Application of pyroglutamic acid butyl ester in preparation of antihypertensive drugs

Abstract

The invention discloses application of pyroglutamic acid butyl ester in preparation of antihypertensive drugs, experiments prove that the pyroglutamic acid butyl ester has a remarkable antihypertensive effect on spontaneously hypertensive rats, and the effect of the pyroglutamic acid butyl ester is obviously superior to that of a positive control drug olmesartan medoxomil; particularly, along with the prolonging of the treatment time, the advantage of the antihypertensive effect is more obvious than that of olmesartan medoxomil; and meanwhile, the safety is good. Therefore, the pyroglutamic acid butyl ester has a remarkable antihypertensive effect, can be applied to preparation of antihypertensive drugs, and has a good market prospect in development of clinical antihypertensive drugs.

Description

Technical Field

[0001] The invention relates to the field of biomedicine, and in particular to application of butyl pyroglutamate in the preparation of antihypertensive drugs. Background Art

[0002] Hypertension is a systemic disease characterized by an increase in arterial pressure above the normal limit (140 / 90 mmHg), accompanied by damage to target organs such as the heart, brain, kidneys and blood vessels. Studies have found that hypertension is a major risk factor for stroke, heart failure and renal failure, and is closely related to coronary heart disease and diabetes. A small number of patients (about 5%) with secondary hypertension with clear etiology can be effectively treated by eliminating the cause. The etiology of most patients with hypertension is still unclear, and long-term intervention with antihypertensive drugs is required to control the appropriate blood pressure level. Currently, the commonly used antihypertensive drugs in clinical practice are mainly diuretics, β-receptor blockers, calcium channel blockers, angiotensin converting enzyme inhibitors and angiotensin II receptor antagonists. However, most drugs have side effects such as hypotension, dry cough and hyperkalemia, and some patients have developed tolerance to some existing drugs. Therefore, finding new antihypertensive drugs has important clinical value and practical significance.

[0003] Fresh bamboo juice is the liquid that naturally drips from the fresh stems of Phyllostrachys glauca, P. nuda and several other plants of the same genus after being heated or roasted. It is sweet and bitter in nature, cold in nature, enters the heart and stomach meridians, and has the effects of clearing away heat and resolving phlegm, calming the nerves and opening the orifices. It is often used clinically to treat lung heat, cough with excessive phlegm, asthma, chest tightness, tongue stiffness due to stroke, and excessive phlegm. [1] , which has obvious cough-relieving effect. Studies have shown that fresh bamboo sap mainly contains flavonoids, phenylpropanoids, amino acids, phenolic acids, inorganic elements and their glycoside components. [1,2] .

[0004] In the study of the chemical composition and activity of fresh bamboo sap, the present invention isolated L-butyl pyroglutamate (Butyl5-oxoprolinate or butyl pyroglutamate), the molecular formula of which is C9H 15 NO3. Literature shows that this compound can enhance melanin production and tyrosinase activity and has the potential to treat melanoma and vitiligo skin diseases [3] ; It can also reduce the toxicity of the anti-tumor drug cisplatin to the kidneys [4] However, there is no report on other pharmacological activities of this component. Therefore, the present invention studies the pharmacodynamics of this compound on a variety of model animals and finds that this compound has a significant antihypertensive effect.

[0005] The compound can be isolated from a variety of Chinese herbal medicines such as fresh bamboo sap, silkworm, dandelion, northern bupleurum, mulberry, etc. [4-6] ; At the same time, according to the literature method [7] , prepared by chemical synthesis; it can also be purchased directly from the market.

[0006] References:

[0007] [1] Feng Huimin, Hu Yunfei, Zeng Qianyao, et al. Research progress on chemical composition and quality control methods of fresh bamboo sap[J]. Chinese Journal of Traditional Chinese Medicine, 2021, 46(11): 2753-2759.

[0008] [2] Lin Kaipeng. Study on chemical composition and quality standard of fresh bamboo sap[D]. Nanchang University, 2020.08.

[0009] [3]Lee CL, Gao ZA, Jhan YL, et al. Tuliposide H–J and bioactive components from the bulb of Amana edulis[J]. Molecules, 2021,26:5907.

[0010] [4]Lee D,Yu JS,Lee SR,et al.Beneficial effects of bioactive compounds in mulberry fruits against cisplatin-induced nephrotoxicity[J].Int.J.Mol.Sci.,2018,19:1117.

[0011] [5] Huang Jin, Liu Yan, Pan Juan, et al. Study on the chemical composition of the n-butanol layer of the aerial part of Bupleurum chinense[J]. Journal of Traditional Chinese Medicine, 2020, 48(1): 38-41.

[0012] [6] Yan Mengqi, Wei Yishen, Huang Wanyi, et al. Study on the chemical composition of silkworm[J]. Journal of Guangxi Normal University (Natural Science Edition), 2023, 41(2): 124-130.

[0013] [7] Peng Xiaohai, Luo Jin, Zhong Min, et al. Synthesis and application of L-pyroglutamate [J]. Chemistry and Bioengineering, 2011, 28, 10: 79-80. Summary of the invention

[0014] The invention aims to provide application of butyl pyroglutamate in preparing antihypertensive drugs.

[0015] The molecular formula of the butyl pyroglutamate of the present invention is C9H 15 NO3, its chemical structure is as follows:

[0016]

[0017] Its chemical name is Butyl 5-oxoprolinate or Butyl 5-oxopyrrolidine-2-carboxylate; it can be one of the chiral isomers L-butyl pyroglutamate or D-butyl pyroglutamate, or it can be the racemic DL-butyl pyroglutamate.

[0018] The butyl pyroglutamate of the present invention can be prepared by separation and purification from fresh bamboo sap, silkworm, dandelion, bupleurum chinense, mulberry and other traditional Chinese medicines, and can also be prepared by a synthetic method or obtained by commercial purchase.

[0019] The present invention adopts spontaneous hypertension model rats, takes Olmesartan medoxomil as a positive control drug, and measures the antihypertensive effect of butyl pyroglutamate by single-dose single-time and single-dose multiple-time oral administration. Experiments have shown that butyl pyroglutamate has a significant antihypertensive effect on spontaneously hypertensive rats, especially after single-dose multiple administration for 15 days, its effect is significantly better than the positive control drug Olmesartan medoxomil, and the blood pressure drops by 30-36% compared with before administration, and drops by 45-51% compared with the blood pressure of blank group rats. This shows that butyl pyroglutamate has a significant antihypertensive effect, and its antihypertensive effect is better than the positive control drug Olmesartan medoxomil. At the same time, the cytotoxicity IC of the compound on the cell line HCT-116 is 50 It is greater than 2048 μg / mL, which is much less than the positive control drug doxorubicin (0.2 μg / mL), indicating that it has good safety. The above indicates that butyl pyroglutamate not only has a significant blood pressure lowering effect, but also indicates good safety. Therefore, butyl pyroglutamate can be used to prepare antihypertensive drugs and shows good market prospects for its development as a clinical antihypertensive drug. DETAILED DESCRIPTION

[0020] Example 1: Synthesis of D-pyroglutamate butyl ester

[0021] Add 25.8g (0.2mol) of D-pyroglutamic acid and 274mL (3.0mol) of butanol to a 500mL four-necked flask, stir to dissolve, cool to 0°C in an ice-salt bath, slowly drop 17.4mL (0.24mol) of freshly distilled thionyl chloride, and control the reaction temperature at 0-5°C during the dropwise addition. Continue stirring and reacting for 30min after the dropwise addition is completed, add triethylamine to adjust the pH value to 7-8, then remove the ice-salt bath and slowly heat to 30°C to react until the raw material spots on TLC disappear. Remove the solvent butanol, unreacted dichlorosulfuron and triethylamine in the system by vacuum distillation to obtain white crystals. Dissolve with 100mL of ethyl acetate, filter to remove triethylamine hydrochloride, add saturated brine to the filtrate, wash 3 times, separate, dry the ethyl acetate layer with anhydrous sodium sulfate, filter, and vacuum distill the filtrate to obtain a light yellow oily substance, i.e., 31.4g of D-pyroglutamic acid butyl ester, with a yield of 84.8% and a purity of 99.2%. HR ESI-MS m / z:186.1137[M+H] + ,208.0959[M+Na] + , molecular formula is C9H 15 NO3; 13 C NMR (150MHz, DMSO-d6))δ:180.6(C-5),172.6(-COO),65.6(C-1'),56.9(C-2),31.2(C-2'),30.5(C-4),25.6(C-3),19.7(C-3'),13.9(C-4').By comparing with the corresponding data of L-pyroglutamic acid butyl ester, it was identified as D-pyroglutamic acid n-butyl ester.

[0022] Example 2: Evaluation of the efficacy of butyl pyroglutamate on spontaneously hypertensive rats

[0023] 1. Experimental animals and test drugs

[0024] Experimental animals: 180 SPF spontaneously hypertensive rats (SHR), weighing 160-180 g, were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd. The animals were raised to a systolic blood pressure of about 180 mmHg and randomly divided into 18 groups a-i (single dose single administration) and A-I (single dose multiple administration), with 10 rats in each group.

[0025] Test drugs: L-pyroglutamic acid, D-pyroglutamic acid, L-pyroglutamic acid butyl ester were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; D-pyroglutamic acid butyl ester was prepared according to the method of Example 1; DL-pyroglutamic acid butyl ester was obtained by mixing D- and L-pyroglutamic acid butyl esters in a ratio of 1:1; Olmesartan medoxomil (positive control drug) was purchased from Hubei Huazhong Pharmaceutical Co., Ltd. The drugs were dissolved in an appropriate amount of 0.3% CMC-Na solution and suspended before use; an equal volume of 0.3% CMC-Na solution was used as a blank control.

[0026] 2. Dosage and method

[0027] Single dose single administration amount: referring to the clinical use of the positive drug Olmesartan, the Olmesartan medoxomil group (2 mg / kg, group a) was administered by gavage; the L-pyroglutamic acid group (40 mg / kg, b), the D-pyroglutamic acid group (40 mg / kg, c), the L-pyroglutamate butyl ester low-dose group (20 mg / kg, group d), the L-pyroglutamate butyl ester high-dose group (40 mg / kg, group e), the D-pyroglutamate butyl ester low-dose group (20 mg / kg, group f), the D-pyroglutamate butyl ester low-dose group (40 mg / kg, group g), and the DL-pyroglutamate butyl ester group (40 mg / kg, group h) were administered by gavage, and the blank group (group i) was administered with the same volume of 0.3% CMC-Na solution by gavage.

[0028] Single dose multiple administration: Olmesartan medoxomil group (2mg.kg -1 .d -1 , Group A) was administered by gavage; L-pyroglutamate group (40 mg.kg -1 .d -1 , group B), D-pyroglutamate group (40 mg.kg -1 .d -1 , C group), L-pyroglutamate butyl ester low-dose group (20 mg.kg -1 .d -1 , group D), high-dose L-pyroglutamate butyl ester group (40 mg.kg -1 .d -1 , Group E), D-pyroglutamate low-dose group (20 mg.kg -1 .d -1 , Group F), D-pyroglutamate low-dose group (40 mg.kg -1 .d -1 , G group), DL-pyroglutamate butyl ester group (40 mg.kg -1 .d -1 , H group), once a day, by gavage for 15 days; the blank group (I group) was given the same volume of 0.3% CMC-Na solution.

[0029] 3. Blood pressure test

[0030] The tail vein blood pressure of SHR rats was measured using the BP-2000SERTES II blood pressure analysis system. Single-dose single administration: Blood pressure was measured before administration and 2.5 h after administration. The blood pressure before administration was recorded as P0, and the blood pressure after administration was recorded as P 2.5 , according to the formula (P0-P 2.5) / P0×100% to calculate the percentage of blood pressure drop before and after administration. Single dose multiple administration: Measure blood pressure before administration and 2.5h after the last administration. The blood pressure before administration is recorded as P0 and the blood pressure after administration is recorded as P 15 , according to the formula (P0-P 15 ) / P0×100% to calculate the percentage of blood pressure decrease before and after administration.

[0031] 4. Test results:

[0032] (1) Single dose administration

[0033] Table 1 Percentage decrease in blood pressure of experimental rats before and after single dose administration (n=10)

[0034]

[0035]

[0036] Note: Compared with the blank control group i, *P<0.05 indicates a significant difference; **P<0.01 indicates a very significant difference. Compared with the positive control olmesartan medoxomil group a, # P < 0.05 indicated a significant difference; ## P<0.01 showed a highly significant difference.

[0037] As shown in Table 1, after a single dose administration, compared with the blank group i, the positive control drug Olmesartan medoxomil group a and all the dose groups d to h of butyl pyroglutamate all had extremely significant hypotensive effects (P < 0.01), while the 40 mg / kg pyroglutamic acid groups b and c did not show significant hypotensive effects (P > 0.05). At the same time, compared with the positive control Olmesartan medoxomil group a, it can be seen that the hypotensive effects of the high-dose butyl pyroglutamate groups e, g, and h were significantly better than those of the positive control drug Olmesartan medoxomil group i (P < 0.01). This shows that a single dose of butyl pyroglutamate has a significant hypotensive effect, and the hypotensive effect of the high-dose group is significantly better than that of the positive control drug Olmesartan medoxomil.

[0038] (2) Single dose multiple administration

[0039] Single doses were given multiple times for 15 days. The changes in tail vein blood pressure and blood pressure of SHR rats before and after treatment are shown in Table 2.

[0040] Table 2 Percentage decrease in blood pressure of experimental rats before and after single dose multiple administration (n=10)

[0041]

[0042]

[0043] Note: Compared with the blank control group I, *P<0.05 indicates a significant difference, **P<0.01 indicates a very significant difference; compared with the positive control olmesartan medoxomil group A, # P<0.05 showed significant difference. ## P < 0.01 indicates a highly significant difference; negative values ​​in the table indicate that the blood pressure 2.5 hours after the last administration was higher than that before administration.

[0044] As shown in Table 2, after 15 days of feeding, the blood pressure of rats in the blank group I without drug administration increased by 14.75%. After single-dose multiple administration, the blood pressure of rats after 15 days of Olmesartan Medoxomil intervention decreased significantly compared with the blank group I (P < 0.01), and there was no significant difference compared with the decrease 15 days ago; the blood pressure of rats in all dose groups of 15 days of butyl pyroglutamate intervention decreased significantly (P < 0.01), and was significantly better than the positive control drug Olmesartan Medoxomil (P < 0.01). Compared with the blank group, the blood pressure of rats in the pyroglutamic acid group decreased, but there was no significant difference (P>0.05); at the same time, the blood pressure of rats in the high-dose group of pyroglutamic acid butyl ester decreased to a greater extent than that of rats in the low-dose group, but there was no significant difference between the two (P>0.05). Combined with Table 1, it can be seen that the antihypertensive effect of rats after 15 days of intervention with 20mg / kg and 40mg / kg was significantly improved compared with that before administration, which was significantly better than the unchanged antihypertensive effect of Olmesartan medoxomil, but there was no significant difference between the low-dose and high-dose groups (P>0.05). This shows that pyroglutamic acid butyl ester not only has a significant antihypertensive effect, but also after a period of drug intervention, the antihypertensive effect of both the low-dose and high-dose groups is significantly better than that of Olmesartan medoxomil, and the antihypertensive effect is also significantly improved compared with Olmesartan medoxomil.

[0045] 4. Conclusion

[0046] It can be seen from the above that butyl glutamate has a significant antihypertensive effect, and its effect is significantly better than the positive control drug Olmesartan Medoxomil, especially after a period of treatment, its further improvement in antihypertensive effect is significantly better than Olmesartan Medoxomil. Therefore, the butyl glutamate described in the present invention can be used for the preparation of antihypertensive drugs, and shows excellent development and application prospects.

[0047] Example 3: Cytotoxicity assay of butyl pyroglutamate

[0048] The cytotoxicity of butyl pyroglutamate on the cell line HCT-116 was determined by MTT method. The specific process was as follows: the cell line HCT-116 in the logarithmic growth phase was digested with 0.01% trypsin, counted, and the number of cells was 2.0×10 3The cells were seeded at a density of 100 μL / well in a 96-well plate, and each well was incubated at 37°C in a 5% CO2 incubator overnight. Using doxorubicin as a control, 7 concentration gradients were set for each compound, and 3 replicates were set for each concentration. Each concentration was added to the corresponding wells and cultured in a 5% CO2 37°C incubator for 72 hours. 5 mg·mL -1 20 μL of MTT was added, incubated at 37°C for 3 hours, the supernatant was discarded, 100 μL / well DMSO was added, mixed and oscillated to dissolve, and the absorbance OD of each well was measured using a Spectra MAX 340 spectrophotometer (the measurement wavelength L1 was 550 nm, and the reference wavelength L2 was 690 nm). The growth inhibition rate of the compound on the cell line HCT-116 was calculated according to the following formula:

[0049] Growth inhibition rate = (1-average OD value of the drug group / average OD value of the control group) × 100% Where, average OD value = [(OD L1 -OD L2 )1+(OD L1 -OD L2 )2+(OD L1 -OD L2 )3] / 3.

[0050] Then the growth inhibition rate was plotted against different concentrations of the compound, and the half-maximal inhibitory concentration (IC 50 ), which is used as the basis for judging the cytotoxicity of the compound: IC 50 The smaller the value, the greater the cytotoxicity of the compound, indicating that the compound is more toxic to human cells. The results showed that whether it is L-butyl pyroglutamate, D-butyl pyroglutamate, or LD-butyl pyroglutamate, the IC 50 All are greater than 2048 μg / mL, while the positive control drug is 0.2 μg / mL. It can be seen from this that the cytotoxicity of butyl pyroglutamate to the cell line HCT-116 is very small, and is much lower than the positive control drug doxorubicin. Therefore, it is predicted that the butyl pyroglutamate of the present invention is less toxic to human cells and has good safety. Combined with the conclusion of the blood pressure reduction test of butyl pyroglutamate, it is further shown that butyl pyroglutamate can be used for the preparation of antihypertensive drugs, and shows its good development and market application prospects as an antihypertensive drug.

[0051] Example 4: Preparation of DL-pyroglutamate butyl ester capsules

[0052] (1) Prescription: 50 mL of DL-butyl pyroglutamate, 60 g of starch, 40 g of lactose, appropriate amounts of sodium lauryl sulfate, 4% PVP, and magnesium stearate, a total of 1,000 capsules.

[0053] (2) Preparation: Dissolve DL-butyl pyroglutamate in an appropriate amount of 95% ethanol, mix with starch, lactose and sodium dodecyl sulfate in a mixer and stir for 15 minutes. Slowly add 4% PVP as a binder while stirring, stir at high speed for 5 minutes, pass the wet granules through a 16-mesh sieve, dry in an oven at 55-60°C, and then granulate. Add lubricant magnesium stearate and mix well. Fill into capsules to prepare DL-butyl pyroglutamate capsules.

[0054] Example 5: Preparation of L-pyroglutamate butyl ester tablets

[0055] (1) Prescription: 200 mL of L-pyroglutamate butyl ester, 40 g of starch, 24 g of 10% starch slurry, 23 g of dry starch, 3 g of magnesium stearate, made into 1,000 tablets.

[0056] (2) Preparation: Dissolve L-pyroglutamate butyl ester in an appropriate amount of 95% ethanol, mix with starch, add starch slurry to make a soft material, squeeze with a 14-mesh sieve to granulate, dry at 70-80°C, sieve with a 12-mesh sieve to size the granules, add dry starch and magnesium stearate, mix well, and then press into tablets to prepare L-pyroglutamate butyl ester tablets.

[0057] The above description and analysis show that butyl pyroglutamate has a significant antihypertensive effect on spontaneously hypertensive rats, and its effect is significantly better than the positive control drug Olmesartan Medoxomil, especially with the extension of treatment time, its antihypertensive effect is more obvious than Olmesartan Medoxomil; at the same time, it has good safety. This further proves that butyl pyroglutamate can be used for the preparation of antihypertensive drugs, and also shows that it has good development and market application prospects in the preparation of antihypertensive drugs.

[0058] Therefore, the present invention proves through experiments that butyl pyroglutamate has a significant hypotensive effect on spontaneously hypertensive rats, and the hypotensive effect of a single dose of 40 mg / kg butyl pyroglutamate administered by oral gavage is significantly better than that of the positive control drug olmesartan medoxomil (2 mg / kg); after multiple oral gavages for 15 days, the hypotensive effect of all butyl pyroglutamate dosage groups (20 mg / kg and 40 mg / kg) is significantly better than that of the positive control drug olmesartan medoxomil; at the same time, the IC values ​​of all chiral isomers of butyl pyroglutamate in the cytotoxicity test of HCT-116 are 50 All of them are greater than 2048 μg / mL, and have good safety. Therefore, it is proved that butyl pyroglutamate can be used for the preparation of antihypertensive drugs, and it also shows that it has good development and market application prospects in the preparation of antihypertensive drugs.

Claims

1. Application of butyl pyroglutamate in the preparation of antihypertensive drugs.

2. The use according to claim 1, characterized in that: The molecular formula of the butyl pyroglutamate is C9H 15 NO3, the chemical structure is as follows: 。 3. The use according to claim 1, characterized in that: The butyl pyroglutamate is an isomer of the chiral isomers L-butyl pyroglutamate and D-butyl pyroglutamate, or a rotational isomer DL-butyl pyroglutamate.

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