 can confirm that the molecular skeleton is correct, which is Fmoc-L-(4,4-difluorocyclohexyl) glycine.

[0047] Test Example 2 The yield of compound 3 (total yield of steps S1-3) and the yield of the target product (total yield of steps S4-6) and purity of Fmoc-L-(4,4-difluorocyclohexyl) glycine synthesized by the synthesis method of Examples 1-7 were detected and calculated, and the test results are recorded in Table 1, yield = actual reaction product mass / theoretical product mass x 100%.

[0048] Table 1 According to the performance test results in Table 1, it can be seen that the synthesis method of Fmoc-L-(4,4-difluorocyclohexyl) glycine of the present application can efficiently prepare the target product Fmoc-L-(4,4-difluorocyclohexyl) glycine. The synthesis process of the present application has 6 steps, among which 5 steps of compounds 1, 2, 4, 5 and intermediate products do not need to be purified and can be directly used in the next step reaction, compound 3 and the final product must be purified by beating, the yield is higher, the beating purification yield of compound 3 reaches 68-75%, the beating purification yield of the target product reaches 62-69%, and the chemical purity is all ≥99.3%, which meets the requirements of drug research and development and industrial production. The synthesis method of the present application is more low-carbon and environmentally friendly, has less solvent consumption, cheap and easily available raw materials, low equipment requirements, mild reaction conditions, high safety, high yield and high purity of the product, high process stability, simple operation steps, can be operated on a large scale, significantly improves the production efficiency, greatly reduces the process conversion difficulty from small-dose production in the laboratory to industrial production, and is suitable for scale-up production.

[0049] According to the performance test results of Examples 1 and 8-9, it can be seen that the optimal reaction temperature and time in the synthesis method of Fmoc-L-(4,4-difluorocyclohexyl) glycine of the present application are as follows: the reaction temperature of step S1 is 30℃, the reaction time is 8h, the reaction temperature of step S2 is 30℃, the reaction time is 10h, the reaction temperature of step S4 is 30℃, the reaction time is 4h, the reaction temperature of step S6 is 20℃, and the reaction time is 8h.

[0050] The specific embodiments are only an explanation of the present application, and are not a limitation of the present application. Those skilled in the art can make modifications to the embodiments without creative contribution after reading the present specification, as long as the modifications are within the scope of the claims of the present application.

Claims

1. A method for synthesizing Fmoc-L-(4,4-difluorocyclohexyl)glycine, characterized in that, Includes the following steps: S1: 4,4-Difluorocyclohexanone reacts with Z-α-phosphonoglycine trimethyl ester and DBU to generate compound 1. The product does not require purification. S2: Compound 1 is hydrogenated under normal pressure via a palladium-on-carbon catalyst to produce compound 2. The product does not require purification. S3: Compound 2 was acetylated with acetic anhydride, and purified by pulping to obtain compound 3; S4: Compound 3 is given by alkaline hydrolysis to obtain compound 4, and the product does not require purification. S5: Compound 4 is hydrolyzed by acetylase to obtain compound 5, and the product does not require purification. S6: Compound 5 was reacted with Fmoc-Osu, and the crude product was purified by pulping to obtain the target product Fmoc-L-(4,4-difluorocyclohexyl)glycine; the synthetic route is as follows:

2. The method for synthesizing Fmoc-L-(4,4-difluorocyclohexyl)glycine according to claim 1, characterized in that, In step S1, the molar ratio of 4,4-difluorocyclohexanone to Z-α-phosphonoglycine trimethyl ester and DBU is 1:(1-1.05):(1-1.05).

3. The method for synthesizing Fmoc-L-(4,4-difluorocyclohexyl)glycine according to claim 1, characterized in that, The amount of the palladium-on-carbon catalyst is 8-12% of compound 1.

4. The method for synthesizing Fmoc-L-(4,4-difluorocyclohexyl)glycine according to claim 1, characterized in that, In step S5, the mass ratio of compound 4 to acetyltransferase is (235-283):

30.

5. The method for synthesizing Fmoc-L-(4,4-difluorocyclohexyl)glycine according to claim 1, characterized in that, The solvents used for pulping and purification in steps S3 and S6 are mixed solvents, including petroleum ether and ethyl acetate.

6. The method for synthesizing Fmoc-L-(4,4-difluorocyclohexyl)glycine according to claim 1, characterized in that, The reaction temperature in step S1 is 20-50℃, and the reaction time is 4-12h.

7. The method for synthesizing Fmoc-L-(4,4-difluorocyclohexyl)glycine according to claim 1, characterized in that, The reaction temperature in step S2 is 20-50℃, and the reaction time is 3-15h.

8. The method for synthesizing Fmoc-L-(4,4-difluorocyclohexyl)glycine according to claim 1, characterized in that, The reaction temperature in step S4 is 20-60℃, and the reaction time is 2-8h.

9. The method for synthesizing Fmoc-L-(4,4-difluorocyclohexyl)glycine according to claim 1, characterized in that, The reaction temperature in step S6 is 0-40℃, and the reaction time is 4-20h.

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