Ring (L-pyroglutamic acid-L-thioproline) and synthesis and application thereof
The synthesis of L-pyroglutamic acid-L-thioproline through a base-assisted reaction with a dehydrating agent addresses inefficiencies in existing methods, achieving high yield and reduced waste for large-scale production.
Patent Information
- Application Number
- CN202510470021.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-15
- Publication Date
- 2025-07-15
AI Technical Summary
The synthesis method of cyclic dipeptide in the prior art has problems such as poor reaction economy, lots of reaction waste, and low reaction efficiency. The immunomodulating agents of existing vaccines cannot effectively improve the immune protection effect of the vaccine.
Using pidomod as raw material, cyclic dipeptide is synthesized by constructing lactam bonds, organic bases or inorganic bases are used as acid binding agents, and the amount of dehydrating agents such as acetic anhydride is reduced, and the ring (L-pyroglutamate-L-thioproline) is synthesized, and it is added to the vaccine as an immunomodulatory agent for veterinary vaccines.
The large-scale preparation of cyclic dipeptides has been achieved, which has improved the immune protection durability and protection ability of the vaccine, and has significantly enhanced the immune efficacy of the vaccine.
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Figure CN120309635A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of chemical drugs, and particularly relates to a cyclo(L-pyroglutamic acid-L-thioproline) and its synthesis and application. Background Art
[0002] Vaccines are the most effective strategy for preventing and controlling the spread of infectious diseases. Currently, veterinary vaccines include inactivated vaccines, live attenuated vaccines, DNA vaccines, and subunit vaccines, etc. Vaccines with a single antigen component cannot provide effective immune protection through the induced immune response. Therefore, it is necessary to add adjuvants to vaccines to enhance the immune efficacy. Therefore, developing novel immunomodulators to enhance the immune efficacy of subunit vaccines or inactivated vaccines, induce cellular immune responses, and improve the antibody response level is an effective way to improve vaccines.
[0003] Pidotimod, as an immunomodulator, is widely used in the treatment and prevention of various diseases in children and adults, especially in the treatment and prevention of recurrent respiratory tract infections in children and chronic bronchitis and chronic obstructive pulmonary disease in adults. It has been confirmed that pidotimod can achieve immune enhancement by affecting innate immunity and adaptive immunity, and has therapeutic and preventive effects on a variety of immune-related diseases. In addition, pidotimod has great application potential in the prevention and treatment of animal diseases. It has been reported that adding pidotimod (0.5 g / L) to the drinking water of 6-day-old chickens can significantly improve the immune efficacy of Newcastle disease vaccine (Immunology Letters, 2017, 187, 15, 14 - 18). These studies indicate that the unique dipeptide structure of pidotimod plays an important role in immunomodulators.
[0004] Cyclic dipeptides are cyclic compounds formed by the condensation of two amino acids through peptide bonds. Due to their unique rigid structure and the presence of both hydrogen donors and hydrogen acceptors, they exhibit various biological activities. It is known that many natural products of cyclic dipeptides have biological activities such as participating in intercellular information transmission, anti-tumor, antibacterial, antioxidant, hypoglycemic, and improving the nervous system. The synthesis methods of cyclic dipeptides include chemical synthesis routes and biosynthetic routes. The chemical synthesis routes include solid-phase synthesis methods and solution synthesis methods.
[0005] According to literature reports, the methods for constructing imides through intramolecular reactions of dipeptides containing pyroglutamic acid to form cyclic dipeptides are as follows:
[0006] 1) Pyrrolidide - glycine (pGlu - Gly - OH) can undergo lactamization to obtain two mixtures when heated in acetic anhydride and acetic acid (J. Med. Chem. 1987, 30, 3, 498 - 503).
[0007]
[0008] (2) An intermediate obtained by reacting 5-ethoxy-2-pyrrolidone with mercaptoacetic acid or mercaptopropionic acid is heated in a mixed system of acetic acid and acetic anhydride to form a bicyclic structure compound (Pharm. Chem. J. 1996, 30, 9, 562 - 567).
[0009]
[0010] In the above methods, in order to construct the imide structural unit, acetic anhydride and acetic acid that are greatly in excess of the substrate are used in the reaction, or acetic anhydride and acetic acid are directly used as solvents, resulting in a relatively reduced yield. At the same time, these methods have poor reaction economy, a large amount of reaction waste, and low reaction efficiency, and are not suitable for the large-scale preparation of cyclic dipeptides. SUMMARY OF THE INVENTION
[0011] The technical problem to be solved by the present invention is to provide a cyclic (L-pyroglutamic acid-L-thioproline).
[0012] Another technical problem to be solved by the present invention is to provide the synthesis of the cyclic (L-pyroglutamic acid-L-thioproline).
[0013] The third technical problem to be solved by the present invention is to provide the application of the cyclic (L-pyroglutamic acid-L-thioproline).
[0014] To solve the above problems, a cyclic (L-pyroglutamic acid-L-thioproline) according to the present invention is characterized in that: the molecular formula of the cyclic (L-pyroglutamic acid-L-thioproline) is C9H 10 N2O3S, and its structural formula is as follows:
[0015]
[0016] The synthesis of the cyclic (L-pyroglutamic acid-L-thioproline) as described above is characterized in that: Pidotimod is mixed with a base, dissolved in an organic solvent, and stirred for 10 - 20 minutes to obtain a mixed solution; a dehydrating agent is added to the mixed solution, and refluxed for 8 - 12 hours. After the reaction is completely carried out, the reaction system is dried by evaporation to obtain a cyclic dipeptide molecule; the cyclic dipeptide molecule is fully dissolved in dichloromethane, washed with water, washed with saturated brine, dried, and separated and purified to obtain the cyclic (L-pyroglutamic acid-L-thioproline).
[0017] The reaction formula is as follows:
[0018]
[0019] Preferably:
[0020]
[0021] The molar ratio of the pidotimod to the base is 1:1 to 3; the mass-to-volume ratio of the pidotimod to the organic solvent is 1 g:5 to 10 mL; the molar ratio of the pidotimod to the dehydrating agent is 1:1 to 3.
[0022] The base refers to one of triethylamine, pyridine, potassium carbonate, potassium tert-butoxide, sodium tert-butoxide, sodium methoxide, cesium carbonate, and sodium carbonate.
[0023] The organic solvent refers to one of acetonitrile, dichloromethane, dichloroethane, tetrahydrofuran, ethyl acetate, DMF, and DMSO.
[0024] The dehydrating agent refers to one of acetic anhydride, trifluoroacetic anhydride, acetyl chloride, acetyl bromide, propionyl chloride, trichloroacetyl chloride, isobutyryl chloride, 4-chlorobutyryl chloride, methyl chloroformate, isobutyl chloroformate, phosphorus pentoxide, phosphorus oxychloride, phosphorus trichloride, phosphorus tribromide, phosphorus pentachloride, TsCl, sulfonyl chloride, thionyl chloride, oxalyl chloride, polyphosphoric acid, benzoyl chloride, oleoyl chloride, and Burgess reagent.
[0025] An application of a cyclic (L-pyroglutamic acid-L-thioproline) as described above, characterized in that: the cyclic (L-pyroglutamic acid-L-thioproline) is added to the vaccine as a veterinary vaccine immunomodulator.
[0026] The veterinary vaccine refers to one of inactivated vaccines, live attenuated vaccines, subunit vaccines, and nucleic acid vaccines.
[0027] The concentration of the cyclic (L-pyroglutamic acid-L-thioproline) in the veterinary vaccine is 0.25 mg / mL.
[0028] The present invention has the following advantages compared with the prior art:
[0029] 1. The present invention directly uses pidotimod as a raw material to synthesize cyclic dipeptides by constructing an intramolecular amide bond.
[0030] 2. The present invention uses organic bases, inorganic bases, etc. as acid-binding agents, with low cost.
[0031] 3. The raw materials of the present invention are easily available, with high yield, and at the same time reduce the usage amount of dehydrating agents such as acetic anhydride. The reaction economy is good and it is suitable for large-scale preparation.
[0032] 4. The cyclic (L-pyroglutamic acid-L-thioproline) of the present invention can be added to the vaccine as a veterinary vaccine immunomodulator, especially as an immunomodulator for foot-and-mouth disease antigen vaccines. It has a good antibody response compared with 206VG, improves the persistence of vaccine immune protection, and significantly enhances the protection ability. Brief Description of the Drawings
[0033] The following further describes in detail the specific embodiments of the present invention in conjunction with the accompanying drawings.
[0034] Figure 1 It is the single crystal structure diagram of Embodiment 1 of the present invention.
[0035] Figure 2 It is the nuclear magnetic resonance spectrum of Embodiment 1 of the present invention ( 1 1H NMR).
[0036] Figure 3 It is the nuclear magnetic resonance spectrum of Embodiment 1 of the present invention ( 13 13C NMR).
[0037] Figure 4 It is the evaluation of the compatibility of the immunomodulator and foot-and-mouth disease antigen on pig immunity in Embodiment 14 of the present invention.
[0038] Figure 5 It is the evaluation of the compatibility of the immunomodulator and foot-and-mouth disease antigen on pig immunity in Embodiment 15 of the present invention. Specific Embodiments
[0039] A cyclo(L-pyroglutamic acid-L-thioproline), the molecular formula of the cyclo(L-pyroglutamic acid-L-thioproline) is C9H 10 N2O3S, and its structural formula is as follows:
[0040]
[0041] The synthesis of the cyclo(L-pyroglutamic acid-L-thioproline): Mix pidotimod with a base, dissolve it in an organic solvent, and stir for 10 - 20 minutes to obtain a mixed solution; add a dehydrating agent to the mixed solution, reflux for 8 - 12 hours, after fully reacting completely, spin-dry the reaction system to obtain a cyclic dipeptide molecule; after the cyclic dipeptide molecule is fully dissolved in dichloromethane, it is successively washed with water, saturated brine, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by column chromatography to obtain cyclo(L-pyroglutamic acid-L-thioproline).
[0042] Among them: The molar ratio of pidotimod to the base is 1:1 - 3; the mass-volume ratio of pidotimod to the organic solvent is 1 g:5 - 10 mL; the molar ratio of pidotimod to the dehydrating agent is 1:1 - 3.
[0043] The base refers to one of triethylamine, pyridine, potassium carbonate, potassium tert-butoxide, sodium tert-butoxide, sodium methoxide, cesium carbonate, sodium carbonate, and triethylamine is preferred.
[0044] The organic solvent refers to one of acetonitrile, dichloromethane, dichloroethane, tetrahydrofuran, ethyl acetate, DMF, DMSO, and acetonitrile is preferred.
[0045] The dehydrating agent refers to one of acetic anhydride, trifluoroacetic anhydride, acetyl chloride, acetyl bromide, propionyl chloride, trichloroacetyl chloride, isobutyryl chloride, 4-chlorobutyryl chloride, methyl chloroformate, isobutyl chloroformate, phosphorus pentoxide, phosphorus oxychloride, phosphorus trichloride, phosphorus tribromide, phosphorus pentachloride, TsCl, sulfonyl chloride, thionyl chloride, oxalyl chloride, polyphosphoric acid, benzoyl chloride, oleoyl chloride, Burgess reagent, and preferably acetic anhydride.
[0046] Example 1:
[0047] Take 0.5 g of pidotimod, 5 mL of acetonitrile (analytical grade), and 0.28 mL of triethylamine and place them in a 25 mL round-bottom flask, and stir for 5 minutes. Then add acetic anhydride (1 eq), and reflux at 80 °C for 8 hours. After the reaction system is dried by rotation, add dichloromethane to dissolve completely, wash successively with water and saturated brine, dry, separate and purify to obtain 0.39 g of a white solid with a yield of 84%.
[0048] This white solid is cyclo(L-pyroglutamic acid-L-thioproline), (R f = 0.27, DCM / CH3OH = 10 / 1), m.p.: 219-221 °C, (c = 2.0 mg / ml, CH3OH); its single crystal structure diagram is as Figure 1 shown. The verification report of the single crystal data is generated by the website https: / / checkcif.iucr.org / , as shown in Table 1 for details.
[0049] Table 1 Verification Report of Single Crystal Data
[0050]
[0051] Nuclear magnetic resonance spectroscopy and high-resolution mass spectrometry information:
[0052] 1 H NMR (600 MHz, DMSO-d6): δ 4.84 (t, J = 8.7 Hz, 1H), 4.73 (t, J = 6.6 Hz, 1H), 4.67 (d, J = 9.9 Hz, 1H), 4.43 (d, J = 9.9 Hz, 1H), 3.42 - 3.35 (m, 2H), 2.63 - 2.57 (m, 1H), 2.46 - 2.41 (m, 1H), 2.22 - 2.15 (m, 2H).
[0053] 13 C NMR (151 MHz, DMSO-d6): δ 173.3, 165.5, 165.3, 62.8, 58.2, 48.6, 32.1, 31.3, 20.1.
[0054] HRMS (ESI): [M+H]+ Calculated for [C9H 11 N2O3S] + : 227.0485, found: 227.0476.
[0055] [M + Na] + Calculated for [C9H 10 N2O3SNa] + : 249.0304, found: 249.0296.
[0056] The nuclear magnetic resonance spectrum is as Figures 2 - 3 shown.
[0057] Example 2:
[0058] Take 2 g of pidotimod, 20 mL of acetonitrile (analytical grade), and 3.3 mL of triethylamine and place them in a 50 mL round-bottom flask, and stir for 10 - 20 minutes. Subsequently, add acetic anhydride (3 eq). After the slow addition is complete, transfer it to an oil bath, heat up, and reflux for 8 - 10 hours. After the reaction system is dried by rotation, add dichloromethane to dissolve it completely, wash it three times with water and then with saturated brine, dry it, separate and purify it to obtain 1.74 g of a white solid with a yield of 94%.
[0059] Example 3:
[0060] Take 2 g of pidotimod, 20 mL of acetonitrile (analytical grade), and 3.3 mL of triethylamine and place them in a 50 mL round-bottom flask, and stir for 10 - 20 minutes. Subsequently, add acetyl chloride (3 eq). After the slow addition is complete, transfer it to an oil bath, heat up, and reflux for 8 - 10 hours. After the reaction system is dried by rotation, add dichloromethane to dissolve it completely, wash it three times with water and then with saturated brine, dry it, separate and purify it to obtain 1.61 g of a white solid with a yield of 87%.
[0061] Example 4:
[0062] Take 2 g of pidotimod, 20 mL of acetonitrile (analytical grade), and 3.3 mL of triethylamine and place them in a 50 mL round-bottom flask, and stir for 10 - 20 minutes. Subsequently, add trifluoroacetic anhydride (3 eq). After the slow addition is complete, transfer it to an oil bath, heat up, and reflux for 8 - 10 hours. After the reaction system is dried by rotation, add dichloromethane to dissolve it completely, wash it three times with water and then with saturated brine, dry it with anhydrous sodium sulfate, separate and purify it to obtain 1.55 g of a white solid with a yield of 84%.
[0063] Example 5:
[0064] Take 0.5 g of pidotimod, 5 mL of tetrahydrofuran (analytical grade), and 0.28 mL of triethylamine and place them in a 25 mL round-bottom flask. Stir for 5 minutes. Then add acetic anhydride (0.38 mL, 2 eq), and reflux at 80 °C for 8 hours. After the reaction system is dried by evaporation, add dichloromethane to dissolve completely, wash successively with water and saturated brine, dry, separate and purify to obtain 0.4112 g of white solid, with a yield of 89%.
[0065] Example 6:
[0066] Take 0.5 g of pidotimod, 5 mL of dichloromethane (analytical grade), and 0.28 mL of triethylamine and place them in a 25 mL round-bottom flask. Stir for 5 minutes. Then add acetic anhydride (0.38 mL, 2 eq), and reflux at 80 °C for 8 hours. After the reaction system is dried by evaporation, add dichloromethane to dissolve completely, wash successively with water and saturated brine, dry, separate and purify to obtain 0.3097 g of white solid, with a yield of 67%.
[0067] Example 7:
[0068] Take 0.5 g of pidotimod, 5 mL of dichloroethane (analytical grade), and 0.28 mL of triethylamine and place them in a 25 mL round-bottom flask. Stir for 5 minutes. Then add acetic anhydride (0.38 mL, 2 eq), and reflux at 80 °C for 8 hours. After the reaction system is dried by evaporation, add dichloromethane to dissolve completely, wash successively with water and saturated brine, dry, separate and purify to obtain 0.3278 g of white solid, with a yield of 71%.
[0069] Example 8:
[0070] Take 0.5 g of pidotimod, 5 mL of ethyl acetate (analytical grade), and 0.28 mL of triethylamine and place them in a 25 mL round-bottom flask. Stir for 5 minutes. Then add acetic anhydride (0.38 mL, 2 eq), and reflux at 80 °C for 8 hours. After the reaction system is dried by evaporation, add dichloromethane to dissolve completely, wash successively with water and saturated brine, dry, separate and purify to obtain 0.3944 g of white solid, with a yield of 85%.
[0071] Example 9:
[0072] Take 0.5 g of pidotimod, 5 mL of acetonitrile (analytical grade), and potassium carbonate (0.28 g, 1 eq) and place them in a 25 mL round-bottom flask. Stir for 5 minutes. Then add acetic anhydride (0.38 mL, 2 eq), and reflux at 80 °C for 8 hours. After the reaction system is dried by evaporation, add dichloromethane to dissolve completely, wash successively with water and saturated brine, dry, separate and purify to obtain 0.3272 g of white solid, with a yield of 71%.
[0073] Example 10:
[0074] Take 0.5 g of pidotimod, 5 mL of acetonitrile (analytical grade), and sodium methoxide (0.11 g, 1 eq) and place them in a 25 mL round-bottom flask. Stir for 5 minutes. Then add acetic anhydride (0.38 mL, 2 eq) and reflux at 80 °C for 8 hours. After the reaction system is dried by rotation, add dichloromethane to dissolve completely, wash successively with water and saturated brine, dry, separate and purify to obtain 0.3353 g of a white solid with a yield of 72%.
[0075] Example 11:
[0076] Take 0.5 g of pidotimod, 5 mL of acetonitrile (analytical grade), and triethylamine (0.28 mL, 1 eq) and place them in a 25 mL round-bottom flask. Stir for 5 minutes. Then add acetic anhydride (0.38 mL, 2 eq) and reflux at 80 °C for 8 hours. After the reaction system is dried by rotation, add dichloromethane to dissolve completely, wash successively with water and saturated brine, dry, separate and purify to obtain 0.4069 g of a white solid with a yield of 88%.
[0077] Example 12:
[0078] Take 0.5 g of pidotimod, 5 mL of acetonitrile (analytical grade), and triethylamine (0.28 mL, 1 eq) and place them in a 25 mL round-bottom flask. Stir for 5 minutes. Then add acetic anhydride (0.57 mL, 3 eq) and reflux at 80 °C for 8 hours. After the reaction system is dried by rotation, add dichloromethane to dissolve completely, wash successively with water and saturated brine, dry, separate and purify to obtain 0.4216 g of a white solid with a yield of 91%.
[0079] Comparative Example 1: Take 0.5 g of pidotimod, acetic anhydride (1 equivalent), and 5 ml of acetonitrile and place them in a 25 ml round-bottom flask. Reflux at 80 °C for 8 hours. After the reaction system is dried by rotation, add dichloromethane to dissolve completely, wash successively with water and saturated brine, dry, separate and purify to obtain 0.1066 g of a white solid with a yield of 23%.
[0080] Comparative Example 2: Take 0.5 g of pidotimod and acetic anhydride (10 equivalents) and place them in a 25 ml round-bottom flask. Reflux at 80 °C for 8 hours. After the reaction system is dried by rotation, add dichloromethane to dissolve completely, wash successively with water and saturated brine, dry, separate and purify to obtain 0.3499 g of a white solid with a yield of 75%.
[0081] Comparative Example 3: Take 0.5 g of pidotimod, acetic anhydride (10 equivalents) and acetic acid (equal volume to acetic anhydride) and place them in a 25 ml round-bottom flask. Reflux at 80 °C for 8 hours. After the reaction system is dried by rotation, add dichloromethane to dissolve completely, wash successively with water and saturated brine, dry, separate and purify to obtain 0.2631 g of a white solid with a yield of 57%.
[0082] Table 2 shows the reaction yields of Comparative Examples 1-3, Example 1, and Examples 11-12.
[0083] Table 2 Reaction yields under different conditions
[0084] Sample Condition Solvent Yield / % Comparative Example 1 Acetic anhydride (1 eq) Acetonitrile (5 mL) 23 Comparative Example 2 Acetic anhydride (10 eq) \ 75 Comparative Example 3 Acetic anhydride (10 eq), acetic acid of equal volume \ 57 Example 1 Triethylamine (1 eq), acetic anhydride (1 eq) Acetonitrile (5 mL) 84 Example 11 Triethylamine (1 eq), acetic anhydride (2 eq) Acetonitrile (5 mL) 88 Example 12 Triethylamine (1 eq), acetic anhydride (3 eq) Acetonitrile (5 mL) 91
[0085] As can be seen from Table 2, compared with the methods described in Comparative Examples 1-3, the method using triethylamine (1 equivalent), acetic anhydride (1 equivalent), and acetonitrile as the solvent increased the yield from 23%, 75%, and 57% to 84%, with a significant increase in yield and a reduction in the amount of acetic anhydride used. In the reaction system of the comparative example, pimodimod and acetic anhydride first form a mixed anhydride intermediate, and then an intramolecular amide cyclization reaction is initiated through the deprotonation of the amino group to finally obtain the target product. When triethylamine is added to the system, triethylamine significantly enhances the cyclization reaction activity of the mixed anhydride intermediate by promoting the deprotonation process of the amino group. This promoting effect makes the intramolecular amidation reaction path more dominant, resulting in a 61% increase in the yield of the target product compared to the comparative example (yield comparison between Example 1 and Comparative Example 1).
[0086] Example 13:
[0087] Take 0.2 g of pimodimod, 5 mL of acetonitrile (analytical grade), and triethylamine (0.33 mL, 3 eq) and place them in a 25 mL round-bottom flask, and stir for 5 minutes. Then add different dehydrating agents (3 eq) and reflux at 80 °C for 8 hours. After the reaction system is dried by rotation, add dichloromethane to dissolve completely, wash successively with water and saturated brine, dry, separate and purify, and the results are shown in Table 3.
[0088] Table 3 Yields when adding different dehydrating agents
[0089] Dehydrating agent Product mass (g) Yield / % Dehydrating agent Product mass (g) Yield / % Acetyl chloride 0.1611 87 Phosphorus oxychloride 0.1148 62 Propionyl chloride 0.1259 68 Phosphorus pentoxide 0.1333 72 Acetyl bromide 0.0963 52 Trichloroacetyl chloride 0.1463 79 Acetic anhydride 0.1741 94 Isobutyryl chloride 0.1352 73 Trifluoroacetic anhydride 0.1555 84 4 - Chlorobutyryl chloride 0.1537 83 Thionyl chloride 0.0333 18 Oxalyl chloride 0.0407 22
[0090] As can be seen from Examples 1-13: When the dehydrating agents are acetic anhydride, acetyl chloride, trifluoroacetic anhydride, trichloroacetyl chloride, 4-chlorobutyryl chloride, isobutyryl chloride, phosphorus pentoxide, and phosphorus oxychloride, the yields are good. When the solvents are acetonitrile, tetrahydrofuran, dichloromethane, dichloroethane, and ethyl acetate, the yields are good. When the bases are triethylamine, sodium methoxide, and potassium carbonate, the yields are good. Finally, it is determined that acetic anhydride is preferably used as the dehydrating agent, triethylamine is used as the base, and acetonitrile is used as the solvent.
[0091] Application of a cyclic (L-pyroglutamic acid-L-thioproline): This cyclic (L-pyroglutamic acid-L-thioproline) is added to a vaccine as a veterinary vaccine immunomodulator to treat or prevent infectious diseases, including foot-and-mouth disease, etc.
[0092] Veterinary vaccines refer to one of inactivated vaccines, live attenuated vaccines, subunit vaccines, nucleic acid vaccines (mRNA or DNA vaccines), including intramuscular, intradermal or inhaled vaccines.
[0093] The concentration of cyclo(L-pyroglutamic acid-L-thioproline) in veterinary vaccines is 0.25 mg / mL.
[0094] The following Example 14 and Example 15 are two batches of experiments carried out simultaneously, with the same experimental procedures but different data sources.
[0095] In Example 14, cyclo(L-pyroglutamic acid-L-thioproline) was dissolved in ISA 206VG at a concentration of 0.25 mg / mL and formulated into a vaccine in combination with foot-and-mouth disease antigen.
[0096]
Experimental pig immunization experiment
[0097] Ten pigs were randomly divided into 2 groups (n = 5), namely the experimental group (OAIP) and the control group (206VG), and each group was intramuscularly injected with an equal amount of vaccine (2 mL / head).
[0098]
Detection of antibodies in serum
[0099] Porcine serum samples were collected on the 7th, 14th, 21st, 28th, 35th, 42nd, 49th, and 56th days respectively to detect the antibody levels.
[0100]
Results
[0101] As Figure 4 shown, on the 14th day after vaccination, the antibody levels in the experimental group and the control group increased rapidly. The antibody level in the experimental group remained at a relatively high level all the time, and the uniformity was higher than that of ISA 206VG.
[0102] In Example 15, cyclo(L-pyroglutamic acid-L-thioproline) was dissolved in ISA 206VG at a concentration of 0.25 mg / mL and formulated into a vaccine in combination with foot-and-mouth disease antigen.
[0103]
Experimental pig immunization experiment
[0104] Ten pigs were randomly divided into 2 groups (n = 5), namely the experimental group (OAIP) and the control group (206VG), and each group was intramuscularly injected with an equal amount of vaccine (2 mL / head).
[0105]
Detection of antibodies in serum
[0106] Porcine serum samples were collected on the 7th, 14th, 21st, 28th, 35th, 42nd, and 49th days respectively to detect the antibody levels.
[0107]
Results
[0108] AsFigure 5 As shown, on the 14th day after vaccination, the antibody levels in the experimental group and the control group increased rapidly. The antibody level in the experimental group was always maintained at a high level, and the homogeneity was higher than that of ISA 206VG.
[0109] In summary, after the cyclic (L-pyroglutamic acid-L-thioproline) of the present invention is combined with the foot-and-mouth disease antigen and applied to immunized pigs with foot-and-mouth disease vaccine, the antibody response homogeneity is superior to that of the control group.
Claims
1. A ring (L-pyroglutamic acid-L-thioproline), characterized in that: The molecular formula of the ring (L-pyroglutamyl-L-thioproline) is C9H 10 N2O3S, and its structural formula is as follows:
2. The synthesis of a ring (L-pyroglutamic acid-L-thioproline) as described in claim 1, characterized in that: Mix pidotimod with an alkali, dissolve it in an organic solvent, and stir for 10 - 20 minutes to obtain a mixed solution; add a dehydrating agent to the mixed solution, reflux for 8 - 12 hours, and after the reaction is complete, spin-dry the reaction system to obtain a cyclic dipeptide molecule; after the cyclic dipeptide molecule is fully dissolved in dichloromethane, wash it with water, wash it with saturated brine, dry it, and purify it to obtain cyclo(L-pyroglutamic acid-L-thioproline).
3. The synthesis of a ring (L-pyroglutamic acid-L-thioproline) as claimed in claim 2, characterized in that: The molar ratio of pidotimod to the alkali is 1:1 - 3; the mass-volume ratio of pidotimod to the organic solvent is 1 g:5 - 10 mL; the molar ratio of pidotimod to the dehydrating agent is 1:1 - 3.
4. The synthesis of a ring (L-pyroglutamic acid-L-thioproline) according to claim 3, characterized in that: The alkali refers to one of triethylamine, pyridine, potassium carbonate, potassium tert-butoxide, sodium tert-butoxide, sodium methoxide, cesium carbonate, and sodium carbonate.
5. The synthesis of a ring (L-pyroglutamic acid-L-thioproline) according to claim 3, characterized in that: The organic solvent refers to one of acetonitrile, dichloromethane, dichloroethane, tetrahydrofuran, ethyl acetate, DMF, and DMSO.
6. The synthesis of a ring (L-pyroglutamic acid-L-thioproline) according to claim 3, characterized in that: The dehydrating agent refers to one of acetic anhydride, trifluoroacetic anhydride, acetyl chloride, acetyl bromide, propionyl chloride, trichloroacetyl chloride, isobutyryl chloride, 4-chlorobutyryl chloride, methyl chloroformate, isobutyl chloroformate, phosphorus pentoxide, phosphorus oxychloride, phosphorus trichloride, phosphorus tribromide, phosphorus pentachloride, TsCl, sulfonyl chloride, thionyl chloride, oxalyl chloride, polyphosphoric acid, benzoyl chloride, oleoyl chloride, and Burgess reagent.
7. Use of a ring (L-pyroglutamic acid-L-thioproline) as described in claim 1, characterized in that: This cyclo(L-pyroglutamic acid-L-thioproline) is added to the vaccine as an immunomodulator for veterinary vaccines.
8. Use of a ring (L-pyroglutamic acid-L-thioproline) according to claim 7, characterized in that: The veterinary vaccine refers to one of inactivated vaccines, live attenuated vaccines, subunit vaccines, and nucleic acid vaccines.
9. Use of a ring (L-pyroglutamic acid-L-thioproline) as claimed in claim 7, characterized in that: The concentration of cyclo(L-pyroglutamic acid-L-thioproline) in the veterinary vaccine is 0.25 mg / mL.