Preparation method of N-Cbz-L-histidine hydrazide

Abstract

The invention belongs to the technical field of chemical synthesis, and particularly provides a preparation method of N-Cbz-L-histidine hydrazide, which comprises the following step: carrying out hydrazide reaction on N-Cbz-L-histidine and hydrazine hydrate to generate the N-Cbz-L-histidine hydrazide. According to the preparation method disclosed by the embodiment of the invention, the N-Cbz-L-histidine is taken as a starting raw material and is directly subjected to hydrazide reaction with the hydrazine hydrate to generate the N-Cbz-L-histidine hydrazide, an esterification intermediate step is not needed, the synthesis route is simple, and the yield is higher.

Description

Technical Field

[0001] The present invention belongs to the technical field of compound preparation, and more specifically, relates to a method for preparing N-Cbz-L-histidylhydrazide. Background Art

[0002] In the field of peptide synthesis, L-histidine is often listed as a "challenging" amino acid due to its unique structural properties. The main side reactions arise from diastereoisomerization and the side chain imidazole ring, which complicates many syntheses. The most promising approach to addressing this problem is to react via histidyl azide, which effectively avoids reactions on the imidazole ring. Furthermore, histidine hydrazide is an effective scavenger of HOHA lactone, protecting human retinal pigment epithelial (ARPE-19) cells from HOHA lactone-induced cytotoxicity, making it a potential therapeutic agent for ocular diseases.

[0003] N-Cbz-L-histidine hydrazide is the precursor of acyl azide and one of the most commonly used derivatives of L-histidine. Therefore, N-Cbz-L-histidine hydrazide plays an important role in peptide synthesis and disease treatment.

[0004] Currently, the reported synthesis methods of N-Cbz-L-histidine hydrazide are generally obtained through two synthetic ideas.

[0005] Idea 1: Histidine is first methylated, then a Cbz protecting group is introduced, and finally hydrazide is performed to obtain N-Cbz-L-histidine hydrazide (J.Am.Chem.Soc.,1954,76,1326). The synthetic route is shown in the following formula (1):

[0006]

[0007] However, the total yield of these two steps is less than 50%. Although there have been related syntheses reported later (Hoppe-Seyler's Zeitschrift fur Physiologische Chemie, 1960, 320, 82-91; Bioorg. and Med. Chem. 1996, 4(2), 179-193.), the yield has not improved much.

[0008] Furthermore, the synthesis method of the first approach mentioned above employs water recrystallization in the synthesis and purification of N-Cbz-L-histidine hydrazide. However, under the influence of high temperature and water, ring closure is easily achieved to produce a byproduct as shown in Formula (2), resulting in product deterioration and reduced yield and purity.

[0009]

[0010] Idea 2: First, introduce a Cbz protecting group into L-histidine, then methylate it, and finally hydrazide it to obtain N-Cbz-L-histidine hydrazide (J. Chem. Soc., Perkin Trans. I 1979, 2261). The synthetic route is shown in the following formula (3):

[0011]

[0012] However, when Cbz was protected first and then esterified, the esterification yield was only 49%.

[0013] In addition, these two synthetic approaches have some common problems, namely, the preparation of hydrazide requires conversion of the ester group into hydrazide, which is a cumbersome process.

[0014] Therefore, it is urgent to propose a preparation method of N-Cbz-L-histidine hydrazide with simple steps, high yield and high purity. Summary of the Invention

[0015] In view of this, the object of the present invention is to provide a method for preparing N-Cbz-L-histidine hydrazide with a short route, high yield and high purity.

[0016] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0017] The preparation method of N-Cbz-L-histidine hydrazide according to an embodiment of the present invention comprises:

[0018] N-Cbz-L-histidine reacts with hydrazine hydrate to generate N-Cbz-L-histidine hydrazide.

[0019] In some embodiments of the present invention, the reaction is carried out under the action of a condensing agent, which includes one or more of N,N'-carbonyldiimidazole, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and 1-cyclohexyl-2-morpholinoethylcarbodiimide p-toluenesulfonate.

[0020] In some embodiments of the present invention, the condensing agent is N,N'-carbonyldiimidazole, and the preparation method specifically comprises:

[0021] dispersing the N-Cbz-L-histidine in an organic solvent;

[0022] adding the condensing agent therein to activate the carboxyl group of N-Cbz-L-histidine;

[0023] After the condensing agent is added dropwise, the hydrazine hydrate is continued to be added dropwise to generate the N-Cbz-L-histidyl hydrazide.

[0024] In other embodiments of the present invention, the condensing agent is 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, 1-cyclohexyl-2-morpholinoethylcarbodiimide p-toluenesulfonate, or a mixture thereof, and the preparation method specifically comprises:

[0025] dispersing the N-Cbz-L-histidine in an organic solvent;

[0026] Adding the condensing agent therein to generate the N-Cbz-L-histidylhydrazide,

[0027] The hydrazine hydrate is added together with the N-Cbz-L-histidine, or after the N-Cbz-L-histidine is added and before the condensing agent is added, or together with the condensing agent, or after the condensing agent is added.

[0028] Furthermore, the organic solvent includes any one or more of acetonitrile, ethyl acetate, dichloromethane, and tetrahydrofuran.

[0029] In some embodiments of the present invention, the molar ratio of N-Cbz-L-histidine:hydrazine hydrate:condensing agent is 1:(2.0-5.0):(1.0-1.5).

[0030] In some embodiments of the present invention, the reaction temperature is -5 to 40° C., and the reaction time is 5 to 12 hours.

[0031] In some embodiments of the present invention, the preparation method further comprises:

[0032] After the reaction is completed, filter and wash the filter cake with an alcohol reagent;

[0033] After washing, the filter cake is recrystallized with an alcohol reagent and dried to obtain the N-Cbz-L-histidylhydrazide.

[0034] In some embodiments of the present invention, the preparation method further comprises:

[0035] Dispersing L-histidine in dichloromethane to obtain a suspension;

[0036] Triethylamine and a catalyst are continuously added thereto, and N-benzyloxycarbonyloxysuccinimide is added thereto for reaction to generate bisbenzyloxycarbonyl-L-histidine, wherein the catalyst is one or more of (3-bromopropyl)trimethylammonium bromide, hexadecyldimethylethylammonium bromide, dimethyldioctylammonium bromide, and didodecyldimethylammonium bromide;

[0037] Ammonia gas is introduced into the reaction solution until the solution is saturated with ammonia gas to remove the benzyloxycarbonyl group on the imidazole group in the bis-benzyloxycarbonyl-L-histidine to obtain the N-Cbz-L-histidine.

[0038] The above technical solution of the present invention has at least one of the following beneficial effects:

[0039] According to the preparation method of the embodiment of the present invention, N-Cbz-L-histidine is used as the starting material, and a hydrazide reaction is directly carried out with hydrazine hydrate to produce N-Cbz-L-histidine hydrazide without the need for an intermediate esterification step. The synthetic route is simple and the yield is higher.

[0040] Furthermore, according to the preparation method of the present invention, alcohols are used for actual recrystallization, which does not generate unnecessary by-products, and the yield and purity are guaranteed;

[0041] Furthermore, according to the preparation method of the present invention, during the homemade N-Cbz-L-histidine, ammonia is used instead of common potassium hydroxide, sodium ethoxide, etc. in the second step of removing the benzyloxycarbonyl group on the imidazole group. The reaction conditions are mild, which not only avoids the loss of the Cbz protecting group on the α-amino group, but also eliminates the problem of damaging the optical rotation of N-Cbz-L-histidine, thereby ensuring that the optical rotation of the final product is not damaged. DETAILED DESCRIPTION

[0042] To make the objectives, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the described embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field are within the scope of protection of the present invention.

[0043] First, the preparation method of N-Cbz-L-histidine hydrazide according to an embodiment of the present invention is described in detail below.

[0044] The preparation method of N-Cbz-L-histidine hydrazide according to an embodiment of the present invention comprises:

[0045] N-Cbz-L-histidine reacts with hydrazine hydrate to form N-Cbz-L-histidine hydrazide.

[0046] That is, according to the preparation method of the embodiment of the present invention, N-Cbz-L-histidine is used as the starting material, and a hydrazide reaction is directly performed with hydrazine hydrate to produce N-Cbz-L-histidine hydrazide without the need for an intermediate esterification step. The synthetic route is simple and the yield is higher.

[0047] Among them, as N-Cbz-L-histidine, either a commercial product can be used or it can be prepared by the following steps:

[0048] Dispersing L-histidine in dichloromethane to obtain a suspension;

[0049] Triethylamine and a catalyst are continuously added thereto, and N-benzyloxycarbonyloxysuccinimide is added thereto for reaction to generate bisbenzyloxycarbonyl-L-histidine, wherein the catalyst is one or more of (3-bromopropyl)trimethylammonium bromide, hexadecyldimethylethylammonium bromide, dimethyldioctylammonium bromide, and didodecyldimethylammonium bromide;

[0050] Ammonia gas is introduced into the reaction solution until the solution is saturated with ammonia gas to remove the benzyloxycarbonyl group on the imidazole group in the bis-benzyloxycarbonyl-L-histidine to obtain the N-Cbz-L-histidine.

[0051] Therefore, in the process of preparing N-Cbz-L-histidine, ammonia is used instead of common potassium hydroxide, sodium ethoxide, etc. in the second step of removing the benzyloxycarbonyl group on the imidazole group. The mild reaction conditions not only avoid the loss of the Cbz protecting group on the α-amino group, but also eliminate the problem of damaging the optical rotation of N-Cbz-L-histidine, thereby ensuring that the optical rotation of the final product will not be damaged.

[0052] In some embodiments of the present invention, to promote the hydrazide reaction, the hydrazide reaction can be carried out in the presence of a condensing agent. The condensing agent can be, for example, one or more of N,N'-carbonyldiimidazole (CDI), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl), and 1-cyclohexyl-2-morpholinoethylcarbodiimide p-toluenesulfonate.

[0053] Different types of condensation reagents have different uses.

[0054] For example, when CDI is used as a condensing agent, the following operations can be performed:

[0055] dispersing the N-Cbz-L-histidine in an organic solvent;

[0056] Adding a condensing agent (i.e., CDI) to activate the carboxyl group of N-Cbz-L-histidine;

[0057] After the condensing agent is added dropwise, the hydrazine hydrate is continued to be added dropwise to generate the N-Cbz-L-histidyl hydrazide.

[0058] That is, a condensing agent is first added to the solution to fully activate the carboxyl group of N-Cbz-L-histidine, and then hydrazine hydrate is added dropwise thereto to cause a hydrazide reaction to generate N-Cbz-L-histidine hydrazide.

[0059] When 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, 1-cyclohexyl-2-morpholinoethylcarbodiimide p-toluenesulfonate, or a mixture thereof is used as the mixture, the following operation can be performed:

[0060] dispersing the N-Cbz-L-histidine in an organic solvent;

[0061] Adding the condensing agent therein to generate the N-Cbz-L-histidylhydrazide,

[0062] The hydrazine hydrate is added together with the N-Cbz-L-histidine, or after the N-Cbz-L-histidine is added and before the condensing agent is added, or together with the condensing agent, or after the condensing agent is added.

[0063] That is, when using 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, 1-cyclohexyl-2-morpholinoethylcarbodiimide p-toluenesulfonate, or a mixture thereof as the mixture, there is no particular restriction on the time of adding the condensing agent, and the condensing agent can be added together with hydrazine hydrate, after hydrazine hydrate, or at the same time.

[0064] The condensing agent is preferably CDI, which has good atom economy, can better activate carboxylic acid, and reduce the occurrence of side reactions.

[0065] In addition, the organic solvent used in the reaction system can be selected from any one or more of acetonitrile, ethyl acetate, dichloromethane, and tetrahydrofuran. Preferably, acetonitrile is selected as the organic solvent. Acetonitrile has a high polarity and a higher solubility for the by-products produced by the raw materials and the condensation reagent, which is conducive to the reaction and purification.

[0066] In some embodiments of the present invention, the molar ratio of N-Cbz-L-histidine: hydrazine hydrate: condensing agent is 1: (2.0-5.0): (1.0-1.5). Excess hydrazine hydrate can increase the concentration of hydrazine hydrate in the solution, accelerate the reaction process, and make the N-Cbz-L-histidine react more completely. Preferably, the molar ratio of N-Cbz-L-histidine: hydrazine hydrate: condensing agent is 1: 4.0: 1.05.

[0067] The hydrazide reaction is carried out at a temperature of -5°C to 40°C and for a reaction time of 5 to 12 hours. The reaction is mild and easy to operate, requires no special equipment, and is suitable for industrial production. Preferably, the reaction temperature is 10-20°C and the reaction time is 8 hours.

[0068] In some embodiments of the present invention, a purification treatment is performed after the hydrazide reaction is completed.

[0069] Specifically, the purification process may be, for example:

[0070] After the hydrazide reaction is completed, the filter is filtered and the filter cake is washed with an alcohol reagent;

[0071] After washing, the filter cake is recrystallized with an alcohol reagent (such as methanol, ethanol, etc.) and dried to obtain the N-Cbz-L-histidyl hydrazide.

[0072] That is to say, by using alcohol reagents instead of water for recrystallization, not only does it not require excessively high temperatures, but it also avoids the generation of unnecessary by-products.

[0073] The preparation method of N-Cbz-L-histidine hydrazide of the present invention is further described in detail below through specific examples.

[0074] Example 1

[0075] Synthesis of N-Cbz-L-histidine

[0076] To a 500mL three-necked flask, add 200mL of dichloromethane and 17.5g of L-histidine. Then, cool the system to 5°C and, while maintaining the temperature at 5-10°C, add 34.3g of triethylamine. Add 0.1g of the catalyst, didodecyldimethylammonium bromide, and, while maintaining the temperature at 5°C, add 64.7g of N-benzyloxycarbonyloxysuccinimide in batches. After additions are complete, slowly warm the system to room temperature and react for 8 hours.

[0077] After the reaction is completed, the system temperature is lowered to 10°C, the pH is adjusted to 1 with concentrated hydrochloric acid, and the mixture is allowed to stand for separation. The obtained organic phase is washed once with 200 mL of water, dried over anhydrous sodium sulfate, and filtered to obtain a dichloromethane solution of the intermediate bis-Cbz-L-histidine.

[0078] The dichloromethane solution of the intermediate was cooled to about 15°C, and ammonia was introduced while controlling the system temperature not to exceed 30°C until the ammonia was saturated. The temperature was then raised and refluxed for reaction for 6 hours.

[0079] After the reaction was complete, the product was filtered and the filter cake was recrystallized with 100 mL of isopropanol. The resulting solid was dried to obtain 24.6 g of a white solid with a yield of 75.4%, a melting point of 163.2-165.1°C, and an optical rotation of -23.3° (C = 66N HCl).

[0080] Synthesis of N-Cbz-L-histidylhydrazide

[0081] In a 500 mL three-necked flask, add 150 mL of acetonitrile and 30 g of N-Cbz-L-histidine, then add 17.66 g of CDI in batches at 20°C, and stir at this temperature for 1 h.

[0082] The reaction solution was cooled to 10°C, and 150 mL of acetonitrile solution of hydrazine hydrate (80%, 25.96 g) was added dropwise while maintaining the temperature at 10-15°C. After the addition was complete, the mixture was kept at 20°C for 7 h.

[0083] Post-treatment: Filtration, washing the filter cake with 50 mL of methanol, recrystallization with 200 mL of methanol, and drying to obtain 25.98 g of white solid N-Cbz-L-histidine hydrazide. Yield: 82.6%, melting point: 170.2-172.1°C, optical rotation: -36.2° (C = 1 1N HCl).

[0084] The NMR data of the obtained product are as follows:

[0085] 1 H NMR (400MHz, DMSO) δ11.77(s,1H),9.15(s,1H),7.52(s,1H),7.45–7.20(m,6H),6.80(br,1H),4.98(s,2H),4.28–4.08(m,3H),2.95–2.68(m,2H).

[0086] The test results were consistent with the structure of N-Cbz-L-histidine hydrazide.

[0087] Example 2

[0088] In a 1L three-necked flask, add 300mL of acetonitrile, 30g of N-Cbz-L-histidine, and 25.96g of hydrazine hydrate (80%). Then cool to 10°C, control the temperature at 10-15°C, add 20.87g of EDC·HCl, and after addition, heat to 20°C and stir for 8h.

[0089] Post-treatment: Filtration, washing the filter cake with 50 mL of methanol, recrystallization with 200 mL of methanol, and drying to obtain 25.20 g of white solid N-Cbz-L-histidine hydrazide. Yield: 80.1%, melting point: 168.3-170.0°C, optical rotation: -35.7° (C = 1 1N HCl).

[0090] Example 3

[0091] In a 500 mL three-necked flask, add 150 mL of dichloromethane and 30 g of N-Cbz-L-histidine, then add 17.66 g of CDI in batches at 20°C, and stir at this temperature for 1 h.

[0092] The reaction solution was cooled to 10°C, and 150 mL of a dichloromethane solution of hydrazine hydrate (80%, 25.96 g) was added dropwise while maintaining the temperature at 10-15°C. After the addition was complete, the mixture was kept at 20°C for 7 h.

[0093] Post-treatment: Filtration, washing the filter cake with 50 mL of methanol, recrystallization with 200 mL of methanol, and drying to obtain 25.57 g of white solid N-Cbz-L-histidine hydrazide. Yield: 81.3%, melting point: 169.5-172.0°C, optical rotation: -36.5° (C = 1 1N HCl).

[0094] Example 4

[0095] In a 500 mL three-necked flask, add 150 mL of acetonitrile and 30 g of N-Cbz-L-histidine, then add 17.66 g of CDI in batches at 20°C, and stir at this temperature for 1 h.

[0096] The reaction solution was cooled to 10°C, and 150 mL of acetonitrile solution of hydrazine hydrate (80%, 19.47 g) was added dropwise while maintaining the temperature at 10-15°C. After the addition was complete, the mixture was kept at 20°C for 7 h.

[0097] Post-treatment: Filtration, washing the filter cake with 50 mL of methanol, recrystallization with 200 mL of methanol, and drying to obtain 24.72 g of white solid N-Cbz-L-histidine hydrazide. Yield: 78.6%, melting point: 168.9-171.1°C, optical rotation: -35.5° (C = 1 1N HCl).

[0098] In summary, according to the preparation method of the present invention, N-Cbz-L-histidine is used as the starting raw material, and a hydrazide reaction is directly carried out with hydrazine hydrate to generate N-Cbz-L-histidine hydrazide, without the need for an intermediate esterification step, the synthesis route is simple, and the yield is higher; in addition, the actual recrystallization is carried out using alcohols, and unnecessary by-products are not generated, and the yield and purity are guaranteed.

[0099] The above is a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for preparing N-Cbz-L-histidylhydrazide, characterized in that: include: N-Cbz-L-histidine reacts with hydrazine hydrate to form N-Cbz-L-histidine hydrazide.

2. The preparation method according to claim 1, characterized in that The hydrazide reaction is carried out under the action of a condensing agent, and the condensing agent is selected from one or more of N,N'-carbonyldiimidazole, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and 1-cyclohexyl-2-morpholinoethylcarbodiimide p-toluenesulfonate.

3. The preparation method according to claim 2, characterized in that The condensing agent is N,N'-carbonyldiimidazole, and the preparation method specifically includes: dispersing the N-Cbz-L-histidine in an organic solvent; adding the condensing agent therein to activate the carboxyl group of N-Cbz-L-histidine; After the condensing agent is added dropwise, the hydrazine hydrate is continued to be added dropwise to generate the N-Cbz-L-histidyl hydrazide.

4. The preparation method according to claim 2, characterized in that The condensing agent is 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, 1-cyclohexyl-2-morpholinoethylcarbodiimide p-toluenesulfonate, or a mixture thereof. The preparation method specifically comprises: dispersing the N-Cbz-L-histidine in an organic solvent; Adding the condensing agent therein to generate the N-Cbz-L-histidylhydrazide, The hydrazine hydrate is added together with the N-Cbz-L-histidine, or after the N-Cbz-L-histidine is added and before the condensing agent is added, or together with the condensing agent, or after the condensing agent is added.

5. The preparation method according to claim 3 or 4, characterized in that The organic solvent is selected from any one or more of acetonitrile, ethyl acetate, dichloromethane and tetrahydrofuran.

6. The preparation method according to claim 1, characterized in that The molar ratio of the N-Cbz-L-histidine:hydrazine hydrate:condensing agent is 1:(2.0-5.0):(1.0-1.5).

7. The preparation method according to claim 1, characterized in that The reaction temperature of the hydrazide reaction is -5 to 40° C., and the reaction time is 5 to 12 hours.

8. The preparation method according to claim 1, characterized in that Also includes: After the hydrazide reaction is completed, the filter is filtered and the filter cake is washed with an alcohol reagent; After washing, the filter cake is recrystallized with an alcohol reagent and dried to obtain the N-Cbz-L-histidylhydrazide.

9. The preparation method according to claim 1, characterized in that Also includes: Dispersing L-histidine in dichloromethane to obtain a suspension; Triethylamine and a catalyst are continuously added thereto, and N-benzyloxycarbonyloxysuccinimide is added thereto for reaction to generate bisbenzyloxycarbonyl-L-histidine, wherein the catalyst is one or more of (3-bromopropyl)trimethylammonium bromide, hexadecyldimethylethylammonium bromide, dimethyldioctylammonium bromide, and didodecyldimethylammonium bromide; Ammonia gas is introduced into the reaction solution until the solution is saturated with ammonia gas to remove the benzyloxycarbonyl group on the imidazole group in the bis-benzyloxycarbonyl-L-histidine to obtain the N-Cbz-L-histidine.

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