CN103193780B - Use of 4,7-dihydrotetrazyl[1,5-alpha]pyrimidine compound and its derivatives in preparation of drug for prevention or treatment on cerebral hemorrhage - Google Patents
Use of 4,7-dihydrotetrazyl[1,5-alpha]pyrimidine compound and its derivatives in preparation of drug for prevention or treatment on cerebral hemorrhage Download PDFInfo
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- CN103193780B CN103193780B CN201210123287.8A CN201210123287A CN103193780B CN 103193780 B CN103193780 B CN 103193780B CN 201210123287 A CN201210123287 A CN 201210123287A CN 103193780 B CN103193780 B CN 103193780B
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- dihydrotetrazyl
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- TZZDTWNOFOIPPS-UHFFFAOYSA-N CC(Nc1nnn[n]1C1c(cc2)cc3c2OCO3)=C1C(Nc1ccccc1OC)=O Chemical compound CC(Nc1nnn[n]1C1c(cc2)cc3c2OCO3)=C1C(Nc1ccccc1OC)=O TZZDTWNOFOIPPS-UHFFFAOYSA-N 0.000 description 1
- JYEZBNGAHUDAKL-UHFFFAOYSA-N CCOc1ccc(C2[n]3nnnc3NC(C)=C2C(Nc(cccc2)c2OC)=O)cc1OCC Chemical compound CCOc1ccc(C2[n]3nnnc3NC(C)=C2C(Nc(cccc2)c2OC)=O)cc1OCC JYEZBNGAHUDAKL-UHFFFAOYSA-N 0.000 description 1
Abstract
The invention discloses a use of 4,7-dihydrotetrazyl[1,5-alpha]pyrimidine compound and its derivatives in preparation of a drug for prevention or treatment on cerebral hemorrhage. Through a self-created computer-assisted drug molecule design program and virtual screening, it is shown that the 4,7-dihydrotetrazyl[1,5-alpha]pyrimidine compound and its derivatives have a ROCK1 protein inhibitory activity and can be used for treating cerebral hemorrhage. Through further biological activity detection of the screened 4,7-dihydrotetrazyl[1,5-alpha]pyrimidine compound, it is shown that the screened 4,7-dihydrotetrazyl[1,5-alpha]pyrimidine compound has an obvious ROCK1 protein inhibitory activity and has good prevention and treatment effects in an atorvastatin-induced zebra fish cerebral hemorrhage model.
Description
Technical field
The novelty teabag of the present invention one compounds, particularly 4,7-dihydro tetrazoliums [1,5-α] pyrimidines and the application of derivative in preparation treatment hematencephalon medicine thereof.
Background technology
Hematencephalon refers to non-traumatic cerebral intraparenchymal hemorrhage, can be caused by many reasons.Hemorrhage the most common with capsula interna district arteriole clinically.Hemorrhagic hemotoncus (or clot) can be isolated, be oppressed neighbouring cerebral tissue, destroys or affects their normal function (motion, sensation, memory, language, cerebration etc.) and occur the symptoms such as hemiplegia, hemianesthesia, speech be unclear; Intracranial pressure rising, Brain shift unit even hernia cerebri is caused when amount of bleeding is large.Disability rate, mortality ratio are all higher, are one of principal diseases causing human death.
Rho kinases (ROCKs) belongs to the serine/threonine protein kitase of AGC family, and it is first Rho downstream effect factor be found, by have adjusted the change of the myosin cytoskeleton of RhoA induction to the impact of phosphorylation light chain.Research finds, the overexpression in many pathophysiological processes of RhoA/ROCK signal path, as atherosclerosis, cerebral vasospasm, hematencephalon, hypertension, myocardial hypertrophy, heart ischemia reperfusion damage, vascular remodeling etc.Therefore treat by blocking ROCK signal the focus that some disease that there is Rho signal excessive activation becomes people's concern.ROCK is divided into ROCK I and ROCK II two kinds.The height of ROCK is expressed or/and high level activation all can cause the generation of disease, and associated disease mainly comprises cardiovascular disorder, nervous system disorders, fibrotic disease and tumour etc.
External many pharmaceuticals and scientific research institution are developing new ROCK inhibitor.At present, although there are some ROCK inhibitor to enter I phase or II phase clinical investigation phase (as BA-210), the ROCK inhibitor that success has been gone on the market only has one, i.e. method Soviet Union ground that (Fasudil), be mainly used in pulmonary hypertension, and be only limitted to Japanese market.
4,7-dihydro tetrazolium [1,5-α] pyrimidines was once in the news as sodium-ion channel inhibitor (blockers), for the treatment of cardiovascular and cerebrovascular diseases and diabetes, but from having no about in the report Yao for prevention and therapy Cerebral Hemorrhage Disease, C.Heterocyl.Chem.2008,45,1609; Gein, V L Russ.J.Org.Chem.2010,46,699; Zeng, LY J.Comb.Chem.2010,12,35 synthetic methods disclosing this compounds, also can buy and obtain this compounds, and such as Chembridge company buys and obtains.
Summary of the invention
The object of the present invention is to provide 4,7-dihydro tetrazolium [1,5-α] pyrimidines and the application of derivative in preparation treatment hematencephalon medicine thereof
The present inventor found through experiments, and 4,7-dihydro tetrazolium [1,5-α] pyrimidines and derivative thereof have ROCK1 protein inhibiting activity, can be used for treating hematencephalon.Wherein, the general structure of 4,7-dihydro tetrazolium [1,5-α] pyrimidines and derivative thereof is:
In formula, R1, R2 and R3 be H, containing the straight chain of C1-6 or branched-chain alkyl, C1-C4 alkoxyl group or C1-C4 aryloxy,
Aryl, substituted aryl, X is C, O or N;
Or R3 is straight chain or an alkane of halogen or halogen substiuted C1-6;
Or R1 and R2 is 5 yuan or 6 yuan of N heterocycles or containing N heterocycle or benzheterocycle;
Or R1 and R2 is single or multiple substituted aryl, and the contraposition be in respectively on aromatic ring or ortho position or a position.
Preferably, R1 ~ R3 is respectively C1-C4 alkyl.
Preferably, C1-C4 aryloxy is benzyloxy, benzene oxyethyl group or benzene propoxy-.
Preferably, R1 and R2 is single or multiple substituting group, and described substituting group is phenyl, chloro-phenyl-, phenylol or anilino.
Especially, 4,7-dihydro tetrazolium [1,5-α] pyrimidines is:
7-(4-hydroxy 3-methoxybenzene base)-5-methyl-6-carboxylic acid benzyl ester-4,7-dihydro tetrazolium (1,5-α) pyrimidine,
7-(3,4-diethoxy phenyl)-6-[N-(2-p-methoxy-phenyl) formamido-]-5-methyl-4,7-dihydro tetrazolium (1,5-α) pyrimidine,
7-(4-benzyloxy-3-p-methoxy-phenyl)-6-(N-phenyl formamide base)-5-methyl-4,7-dihydro tetrazolium, (1,5-α) pyrimidine,
7-(2-N-pyrimidine)-5-fluoro-6-carboxylic acid benzyl ester-4,7-dihydro tetrazolium (1,5-α) pyrimidine,
7-phenyl-6-(N-phenmethyl) formamido--5-methyl-4,7-dihydro tetrazolium (1,5-α) pyrimidine,
7-(1,3-benzodioxole)-6-[N-(2-p-methoxy-phenyl) formamido-]-5-methyl-4,7-dihydro tetrazolium (1,5-α) pyrimidine,
7-(3-methoxyl group-3 '-nitro-4-hydroxy phenyl)-6-[N-(2-p-methoxy-phenyl) formamido-]-5-methyl-4,7-dihydro tetrazolium (1,5-α) pyrimidine,
7-(4-benzyloxy phenyl)-6-[N-(2-pyridine) formamido-]-4,7-dihydro tetrazoliums (1,5-α) pyrimidine or
7-(4-p-methoxy-phenyl)-6-carboxylic acid isopropyl esters-4,7-dihydro tetrazolium (1,5-α) pyrimidine.
Preferably, 4,7-dihydro tetrazolium [1,5-α] pyrimidines is its pharmacy acceptable salt.As its hydrochloride, phosphoric acid salt, vitriol, acetate, maleate, citrate, benzene sulfonate, toluenesulfonate, fumarate, tartrate.
The present inventor is by own Computeraided drug design program, and virtual screening finds that 4,7-dihydro tetrazolium [1,5-α] pyrimidines and derivative thereof may have ROCK1 protein inhibiting activity, can be used for treating hematencephalon.By carrying out further Determination of biological activity to screening the compound obtained, finding that it all has obvious inhibit activities to ROCK 1 albumen, in prevention and therapy atorvastatin induction zebra fish cerebral hemorrhage mold, all showing good effect.
Accompanying drawing explanation
Fig. 1 is the suppression curve of the present invention one particular compound to ROCK 1;
Fig. 2 is the impact of the present invention one particular compound on the hematencephalon of atorvastatin induction zebra fish.(A). processing zebra fish with 0.2%DMSO (solvent) is blank group; (B). large zebra fish process 1 μM of atorvastatin of after fertilization 24 hours 24 hours; (C). the large zebra fish pre-treatment compound of after fertilization 22.5 hours 2.5 hours, reprocessing 1 μM of atorvastatin 24 hours after cleaning.Double transgenics zebra fish, wherein green fluorescence is Tg (fli1a:EGFP) y1, and red fluorescence is Tg (gatal:dsRed) sd2, the 3rd overlapping photographs being classified as first two columns;
Fig. 3 is the result for the treatment of figure of the present invention one particular compound to the hematencephalon of atorvastatin induction zebra fish;
Fig. 4 is that Mouse Weight is schemed over time.
Embodiment
Below in conjunction with experiment and experimental data, further illustrate the present invention.
4,7-dihydro tetrazoliums [1, the 5-α] pyrimidines used in following examples and derivative thereof are all purchased from Chembridge company (U.S., San Diego).
During the enzyme inhibition activity experiment of following ROCK1, the experiment of zebra fish hematencephalon evaluating drug effect, Mouse Acute Toxicity are tested, the medicable compound of tool used is 7-(4-hydroxy 3-methoxybenzene base)-5-methyl-6-carboxylic acid benzyl ester-4,7-dihydro tetrazolium (1,5-α) pyrimidine, its structural formula is:
The enzyme inhibition activity experiment of ROCK1
Experimental principle: the measurement of compound R OCK1 inhibit activities have employed the Z '-LYTETM technology of Invitrogen company, this technology based on FRET (fluorescence resonance energy transfer) (FRET) principle, based on the sensitivity differences that phosphorylation and non-phosphorylating polypeptide are cut proteolysis.
Experimental technique:
(1) preparation of reagent.Kinase buffer liquid: the 5X kinase buffer liquid of 2ml is diluted with water to 10ml; Compound: the compound that test is diluted with water to a concentration gradient; The mixing solutions of ROCK kinases/substrate: the mixing solutions preparing 2250 μ LROCK/ substrates; The concentration of enzyme is 10ng/ml, and the concentration of substrate is 4 μMs, and solvent is kinase buffer liquid; MALDI-PSD solution: serine/threonine phosphorylation 7 peptide of 2 μ L is added in the kinase buffer liquid of 498 μ L, fully mixes; ATP solution: the ATP solution preparing 1110 μ L.Concentration is 50 μMs, and solvent is kinase buffer liquid; Developer: by the dilution proportion developer A of 1: 32768.
(2) experimental procedure:
1) compound solution that 2.5 μ L prepare is added to black 384 orifice plate;
2) mixing solutions of 5 μ LROCK kinases/substrates is added;
3) 2.5 μ LATP solution are added;
4) 384 orifice plates at room temperature shaken and cultivate 1 hour;
5) 5 μ L developers are joined in orifice plate, continue reaction 1 hour;
6) orifice plate is placed in microplate reader reading.
Experimental result: 7-(4-hydroxy 3-methoxybenzene the base)-5-methyl-6-carboxylic acid benzyl ester-4 using different concns, 7-dihydro tetrazolium (1,5-α) pyrimidine carry out ROCK1 enzyme inhibition activity experiment, find that this compound has good inhibit activities, its 503nhibiting concentration IC
50be 13.7 μMs/L, experimental result as shown in Figure 1.
Zebra fish hematencephalon evaluating drug effect is tested
Experimental principle:
Atorvastatin has the effect of induction hematencephalon to zebra fish, by contrast through compound treatment and undressed cerebral hemorrhage mold, evaluates the effect of medicine in the hematencephalon of prevention and therapy atorvastatin induction zebra fish.
Experimental procedure:
1) 21 hours large transgenosiss and wild-type zebrafish pre-treatment compound about 2.5 hours after selective fertilization, process 24 hours again after cleaning, observe finally by confocal microscope under 1 μM of atorvastatin concentration;
2) 1 μM of atorvastatin and 5 μMs of co-treatment zebra fishs 24 hours, observe finally by confocal microscope.
Experimental result:
Experimental result is (in figure, A) as shown in Figure 2. and processing zebra fish with 0.2%DMSO (solvent) is blank group; (B). large zebra fish process 1 μM of atorvastatin of after fertilization 24 hours 24 hours; (C). the large zebra fish pre-treatment compound of after fertilization 22.5 hours 2.5 hours, reprocessing 1 μM of atorvastatin 24 hours after cleaning.Double transgenics zebra fish, wherein green fluorescence is Tg (fli1a:EGFP) y1, and red fluorescence is Tg (gata1:dsRed) sd2, the 3rd overlapping photographs being classified as first two columns).
1 μM of atorvastatin process after 24 hours, significantly red patch is had at zebra fish encephalic, it is the transgenic zebrafish of Tg (fli1a:EGFP) y1 and Tg (gata1:dsRed) sd2 double cross herein, the patch of its redness represents zebra fish encephalic and have accumulated blood, namely zebra fish hematencephalon.But be through compound pre-treatment after 2.5 hours, the phenomenon of zebra fish hematencephalon does not occur completely, prove that compound has good effect in the hematencephalon of prevention atorvastatin induction zebra fish.In addition, at compound to (as Fig. 3) in the treatment effectiveness evaluation of zebra fish hematencephalon, the phenomenon of zebra fish hematencephalon does not occur completely, proves that compound has good curative effect in the hematencephalon for the treatment of atorvastatin induction zebra fish.Therefore, no matter be prevention hematencephalon or treatment hematencephalon, compound all shows good effect.
Mouse Acute Toxicity is tested
Experimental principle:
Acute toxicity test mainly study single or multiple in certain hour give chemical substance after the toxic reaction that produces of animal and death condition.This experiment can obtain mld (LD
50), the tested material dosage of laboratory animal 50% death can be caused.According to OECD chemical toxicity grade scale in 1998, with 5,50,300,2000,5000mg/kg for dividing point, substance toxicity is divided into 5 grades.Work as LD
50during > 2000mg/kg, this kind of compound can be considered to substantially nontoxic.
Experimental procedure:
Experiment adopts balb/c mouse 10, about body weight 20g.Adopt the mode of oral administration, dosage was 2000mg/kg, to observation post administration 14 days.
Experimental result: experimental result shows, mouse there is no any unusual condition, without dead.As shown in Figure 4, upon administration, the body weight of mouse is steadily increasing, illustrates that this compound is very safe, substantially can assert nontoxic, can carry out follow-up research and development as a kind of potential medicine.
Claims (2)
1.7-(4-hydroxy 3-methoxybenzene base)-5-methyl-6-carboxylic acid benzyl ester-4,7-dihydro tetrazolium (1,5-α) application in preparation prevention or treatment hematencephalon medicine of pyrimidine and medicinal derivative thereof, described 7-(4-hydroxy 3-methoxybenzene base)-5-methyl-6-carboxylic acid benzyl ester-4,7-dihydro tetrazolium (1,5-α) pyrimidine medicinal derivative is its pharmacy acceptable salt.
2. application according to claim 1, it is characterized in that: pharmaceutically acceptable 7-(4-hydroxy 3-methoxybenzene base)-5-methyl-6-carboxylic acid benzyl ester-4,7-dihydro tetrazolium (1,5-α) pyrimidine salt is its hydrochloride, phosphoric acid salt, vitriol, acetate, maleate, citrate, benzene sulfonate, toluenesulfonate, fumarate, tartrate.
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| CN201210123287.8A CN103193780B (en) | 2012-04-24 | 2012-04-24 | Use of 4,7-dihydrotetrazyl[1,5-alpha]pyrimidine compound and its derivatives in preparation of drug for prevention or treatment on cerebral hemorrhage |
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| CN201210123287.8A CN103193780B (en) | 2012-04-24 | 2012-04-24 | Use of 4,7-dihydrotetrazyl[1,5-alpha]pyrimidine compound and its derivatives in preparation of drug for prevention or treatment on cerebral hemorrhage |
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| CN103193780B true CN103193780B (en) | 2015-07-08 |
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| US20160113931A1 (en) * | 2014-10-24 | 2016-04-28 | University Of Macau | Method of treating and/or preventing neurodegenerative diseases |
| TWI800498B (en) * | 2016-12-21 | 2023-05-01 | 義大利商吉斯藥品公司 | Bicyclic dihydropyrimidine-carboxamide derivatives as rho-kinase inhibitors |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2007139856A2 (en) * | 2006-05-22 | 2007-12-06 | Alantos Pharmaceuticals, Inc. | Heterobicyclic metalloprotease inhibitors |
| WO2009006580A1 (en) * | 2007-07-05 | 2009-01-08 | Cv Therapeutics, Inc. | Optionally condensed dihydropyridine, dihydropyrimidine and dihydropyrane derivatives acting as late sodium channel blockers |
| CA2749040A1 (en) * | 2009-01-09 | 2010-07-15 | Franz Von Nussbaum | Triazolo and tetrazolo pyrimidine derivatives as hne inhibitors for treating copd |
| CN102432612A (en) * | 2011-09-07 | 2012-05-02 | 苏州大学 | 4,7-dihydrotetrazole[1,5-a]pyrimidine derivative and application thereof to preparation of antitumor medicine |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2007139856A2 (en) * | 2006-05-22 | 2007-12-06 | Alantos Pharmaceuticals, Inc. | Heterobicyclic metalloprotease inhibitors |
| WO2009006580A1 (en) * | 2007-07-05 | 2009-01-08 | Cv Therapeutics, Inc. | Optionally condensed dihydropyridine, dihydropyrimidine and dihydropyrane derivatives acting as late sodium channel blockers |
| CA2749040A1 (en) * | 2009-01-09 | 2010-07-15 | Franz Von Nussbaum | Triazolo and tetrazolo pyrimidine derivatives as hne inhibitors for treating copd |
| CN102432612A (en) * | 2011-09-07 | 2012-05-02 | 苏州大学 | 4,7-dihydrotetrazole[1,5-a]pyrimidine derivative and application thereof to preparation of antitumor medicine |
Non-Patent Citations (1)
| Title |
|---|
| V. L. Gein,等.Synthesis of Alkyl 5-Aryl-7-methyl-1,5-dihydrotetrazolo-[1,5-a]pyrimidine-6-carboxylates.《Russian Journal of Organic Chemistry》.2010,第46卷(第5期),第699-705页. * |
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