CN107963987A - A kind of wood alkali derivant and preparation method thereof and the application in the medicine for preparing treatment cardio-cerebralvascular diseases - Google Patents

A kind of wood alkali derivant and preparation method thereof and the application in the medicine for preparing treatment cardio-cerebralvascular diseases Download PDF

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CN107963987A
CN107963987A CN201610907125.1A CN201610907125A CN107963987A CN 107963987 A CN107963987 A CN 107963987A CN 201610907125 A CN201610907125 A CN 201610907125A CN 107963987 A CN107963987 A CN 107963987A
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acid
alkali derivant
wood alkali
medicine
wood
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CN107963987B (en
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李文保
李峰
张良
张鹏
代龙
管华诗
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Qingdao Marine Biomedical Research Institute Co Ltd
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    • 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

Application the present invention provides a kind of wood alkali derivant and preparation method thereof and in the medicine for preparing treatment cardio-cerebralvascular diseases, the wood alkali derivant have the structure of logical formula (I), its synthetic method includes：LThe hydrogenation reaction under catalyst effect is made proline a with formaldehydeNMethylLProline b；NMethylLProline b respectively with ortho-aminobenzoic acid,γAmino-butanamide, glutamine and glycine are in dicyclohexylcarbodiimide（DCC）Wood alkali derivant B 1, B 2, B 3 and B 4 is made in the lower condensation reaction of effect.The present invention is proved through pharmacological experiment; wood alkali derivant provided by the invention has protection effects on neural system; have the function that to treat cardio-cerebralvascular diseases, present invention also offers application of the wood alkali derivant in the medicine for preparing treatment cardio-cerebralvascular diseases.

Description

A kind of wood alkali derivant and preparation method thereof and prepare treat cardio-cerebralvascular disease Application in the medicine of disease
Technical field
The invention belongs to pharmaceutical technology field, and in particular to a kind of wood alkali derivant and preparation method thereof and be controlled in preparation Treat the application in the medicine of cardio-cerebralvascular diseases.
Background technology
Cerebral apoplexy is the refractory disease for seriously endangering human life's safety, dead with high incidence, high disability rate and height The characteristics of dying rate.In the Proportional Death Rate that the Ministry of Public Health of China in 2008 announces, cerebral apoplexy is the lethal original of the 3rd in city Cause, is the deputy cause of death in rural area.Epidemiology survey shows the incidence of young stroke in recent years increasingly Height, wherein the cerebral apoplexy that less than 45 years old adult occurs, is respectively 5% and 13.44% in the ratio of western countries and domestic report. Cerebral apoplexy is divided into by its property in ischemic cerebral apoplexy and hemorrhagic apoplexy, and wherein cerebral arterial thrombosis accounts for patients with stroke sum 75%~85%.
Cerebral arterial thrombosis treatment has a variety of therapeutic modalities, and drug therapy is the primary treatment that current Treatment of Cerebral Stroke is taken One of mode.The medicine of Clinical practice is broadly divided into following a few classes at present：Thrombolytic drug, medicament for resisting platelet aggregation, drop Fine medicine, anticoagulant and nerve protection medicine etc..By untiringly making great efforts, in the research of cerebral arterial thrombosis medicine It has been had made great progress that, but also many problems are still unresolved.Application range, the anti-freezing drop extended such as thrombolysis " time window " is fine The medicament selection for the treatment of and it is used in combination, the Clinical efficacy of nerve protection medicine and security etc..With to stroke onset Reason and disease mechanisms research are goed deep into, our understanding to therapy target are also gradually clear, comprehensively, this is Treatment of Cerebral Stroke medicine The research of thing and Clinical practice provide more research directions.
Motherwort toil, be slightly cold, and Return liver, pericardium channel, there is promoting blood circulation, menstruation regulating, removing blood stasis for promoting tissue regeneration, inducing diuresis to remove edema and other effects.Cure mainly The diseases such as irregular menstruation, uterine bleeding difficult labour, dysmenorrhoea, postpartum stasis, are known as " blood man panacea ", the title of " through producing good medicine ".Stachydrine (Stachydrine) proline glycine betaine (Proline Betaine) or N, N- dimethylproline (N, N-Dimethyl are also known as Proline), it is simplest pyrrolidine alkaloid, and one of principle active component of Chinese medicine motherwort.The chemistry knot of stachydrine Structure is as follows：
Modern pharmacology research shows that stachydrine has many physiologically actives：Expansible peripheral blood vessel, increases blood Flow, platelet aggregation-against, reduces blood viscosity；Coronary artery and cardiac muscle nutritional blood flow can be improved, it is bad to reduce cardiac muscle cell Dead amount, reduces vascular resistence, improves microcirculation, reducing heart rate, reduce the effect such as cardiac output, is expected to become good heart and brain blood The medicine of guard system disease；It can suppress the generation of breast cancer and endometriosis interna；With eliminating the phlegm, antibechic, relaxation bronchus The effect of smooth muscle.
The researchs such as poplar solution people find that stachydrine has protective effect (horse rubine to Acute Myocardial Ischemia in Rats reperfusion injury Deng Chinese experimental study on science of prescription, 2006,12,40-42.).It is acute that the researchs such as Long Zijiang find that stachydrine can be reduced substantially Rat's blood stasis model whole blood viscosity and hematocrit value (HCT), extend PT and APTT, reduce plasma substance P level, raise 6-K-PGF1 α/TXB2 ratios (the Anhui Chinese Medicine College journals such as Zhu Hong, 2007,26,38-40.).Wei Hongchang etc. has found that certain density benefit is female Careless stachydrine can reduce the body surface area of hypertrophic cardiomyocytes, Protein/DNA ratios and ROS positive cell ratios；Benefit Brittle Falsepimpernel Herb stachydrine intervention reduces the expression water of NF- κ B (p65) albumen in p-I κ B α (ser32) and core in hypertrophic cardiomyocytes slurry Flat (the .Journal of Chinese Medicinal such as Guo Wei Materials, 2012,35,940-943.).King's equality is found Stachydrine hydrochloride to uterine contractile caused by oxytocin have enhancing or synergistic effect (the .Pharmacy Today such as Qin Meirong, 2013,23,410-412.) etc..
But more and more evidences show that the toxicity of stachydrine hydrochloride is larger, its further research and development is limited, New compound is thus designed on stachydrine architecture basics reduces its toxicity, and then develops the preventing angiocardiopathy or cerebrovascular diseases of high-efficiency low-toxicity Medicine is of great significance.
So far, the patent or other document reports of the cardio-cerebralvascular drug research based on stachydrine structure are not yet found Road.
The content of the invention
The present invention seeks in view of the deficiencies of the prior art, there is provided a series of derivatives based on stachydrine structure and its Application in the medicine for preparing treatment cardio-cerebralvascular diseases.The present invention is proved through pharmacological experiment, of the present invention to be based on The derivative of stachydrine structure has protection effects on neural system, all has good therapeutic effect for ischemic cerebral apoplexy is medium.
It is a further object of the present invention to provide the preparation method of wood alkali derivant.
For achieving the above object, technical scheme is as follows：
The present invention provides a kind of wood alkali derivant, its general structure such as formula (I) are shown：
In formula：
R1For natural amino acid and its acid amides, ester group analog；Or gamma-amino butyramide, ortho-aminobenzoic acid and its acyl Amine, ester group analog；
R2For H, CF3、OCF3、OH、C1-12Alkyl or C1-12Aryl；
R3、R4And R5It is halogen atom, H, CF3、OCF3、OH、SH、NH2, carboxyl, ester group, sulfuryl, sulfoxide group, sulfonic acid Base, sulfonate group, sulfoamido, ketone group, aldehyde radical, nitro, nitroso, C1-12Alkyl or aryl；
The halogen atom includes F, Cl, Br or I, and the alkyl includes saturation or undersaturated open chain alkyl, satisfies And/or undersaturated cyclic hydrocarbon group.
Further：The natural amino acid is glycine, alanine, valine, leucine, isoleucine, asparagus fern ammonia Acid, asparagine, glutamine, phenylalanine, tryptophan, methionine, lysine, arginine, histidine and glutamic acid.
Further：The wood alkali derivant is compound B-1, B-2, B-3 and B-4：
Present invention also offers the preparation method of the wood alkali derivant, it includes the following steps：
(1) N- methyl-L-prolines b is made with formaldehyde hydrogenation reaction under palladium carbon catalytic action in L-PROLINE a：
(2) the N- methyl-L-prolines b respectively with ortho-aminobenzoic acid, gamma-amino butyramide, glutamine and sweet Propylhomoserin occurs condensation reaction under dicyclohexylcarbodiimide effect and wood alkali derivant B-1, B-2, B-3 and B-4 is made respectively：
Present invention also offers the wood alkali derivant and its pharmaceutically acceptable salt to prepare treatment heart and brain Application in the medicine of vascular diseases.
Further：The wood alkali derivant includes compound A-1, A-2, A-3, A-4, B-1, B-2, B-3 and B-4：
Further：It is hard that the cardio-cerebralvascular diseases include cerebral arterial thrombosis, cerebral infarction, headstroke and cerebral artery Change.
Further：The pharmaceutically acceptable salt includes salt derived from inorganic acid and organic acid.
Further：The inorganic acid includes hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid；The organic acid includes methanesulfonic acid, three Fluorine methanesulfonic acid, benzene sulfonic acid, p-methyl benzenesulfonic acid, 1-naphthalene sulfonic aicd, 2- naphthalene sulfonic acids, acetic acid, trifluoroacetic acid, malic acid, tartaric acid, lemon Acid, lactic acid, oxalic acid, butanedioic acid, fumaric acid, maleic acid, benzoic acid, salicylic acid, phenylacetic acid and tussol.
Compared with prior art, the wood alkali derivant synthesized designed by the present invention is in neuroprotection, anti-oxidant, suppression is thin Cellular toxicity, anti-freezing etc. have the activity better than stachydrine and positive drug, have exploitation for treatment cardio-cerebralvascular diseases medicine The bright prospects of thing.
Brief description of the drawings
Fig. 1 is blank control group dye image in embodiment 8；
Fig. 2 is model group dye image in embodiment 8；
Fig. 3 is positive drug group dye image in embodiment 8；
Fig. 4 is compound B-1 group dye images in embodiment 8；
Fig. 5 is compound A-3 group dye images in embodiment 8；
Fig. 6 is blank control group colour developing figure picture in embodiment 10；
Fig. 7 is model group colour developing figure picture in embodiment 10；
Fig. 8 is positive drug group colour developing figure picture in embodiment 10；
Fig. 9 is compound B-1 groups colour developing figure picture in embodiment 10；
Figure 10 is compound A-3 groups colour developing figure picture in embodiment 10.
Embodiment
Present invention is better described in following embodiments.But the invention is not restricted to following embodiments.
Embodiment 1：The synthetic route of N- methyl-L-prolines b is as follows：
L-PROLINE (4.0g, 34.8mmol) is dissolved in methanol (40mL), sequentially add 40% formalin (2.8mL, 38.2mmol) with 10% palladium carbon (1g), finish, be hydrogenated with, when reaction 24 is small at room temperature, after the reaction was complete, filtering, mother liquor concentrations are done 4g white solids are obtained, yield 89%, is N- methyl-L-prolines (compound b).ESI-MS：[the M+H of (m/z, %)=130 ]+。
Embodiment 2：(S) synthetic route of -2- (1- methylpyrrole -2- formamides)-benzoic acid (compound B-1) is as follows：
The N- methyl-L-prolines b (1.29g, 10mmol) made from embodiment 1 is added into dichloromethane (20mL), Dicyclohexylcarbodiimide DCC (2.47g, 12mmol) and ortho-aminobenzoic acid (1.37g, 10mmol) are sequentially added, at room temperature React 24 it is small when, filter off white solid, mother liquor concentrations do to obtain white solid 1.6g, and yield 61%, is compound B-1.
1H NMR(500MHz,CD3OD) δ=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)。
Embodiment 3：(S) conjunction of-N- (4- amino -4- oxos butyl) -1- methylpyrrole -2- formamides (compound B-2) It is as follows into route：
The N- methyl-L-prolines b (1.29g, 10mmol) is added into dichloromethane (20mL), sequentially adds DCC (2.47g, 12mmol) and gamma-amino butyramide (1.02g, 10mmol), when reaction 24 is small at room temperature, filtering, mother liquor concentrations are done White solid 1.6g is obtained, yield 75.1%, is compound B-2.
1H NMR(500MHz,CD3OD) δ=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).
Embodiment 4：(R) -5- amino -2- ((S) -1- methylpyrrole -2- formamides) -5- oxopentanoic acids (compound B-3) Synthetic route it is as follows：
The N- methyl-L-prolines b (1.29g, 10mmol) is added into dichloromethane (20mL), sequentially adds DCC (2.47g, 12mmol) and L-Glutamine (1.46g, 10mmol), when reaction 24 is small at room temperature, filtering, mother liquor concentrations are done in vain Color solid 1.5g, yield 58.4%, it is compound B-3.
1H NMR(500MHz,CD3OD) δ=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).
Embodiment 5：(S) synthetic route of -2- (1- methylpyrrole -2- formamides)-acetic acid (compound B-4) is as follows：
The N- methyl-L-prolines b (1.29g, 10mmol) is added into dichloromethane (20mL), sequentially adds DCC (2.47g, 12mmol) and glycine (0.75g, 10mmol), when reaction 24 is small at room temperature, filtering, mother liquor concentrations obtain white solid 1g, yield 54%, is compound B-4.
1H NMR(500MHz,CD3OD) δ=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)。
Embodiment 6：Measure effect of the wood alkali derivant of the present invention to external blood coagulation
Experiment packet：Blank control group, razaxaban low dose group (50nmol), razaxaban middle dose group (100nmol), razaxaban high dose group (500nmol), test compound low dosage (50nmol), test compound middle dosage Group (100nmol), test compound high dose group (500nmol)；
Sample treatment：Using physiological saline solution.
Experimental method：Test tube is taken to be separately added into corresponding solution 0.2mL.Rabbit is according to 5mLkg-1Dosage auricular vein is injected 20% urethanes (urethane) is anaesthetized, and arteria carotis intubation takes blood, is separately added into above-mentioned each test tube fresh Blood 1mL, mixing are placed on 37 DEG C of water-baths.Start stopwatch record blood coagulation time immediately, do not have when test tube can be to be fully inverted When having blood outflow, represent that blood solidifies completely, record blood coagulation time.Experimental result is as shown in table 1.
1 blood coagulation time of table
From table 1：Involved compound shows anticoagulation in table 1, and wherein compound B-1, A-3, A-4 exists More significant anticoagulant effect is shown in ex vivo whole blood anti-freezing experiment.
Embodiment 7：The wood alkali derivant is measured to glutamate induction neural cell injury model cell survival rate Effect
Experiment packet：Blank control group, model group, positive drug group (MK-801), test compound low dose group (10 μ Mol), test compound middle dose group (20 μm of ol), test compound high dose group (40 μm of ol)；
Sample treatment：Using physiological saline solution.
Experimental method：Rat suckling mouse volume fraction is placed in the plate of the liquid containing dissection after 75% ethanol disinfection, to break end, Full brain is isolated, hippocampus is separated under disecting microscope, digests 30min in 0.25% 37 DEG C of trypsase incubator.Suction centrifugation Guan Zhong, adds 2~3mL kinds and plants the digestion that liquid terminates pancreatin, and the thin suction pipe to be successively decreased with bore gently blows and beats hippocampus fragment for several times, will Sediment is sucked after another centrifuge tube plus plantation liquid continues to blow and beat, until hippocampus fragment is all scattered by piping and druming.200 mesh metallic screens Net filtration, adjustment cell concentration are 1 × 109A L-1, it is inoculated in and uses 0.1gL in advance-1Processed 6 orifice plate of poly-D-lysine In, 1mL holes-1, 24h is cultivated in incubator, and full dose changes liquid after cell attachment, to remove non-viable non-apoptotic cell, after cultivating 72h, adds It is 3mgL to enter concentration-1Cytarabine, to suppress the growth of non-neuronal cells, full dose changes fresh culture into after acting on 24h Liquid.Later every 3 days half amounts change liquid 1 time.Primary rat cerebellar granule nerve cell is completed to add for examination for the 6th day after being separately cultured Compound (answers 1000 times of dilutions) when adding 96 orifice plate.The glutamic acid that 7th day addition concentration is 200 μm of ol is (when adding 96 orifice plate Answer 100 times of dilutions).With MTT, (final concentration should be 0.5mgmL when adding 96 orifice plate within 8th day-1) detection cell survival rate.Experiment The results are shown in Table 2.
2 Neuronal Survival rate testing result of table
From table 2：Involved compound is compared with glutamic acid (Glu) inducing nerve cell damage model group in table 2 Variant, wherein B-1, B-2, B-4, A-3 compound have aobvious compared with glutamic acid (Glu) inducing nerve cell damage model group Sex differernce is write, illustrates that there is certain neuroprotective activity.
Embodiment 8：TUNEL methods measure effect of the wood alkali derivant to Apoptosis
Experiment packet：Blank control group, model group, positive drug group (MK-801), test compound group (20 μm of ol)；
Sample treatment：Using physiological saline solution.
Experimental method：Primary rat cerebellar granule nerve cell is completed to add test compound in the 6th day after being separately cultured (answering 1000 times of dilutions when adding 96 orifice plate).Add within 7th day concentration and (answer 100 when adding 96 orifice plate for the glutamic acid of 200 μm of ol Dilute again).8th day creep plate, is dyed using TUNEL kits, and Apoptosis situation is detected using laser confocal microscope. Stained positive cell is apoptotic cell.Experimental result is as shown in table 3.
3 Apoptosis testing result of table
From table 3：Compound B-1, A-3 can suppress the nerve cell apoptosis of glutamate induction in 20 μm of ol, to god There is protective effect through cell.
Fig. 1-5 is laser confocal microscope image, and positive signal represents apoptotic cell, and positive signal is more, generation Table Apoptosis number is more.As seen from the figure, Fig. 2 model groups positive signal is more, represents a large amount of apoptosis of cell；Fig. 1 blank groups, figure 3 positive drug groups, Fig. 4 compound B-1 groups, Fig. 5 compound A-3 group positive signals are less, represent Apoptosis negligible amounts, explanation Compound B-1, A-3 can suppress the nerve cell apoptosis of glutamate induction in 20 μm of ol.
Embodiment 9：Wood alkali derivant is measured to glutamate induction neural cell injury model cell lactic dehydrogenase (LDH) active effect
Experiment packet：Blank control group, model group, positive drug group (MK-801), test compound group (20 μm of ol)；
Sample treatment：Using physiological saline solution.
Experimental method：Primary rat cerebellar granule nerve cell is completed to add test compound in the 6th day after being separately cultured (answering 1000 times of dilutions when adding 96 orifice plate).Add within 7th day concentration and (answer 100 when adding 96 orifice plate for the glutamic acid of 200 μm of ol Dilute again).Cell is measured using LDH kits within 8th day.Experimental result is as shown in table 4.
4 lactic dehydrogenase of table (LDH) determination of activity result
Packet Lactic dehydrogenase (LDH) release rate (%) (n=3)
Blank control 100.0±4.00
Model group 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 shown in Table 4：Compound B-1, A-3 has significant difference, Neng Gouxian under 20 μm of ol concentration compared with model group Write and suppress LDH activity, illustrate with the effect for suppressing cytotoxicity.
Embodiment 10：Wood alkali derivant is measured to active oxygen in glutamate induction neural cell injury model cell (ROS) effect of content
Experiment packet：Blank control group, model group, positive drug group (MK-801), test compound group (20 μm of ol)；
Sample treatment：Using physiological saline solution.
Experimental method：Primary rat cerebellar granule nerve cell is completed to add test compound in the 6th day after being separately cultured (answering 1000 times of dilutions when adding 96 orifice plate).Add within 7th day concentration and (answer 100 when adding 96 orifice plate for the glutamic acid of 200 μm of ol Dilute again).Probe is loaded using DCF kits within 8th day, taken pictures using laser confocal microscope, detect.Positive signal table Show that reactive oxygen species respond.Experimental result is as shown in table 5.
5 reactive oxygen species of table (ROS) testing result
Packet Intracellular reactive oxygen content (%) (n=3)
Blank control 100.00±6.71
Model group 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
From table 5：Compound B-1, A-3 can reduce glutamate induction damage under 20 μm of ol concentration compared with model group Hinder the active o content in nerve cell, illustrate that the neuroprotection of B-1, A-3 may be by inhibitory activity oxygen activity.
Fig. 6-10 is laser confocal microscope image, and positive signal represents reactive oxygen species response, positive letter It is number strong, it is higher to represent intracellular reactive oxygen content.As seen from the figure, Fig. 7 model groups positive signal is stronger, represents reactive oxygen species Content is high；Fig. 6 blank groups, Fig. 8 positive drugs group, Fig. 9 compound B-1 groups, Figure 10 compound A-3 group positive signals are weaker, represent Intracellular reactive oxygen content is less, illustrates that compound B-1, A-3 can reduce glutamate induction damaged nerve cells in 20 μm of ol Middle active o content.
Embodiment 11：Wood alkali derivant is measured to glutamate induction neural cell injury model superoxide dismutase (SOD) active effect
Experiment packet：Blank control group, model group, positive drug group (MK-801), test compound group (20 μm of ol)；
Sample treatment：Using physiological saline solution.
Experimental method：It is 200 μ that Primary rat cerebellar granule nerve cell, which is completed to add concentration in the 7th day after being separately cultured, Glutamic acid (answering 100 times of dilutions when adding 96 orifice plate) modeling of mol.Corresponding concentration is added after the completion of modeling and supplies reagent thing, medicine After acting on 48h, protein sample is collected.SOD contents in sample are detected using SOD reagent box for detecting content.Experimental result such as table 6 It is shown.
Table 6SOD content detection results
Packet SOD relative amounts (%) (n=3)
Blank control 100.00±0.00
Model group 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
From table 6：In glutamate induction neural cell injury model, stachydrine, compound B-1, A-3 can increase The expression of superoxide dismutase (SOD), illustrates its Wheat Protein.
Embodiment 12：Wood alkali derivant is measured to glutamate induction neural cell injury model malonaldehyde (MDA) content Effect
Experiment packet：Blank control group, model group, positive drug group (MK-801), test compound group (20 μm of ol)；
Sample treatment：Using physiological saline solution.
Experimental method：It is 200 μ that Primary rat cerebellar granule nerve cell, which is completed to add concentration in the 7th day after being separately cultured, Glutamic acid (answering 100 times of dilutions when adding 96 orifice plate) modeling of mol.Corresponding concentration is added after the completion of modeling and supplies reagent thing, medicine After acting on 48h, protein sample is collected.MDA contents in sample are detected using MDA reagent box for detecting content.Experimental result such as table 7 It is shown.
Table 7MDA content detection results
From table 7：In glutamate induction neural cell injury model, stachydrine, compound B-1, A-3 can reduce Malonaldehyde (MDA) content, illustrates that it has the function that lipid peroxidation inhibition in cell.
The above embodiments are merely illustrative of the technical solutions of the present invention, rather than is limited；Although with reference to foregoing reality Example is applied the present invention is described in detail, for those of ordinary skill in the art, still can be to foregoing implementation Technical solution described in example is modified, or carries out equivalent substitution to which part technical characteristic；And these are changed or replace Change, the essence of appropriate technical solution is departed from the spirit and scope of claimed technical solution of the invention.

Claims (9)

  1. A kind of 1. wood alkali derivant, it is characterised in that its general structure such as formula（I）It is shown：
    (Ⅰ)
    In formula：
    R1For natural amino acid and its acid amides, ester group analog；Orγ- amino-butanamide, ortho-aminobenzoic acid and its acid amides, ester Base analog；
    R2For H, CF3、OCF3、OH、C1-12Alkyl or C1-12Aryl；
    R3、R4And R5It is halogen atom, H, CF3、OCF3、OH、SH、NH2, carboxyl, ester group, sulfuryl, sulfoxide group, sulfonic group, sulphur Perester radical, sulfoamido, ketone group, aldehyde radical, nitro, nitroso, C1-12Alkyl or aryl；
    The halogen atom includes F, Cl, Br or I；The alkyl include saturation or undersaturated open chain alkyl, saturation or Undersaturated cyclic hydrocarbon group.
  2. 2. wood alkali derivant according to claim 1, it is characterised in that：The natural amino acid is glycine, the third ammonia Acid, valine, leucine, isoleucine, aspartic acid, asparagine, glutamine, phenylalanine, tryptophan, methionine, Lysine, arginine, histidine and glutamic acid.
  3. 3. wood alkali derivant according to claim 1, it is characterised in that：The wood alkali derivant for compound B-1, B-2, B-3 and B-4：
    。
  4. 4. the preparation method of the wood alkali derivant described in claim 3, it is characterised in that：It includes the following steps：
    （1）LThe hydrogenation reaction under palladium carbon catalytic action is made-proline a with formaldehydeN- methyl-L- proline b：
    ；
    （2）It is describedN- methyl-L- proline b respectively with ortho-aminobenzoic acid,γ- amino-butanamide, glutamine and glycine Condensation reaction occurs under dicyclohexylcarbodiimide effect described wood alkali derivant B-1, B-2, B-3 and B-4 are made respectively：
    。
  5. 5. the wood alkali derivant and its pharmaceutically acceptable salt described in claim 1 are preparing treatment cardiovascular and cerebrovascular disease Medicine in application.
  6. 6. wood alkali derivant according to claim 5 and its pharmaceutically acceptable salt are preparing treatment cardiovascular and cerebrovascular Application in the medicine of disease, it is characterised in that：The wood alkali derivant include compound A-1, A-2, A-3, A-4, B-1, B-2, B-3 and B-4：
    。
  7. 7. wood alkali derivant according to claim 5 and its pharmaceutically acceptable salt are preparing treatment cardiovascular and cerebrovascular Application in the medicine of class disease, it is characterised in that：The cardio-cerebralvascular diseases include cerebral arterial thrombosis, cerebral infarction, brain Apoplexy and cerebral arteriovenous malformation.
  8. 8. wood alkali derivant according to claim 5 and its pharmaceutically acceptable salt are preparing treatment cardiovascular and cerebrovascular Application in the medicine of disease, it is characterised in that：The pharmaceutically acceptable salt includes salt derived from inorganic acid and organic acid.
  9. 9. wood alkali derivant according to claim 8 and its pharmaceutically acceptable salt are preparing treatment cardiovascular and cerebrovascular Application in the medicine of disease, it is characterised in that：The inorganic acid includes hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid；The organic acid Including methanesulfonic acid, trifluoromethanesulfonic acid, benzene sulfonic acid, p-methyl benzenesulfonic acid, 1-naphthalene sulfonic aicd, 2- naphthalene sulfonic acids, acetic acid, trifluoroacetic acid, apple Acid, tartaric acid, citric acid, lactic acid, oxalic acid, butanedioic acid, fumaric acid, maleic acid, benzoic acid, salicylic acid, phenylacetic acid and almond Acid.
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CN114010843A (en) * 2021-11-16 2022-02-08 四川大学 New application of stachydrine

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CN112409443A (en) * 2021-01-25 2021-02-26 潍坊科技学院 Proteborl derivative, preparation method thereof and application thereof in preparation of medicines for treating cardiovascular and cerebrovascular diseases
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
CN114010843A (en) * 2021-11-16 2022-02-08 四川大学 New application of stachydrine

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