Method for preparing cyclic (L-proline-L-tryptophan) dipeptide by one-pot method
The one-pot synthesis of cyclic (L-proline-L-tryptophan) dipeptides solves the problems of long synthesis cycle, large solvent consumption and low yield in existing technologies, and achieves high-purity and high-yield production of cyclic dipeptides, which is suitable for industrial applications.
Patent Information
- Application Number
- CN202511032737.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-25
- Publication Date
- 2025-11-21
AI Technical Summary
Existing cyclic dipeptide synthesis processes are characterized by long cycles, a wide variety of solvents, large quantities of solvents used, low yields, and complex operations, making them unsuitable for industrial production.
Cyclic (L-proline-L-tryptophan) dipeptides were prepared using a one-pot method. The linear dipeptide condensation and intramolecular cyclization reactions were carried out in the same reactor. Specific solvents, amines, condensing agents, deprotecting agents, and cyclizing agents were used to avoid intermediate separation. Purification was achieved by a mixed solvent crystallization method.
It significantly improved product purity and yield, reduced production costs and operational complexity, and achieved green and environmentally friendly industrial production.
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Figure CN120987948A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of cyclic dipeptide synthesis technology, specifically relating to a one-pot method for preparing cyclic (L-proline-L-tryptophan) dipeptides. Background Technology
[0002] The statements herein provide only background information in relation to this invention and do not necessarily constitute prior art.
[0003] Cyclic dipeptides, as a special class of bioactive molecules, exhibit excellent stability and targeted binding ability due to their unique cyclic structure, and have significant application potential in drug development (such as anti-tumor and immunomodulatory applications) and functional materials. Traditional cyclic dipeptide synthesis often employs a stepwise method, first preparing a linear dipeptide and then achieving cyclization through intramolecular condensation. The synthesis process requires multiple separations of intermediates and solvent changes. Intramolecular condensation requires a palladium-on-carbon catalyst, which is costly, requires recycling, and improperly disposed of waste can cause environmental pollution.
[0004] In summary, existing technologies have long overall process preparation cycles, require a variety of solvents in large quantities, have low yields, are complex to operate, and are not economically viable, making them unsuitable for industrial production. Summary of the Invention
[0005] To address the shortcomings of existing technologies, this invention provides a one-pot method for preparing cyclic (L-proline-L-tryptophan) dipeptides, comprising the following steps: S1, add carboxyl-protected starting material 1 to the solvent, stir until dissolved, add free organic amine, then add condensing agent and amino-protected starting material 2, and control the temperature to react until the reaction is complete.
[0006] The starting material 1 is an ester or salt of L-proline and L-tryptophan; Furthermore, the starting material 1 is one of L-proline methyl ester hydrochloride, L-proline ethyl ester hydrochloride, L-tryptophan methyl ester hydrochloride, and L-tryptophan ethyl ester hydrochloride, preferably L-proline methyl ester hydrochloride.
[0007] The molar ratio of starting material 1 to starting material 2 in this invention is any ratio between 1:2 and 2:1, preferably 1:1.
[0008] Furthermore, the reaction solvent is one of dichloromethane, chloroform, methanol, ethanol, isopropanol, and ethyl acetate, with dichloromethane being preferred.
[0009] The amount of solvent used in step S1 of the present invention is 2 to 20 times the volume of the starting material 1, preferably 10 times the volume.
[0010] Furthermore, the organic amine is selected from diethylamine and triethylamine, with triethylamine being preferred.
[0011] The amount of organic amine used in step S1 of this invention is 1 to 5 times the equivalent of starting material 1, preferably 2 equivalents; Furthermore, the condensing agent is one or a combination of several of HOBT, HOAT, HATU, HBTU, EDCI, CDI, DCC, and DIC, with CDI being preferred.
[0012] The amount of condensing agent used in step S1 of the present invention is 0.5 to 5 times the equivalent of the starting material 1, preferably 2 equivalents.
[0013] Furthermore, the amino-protected starting material 2 is one of Boc-L-proline, Fmoc-L-proline, Boc-L-tryptophan, and Fmoc-L-tryptophan. The corresponding material is selected according to the starting material 1, with Boc-L-tryptophan being preferred. Furthermore, the temperature control reaction temperature is -10~90℃, preferably room temperature.
[0014] Furthermore, after the reaction is complete, the reaction solution is washed once with an acid solution and once with an alkaline solution.
[0015] The acid-water solution described in this invention is an aqueous solution of one of the following acids: dilute hydrochloric acid, dilute sulfuric acid, and glacial acetic acid, preferably dilute hydrochloric acid; The alkaline aqueous solution of the present invention is an aqueous solution of one of the following bases: sodium bicarbonate, sodium carbonate, sodium hydroxide, potassium carbonate, potassium hydroxide, concentrated ammonia, diethylamine, and triethylamine, preferably sodium hydroxide.
[0016] S2, after washing the organic layer in step S1, add the deprotecting agent directly. After the reaction is complete under controlled temperature, add an alkaline aqueous solution to wash away the deprotecting agent. Furthermore, the deprotection reagent includes a Boc deprotection reagent and an Fmoc deprotection reagent; the Boc deprotection reagent is selected from one of trifluoroacetic acid, hydrochloric acid / dioxane, and hydrochloric acid / ethyl acetate, preferably hydrochloric acid / ethyl acetate or a mixture of multiple types; the Fmoc deprotection reagent is selected from one or more of piperidine and diethylamine, preferably piperidine; Furthermore, the amount of the deprotecting agent is 1 to 20 times the equivalent of the starting material 2, preferably 15 equivalents.
[0017] S3, after washing the organic layer, a cyclizing agent is added, and the reaction is carried out under controlled temperature; Furthermore, the cyclizing agent is one or more of sodium bicarbonate, sodium carbonate, sodium hydroxide, potassium carbonate, potassium hydroxide, concentrated ammonia, diethylamine, and triethylamine, preferably sodium hydroxide.
[0018] Furthermore, the amount of the cyclizing agent is 1 to 20 times the equivalent of the starting material 1, preferably 10 equivalents.
[0019] S4. After step S3 is complete, wash once with water. After washing, concentrate the organic layer and add an inert solvent to control the temperature for crystallization for more than 2 hours. Filter and dry to obtain cyclic (L-proline-L-tryptophan) dipeptide.
[0020] Furthermore, the inert solvent is one or a mixture of n-heptane, n-hexane, cyclohexane, diethyl ether, petroleum ether, methyl tert-butyl ether, and toluene, preferably methyl tert-butyl ether.
[0021] Furthermore, the amount of inert solvent used in step S4 is 0.5 to 10 times the equivalent of the starting material 1, preferably 5 equivalents; The beneficial effects achieved by one or more embodiments of the present invention described above are as follows: 1. Based on the condensing agent, solvent system, and reaction temperature provided by this invention, the condensation and intramolecular cyclization reactions of linear dipeptides are completed sequentially in the same reactor, avoiding the separation and purification steps of intermediate products. After the reaction, the mixed solvent crystallization method significantly improves the product purity (>95%) and yield (≥85%).
[0022] 2. The synthesis method provided by this invention does not require intermediate separation, and the process does not require changing the solvent, which reduces production costs and operational complexity, saves solvent consumption, and is more green and environmentally friendly. Attached Figure Description
[0023] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.
[0024] Figure 1 The image shows a positive ion MS diagram of the cyclic (L-proline-L-tryptophan) dipeptide produced in Example 1. Figure 2 MS image of the cyclic (L-proline-L-tryptophan) dipeptide produced in Example 1 with negative ions; Figure 3 MS chromatogram of the cyclic (L-proline-L-tryptophan) dipeptide produced in Example 1 1 HNMR spectrum; Figure 4 The HPLC chromatogram of the cyclic (L-proline-L-tryptophan) dipeptide produced in Example 1 is shown below. Figure 5 This is an HPLC chromatogram of the cyclic (L-proline-L-tryptophan) dipeptide produced in Example 2. Detailed Implementation
[0025] It should be noted that the following detailed description is illustrative and intended to provide further explanation of the invention. Unless otherwise specified, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.
[0026] This invention provides a one-pot method for preparing cyclic (L-proline-L-tryptophan) dipeptides, comprising the following steps: S1, add carboxyl-protected starting material 1 to the solvent, stir until dissolved, add free organic amine, then add condensing agent and amino-protected starting material 2, and control the temperature to react until the reaction is complete.
[0027] The starting material 1 is an ester or salt of L-proline and L-tryptophan; Furthermore, the starting material 1 is one of L-proline methyl ester hydrochloride, L-proline ethyl ester hydrochloride, L-tryptophan methyl ester hydrochloride, and L-tryptophan ethyl ester hydrochloride, preferably L-proline methyl ester hydrochloride.
[0028] The molar ratio of starting material 1 to starting material 2 in this invention is any ratio between 1:2 and 2:1, preferably 1:1.
[0029] Furthermore, the reaction solvent is one of dichloromethane, chloroform, methanol, ethanol, isopropanol, and ethyl acetate, with dichloromethane being preferred.
[0030] The amount of solvent used in step S1 of the present invention is 2 to 20 times the volume of the starting material 1, preferably 10 times the volume.
[0031] Furthermore, the organic amine is selected from diethylamine and triethylamine, with triethylamine being preferred.
[0032] The amount of organic amine used in step S1 of this invention is 1 to 5 times the equivalent of starting material 1, preferably 2 equivalents; Furthermore, the condensing agent is one or a combination of several of HOBT, HOAT, HATU, HBTU, EDCI, CDI, DCC, and DIC, with CDI being preferred.
[0033] The amount of condensing agent used in step S1 of the present invention is 0.5 to 5 times the equivalent of the starting material 1, preferably 2 equivalents.
[0034] Furthermore, the amino-protected starting material 2 is one of Boc-L-proline, Fmoc-L-proline, Boc-L-tryptophan, and Fmoc-L-tryptophan. The corresponding material is selected according to the starting material 1, with Boc-L-tryptophan being preferred. Furthermore, the temperature control reaction temperature is -10~90℃, preferably room temperature.
[0035] Furthermore, after the reaction is complete, the reaction solution is washed once with an acid solution and once with an alkaline solution.
[0036] The acid-water solution described in this invention is an aqueous solution of one of the following acids: dilute hydrochloric acid, dilute sulfuric acid, and glacial acetic acid, preferably dilute hydrochloric acid; The alkaline aqueous solution described in this invention is one of the following: sodium bicarbonate, sodium carbonate, sodium hydroxide, potassium carbonate, potassium hydroxide, concentrated ammonia, diethylamine, and triethylamine, preferably sodium hydroxide.
[0037] S2, after washing the organic layer, add the deprotecting agent directly, and after the reaction is complete under controlled temperature, add an alkaline aqueous solution to wash away the deprotecting agent; Furthermore, the deprotection reagent includes a Boc deprotection reagent and an Fmoc deprotection reagent; the Boc deprotection reagent is selected from one of trifluoroacetic acid, hydrochloric acid / dioxane, and hydrochloric acid / ethyl acetate, preferably hydrochloric acid / ethyl acetate; the Fmoc deprotection reagent is selected from one of piperidine and diethylamine, preferably piperidine; S3, after washing the organic layer, a cyclizing agent is added, and the reaction is carried out under controlled temperature; Furthermore, the cyclizing agent is one of the following: sodium bicarbonate, sodium carbonate, sodium hydroxide, potassium carbonate, potassium hydroxide, concentrated ammonia, diethylamine, and triethylamine, preferably sodium hydroxide.
[0038] S4. After step S3 is complete, wash once with water. After washing, concentrate the organic layer and add an inert solvent to control the temperature for crystallization for more than 2 hours. Filter and dry to obtain cyclic (L-proline-L-tryptophan) dipeptide.
[0039] Furthermore, the inert solvent is one of the following: n-heptane, n-hexane, cyclohexane, diethyl ether, petroleum ether, methyl tert-butyl ether, and toluene, preferably methyl tert-butyl ether.
[0040] The amount of the deprotecting agent used in step S2 of the present invention is 1 to 20 times the equivalent of the starting material 2, preferably 15 equivalents; The amount of cyclizing reagent used in step S3 of the present invention is 1 to 20 times the equivalent of starting material 1, preferably 10 equivalents; The amount of inert solvent used in step S4 of the present invention is 0.5 to 10 times the equivalent of the starting material 1, preferably 5 equivalents; The temperature for controlled crystallization in step S4 of this invention is -15~40℃, preferably room temperature.
[0041] In some embodiments, the reaction process is as follows: The present invention will be further described below with reference to the embodiments.
[0042] The product was tested using HPLC: High-performance liquid chromatography (HPLC) analysis was used for purity detection, performed on a Shimadzu LC-20AT, using a DAD (diode array detector), and the area was calculated using the area normalization method; the standard chromatographic conditions were: SHIMSEN Superb II C18 (250×4.6 mm, 5μm), phase A was H2O, phase B was CH3CN, volume ratio A:B = 60:40, isocratic elution for 30 min, flow rate 1.0 mL / min, detection wavelength 218 nm, injection volume 10 μl, and column temperature 40℃.
[0043] Example 1 Add L-proline methyl ester hydrochloride (5.5 g, 33 mmol), 55 mL of dichloromethane, triethylamine (6.7 g, 66 mmol), CDI (10.9 g, 66 mmol), and Boc-L-tryptophan (10.1 g, 33 mmol) to a 250 mL single-necked flask. Stir the mixture at room temperature for 4 h, and monitor the reaction for completion by TLC. Wash once with 55 mL of 1 N hydrochloric acid solution and once with 55 mL of saturated sodium bicarbonate solution. Discard the aqueous layer, and use the dichloromethane layer directly for the next step.
[0044] Transfer the dichloromethane layer obtained in the previous step to a 1000 ml single-necked flask, add 500 ml of 1 N hydrochloric acid / ethyl acetate solution, and react at room temperature for 1 h. Monitor the reaction completion by TLC. Add 400 ml of saturated sodium bicarbonate solution, wash the organic layer once, discard the aqueous layer, and use the organic layer directly for the next step.
[0045] Sodium hydroxide (13.2 g, 330 mmol) was added to the organic layer, and the reaction was carried out at room temperature for 6 h. The reaction was monitored by TLC until completion. The mixture was washed once with 400 ml of water, the aqueous layer was discarded, and the organic layer was concentrated under reduced pressure to about 100 ml. 500 ml of methyl tert-butyl ether was added, and the mixture was allowed to crystallize at room temperature for 2 h. The crystals were filtered and dried to give about 8.5 g of white solid.
[0046] Yield: 90.91%, Purity: 98.056% Figure 4 ).
[0047] MS diagrams of the product 1 HNMR and HPLC chromatograms are as follows: Figures 1-4 As shown.
[0048] Example 2 Add Boc-L-proline (7.15 g, 33 mmol), 55 mL of dichloromethane, triethylamine (6.7 g, 66 mmol), CDI (10.9 g, 66 mmol), and L-tryptophan methyl ester hydrochloride (8.46 g, 33 mmol) to a 250 mL single-necked flask. Stir the mixture at room temperature for 4 h, and monitor the reaction for completion by TLC. Wash once with 55 mL of 1 N hydrochloric acid solution and once with 55 mL of saturated sodium bicarbonate solution. Discard the aqueous layer, and use the dichloromethane layer directly for the next step.
[0049] The dichloromethane layer obtained in the previous step was added to trifluoroacetic acid (56.2 g, 495 mmol), and the reaction was carried out at room temperature for 2 h. The reaction was monitored by TLC until completion. 100 ml of 20% sodium hydroxide solution was added to wash the organic layer once. The aqueous layer was discarded, and the organic layer was used directly for the next step.
[0050] Sodium hydroxide (13.2 g, 330 mmol) was added to the organic layer, and the reaction was carried out at room temperature for 6 h. The reaction was monitored by TLC until completion. The mixture was washed once with 100 ml of water, the aqueous layer was discarded, and the organic layer was transferred to a 500 ml single-necked flask. 275 ml of methyl tert-butyl ether was added, and the mixture was allowed to crystallize at room temperature for 2 h. The crystals were then filtered and dried to give about 8.4 g of white solid.
[0051] Yield: 89.84%, Purity: 96.816% Figure 5 ).
[0052] Example 3 Comparison of different conditions 1. Based on Example 1, with other conditions unchanged, the starting material ratio was adjusted, and the yield and product purity were compared. The results are summarized in the table below: Table 1 Comparison of yield and quality under different starting material ratios
[0053] As can be seen from Table 1, the yield and quality do not differ much under different combinations and ratios of starting materials. L-proline methyl ester and Boc-L-tryptophan are preferred as starting materials, and the equivalent ratio of 1:1 is due to their low price and strong economic efficiency.
[0054] 2. Based on Example 1, with other conditions unchanged, the type and amount of reaction solvent were adjusted, and the yield and product purity were compared. The results are summarized in the table below: Table 2 Comparison of yield and mass under different reaction solvents and their dosage ratios
[0055] Table 2 shows that dichloromethane and trichloromethane have better yields than other solvents, while maintaining essentially the same quality. Since trichloromethane is a precursor chemical for toxic substances, dichloromethane is preferred.
[0056] 3. Based on Example 1, with other conditions unchanged, the type and amount of condensing agent were adjusted, and the reaction time was compared. The results are summarized in the table below: Table 3 Comparison of reaction completion time under different condensing agents and dosages
[0057] As can be seen from Table 3, the reaction time of condensing agent CDI at 5 times the equivalent is much shorter than that of other condensing agents. Therefore, CDI is preferred as the condensing agent.
[0058] 4. Based on Example 1, with other conditions unchanged, the type and amount of the deprotection reagent were adjusted, and the reaction time was compared. The results are summarized in the table below: Table 4 Comparison of reaction completion time under different deprotection reagents and dosages
[0059] As can be seen from the table, the reaction time of the Boc-deprotecting agent hydrochloric acid / ethyl acetate at 15 equivalents is much shorter than that of other condensing agents. Therefore, hydrochloric acid / ethyl acetate is preferred as the Boc-deprotecting agent. Similarly, piperidine at 15 equivalents is preferred as the Fmoc-deprotecting agent.
[0060] 5. Based on Example 1, with other conditions unchanged, the type and amount of cyclizing reagent were adjusted, and the reaction times were compared. The results are summarized in the table below: Table 5 Comparison of reaction completion time under different cyclizing reagents and dosages
[0061] The table shows that sodium hydroxide reacts significantly less than other reagents at 10 times the equivalent dosage.
[0062] 6. Based on Example 1, with other conditions unchanged, adjust the type and amount of inert solvent, compare the yield and product purity, and summarize the results in the table below: Table 6. Comparison of yield and mass under different inert solvents and dosage ratios
[0063] The data in the table show that when methyl tert-butyl ether is used as the inert solvent, the total product yield is high and the product purity is the highest when the amount used is 5 times.
[0064] 7. Based on Example 1, with other conditions unchanged, adjust the type and amount of inert solvent, compare the yields, and summarize the results in the following table: Table 7. Comparison of yield and mass at different crystallization temperatures
[0065] The data in the table show that the crystallization temperature at or below 25℃ has virtually no effect on the yield, so room temperature is the preferred crystallization temperature.
[0066] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.
Claims
1. A process for the "one-pot" preparation of the cyclic (L-proline-L-tryptophan) dipeptide characterized in that, The method comprises the following steps: S1, adding carboxyl-protected starting material 1 to a solvent, after stirring and dissolving, adding an organic amine, then adding a condensing agent and an amino-protected starting material 2, and controlling the temperature to react until the reaction is complete; after the reaction is complete, the reaction solution is washed with an acid aqueous solution and a base aqueous solution respectively; The starting material 1 is an ester or an acid salt of an ester of L-proline and L-tryptophan; S2, directly adding a deprotection reagent to the organic layer after washing in step S1, and adding a base aqueous solution to wash away the deprotection reagent after the reaction is complete under temperature control; S3, adding a cyclization reagent to the organic layer after washing in step S2, and controlling the temperature to react; S4, after the reaction in step S3 is complete, washing with water, concentrating the organic layer after washing, adding an inert solvent, controlling the temperature to crystallize for more than 2 hours, filtering, and drying to obtain a cyclic (L-proline-L-tryptophan) dipeptide.
2. The method according to claim 1, in step S1, the starting material 1 is one of L-proline methyl ester hydrochloride, L-proline ethyl ester hydrochloride, L-tryptophan methyl ester hydrochloride and L-tryptophan ethyl ester hydrochloride, preferably L-proline methyl ester hydrochloride; The molar ratio of the starting material 1 to the starting material 2 is 1:2-2:
1.
3. The method according to claim 1, in step S1, the reaction solvent is one of dichloromethane, chloroform, methanol, ethanol, isopropanol, ethyl acetate, preferably dichloromethane; The amount of the solvent is 2-20 times the volume of the starting material 1.
4. The method according to claim 1, in step S1, the organic amine is selected from one of diethylamine and triethylamine; The amount of the organic amine is 1-5 equivalents of the starting material 1.
5. The method according to claim 1, in step S1, the condensing agent is one or a combination of several of HOBT, HOAT, HATU, HBTU, EDCI, CDI, DCC and DIC; The amount of the condensing agent is 0.5-5 equivalents of the starting material 1.
6. The method according to claim 1, in step S1, the amino-protected starting material 2 is one of Boc-L-proline, Fmoc-L-proline, Boc-L-tryptophan and Fmoc-L-tryptophan, and the corresponding material is selected according to the starting material 1.
7. The method according to claim 1, in step S1, the acid aqueous solution is an aqueous solution of one of dilute hydrochloric acid, dilute sulfuric acid and glacial acetic acid; The base aqueous solution is an aqueous solution of one of sodium bicarbonate, sodium carbonate, sodium hydroxide, potassium carbonate, potassium hydroxide, concentrated ammonia and triethylamine.
8. The method according to claim 1, in step S2, the deprotection reagent comprises a de-Boc reagent and a de-Fmoc reagent; the de-Boc reagent is selected from one or a combination of several of trifluoroacetic acid, hydrochloric acid / dioxane and hydrochloric acid / ethyl acetate, preferably hydrochloric acid / ethyl acetate; the de-Fmoc reagent is selected from one or a combination of several of piperidine and diethylamine; The amount of the deprotection reagent is 1-20 equivalents of the starting material 2.
9. The method of claim 1, wherein in step S3, the cyclization reagent is one or more of sodium bicarbonate, sodium carbonate, sodium hydroxide, potassium carbonate, potassium hydroxide, concentrated ammonia, diethylamine, and triethylamine; and the amount of the cyclization reagent is 1 to 20 times the amount of the starting material 1.
10. The method of claim 1, wherein in step S4, the inert solvent is one or more of n-heptane, n-hexane, cyclohexane, diethyl ether, petroleum ether, methyl tert-butyl ether, and toluene; and the amount of the inert solvent is 0.5 to 10 times the amount of the starting material 1.