CN107963987B - A stachydrine derivative, its preparation method and application in preparing medicine for treating cardiovascular disease and cerebrovascular disease - Google Patents

A stachydrine derivative, its preparation method and application in preparing medicine for treating cardiovascular disease and cerebrovascular disease Download PDF

Info

Publication number
CN107963987B
CN107963987B CN201610907125.1A CN201610907125A CN107963987B CN 107963987 B CN107963987 B CN 107963987B CN 201610907125 A CN201610907125 A CN 201610907125A CN 107963987 B CN107963987 B CN 107963987B
Authority
CN
China
Prior art keywords
acid
stachydrine
derivative
cardiovascular
group
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN201610907125.1A
Other languages
Chinese (zh)
Other versions
CN107963987A (en
Inventor
李文保
李峰
张良
张鹏
代龙
管华诗
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Qingdao Marine Biomedical Research Institute Co Ltd
Original Assignee
Qingdao Marine Biomedical Research Institute Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Qingdao Marine Biomedical Research Institute Co Ltd filed Critical Qingdao Marine Biomedical Research Institute Co Ltd
Priority to CN201610907125.1A priority Critical patent/CN107963987B/en
Priority to PCT/CN2017/106555 priority patent/WO2018072689A1/en
Publication of CN107963987A publication Critical patent/CN107963987A/en
Application granted granted Critical
Publication of CN107963987B publication Critical patent/CN107963987B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D207/00—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D207/02—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D207/04—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members
    • C07D207/10—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D207/16—Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/401—Proline; Derivatives thereof, e.g. captopril
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/05—Dipeptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06139—Dipeptides with the first amino acid being heterocyclic
    • C07K5/06165—Dipeptides with the first amino acid being heterocyclic and Pro-amino acid; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Abstract

The invention provides a stachydrine derivative, a preparation method thereof and application thereof in preparing medicaments for treating cardiovascular and cerebrovascular diseases, wherein the stachydrine derivative has a structure shown in a general formula (I), and the synthesis method comprises the following steps:L-proline a and formaldehyde are subjected to hydrogenation reaction under the catalytic action of a catalyst to prepare the compoundN-methyl-L-proline b;N-methyl-LProline b with anthranilic acid,γCondensation reaction of aminobutanamide, glutamine and glycine under the action of Dicyclohexylcarbodiimide (DCC) to obtain stachydrine derivatives B-1, B-2, B-3 and B-4. Pharmacological experiments prove that the stachydrine derivative provided by the invention has the effect of protecting nerves and treating cardiovascular and cerebrovascular diseases, and the invention also provides application of the stachydrine derivative in preparing a medicament for treating the cardiovascular and cerebrovascular diseases.

Description

A stachydrine derivative, its preparation method and application in preparing medicine for treating cardiovascular disease and cerebrovascular disease
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to a stachydrine derivative, a preparation method thereof and application thereof in preparing medicines for treating cardiovascular and cerebrovascular diseases.
Background
Stroke is an intractable disease seriously harming the life safety of human beings, and has the characteristics of high morbidity, high disability rate and high mortality rate. In the antecedent of death cause published by the ministry of health of China in 2008, cerebral apoplexy is the third cause of death in cities and the second cause of death in rural areas. Epidemiological investigations have shown that the incidence of stroke in young adults has been increasing in recent years, with stroke occurring in adults under the age of 45 years being reported in proportions of 5% and 13.44% in western and domestic countries, respectively. The cerebral apoplexy is divided into cerebral arterial thrombosis and hemorrhagic apoplexy according to the properties, wherein the cerebral arterial thrombosis accounts for 75 to 85 percent of the total number of stroke patients.
There are many treatment modes for treating cerebral arterial thrombosis, and drug therapy is one of the main treatment modes adopted for treating cerebral arterial thrombosis at present. The current clinical therapeutic drugs are mainly divided into the following categories: thrombolytic drugs, anti-platelet aggregation drugs, defibrotizing drugs, anticoagulant drugs, neuroprotective drugs, and the like. Through continuous efforts, great progress has been made in the research of drugs for treating ischemic stroke, but many problems remain unsolved. Such as the expanded application range of the thrombolysis 'time window', the drug selection and the combined use of the anticoagulation and the defibrination treatment, the clinical effectiveness and the safety of the neuroprotective drugs, and the like. With the deep research on the pathogenesis and the disease mechanism of the stroke, the understanding of the treatment target is gradually clear and comprehensive, so that more research directions are provided for the research and the clinical use of the stroke treatment medicine.
The motherwort herb is bitter and pungent and is slightly cold, enters liver and pericardium channels, and has the effects of activating blood, regulating menstruation, removing blood stasis, promoting tissue regeneration, inducing diuresis, reducing swelling and the like. Mainly treats irregular menstruation, metrorrhagia, dystocia, dysmenorrheal, postpartum stasis and other symptoms, and is called as 'Xue Jia Sheng Yao' and 'Jing Yu Liang Yao'. Stachydrine (Stachydrine) is also called Proline Betaine (Proline Betaine) or N, N-Dimethylproline (N, N-Dimethylproline), is the simplest pyrrole alkaloid and is one of the main effective components of the Chinese medicine motherwort. The chemical structure of stachydrine is as follows:
Figure BDA0001133597150000011
modern pharmacological studies have shown that stachydrine has a wide range of physiological activities: peripheral blood vessel dilatation, blood flow increase, platelet aggregation resistance and blood viscosity reduction; can improve the blood flow of coronary artery and cardiac muscle trophism, reduce the necrotic amount of cardiac muscle cells, reduce the vascular resistance, improve microcirculation, slow down heart rate, reduce cardiac output and so on, and is expected to become a good therapeutic drug for cardiovascular and cerebrovascular system diseases; can inhibit the occurrence of breast cancer and uterine myopathy; has expectorant, antitussive, and bronchial smooth muscle relaxing effects.
Studies of Yangjiehen and the like find that the stachydrine has a protective effect on acute myocardial ischemia-reperfusion injury of rats (Mayuhong and the like, Chinese experimental prescriptions research, 2006,12, 40-42.). Studies of Longzijiang and the like find that stachydrine can obviously reduce the whole blood viscosity and the Hematocrit (HCT) of acute blood stasis rats, prolong PT and APTT, reduce the level of blood plasma TXB2 and increase the ratio of 6-K-PGF1 alpha to TXB2 (Zhuhong et al, proceedings of Anhui traditional Chinese medicine academy, 2007,26 and 38-40.). Weihongchang and the like find that leonurus stachydrine with a certain concentration can reduce the body surface area, the Protein/DNA ratio and the ROS positive cell ratio of hypertrophic myocardial cells; leonurus stachydrine intervention reduced the expression levels of p-I κ B α (ser32) and NF- κ B (p65) proteins in hypertrophic myocardial cytoplasm (Guo Wei et al, Journal of Chinese Medicinal Materials,2012,35, 940-943.). Wanping et al found that stachydrine hydrochloride had an enhancing or synergistic effect on uterine contraction caused by oxytocin (Qin Meirong et al pharmaceutical Today,2013,23,410-412.) and the like.
However, more and more evidences show that the toxicity of the stachydrine hydrochloride is high, so that the further research and development of the stachydrine hydrochloride are limited, and therefore, the design of a new compound on the basis of the structure of the stachydrine hydrochloride to reduce the toxicity of the stachydrine hydrochloride is of great significance in developing a novel high-efficiency and low-toxicity cardiovascular and cerebrovascular medicine.
So far, no patent or other literature report about the study of cardiovascular and cerebrovascular medicines based on the stachydrine structure is found.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a series of derivatives based on stachydrine structures and application thereof in preparing medicines for treating cardiovascular and cerebrovascular diseases. Pharmacological experiments prove that the derivative based on the stachydrine structure has a nerve protection effect and has a good treatment effect on cerebral arterial thrombosis and the like.
Another object of the present invention is to provide a process for the preparation of stachydrine derivatives.
In order to achieve the purpose, the technical scheme of the invention is as follows:
the invention provides a stachydrine derivative, which has a structural general formula shown as a formula (I):
Figure BDA0001133597150000031
in the formula:
R1natural amino acids and amide and ester analogues thereof; or gamma-aminobutanamide, anthranilic acid, and amide, ester-based analogs thereof;
R2is H, CF3、OCF3、OH、C1-12Or C is a hydrocarbon group1-12Aryl of (a);
R3、R4and R5Are each a halogen atom, H, CF3、OCF3、OH、SH、NH2Carboxyl, ester group, sulfone group, sulfoxide group, sulfonic group, sulfonate group, sulfonamide group, ketone group, aldehyde group, nitro group, nitroso group, C1-12An alkyl or aryl group of (a);
the halogen atom comprises F, Cl, Br or I, and the alkyl group comprises saturated or unsaturated open-chain alkyl group and saturated or unsaturated cyclic alkyl group.
Further: the natural amino acids are glycine, alanine, valine, leucine, isoleucine, aspartic acid, asparagine, glutamine, phenylalanine, tryptophan, methionine, lysine, arginine, histidine and glutamic acid.
Further: the stachydrine derivatives are compounds B-1, B-2, B-3 and B-4:
Figure BDA0001133597150000032
the invention also provides a preparation method of the stachydrine derivative, which comprises the following steps:
(1) carrying out hydrogenation reaction on the L-proline a and formaldehyde under the catalytic action of palladium carbon to obtain N-methyl-L-proline b:
Figure BDA0001133597150000033
(2) the N-methyl-L-proline B respectively carries out condensation reaction with anthranilic acid, gamma-aminobutanamide, glutamine and glycine under the action of dicyclohexylcarbodiimide to respectively prepare stachydrine derivatives B-1, B-2, B-3 and B-4:
Figure BDA0001133597150000041
the invention also provides the application of the stachydrine derivative and the pharmaceutically acceptable salt thereof in preparing the medicines for treating the cardiovascular and cerebrovascular diseases.
Further: the stachydrine derivative comprises compounds A-1, A-2, A-3, A-4, B-1, B-2, B-3 and B-4:
Figure BDA0001133597150000042
further: the cardiovascular and cerebrovascular diseases comprise ischemic stroke, cerebral infarction, cerebral apoplexy and cerebral arteriosclerosis.
Further: the pharmaceutically acceptable salts include salts derived from inorganic and organic acids.
Further: the inorganic acid comprises hydrochloric acid, hydrobromic acid, sulfuric acid and phosphoric acid; the organic acids include methanesulfonic acid, trifluoromethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, 1-naphthalenesulfonic acid, 2-naphthalenesulfonic acid, acetic acid, trifluoroacetic acid, malic acid, tartaric acid, citric acid, lactic acid, oxalic acid, succinic acid, fumaric acid, maleic acid, benzoic acid, salicylic acid, phenylacetic acid, and mandelic acid.
Compared with the prior art, the stachydrine derivative designed and synthesized by the invention has better activity than the stachydrine and positive drugs in the aspects of neuroprotection, antioxidation, cytotoxicity inhibition, anticoagulation and the like, and has wide prospect of being developed into drugs for treating cardiovascular and cerebrovascular diseases.
Drawings
FIG. 1 is a stained image of the white control group of example 8;
FIG. 2 is a dye image of the model set of example 8;
FIG. 3 is a staining image of the actinolite of example 8;
FIG. 4 is a dye image of the compound B-1 group in example 8;
FIG. 5 is a dye image of Compound A-3 group of example 8;
FIG. 6 is a developed image of an empty white control group in example 10;
FIG. 7 is a color image of the model group in example 10;
FIG. 8 is a developed image of the actinolite set in example 10;
FIG. 9 is a color image of the compound B-1 group in example 10;
FIG. 10 is a color image of Compound A-3 group in example 10.
Detailed Description
The following embodiments better illustrate the present invention. However, the present invention is not limited to the following examples.
Example 1: the synthetic route of N-methyl-L-proline b is as follows:
Figure BDA0001133597150000051
dissolving L-proline (4.0g,34.8mmol) in methanol (40mL), sequentially adding 40% formaldehyde water solution (2.8mL, 38.2mmol) and 10% palladium-carbon (1g), adding, hydrogenating, reacting at room temperature for 24 hr, filtering, concentrating the mother liquor, and drying to obtain the final product4g of a white solid, in a yield of 89%, was N-methyl-L-proline (compound b). ESI-MS: (M/z,%) > 130[ M + H]+。
Example 2: the synthetic route of (S) -2- (1-methylpyrrole-2-carboxamide) -benzoic acid (compound B-1) is as follows:
Figure BDA0001133597150000052
the N-methyl-L-proline B (1.29g,10mmol) prepared in example 1 was added to dichloromethane (20mL), dicyclohexylcarbodiimide DCC (2.47g, 12mmol) and anthranilic acid (1.37g,10mmol) were sequentially added, and the mixture was reacted at room temperature for 24 hours, after which a white solid was filtered off, and the mother liquor was concentrated to dryness to obtain a white solid (1.6 g, yield 61%) which was the compound B-1.
1H NMR(500MHz,CD3OD)＝8.45(d,J＝8.3Hz,1H),8.10(dd,J＝7.9,1.3Hz,1H),7.61-7.53(m,1H),7.22(t,J＝7.6Hz,1H),4.43(t,J＝8.4Hz,1H),3.82-3.72(m,1H),3.36-3.22(m,1H),3.02(s,3H),2.77-2.67(m,1H),2.25(ddd,J＝18.2,9.2,3.9Hz,2H),2.17-2.06(m,1H)。
Example 3: the synthetic route for (S) -N- (4-amino-4-oxobutyl) -1-methylpyrrole-2-carboxamide (Compound B-2) is as follows:
Figure BDA0001133597150000061
adding dichloromethane (20mL) into the N-methyl-L-proline B (1.29g,10mmol), sequentially adding DCC (2.47g, 12mmol) and gamma-aminobutanamide (1.02g,10mmol), reacting at room temperature for 24 hours, filtering, and concentrating the mother liquor to obtain a white solid 1.6g, wherein the yield is 75.1%, and the compound B-2 is obtained.
1H NMR(500MHz,CD3OD)＝3.94(t,J＝7.9Hz,1H),3.71-3.59(m,1H),3.28(d,J＝7.1Hz,2H),3.13(dt,J＝20.8,10.5Hz,1H),2.88(s,3H),2.59-2.46(m,1H),2.29-2.22(m,2H),2.22-2.10(m,1H),2.08-1.97(m,2H),1.88-1.77(m,2H).
Example 4: the synthetic route for (R) -5-amino-2- ((S) -1-methylpyrrole-2-carboxamide) -5-oxopentanoic acid (compound B-3) is as follows:
Figure BDA0001133597150000062
adding dichloromethane (20mL) into the N-methyl-L-proline B (1.29g,10mmol), sequentially adding DCC (2.47g, 12mmol) and L-glutamine (1.46g,10mmol), reacting at room temperature for 24 hours, filtering, and concentrating the mother liquor to obtain a white solid 1.5g with a yield of 58.4%, thus obtaining the compound B-3.
1H NMR(500MHz,CD3OD)＝4.30(dd,J＝7.4,5.0Hz,1H),3.79-3.69(m,1H),3.61-3.49(m,1H),2.96(dt,J＝18.2,9.1Hz,1H),2.79(d,J＝8.7Hz,3H),2.47(dq,J＝12.3,7.5Hz,1H),2.31-2.23(m,2H),2.23-2.13(m,1H),2.12-2.03(m,2H),1.97(ddd,J＝20.2,13.2,7.2Hz,2H).
Example 5: the synthetic route for (S) -2- (1-methylpyrrole-2-carboxamide) -acetic acid (compound B-4) is as follows:
Figure BDA0001133597150000071
adding dichloromethane (20mL) into the N-methyl-L-proline B (1.29g,10mmol), sequentially adding DCC (2.47g, 12mmol) and glycine (0.75g,10mmol), reacting at room temperature for 24 hours, filtering, and concentrating the mother liquor to obtain a white solid 1g, wherein the yield is 54%, and the compound B-4 is obtained.
1H NMR(500MHz,CD3OD)＝4.14(t,J＝8.3,1H),4.00(s,2H),3.71(ddd,J＝11.4,7.7,4.1,1H),3.22(d,J＝11.1,1H),2.95(d,J＝10.5,3H),2.58(dd,J＝12.2,7.5,1H),2.24-2.02(m,3H)。
Example 6: determination of the Effect of the Stachydrine derivatives of the invention on in vitro coagulation
Grouping experiments: a blank control group, a rivaroxaban low dose group (50nmol), a rivaroxaban middle dose group (100nmol), a rivaroxaban high dose group (500nmol), a test compound low dose group (50nmol), a test compound middle dose group (100nmol), and a test compound high dose group (500 nmol);
sample treatment: dissolving with physiological saline.
The experimental method comprises the following steps: 0.2mL of the corresponding solution was added to each test tube. The rabbit is 5 mL/kg-120% ethyl carbamate (urethane) is injected into the ear margin vein for anesthesia, carotid artery intubation is performed to obtain blood, 1mL of fresh blood is added into each test tube respectively, and the test tubes are placed in a water bath at 37 ℃ after being mixed uniformly. A stopwatch was started immediately to record the blood clotting time, indicating that blood was completely coagulated when the tube could be completely inverted without blood flowing out, and the blood clotting time was recorded. The results of the experiment are shown in table 1.
TABLE 1 blood clotting time
Figure BDA0001133597150000072
Figure BDA0001133597150000081
As can be seen from table 1: the compounds in the table 1 all show an anticoagulation effect, wherein the compounds B-1, A-3 and A-4 show a more remarkable anticoagulation effect in an in vitro whole blood anticoagulation experiment.
Example 7: determining the effect of the stachydrine derivatives on the cell survival rate of a glutamic acid-induced nerve cell injury model
Grouping experiments: blank control group, model group, positive drug group (MK-801), test compound low dose group (10 mu mol), test compound medium dose group (20 mu mol) and test compound high dose group (40 mu mol);
sample treatment: dissolving with physiological saline.
The experimental method comprises the steps of disinfecting a rat suckling mouse by using 75% ethanol in volume fraction, placing the broken end in a plate containing dissection liquid, separating out the whole brain, separating the hippocampus under a dissection microscope, digesting the rat suckling mouse in a 0.25% trypsin 37 ℃ incubator for 30min, sucking the rat suckling mouse into a centrifugal tube, adding 2-3 mL of planting liquid to stop digestion of pancreatin, slightly blowing and beating fragments of the hippocampus for a plurality of times by using a fine suction tube with gradually reduced caliber, sucking sediments into another centrifugal tube, adding the planting liquid to continue blowing and beating until all the hippocampus fragments are blown and scattered, filtering by using a 200-mesh metal screen, adjusting the cell concentration to be 1 × 109L-1Is connected toFor pre-application of 0.1 g.L-11mL of polylysine-treated 6-well plates-1Culturing in incubator for 24 hr, changing liquid to remove necrotic cells after cell adherence, culturing for 72 hr, and adding 3 mg.L-1The cytarabine is used for inhibiting the growth of non-nerve cells, and the total amount of the cytarabine is changed into a fresh culture solution after 24 hours of action. Half the amount of the solution was changed 1 time every 3 days later. Test compounds were added six days after isolation of primary rat cerebellar granule neurons (1000-fold dilution was used for 96-well plates). Glutamic acid was added at a concentration of 200. mu. mol on the seventh day (100-fold dilution was required when adding to 96-well plates). MTT (final concentration of 0.5 mg. mL when added to 96-well plate) was used on day eight-1) And detecting the cell survival rate. The results of the experiment are shown in table 2.
TABLE 2 detection results of the survival rate of nerve cells
Figure BDA0001133597150000082
Figure BDA0001133597150000091
As can be seen from table 2: the compounds related in the table 2 are different from the glutamic acid (Glu) induced nerve cell damage model group, wherein the compounds B-1, B-2, B-4 and A-3 have significant difference from the glutamic acid (Glu) induced nerve cell damage model group, which indicates that the compounds have certain neuroprotective activity.
Example 8: TUNEL assay for the effect of stachydrine derivatives on apoptosis
Grouping experiments: blank control group, model group, positive drug group (MK-801) and test compound group (20 mu mol);
sample treatment: dissolving with physiological saline.
The experimental method comprises the following steps: test compounds were added six days after isolation of primary rat cerebellar granule neurons (1000-fold dilution was used for 96-well plates). Glutamic acid was added at a concentration of 200. mu. mol on the seventh day (100-fold dilution was required when adding to 96-well plates). And climbing the film on the eighth day, adopting a TUNEL kit for staining, and detecting the apoptosis condition by using a laser confocal microscope. Cells staining positive were apoptotic cells. The results of the experiment are shown in table 3.
TABLE 3 apoptosis assay results
Figure BDA0001133597150000092
Figure BDA0001133597150000101
As can be seen from table 3: the compounds B-1 and A-3 can inhibit glutamate-induced nerve cell apoptosis at 20 mu mol, and have protective effect on nerve cells.
Fig. 1-5 are confocal laser scanning microscope images, wherein positive signals represent apoptotic cells, and the more positive signals, the more apoptotic cells. As can be seen, the positive signals of the model group in FIG. 2 are more, indicating that the cells are largely apoptotic; FIG. 1 shows that the blank group, FIG. 3 shows that the positive drug group, FIG. 4 shows that the compound B-1 group and FIG. 5 shows that the compound A-3 group show less positive signals and the apoptosis number is less, which indicates that the compounds B-1 and A-3 can inhibit the glutamate-induced nerve cell apoptosis at 20 mu mol.
Example 9: determination of the Effect of Stachydrin derivatives on the Lactate Dehydrogenase (LDH) Activity of glutamate-induced nerve cell injury model cells
Grouping experiments: blank control group, model group, positive drug group (MK-801) and test compound group (20 mu mol);
sample treatment: dissolving with physiological saline.
The experimental method comprises the following steps: test compounds were added six days after isolation of primary rat cerebellar granule neurons (1000-fold dilution was used for 96-well plates). Glutamic acid was added at a concentration of 200. mu. mol on the seventh day (100-fold dilution was required when adding to 96-well plates). Cells were assayed on day eight using the LDH kit. The results of the experiment are shown in table 4.
TABLE 4 results of Lactate Dehydrogenase (LDH) Activity measurement
Grouping Lactate Dehydrogenase (LDH) release rate (%) (n ═ 3)
Blank control 100.0±4.00
Model set 266.9±14.31
MK-801(10μmol) 107.6±2.29
Stachydrine 242.3±3.90
B-1 221.3±20.18
A-3 181.6±12.58
A-4 281.8±16.36N＝3
As can be seen from Table 4: compared with a model group, the compounds B-1 and A-3 have significant difference at the concentration of 20 mu mol, can significantly inhibit LDH activity, and show that the compounds B-1 and A-3 have the effect of inhibiting cytotoxicity.
Example 10: determination of Effect of stachydrine derivatives on content of Reactive Oxygen Species (ROS) in glutamate-induced nerve cell injury model cells
Grouping experiments: blank control group, model group, positive drug group (MK-801) and test compound group (20 mu mol);
sample treatment: dissolving with physiological saline.
The experimental method comprises the following steps: test compounds were added six days after isolation of primary rat cerebellar granule neurons (1000-fold dilution was used for 96-well plates). Glutamic acid was added at a concentration of 200. mu. mol on the seventh day (100-fold dilution was required when adding to 96-well plates). And (5) loading the probe by using a DCF kit on the eighth day, photographing by using a laser confocal microscope, and detecting. A positive signal indicates an intracellular reactive oxygen response. The results of the experiment are shown in Table 5.
TABLE 5 results of intracellular Reactive Oxygen Species (ROS) detection
Grouping Intracellular active oxygen content (%) (n ═ 3)
Blank control 100.00±6.71
Model set 1648.82±38.25
MK-801(10μmol) 38.75±4.81
Stachydrine 1503.57±64.65
B-1 713.93±67.21
A-3 1038.84±107.27
A-4 1548.21±140.72
As can be seen from table 5: the compounds B-1 and A-3 can reduce the active oxygen content in the glutamic acid induced damage nerve cells compared with the model group at the concentration of 20 mu mol, which indicates that the neuroprotective effect of the compounds B-1 and A-3 can be realized by inhibiting the active oxygen activity.
FIGS. 6-10 are confocal laser scanning microscopy images showing that positive signals indicate intracellular reactive oxygen species responses, and strong positive signals indicate higher intracellular reactive oxygen species content. As can be seen, the positive signal of the model group in FIG. 7 is strong, indicating that the active oxygen content in the cells is high; FIG. 6 shows that the positive signals of the blank group, the group of positive drugs in FIG. 8, the compound B-1 group in FIG. 9 and the compound A-3 group in FIG. 10 are weak, which indicates that the active oxygen content in cells is low, and thus it is demonstrated that the active oxygen content in the glutamate-induced damaged nerve cells can be reduced by 20. mu. mol of the compounds B-1 and A-3.
Example 11: determination of Effect of stachydrine derivatives on glutamate induced nerve cell injury model superoxide dismutase (SOD) Activity
Grouping experiments: blank control group, model group, positive drug group (MK-801) and test compound group (20 mu mol);
sample treatment: dissolving with physiological saline.
The experimental method comprises the following steps: and adding 200 mu mol glutamic acid (which is diluted by 100 times when being added into a 96-well plate) to mold on the seventh day after the primary rat cerebellum granule nerve cells are separated and cultured. After the molding is finished, the test drugs with corresponding concentration are added, and after the drugs act for 48 hours, the protein sample is collected. And detecting the SOD content in the sample by using an SOD content detection kit. The results of the experiment are shown in Table 6.
TABLE 6SOD content test results
Grouping Relative content (%) of SOD (n ═ 3)
Blank control 100.00±0.00
Model set 64.01±3.30
MK-801(10μmol) 93.52±2.45
Stachydrine 70.87±3.03
B-1 74.74±2.10
A-3 73.91±3.11
A-4 62.16±3.23
As can be seen from table 6: in a glutamic acid-induced nerve cell injury model, stachydrine and compounds B-1 and A-3 can increase the expression of superoxide dismutase (SOD), which indicates that the compound has antioxidation.
Example 12: determination of Effect of Stachydrin derivatives on Malondialdehyde (MDA) content in glutamate-induced nerve cell injury model
Grouping experiments: blank control group, model group, positive drug group (MK-801) and test compound group (20 mu mol);
sample treatment: dissolving with physiological saline.
The experimental method comprises the following steps: and adding 200 mu mol glutamic acid (which is diluted by 100 times when being added into a 96-well plate) to mold on the seventh day after the primary rat cerebellum granule nerve cells are separated and cultured. After the molding is finished, the test drugs with corresponding concentration are added, and after the drugs act for 48 hours, the protein sample is collected. And detecting the MDA content in the sample by adopting an MDA content detection kit. The results of the experiment are shown in Table 7.
TABLE 7MDA content test results
Figure BDA0001133597150000121
Figure BDA0001133597150000131
As can be seen from table 7: in the glutamic acid-induced nerve cell injury model, stachydrine and the compounds B-1 and A-3 can reduce the content of Malondialdehyde (MDA) in cells, which indicates that the stachydrine and the compounds B-1 and A-3 have the effect of resisting lipid oxidation.
The above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions.

Claims (6)

1. A stachydrine derivative characterized by: the stachydrine derivative is a compound B-1, B-2 or B-4:
Figure 918957DEST_PATH_IMAGE001
。
2. a process for producing a stachydrine derivative according to claim 1, characterized in that: it comprises the following steps:
（1）L-proline a and formaldehyde are subjected to hydrogenation reaction under the catalytic action of palladium carbon to prepare the compoundN-methyl-L-proline b:
Figure DEST_PATH_IMAGE002
；
(2) the above-mentionedN-methyl-LProline b with anthranilic acid,γ-aminobutanamide and glycine are subjected to condensation reaction under the action of dicyclohexylcarbodiimide to respectively prepare the stachydrine derivatives B-1, B-2 and B-4:
Figure 773780DEST_PATH_IMAGE003
。
3. the use of the stachydrine derivative according to claim 1 and pharmaceutically acceptable salts thereof for the preparation of medicaments for the treatment of cardiovascular and cerebrovascular diseases.
4. The use of a stachydrine derivative according to claim 3 and pharmaceutically acceptable salts thereof for the preparation of a medicament for the treatment of cardiovascular and cerebrovascular diseases, characterized in that: the cardiovascular and cerebrovascular diseases comprise ischemic stroke, cerebral infarction, cerebral apoplexy and cerebral arteriosclerosis.
5. The use of a stachydrine derivative according to claim 3 and pharmaceutically acceptable salts thereof for the preparation of a medicament for the treatment of cardiovascular and cerebrovascular diseases, characterized in that: the pharmaceutically acceptable salts include salts derived from inorganic and organic acids.
6. The use of a stachydrine derivative according to claim 5 and pharmaceutically acceptable salts thereof for the preparation of a medicament for the treatment of cardiovascular and cerebrovascular diseases, characterized in that: the inorganic acid comprises hydrochloric acid, hydrobromic acid, sulfuric acid and phosphoric acid; the organic acids include methanesulfonic acid, trifluoromethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, 1-naphthalenesulfonic acid, 2-naphthalenesulfonic acid, acetic acid, trifluoroacetic acid, malic acid, tartaric acid, citric acid, lactic acid, oxalic acid, succinic acid, fumaric acid, maleic acid, benzoic acid, salicylic acid, phenylacetic acid, and mandelic acid.
CN201610907125.1A 2016-10-19 2016-10-19 A stachydrine derivative, its preparation method and application in preparing medicine for treating cardiovascular disease and cerebrovascular disease Active CN107963987B (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
CN201610907125.1A CN107963987B (en) 2016-10-19 2016-10-19 A stachydrine derivative, its preparation method and application in preparing medicine for treating cardiovascular disease and cerebrovascular disease
PCT/CN2017/106555 WO2018072689A1 (en) 2016-10-19 2017-10-17 Stachydrine derivative, preparation method therefor, and applications thereof in preparation of drugs for treating cardiovascular and cerebrovascular diseases

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201610907125.1A CN107963987B (en) 2016-10-19 2016-10-19 A stachydrine derivative, its preparation method and application in preparing medicine for treating cardiovascular disease and cerebrovascular disease

Publications (2)

Publication Number Publication Date
CN107963987A CN107963987A (en) 2018-04-27
CN107963987B true CN107963987B (en) 2020-09-08

Family

ID=61996866

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201610907125.1A Active CN107963987B (en) 2016-10-19 2016-10-19 A stachydrine derivative, its preparation method and application in preparing medicine for treating cardiovascular disease and cerebrovascular disease

Country Status (2)

Country Link
CN (1) CN107963987B (en)
WO (1) WO2018072689A1 (en)

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110357800B (en) * 2018-04-09 2022-07-26 青岛海生洋润生物科技有限公司 Proline derivative and application thereof in preparing medicine for treating cardiovascular and cerebrovascular diseases
CN109350615A (en) * 2018-08-17 2019-02-19 上海市浦东新区浦南医院 Stachydrine hydrochloride is in the new application for preventing and treating ischemic cerebrovascular disease
CN112409443B (en) * 2021-01-25 2021-04-27 潍坊科技学院 Proteborl derivative, preparation method thereof and application thereof in preparation of medicines for treating cardiovascular and cerebrovascular diseases
CN114010843B (en) * 2021-11-16 2022-04-26 四川大学 Application of stachydrine

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5589182A (en) * 1993-12-06 1996-12-31 Tashiro; Renki Compositions and method of treating cardio-, cerebro-vascular and alzheimer's diseases and depression
CN101596232A (en) * 2009-07-03 2009-12-09 复旦大学 The purposes of the Herba Leonuri extract of purification in the nerve protection medicine of preparation brain injury
CN102670592B (en) * 2012-05-18 2014-08-20 成都第一药业有限公司 Motherwort alkaloid and choline medicinal composition

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
Pharmacology of Casimiroa edulis IV. Hypotensive effects of compounds isolated from methanolic extracts in rats and guinea pigs;Gil A. Magos, et al.;《Journal of Ethnopharmacology》;19991231;第64卷;第35-44页 *
野西瓜化学成分提取分离及抗氧化活性测定;于蕾等;《哈尔滨商业大学学报(自然科学版)》;20110831;第27卷(第4期);第524-527页 *

Also Published As

Publication number Publication date
WO2018072689A1 (en) 2018-04-26
CN107963987A (en) 2018-04-27

Similar Documents

Publication Publication Date Title
CN107963987B (en) A stachydrine derivative, its preparation method and application in preparing medicine for treating cardiovascular disease and cerebrovascular disease
CN100999470B (en) Salvia minium phenolic acid A and process of preparing preparation and use
CN110357800B (en) Proline derivative and application thereof in preparing medicine for treating cardiovascular and cerebrovascular diseases
WO2010111935A1 (en) New salvianolic acid compound l, preparation method and use thereof
EP3838887B1 (en) 2-(1-acyloxypentyl) benzoic acid salt formed by basic amino acid or aminoguanidine, preparation method therefor and uses thereof
KR20080027191A (en) New benzoxazole derivative, process for the preparation thereof and pharmaceutical composition comprising the same
CN103118677A (en) Uses of benzoate and its derivatives
CN105189446A (en) Phloroglucinol derivatives and application thereof in treatment of neurodegenerative disorder
WO2021103749A1 (en) Left-handed bicyclic morpholine and salt thereof, preparation method therefor, pharmaceutical composition, and application
EP2899186B1 (en) New hydroxysafflor yellow pharmaceutical salts
WO2005003146A1 (en) The c-glycosylisoflavones having alkylaminoalkoxyl substituent, the preparation and the use of the same
CN110577583B (en) RGDF modified heptacyclic aldehyde, its synthesis, antithrombotic activity and application
CN107753469B (en) Application of NDGA analogue in preparing antioxidant drugs
US9624254B2 (en) Hydroxysafflor yellow pharmaceutical salts
CN103193642B (en) Carvacrol derivatives and synthetic method and application thereof
CN115364081B (en) Application of 2, 4-dihydroxy-6-methoxyacetophenone in preparation of medicine for regulating blood coagulation function
CN112094255B (en) Scutellarin aglycone derivative, and preparation method and application thereof
WO2012040919A1 (en) Use of baicalein in manufacturing medicament for preventing and treating parkinson's disease
CN112409443B (en) Proteborl derivative, preparation method thereof and application thereof in preparation of medicines for treating cardiovascular and cerebrovascular diseases
CN110577569B (en) RGDV-modified heptacyclic aldehyde, synthesis, antithrombotic activity and application thereof
CN107056877A (en) A kind of steroid compound and application thereof
CN114853715B (en) Organic nitrite donor ketal type prodrug, preparation method and medical application thereof
CN109694395B (en) Compound for ischemia reperfusion injury and preparation method thereof
CN116987091A (en) Medicine for treating epileptic seizure disease and preparation method thereof
EP4247805A1 (en) Compounds as pu. 1 inhibitors

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant