CN112457265A - Tetrazole derivative, preparation method thereof, pharmaceutical composition containing tetrazole derivative and application of pharmaceutical composition - Google Patents

Tetrazole derivative, preparation method thereof, pharmaceutical composition containing tetrazole derivative and application of pharmaceutical composition Download PDF

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CN112457265A
CN112457265A CN202011231596.8A CN202011231596A CN112457265A CN 112457265 A CN112457265 A CN 112457265A CN 202011231596 A CN202011231596 A CN 202011231596A CN 112457265 A CN112457265 A CN 112457265A
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tetrazol
pentan
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董晓武
金一真
庄宇昕
车金鑫
陈斌辉
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Zhejiang University ZJU
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Abstract

The invention discloses a tetrazole derivative which has a structure shown in a general formula I, wherein R1Is H, C1‑C3An alkyl chain or a haloalkyl chain. The invention also discloses the tetrazoleThe composition of the derivative and the application thereof in preparing medicines for preventing and resisting cerebral apoplexy. The inventor of the invention proves that the compound has obvious inhibition effect on platelet aggregation activity induced by ADP, good protection effect on primary mouse nerve cell model with OGD/R injury and good oral pharmacokinetic properties of rats through multiple experiments. Therefore, the compound can be used as a neuroprotective agent in medicaments for treating and preventing cerebral apoplexy.

Description

Tetrazole derivative, preparation method thereof, pharmaceutical composition containing tetrazole derivative and application of pharmaceutical composition
Technical Field
The invention relates to the field of medicaments, in particular to a tetrazole derivative, a preparation method thereof, a pharmaceutical composition containing the tetrazole derivative and application of the tetrazole derivative in preparing medicaments for preventing and resisting cardiovascular and cerebrovascular diseases, and improving cardiovascular and cerebrovascular circulatory disturbance or antithrombotic.
Background
Stroke is one of the common cardiovascular and cerebrovascular diseases, and has become the leading cause of disability and the second leading cause of death in adults worldwide. The incidence of ischemic stroke accounts for about 80% of the total stroke, and seriously threatens the health of human beings. Over the last decade, research has focused primarily on exploring more effective therapeutic strategies to reduce stroke-induced death and disability. A number of drugs have been explored in clinical trials and animal models, but there is still a lack of effective therapeutic strategies. In addition, there is no neuroprotective agent preventive drug against acute ischemic stroke, and therefore, there is a need for a novel compound having a novel structural feature and a novel mechanism of action, which has a wide market and clinical demand in this field. The tetrazole derivative is widely applied to the aspect of drug development, has good drug effects on the aspects of hypertension resistance, inflammation diminishing, bacteria resistance, platelet aggregation inhibitor, asthma treatment and the like, and for example, antihypertensive drugs, namely the first oral active potent non-peptide angiotensin II receptor antagonist Losartan (Losartan), and platelet aggregation inhibitor drug cilostazol are introduced into a tetrazole structure and achieve very good pharmacological effects.
Disclosure of Invention
The invention aims to provide a tetrazole derivative and pharmaceutically acceptable salt or solvate thereof.
The invention also provides a method for preparing the tetrazole derivative.
The invention also provides a pharmaceutical composition containing the tetrazole derivative and pharmaceutically acceptable salts or solvates thereof.
The invention also provides an application of the tetrazole derivative and pharmaceutically acceptable salt or solvate thereof in preparing a medicament for preventing and resisting cerebral apoplexy.
The invention adopts the following technical scheme:
the invention provides a tetrazole derivative which has a structure shown in a general formula (I):
Figure BDA0002765401380000021
or an optical isomer thereof;
or a pharmaceutically acceptable salt or solvate thereof;
wherein: r1Is H, C1-C3An alkyl chain or haloalkyl chain of (a);
R2is H, amino, nitro, hydroxyl, ether bond, methyl, ester group, carbonyl group, -CF3，-OCF3N-butyl, isopropyl, a peptide bond, one or more Cl, one or more F, one or more Br, or a combination of at least two of one or more Cl, one or more F, one or more Br;
R3independently selected from
Figure BDA0002765401380000022
Wherein: r4Is H or C (═ O) C1-C3；
A is benzene ring or six-membered heterocyclic ring containing at least one N atom or five-membered heterocyclic ring containing at least one N atom.
Preferably, R1When H and A is a benzene ring, R2Is not H, F, Cl, Br;
further, preferably, the tetrazole derivative of the present invention has a structure represented by the general formula (II):
Figure BDA0002765401380000023
or an optical isomer thereof; or a pharmaceutically acceptable salt or solvate thereof; or an optical isomer thereof, pharmaceutically acceptable salt or solvate thereof;
wherein R is1、R2、R3The definition is the same as above; x is C or N.
Further, preferably, the tetrazole derivative has a structure represented by general formula III:
Figure BDA0002765401380000031
or an optical isomer thereof; or a pharmaceutically acceptable salt or solvate thereof; or an optical isomer thereof, pharmaceutically acceptable salt or solvate thereof;
preferably, R1H, methyl, ethyl; r2H, Cl, F, Br or amino;
x is C or N.
Preferably, R is1Is methyl or ethyl; preferably, X is N;
further, preferably, the pharmaceutically acceptable salt thereof is a salt with one or more of sodium hydroxide, potassium hydroxide, lithium hydroxide, sodium methoxide, potassium methoxide, sodium ethoxide, potassium ethoxide, triethylamine and tert-butylamine;
still more preferably sodium hydroxide, potassium hydroxide having the general formula Va, Vb:
Figure BDA0002765401380000032
or an optical isomer thereof, wherein X, R2The definition is the same as above.
Preferably, the tetrazole derivative comprises one or more of the following compounds:
1- (4-bromo-2- (1-methyl-1H-tetrazol-5-yl) phenyl) pentan-1-ol
1- (6-bromo-2- (1H-tetrazol-5-yl) pyridin-3-yl) pentan-1-ol
1- (6-bromo-2- (1-methyl-1H-tetrazol-5-yl) pyridin-3-yl) pentan-1-ol
1- (4-chloro-2- (1-methyl-1H-tetrazol-5-yl) phenyl) pentan-1-ol
1- (6-chloro-2- (1H-tetrazol-5-yl) pyridin-3-yl) pentan-1-ol
1- (6-chloro-2- (1-methyl-1H-tetrazol-5-yl) pyridin-3-yl) pentan-1-ol
1- (4-fluoro-2- (1-methyl-1H-tetrazol-5-yl) phenyl) pentan-1-ol
1- (6-fluoro-2- (1H-tetrazol-5-yl) pyridin-3-yl) pentan-1-ol
1- (6-fluoro-2- (1-methyl-1H-tetrazol-5-yl) pyridin-3-yl) pentan-1-ol
1- (2- (1-methyl-1H-tetrazol-5-yl) phenyl) pent-1-ol
1- (2- (1H-tetrazol-5-yl) pyridin-3-yl) pent-1-ol
1- (2- (1-methyl-1H-tetrazol-5-yl) pyridin-3-yl) pentan-1-ol
1- (4-amino-2- (1H-tetrazol-5-yl) phenyl) pentan-1-ol
1- (4-amino-2- (1-methyl-1H-tetrazol-5-yl) phenyl) pentan-1-ol
1- (4-nitro-2- (1H-tetrazol-5-yl) phenyl) pentan-1-ol
1- (4-Nitro-2- (1-methyl-1H-tetrazol-5-yl) phenyl) pentan-1-ol
1- (2- (1H-tetrazol-5-yl) -4- (trifluoromethyl) phenyl) pentan-1-ol
1- (2- (1-methyl-1H-tetrazol-5-yl) -4- (trifluoromethyl) phenyl) pentan-1-ol
Or other optical isomers of the above compounds; or a pharmaceutically acceptable salt or solvate of the above compound; or an optical isomer thereof.
In particular, according to formula I, preferred compounds of the invention are:
Figure BDA0002765401380000041
Figure BDA0002765401380000051
Figure BDA0002765401380000061
and optical isomers of the above compounds; or a pharmaceutically acceptable salt or solvate thereof.
Or other optical isomers of the above compounds; or a pharmaceutically acceptable solvate of the above compound.
Preferably, the pharmaceutically acceptable salt is a salt with one or more of the following bases: sodium hydroxide, potassium hydroxide, lithium hydroxide, triethylamine, tert-butylamine.
Preferably, the tetrazole derivatives include:
1- (6-bromo-2- (1H-tetrazol-5-yl) pyridin-3-yl) pentan-1-ol sodium salt
Potassium 1- (6-bromo-2- (1H-tetrazol-5-yl) pyridin-3-yl) pentan-1-ol salt
Lithium 1- (6-bromo-2- (1H-tetrazol-5-yl) pyridin-3-yl) pentan-1-ol salt
1- (6-bromo-2- (1H-tetrazol-5-yl) pyridin-3-yl) pent-1-ol triethylamine salt
1- (6-bromo-2- (1H-tetrazol-5-yl) pyridin-3-yl) pent-1-ol tert-butylamine salt
Sodium salt of 1- (2- (1H-tetrazol-5-yl) -4- (trifluoromethyl) phenyl) pentan-1-ol
Potassium 1- (2- (1H-tetrazol-5-yl) -4- (trifluoromethyl) phenyl) pent-1-ol salt
Lithium 1- (2- (1H-tetrazol-5-yl) -4- (trifluoromethyl) phenyl) pent-1-olate
1- (2- (1H-tetrazol-5-yl) -4- (trifluoromethyl) phenyl) pent-1-ol triethylamine salt
1- (2- (1H-tetrazol-5-yl) -4- (trifluoromethyl) phenyl) pent-1-ol tert-butylamine salt
Sodium salt of 1- (4-amino-2- (1H-tetrazol-5-yl) phenyl) pentan-1-ol
Potassium salt of 1- (4-amino-2- (1H-tetrazol-5-yl) phenyl) pent-1-ol
Lithium 1- (4-amino-2- (1H-tetrazol-5-yl) phenyl) pent-1-ol salts
1- (4-amino-2- (1H-tetrazol-5-yl) phenyl) pent-1-ol triethylamine salt
1- (4-amino-2- (1H-tetrazol-5-yl) phenyl) pent-1-ol tert-butylamine salt
Or other optical isomers of the above compounds; or a pharmaceutically acceptable solvate of the above compound.
Reacting a compound (1) with an acyl protection reagent to obtain a compound (2), reacting the compound (2) with sodium azide, adding hydrochloric acid, removing a protecting group and eliminating residual sodium azide to obtain a compound (3), and reacting the compound (3) with a Grignard reagent to obtain a compound shown in a formula (I); optionally, the compound shown in the formula (I) is further reacted with an alkylating agent to obtain a tetrazolylalkyl substitution compound shown in the formula (I'):
Figure BDA0002765401380000071
preferably, the acyl protecting agent is ethylene glycol.
The compound (1) can be obtained by taking the compound shown in the formula (1-1) as a raw material through bromination and hydrolysis:
Figure BDA0002765401380000072
when the compound (1-2) is prepared from the compound (1-1), NBS is used as a brominating reagent, AIBN is optionally added as an initiator, carbon tetrachloride is used as a reaction solvent, the reaction temperature is reflux temperature, and the reaction time is 10-24 hours.
Wherein the molar ratio of the brominating agent to the compound (1-1) is 1.5-3: 1. After the reaction is finished, directly adding water and ethanol (1:1) and silver nitrate with the same equivalent weight as the compound, and performing hydrolysis reaction in the second step at the reaction temperature of 60-80 ℃. The reaction time was 4 hours.
Preferably, the temperature for the reaction of the compound (1) and the acyl protective reagent is 70-130 ℃; the reactant can be toluene, and the catalyst can be p-toluenesulfonic acid. Wherein the molar ratio of the ethylene glycol to the compound (1) is 1-10: 1, more preferably 2-8: 1, and still more preferably 4-7: 1. The molar ratio of the p-toluenesulfonic acid to the compound (1) is 0.03-0.2: 1, and more preferably 0.05 to 0.1: 1.
Preferably, the temperature for the reaction of the compound (2) and the sodium azide is 110-170 ℃. Wherein, ammonium chloride is added, the reaction solvent is DMF, and diluted hydrochloric acid is used for quenching after the reaction is finished. The molar ratio of the sodium azide to the compound (2) is 2-10: 1, and more preferably 4-7: 1. The molar ratio of ammonium chloride to the compound (2) is 2 to 10:1, and more preferably 4 to 7: 1.
The format reagent can be a commercial product or prepared on site. Can be prepared from bromo-C3-C5The alkane reacts with magnesium, and iodine particles can be added in the preparation process. Bromo C3-C5The molar ratio of the alkane to the compound (2) is 2 to 10:1, and more preferably 4 to 7: 1.
Preferably, the reaction temperature of the compound (3) and the alkylating reagent (iodotin) is 30-50 ℃, DMF is selected as a reaction solvent, and sodium hydrogen is selected as a catalyst. Wherein the molar ratio of the iodocarbon to the compound (3) is 1-2: 1, and more preferably 1 to 1.5: 1. The molar ratio of sodium hydrogen to the compound (3) is 2-3: 1, and more preferably 2.5 to 3: 1.
A pharmaceutical composition, comprising at least one active ingredient and one or more pharmaceutically acceptable carriers or excipients, wherein the active ingredient is a tetrazole derivative according to any one of the above technical schemes.
An application of the tetrazole derivative in preparing the medicines for preventing and resisting cardiovascular and cerebrovascular diseases, improving cardiovascular and cerebrovascular circulatory disturbance or resisting thrombus.
The term "pharmaceutically acceptable derivative" as used herein refers to salts and solvates of the selected compound.
The term "alkyl" as used herein refers to straight or branched chain alkane-containing groups of carbon atoms, examples of "alkyl" as used herein include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, and the like, and "alkyl" also includes substituted alkyl groups. Alkyl groups may be optionally substituted one or more times with halo.
The term "halogen" as used herein denotes fluorine, chlorine, bromine or iodine.
The term "solvate" as used herein refers to a complex of variable stoichiometry formed by a solute (e.g., formula (I) of the present invention) and a solvent. For the purposes of the present invention, the solvent should not interfere with the biological activity of the solute. Examples of suitable solvents include, but are not limited to, water, methanol, ethanol, and acetic acid. Preferably, the solvent used is a pharmaceutically acceptable solvent. Suitable pharmaceutically acceptable solvents include, but are not limited to, water, ethanol, and acetic acid. More preferably, the solvent used is water.
The salts of the tetrazole compounds of the present invention can be prepared by methods well known to those skilled in the art. The salt can be organic base salt, inorganic base salt and the like, and the organic base salt comprises sodium methoxide, potassium methoxide, sodium ethoxide, potassium ethoxide, triethylamine, tert-butylamine and the like; the inorganic alkali salt includes sodium hydroxide, potassium hydroxide, lithium hydroxide and the like.
The second purpose of the present invention is to provide a pharmaceutical composition, which comprises at least one active ingredient and one or more pharmaceutically acceptable carriers or excipients, wherein the active ingredient can be any one or more of tetrazole compounds with the structure shown in the general formula (I) of the present invention and preferred compounds thereof, optical isomers of the compounds, pharmaceutically acceptable salts of the compounds or the optical isomers thereof, and solvates of the compounds or the optical isomers thereof.
The carrier comprises one or more of conventional diluent, excipient, filler, binder, humectant, disintegrating agent, absorption promoter, surfactant, adsorption carrier, lubricant, etc., and optionally flavoring agent, sweetener, etc. The medicine can be prepared into various forms such as tablets, powder, granules, capsules, oral liquid, injection and the like, and the medicines of the various forms can be prepared according to a conventional method in the pharmaceutical field.
The invention also provides application of the compound of the formula (I) and optical isomers thereof or pharmaceutically acceptable salts or solvates thereof in preparing medicaments for preventing and resisting cardiovascular and cerebrovascular diseases, improving cardiovascular and cerebrovascular circulatory disorders or resisting thrombus.
The invention also provides application of the compound or the medicinal salt thereof in preparing medicaments for preventing and resisting cardiovascular and cerebrovascular diseases, improving cardiovascular and cerebrovascular circulatory disturbance or resisting thrombus, in particular application in preparing medicaments for treating acute ischemic stroke. In other words, the present invention provides the use of a tetrazole compound or a pharmaceutically acceptable salt thereof alone or in combination with other drugs for the treatment of acute ischemic stroke. Anti-stroke agents that can be used in combination with the compounds provided herein or pharmaceutically acceptable salts thereof include, but are not limited to, at least one of the following: free radical scavengers (such as edaravone); neuroprotective agents (e.g., schpulan); antiplatelet agents (e.g., clopidogrel, aspirin); antithrombotic agents (e.g. rivaroxaban).
The inventor of the invention proves that the compound has inhibitory activity on platelet aggregation induced by ADP, good protection effect on mouse primary cortical nerve cells damaged by OGD/R and good oral pharmacokinetic properties of rats through multiple experiments. Therefore, the compound can be used as a neuroprotective agent to prepare medicines for treating and preventing acute ischemic stroke.
Drawings
FIG. 1 shows NBP dose-time curves (IG10mg/kg) in SD rat plasma.
FIG. 2 is a graph showing the plasma time course (IG10mg/kg) of SD rat of compound V-7 of the present invention.
FIG. 3 is a half-life curve of compound V-7 of the present invention in SD rat plasma (IG10 mg/kg).
Detailed Description
The following examples are provided to illustrate the applicability of the present invention, and it will be understood by those skilled in the art that various modifications and substitutions can be made to the corresponding technical features according to the teachings of the prior art, and still fall within the scope of the present invention as claimed.
EXAMPLE 1 Synthesis of intermediates 1-3
Figure BDA0002765401380000101
Raw materials 1-1(13.2g, 0.1mol), NBS (N-bromosuccinimide) (44.5g, 0.25mol), AIBN (azobisisobutyronitrile) (720mg, 3mmol), and carbon tetrachloride (20ml) were added to a three-necked flask, heated under reflux for 16 hours, cooled to room temperature, and concentrated under reduced pressure to remove carbon tetrachloride, to give a mixture containing intermediate 1-2. Adding water and ethanol (100 ml: 100ml) into the mixture, reacting for 4h at 70 ℃, extracting by ethyl acetate to obtain an organic phase, drying by anhydrous sodium sulfate, concentrating under reduced pressure and evaporating the ethyl acetate to dryness, and purifying the obtained crude product by silica gel column chromatography (ethyl acetate: petroleum ether: 1:50-1:10) to obtain white to pale yellow crystal intermediate 1-3 about 8.4g, wherein the yield is 40% and the purity is more than 99%. HRMS m/z (ESI) calcd for C8H6N2O[M+H]+147.05found:147.67。
Example 2: synthesis of intermediates 1 to 5
Figure BDA0002765401380000111
The method comprises the following steps: the intermediates 1-3(2.8g, 20mmol), ethylene glycol (6.2g, 0.1mol), toluene (20ml), p-toluenesulfonic acid (275mg, 1.6mmol) were added to a single-necked flask and heated under reflux, water was removed using a water separator until the amount of water in the water separator did not increase any more, and the flask was cooled to room temperature. Saturated sodium bicarbonate solution was added until the aqueous layer was neutral or basic, the aqueous layer was separated, and the organic layer was concentrated under reduced pressure to give 4.5g of pale yellow oily semi-solid intermediates 1 to 4. The yield thereof was found to be 89%. The purity is more than 99%.
Step two: a single neck flask was charged with intermediate 1-4(0.7g, 3.93mmol), sodium azide (1.28g, 19.65mmol), ammonium chloride (1.05g, 19.65mmol), and DMF (10ml), heated at reflux for 12h, cooled to room temperature, quenched with 1N hydrochloric acidAfter the deprotection was confirmed by TLC, the organic phase was extracted with ethyl acetate, concentrated under reduced pressure to remove ethyl acetate, and the resulting crude product was purified by silica gel column chromatography (dichloromethane: methanol 10:1) to obtain 924mg of intermediate 1-5. Yield 93%, HRMS m/z (ESI) calcd for C8H7N5O[M-H]-189.07found:189.56。
Example 3: synthesis of intermediates 2-3
Figure BDA0002765401380000112
With reference to the procedure of example 1, 5- (trifluoromethyl) -2-methylbenzonitrile 2-1(18.5g, 0.1mol) was used as a starting material to give 15.2g of 2-3 as a solid in a yield of 76% and a purity of greater than 99% HRMS: m/z (ESI) calcd for C9H4F3NO[M+H]+200.02found:200.25。
Example 4: synthesis of intermediates 2 to 5
Figure BDA0002765401380000113
Referring to step one and step two of example 2, intermediate 2-3 was used in place of intermediate 1-3 and intermediate 2-4 was used in place of intermediate 1-4 to give 2.3g of solid 2-5 in 56% yield (two steps) with purity greater than 99% HRMS: m/z (ESI) calcd for C9H5F3N4O[M-H]-241.04found:240.89。
Example 5: synthesis of intermediate 3-1
Figure BDA0002765401380000121
With reference to the procedure of example 1, 5-nitro-2-methylbenzonitrile 3-1(16.2g, 0.1mol) was used as a starting material to give 8.9g of 4-1a-3 as a solid in a yield of 50% and a purity of more than 99%, HRMS: m/z (ESI) calcd for C8H4N2O3[M+H]+177.02found:177.23。
Example 6: synthesis of intermediates 3-5
Figure BDA0002765401380000122
Referring to step one and step two of example 2, intermediate 3-3 was used in place of intermediate 1-3 and intermediate 3-4 was used in place of intermediate 1-4 to give 3.5g of solid 3-5 in 72% yield (two steps) with purity greater than 99% HRMS: m/z (ESI) calcd for C8H5N5O3[M-H]-218.04found:217.92。
Example 7: synthesis of intermediate 4-3
Figure BDA0002765401380000123
With reference to the procedure of example 1, starting from 6-bromo-3-methylpyridine 4-1(19.5g, 0.1mol), 6.2g of solid 4-3 were obtained in a yield of 29% and with a purity of greater than 99%, HRMS: m/z (ESI) calcd for C7H3BrN2O[M+H]+210.94found:211.05。
Example 8: synthesis of intermediates 4 to 5
Figure BDA0002765401380000124
Referring to step one and step two of example 2, intermediate 4-3 was used instead of intermediate 1-3 and intermediate 4-4 was used instead of intermediate 1-4 to give 2.2g of solid 4-5 in 35% yield (two steps) with a purity of greater than 99% HRMS: m/z (ESI) calcd for C7H4BrN5O[M-H]-251.96found:252.05。
Example 9: synthesis of intermediate 5-3
Figure BDA0002765401380000131
With reference to the procedure of example 1, starting from 5-bromo-2-methylbenzonitrile 5-1(19.4g, 0.1mol), 6.2g of a solid 5-3 was obtained in 29% yield and purityGreater than 99% HRMS m/z (ESI) calcd for C8H4BrNO[M+H]+209.95found:210.10。
Example 10: synthesis of intermediates 5-5
Figure BDA0002765401380000132
Referring to step one and step two of example 2, intermediate 5-3 was used in place of intermediate 1-3 and intermediate 5-4 was used in place of intermediate 1-4 to give 2.6g of solid 5-5 in 37% yield (two steps) with a purity of greater than 99% HRMS: m/z (ESI) calcd for C8H5BrN4O[M-H]-250.96found:251.32。
Example 11: synthesis of intermediate 6-3
Figure BDA0002765401380000133
Referring to the procedure of example 1, 5-chloro-2-methylbenzonitrile 6-1(15.1g, 0.1mol) was used as a starting material to give 7.3g of solid 6-3 in 44% yield with a purity of greater than 99% HRMS: m/z (ESI) calcd for C8H4ClNO[M+H]+166.00found:166.23。
Example 12: synthesis of intermediate 6-5
Figure BDA0002765401380000134
Referring to step one and step two of example 2, intermediate 6-3 was used instead of intermediate 1-3 and intermediate 6-4 was used instead of intermediate 1-4 to give 3.2g of white solid 6-5 in 65% yield (two steps) with a purity of greater than 99% HRMS: m/z (ESI) calcd for C8H5ClN4O[M-H]-207.02found:206.95。
Example 13: synthesis of intermediate 7-3
Figure BDA0002765401380000141
Referring to the procedure of example 1, 5-fluoro-2-methylbenzonitrile 7-1(13.5g, 0.1mol) was used as a starting material to give 4.5g of solid 7-3 in 30% yield and 99% purity, HRMS: m/z (ESI) calcd for C8H4FNO[M+H]+150.03found:149.68。
Example 14: synthesis of intermediate 7-5
Figure BDA0002765401380000142
Referring to step one and step two of example 2, intermediate 7-3 was used instead of intermediate 1-3 and intermediate 7-4 was used instead of intermediate 1-4 to give 1.9g of solid 7-5 in 39% yield (two steps) with a purity of greater than 99% HRMS: m/z (ESI) calcd for C8H5FN4O[M-H]-191.04found:191.25。
Example 15: synthesis of intermediate 8-5
Figure BDA0002765401380000143
Referring to the first and second steps of example 2, starting with 2-formylbenzonitrile 8-3(13g, 0.1mol), and intermediate 8-4 replacing intermediate 1-4, 6g of solid 8-5 was obtained in 34% (two steps) yield greater than 99% purity, HRMS: m/z (ESI) calcd for C7H5N5O[M-H]-173.05found:173.55。
Example 16: synthesis of intermediate 9-3
Figure BDA0002765401380000151
With reference to the procedure of example 1, starting from (15.2g, 0.1mol) 6-chloro-3-methylpyridine 9-1, 6.9g of solid 9-3 were obtained in 41% yield with a purity of greater than 99%, HRMS: m/z (ESI) calcd for C7H3ClN2O[M+H]+166.99found:167.20。
Example 17: synthesis of intermediate 9-5
Figure BDA0002765401380000152
Referring to step one and step two of example 2, intermediate 9-3 was used instead of intermediate 1-3 and intermediate 9-4 was used instead of intermediate 1-4 to give 3.5g of solid 9-5 in 69% (two steps) purity greater than 99% HRMS: m/z (ESI) calcd for C7H4BrN5O[M-H]-251.96found:252.05。
Example 18: synthesis of intermediate 10-3
Figure BDA0002765401380000153
With reference to the procedure of example 1, 5-fluoro-2-methylbenzonitrile 10-1(13.6g, 0.1mol) was used as a starting material to give 7.5g of a solid 10-3 in 50% yield with a purity of greater than 99% HRMS: m/z (ESI) calcd for C7H3BrN2O[M+H]+151.02found:151.20。
Example 19: synthesis of intermediate 10-5
Figure BDA0002765401380000154
Referring to step one and step two of example 2, intermediate 10-3 was used instead of intermediate 1-3 and intermediate 10-4 was used instead of intermediate 1-4 to give 2.7g of solid 10-5 in 64% yield (two steps) with a purity of greater than 99% HRMS: m/z (ESI) calcd for C7H4BrN5O[M-H]-192.04found:191.89。
Example 20: synthesis of intermediate 11-5
Figure BDA0002765401380000161
Referring to the first and second steps of example 2, using (13.2g, 0.1mol) 3-formylpyridinoline 11-3 as the starting material and intermediate 11-4 instead of intermediate 1-4, 4.5g of solid 4-1d-5 was obtained with a yield of 25% (two steps), purity greater than 99%, HRMS: m/z (R) ((R))ESI)calcd for C7H5N5O[M-H]-174.05found:173.99。
Example 21: synthesis of target Compound V-1
Figure BDA0002765401380000162
Bromobutane (688.84mg, 5.03mmol), iodine particles (100mg, 0.39mmol), metal magnesium strips (100.56mg, 4.19mmol), 10mL of anhydrous tetrahydrofuran and nitrogen protection are added into a three-necked flask, heating and refluxing are carried out for 12h, 189mg of intermediate 4-1-5 is dissolved in 15mL of anhydrous tetrahydrofuran in the three-necked flask and nitrogen protection is carried out, the prepared Grignard reagent is injected under ice bath conditions and reacts for 10h at room temperature, and the obtained crude product is purified by silica gel column chromatography (dichloromethane: methanol 10:1) to obtain 210mg of target compound V-1 with the purity of more than 99%.1HNMR(CD3OD,500MHz)δ：0.70(t，3H，CH3)，0.96～1.25(m，6H，CH2)，4.23(t，1H，C(OH)H)，7.56(d，1H，ArH)，7.75(dd，1H，ArH)，7.97(d，1H，ArH)。HRMS:m/z(ESI)calcd for C12H17N5O[M-H]-246.14found:246.77。
Example 22: synthesis of target Compound V-2
Figure BDA0002765401380000163
In a single-necked flask, V-1 (100mg, 0.404mmol), sodium hydrogen (30mg, 1.209mmol), 10ml of anhydrous DMF was added, the mixture was reacted at 45 ℃ for 0.5 hour under nitrogen protection, iodomethane (60mg, 0.404mmol) was injected by a syringe, the mixture was reacted at 38 ℃ for 4 hours, and the obtained crude product was purified by silica gel column chromatography (dichloromethane: methanol ═ 10:1) to obtain 30mg of the objective compound V-2. The purity is more than 99%.1HNMR(CDCl3,500MHz)δ：0.71(t，3H，CH3)，0.85～1.35(m，6H，CH2)，4.15(t，1H，C(OH)H)，4.65(s，3H，N-CH3)7.77(d，1H，ArH)，7.85(dd，1H，ArH)，8.01(d，1H，ArH)。HRMS:m/z(ESI)calcd for C13H19N5O[M+H]+262.16found:262.35。
Example 23: synthesis of target Compound V-3
Figure BDA0002765401380000171
Referring to the procedure of example 21, intermediate 2-5 was used instead of intermediate 1-5 to give 120mg of the target compound V-3 in 62% yield and purity greater than 99%.1HNMR(CD3OD,500MHz)δ：0.85(t，3H，CH3)，0.9～1.25(m，6H，CH2)，4.13(t，1H，C(OH)H)，8.25(d，1H，ArH)，8.65(dd，1H，ArH)，8.77(d，1H，ArH)。HRMS:m/z(ESI)calcd for C13H15F3N4O[M-H]-299.12found:299.25。
Example 24: synthesis of target Compound V-4
Figure BDA0002765401380000172
Referring to the procedure of example 22, V-1 was replaced with V-3 to give 30mg of the title compound V-4 in 65% yield and greater than 99% purity.1HNMR(CDCl3,500MHz)δ：0.86(t，3H，CH3)，0.95～1.29(m，6H，CH2)，4.37(t，1H，C(OH)H)，4.72(s，3H，N-CH3)，8.13(d，1H，ArH)，8.77(dd，1H，ArH)，8.85(d，1H，ArH)。HRMS:m/z(ESI)calcd for C14H17F3N4O[M+H]+315.14found:315.26。
Example 25: synthesis of target Compound V-5
Figure BDA0002765401380000181
Referring to the procedure of example 21, intermediates 3-5 were used instead of intermediates 1-5 to give 80mg of the target compound V-5 in 35% yield and purity greater than 99%.1HNMR(CD3OD,500MHz)δ：0.67(t，3H，CH3)，0.72～1.26(m，6H，CH2)，4.51(t，1H，C(OH)H)，8.13(d，1H，ArH)，8.35(dd，1H，ArH)，8.75(d，1H，ArH)。HRMS:m/z(ESI)calcd for C12H15N5O3[M-H]-276.12found:276.25。
Example 26: synthesis of target Compound V-6
Figure BDA0002765401380000182
Referring to the procedure of example 22, V-1 was replaced with V-5 to give 40mg of the title compound V-6 in 78% yield and purity greater than 99%.1HNMR(CDCl3,500MHz)δ：0.69(t，3H，CH3)，0.75～1.27(m，6H，CH2)，4.25(s，3H，N-CH3)，4.37(t，1H，C(OH)H)，7.95(d，1H，ArH)，8.23(dd，1H，ArH)，8.64(d，1H，ArH)。HRMS:m/z(ESI)calcd for C12H17N5O[M+H]+292.13found:292.25。
Example 27: synthesis of target Compound V-7
Figure BDA0002765401380000183
Referring to the procedure of example 21, intermediates 4-5 were used instead of intermediates 1-5 to give 168mg of the title compound V-7 in 69% yield and greater than 99% purity.1HNMR(CD3OD,500MHz)δ：0.69(t，3H，CH3)，1.24～1.45(m，6H，CH2)，4.57(t，1H，C(OH)H)，7.24(d，1H，ArH)，7.69(d，1H，ArH)。HRMS:m/z(ESI)calcd for C11H14BrN5O[M-H]-310.04found:309.88。
Example 28: synthesis of target Compound V-8
Figure BDA0002765401380000191
With reference to the steps of the implementation 22,v-7 is used to replace V-1, and 30mg of a target compound V-8 with the purity of more than 99 percent is obtained.1HNMR(CD3OD,500MHz)δ：0.71(t，3H，CH3)，0.83～1.25(m，6H，CH2)，3.95(s，3H，N-CH3)，4.67(t，1H，C(OH)H)，7.69(d，1H，ArH)，8.13(d，1H，ArH)。HRMS:m/z(ESI)calcd for C12H16BrN5O[M+H]+326.05found:325.69。
Example 29: synthesis of target Compound V-9
Figure BDA0002765401380000192
Referring to the procedures of examples 21 and 22, intermediate 5-5 was used instead of V-1 and V-9a was used instead of V-1 to obtain 30mg of the objective compound V-9 with a purity of more than 99%.1HNMR(CDCl3,500MHz)δ：0.71(t，3H，CH3)，0.83～1.25(m，6H，CH2)，4.35(s，3H，N-CH3)，4.44(t，1H，C(OH)H)，7.24(d，1H，ArH)，7.75(dd，1H，ArH)，7.89(d，1H，ArH)。HRMS:m/z(ESI)calcd for C13H17BrN4O[M+H]+325.06found:325.45。
Example 30: synthesis of target Compound V-10
Figure BDA0002765401380000193
Referring to the procedures of examples 21 and 22, intermediate 6-5 was used instead of V-1 and V-10a was used instead of V-1 to give 45mg of the objective compound V-10 with a purity of more than 99%.1HNMR(CDCl3,500MHz)δ：0.79(t，3H，CH3)，0.85～1.35(m，6H，CH2)，4.39(t，1H，C(OH)H)，4.56(s，3H，N-CH3)，7.35(d，1H，ArH)，7.67(dd，1H，ArH)，7.98(d，1H，ArH)。HRMS:m/z(ESI)calcd for C13H17ClN4O[M+H]+281.11found:281.25。
Example 31: synthesis of target Compound V-11
Figure BDA0002765401380000201
Referring to the procedures of example 21 and example 22, substituting V-1 with 7-5 and V-1 with V-11a gave 35mg of the title compound V-11 in a purity of greater than 99%.1HNMR(CDCl3,500MHz)δ：0.67(t，3H，CH3)，0.75～1.24(m，6H，CH2)，4.36(s，3H，N-CH3)，4.41(t，1H，C(OH)H)，7.68(d，1H，ArH)，7.98(dd，1H，ArH)，8.12(d，1H，ArH)HRMS:m/z(ESI)calcd for C13H17FN4O[M+H]+265.14found:265.39。
Example 32: synthesis of target Compound V-12
Figure BDA0002765401380000202
Referring to the procedures of example 21 and example 22, intermediate 8-5 was used instead of V-1 and V-12a was used instead of V-1 to give 40mg of the objective compound V-12 with a purity of more than 99%.1HNMR(CDCl3,500MHz)δ：0.68(t，3H，CH3)，0.72～1.05(m，6H，CH2)，3.98(s，3H，N-CH3)，4.37(t，1H，C(OH)H)，7.27(d，1H，ArH)，7.48(m，1H，ArH)，7.58(m，1H，ArH)，7.82(d，1H，ArH)。HRMS:m/z(ESI)calcd for C13H18N4O[M+H]+247.15found:247.29。
Example 33: synthesis of target Compound V-13
Figure BDA0002765401380000203
Referring to the procedure of example 21, intermediate 9-5 was used instead of intermediate 1-5 to give 69mg of the target compound V-13 in 38% yield and purity greater than 99%.1HNMR(CD3OD,500MHz)δ：0.72(t，3H，CH3)，0.85～1.12(m，6H，CH2)，4.27(t，1H，C(OH)H)，7.69(d，1H，ArH)，7.98(d，1H，ArH)。HRMS:m/z(ESI)calcd for C11H14ClN5O[M-H]-266.09found:265.79。
Example 34: synthesis of target Compound V-14
Figure BDA0002765401380000211
Referring to the procedure of example 22, V-1 was replaced with V-13 to give 36mg of the title compound V-14 with a purity of greater than 99%.1HNMR(CD3OD,500MHz)δ：0.69(t，3H，CH3)，0.79～1.24(m，6H，CH2)，4.29(t，1H，C(OH)H)，4.35(s，3H，N-CH3)，7.86(d，1H，ArH)，8.02(d，1H，ArH)。HRMS:m/z(ESI)calcd for C12H16ClN5O[M+H]+282.10found:282.64。
Example 35: synthesis of target Compound V-15
Figure BDA0002765401380000212
Referring to the procedure of example 21, intermediate 10-5 was used instead of intermediate 1-5 to give 135mg of the target compound V-15 in a yield of 64% and a purity of more than 99%.1HNMR(CD3OD,500MHz)δ：0.76(t，3H，CH3)，0.87～1.35(m，6H，CH2)，4.39(t，1H，C(OH)H)，7.67(d，1H，ArH)，8.24(d，1H，ArH)。HRMS:m/z(ESI)calcd for C11H14FN5O[M-H]-250.12found:250.15。
Example 36: synthesis of target Compound V-16
Figure BDA0002765401380000213
Referring to the procedure of example 22, V-1 was replaced with V-15 to give 65mg of the title compound V-16 in 64% yield and greater than 99% purity.1HNMR(CD3OD,500MHz)δ：0.75(t，3H，CH3)，0.85～1.31(m，6H，CH2)，4.46(s，3H，N-CH3)，4.55(t，1H，C(OH)H)，7.99(d，1H，ArH)，8.23(d，1H，ArH)。HRMS:m/z(ESI)calcd for C12H16FN5O[M+H]+266.13found:266.36。
Example 37: synthesis of target Compound V-17
Figure BDA0002765401380000221
Referring to the procedure of example 21, intermediate 11-5 was used instead of intermediate 1-5 to give 116mg of the target compound V-17 in 59% yield and purity greater than 99%.1HNMR(CD3OD,500MHz)δ：0.72(t，3H，CH3)，0.85～0.98(m，6H，CH2)，4.43(t，1H，C(OH)H)，7.34(d，1H，ArH)，7.86(m，1H，ArH)，8.42(d，1H，ArH)。HRMS:m/z(ESI)calcd for C11H15N5O[M-H]-232.13found:232.15。
Example 38: synthesis of target Compound V-18
Figure BDA0002765401380000222
Referring to the procedure of example 13, V-1 was replaced with V-17 to give 62mg of the title compound V-18 in 93% yield and greater than 99% purity.1HNMR(CD3OD,500MHz)δ：0.67(t，3H，CH3)，0.82～1.21(m，6H，CH2)，4.35(s，3H，N-CH3)，4.45(t，1H，C(OH)H)，7.45(d，1H，ArH)，7.86(m，1H，ArH)，8.55(d，1H，ArH)。HRMS:m/z(ESI)calcd for C12H17N5O[M+H]+248.14found:247.99。
Example 39: the invention discloses the inhibition effect of compounds on ADP (adenosine diphosphate) induced in-vitro platelet aggregation
A washed platelet sample without any reagent or medicament is taken as a blank control group, a washed platelet sample with ADP and dimethyl sulfoxide (DMSO) is taken as a negative control group, and a washed platelet sample with ADP and a marketed medicament butylphthalide (NBP) is taken as a positive control group. The inhibitory effect of the compounds obtained according to the invention on ADP-induced platelet aggregation was determined using an in vitro platelet aggregation assay.
The experimental method for the inhibition of ADP-induced platelet aggregation by the compounds of the present invention is as follows:
experimental materials:
medicine preparation: the test drugs are: the monomer compounds V-1 to V-18 obtained by the invention. Positive control drug: butylphthalide, purchased from Shanghai Bigdi pharmaceutical science, Inc. Purity > 95%.
Experimental animals: SPF grade SD male mice. Feeding into stainless steel wire cages with volume of 500 × 350 × 200mm (length × width × height), with no more than 5 cages. During the test, animals of other species were not kept in the same room area. License number for experimental animals: SYXK (Zhe) 2012-0178. The temperature is strictly controlled at 18-26 ℃, the humidity is 40-70%, the daily temperature difference is not more than 4 ℃, the ventilation frequency is more than 8 times/hour, and the illumination is controlled for 12 hours/darkness for 12 hours, and the day and night alternation (8:00-20:00 illumination) is carried out. The experimental animals use the drinking bottles to drink water freely and ingest food freely.
Washing platelet samples to obtain methods: SD rats are anesthetized by intraperitoneal injection of 10% chloral hydrate at a dose of 0.3ml/100g, abdominal aorta is subjected to blood collection, each animal takes about 10ml of blood, and the blood is placed in a centrifuge tube containing citrate dextrose Anticoagulant (ACD). Mixing according to the ratio of blood to ACD 9: 1. Centrifuging at 25 deg.C for 20min at 120g to obtain supernatant (PRP). Diluting PRP with ACD to prepare washed platelet, mixing uniformly according to the volume of blood: ACD of 1:3, 800 g; centrifuging at 25 deg.C for 10min to obtain precipitate as platelet. The supernatant was carefully pipetted into a new centrifuge tube, the lower layer liquid was centrifuged at 800g for 10 minutes, and the upper layer liquid was pipetted as platelet-free plasma ready for use (PPP). The pellet was resuspended with a platelet resuspension solution (Tyrode's buffer). After counting the blood cell counting plates, the platelets were diluted to 200X 10 with the resuspension Tyrode's buffer9And (2) per liter.
In vitro platelet aggregation assay: blank control group: a washed platelet sample (290 mu L) is taken and incubated in a disposable sample cup provided with a disposable stirrer for 3min at room temperature, then put in an incubation hole of a prisheng four-channel platelet aggregation tester for incubation for 3min at constant temperature of 37 ℃, after the PPP is used for zero setting, the platelet aggregation rate is detected after adding an inducer ADP10 mu L (the concentration of an ADP system is 10uM, and 10 mu L of physiological saline is added in a blank control group), and the maximum platelet aggregation rate (%) within 300s is recorded. The platelet aggregation-inducing effect of ADP was confirmed. Negative control group: the platelet sample (288. mu.l) is washed and put into a disposable sample cup with a disposable stirrer, 2. mu.L DMSO is added, the mixture is incubated for 3min at room temperature, then put into an incubation hole of a prisoner four-channel platelet aggregation tester and incubated for 3min at constant temperature of 37 ℃, after the mixture is zeroed by PPP, the platelet aggregation rate is detected after adding the inducer ADP 10. mu.L (the concentration of ADP system is 10uM), and the maximum platelet aggregation rate (%) within 300s is recorded. Each group was run in triplicate. Positive control and experimental groups: the platelet sample (288 μ L) is washed and put into a disposable sample cup with a disposable stirrer, 2 μ L butylphthalide or the compound obtained by the invention (the concentration of the compound system is 0.1mM) is added, the mixture is incubated for 3min at room temperature, then the mixture is put into an incubation hole of a prisheng four-channel platelet aggregation tester for incubation for 3min at the constant temperature of 37 ℃, after the PPP is used for zero setting, the platelet aggregation rate is detected after adding the inducer ADP10 μ L (the concentration of the ADP system is 10uM), and the maximum platelet aggregation rate (%) within 300s is recorded. Each group was run in triplicate. Inhibition rate calculation formula:
Figure BDA0002765401380000241
the experimental data were analyzed by data analysis software supplied by Graphpad Prism, where single factor analysis was used. The data of Table 1 were obtained.
TABLE 1 inhibition of ADP-induced platelet aggregation by the compounds disclosed in the present invention
Figure BDA0002765401380000242
Figure BDA0002765401380000251
Note: t-test, P <0.0001 compared to DMSO group; ++: t-test, P <0.001 compared to DMSO group; +: t-test, P <0.05 compared to DMSO group; and N.S. t-test, compared with DMSO group, has no significant difference.
As can be seen from Table 1, 0.1mM of the disclosed compounds V-1 to V-3, V-7 to V10, V-12 to V-14, V-16 to V-18 of the present invention have significant inhibitory effects on ADP-induced platelet aggregation in vitro.
Example 40: the invention discloses a protective effect of a compound on primary cortical neuron cells of an OGD/R fetal mouse
Experiment design:
the protective effect of the compound obtained by the invention on the fetal rat primary cortical neuron cells molded by OGD/R is measured by a CCK8 method by taking normal neuron cells not subjected to OGD/R molding as a control group, neuron cells only subjected to OGD/R molding and not subjected to drug treatment as a model group, neuron cells subjected to OGD/R molding and treated by using a marketed drug butylphthalide (NBP) as a positive control, and neuron cells subjected to OGD/R molding and treated by using the compound as an experimental group.
The pharmacological experimental method for the neuron cytoprotective effect of OGD/R modeling of the compound of the invention comprises the following steps:
experimental materials:
medicine preparation: the test drugs are: the monomer compounds V-1, V-2, V-7, V-8, V-9 and V-12 are obtained by the invention. Positive control drug: butylphthalide, purchased from Shanghai Bigdi pharmaceutical science, Inc. Purity > 95%.
Cell lines: day 15 ICR fetal rat cerebral cortical neurons.
Culture medium and additives: neurobasal (NB broth), B27 additive, GlutaMax additive, DMEM sugarless, FBS, manufactured by Gibco, USA.
The preparation method of the culture medium comprises the following steps:
NBM/B27 medium-low glutamine version: if a 50mL centrifuge tube is used, 45mLNB culture solution, 0.9mLB27 additive, 1% double antibody and 11.25. mu.L Glutamax additive are added;
NBM/B27 medium-high glutamine version: if a 50mL centrifuge tube is used, 45mLNB culture solution, 0.9mLB27 additive, 1% double antibody and 27 μ L Glutamax additive are added;
the preparation method of the medicine comprises the following steps: the drug (compound V-1) was dissolved in DMSO to prepare a stock solution of the corresponding concentration, and diluted with a medium in a certain ratio.
Cell obtaining and culturing:
the pregnant mouse was sacrificed by cervical dislocation, placed in a supine position with an opening in the abdomen, and the uterus was removed. The resulting fetal rat was placed in HBSS, and its brain was removed using surgical scissors and forceps and the superficial vascular membranes were separated.
Cells were digested with pancreatin and DNase at 37 ℃ for 25-30 min. After completion of digestion, digestion was stopped with 10% FBS + DMEM, followed by transfer to DMEM. After the cells were blown free with a pipette tip from large to small calibers, the supernatant was collected and centrifuged. After centrifugation, the supernatant was discarded, and the pellet was diluted to 50 ten thousand cells/mL with a medium (NBM/B27 medium-high glutamine plate) and seeded in a 96-well plate (10 ten thousand/well, 100. mu.L/well).
The resulting cells were incubated at 37 ℃ with 5% CO2Incubation in cell culture incubator, half a change of medium every three days (NBM/B27 medium-low glutamine version), and administration 24h before OGD on the seventh day.
Administration: dissolving the compound in dimethyl sulfoxide (DMSO) to obtain 20000 μ M stock solution, diluting with culture medium (NBM/B27 medium-low-glutamine plate) to obtain culture medium (20 μ M) containing medicinal liquid, sucking out 50 μ L of culture medium per well, adding 50 μ L of culture medium containing medicinal liquid to a final concentration of 10 μ M at 37 deg.C and 5% CO2Incubate in cell incubator for 2 hours, blank and model groups were not changed.
Molding:
OGD: after 2 hours, the cell culture medium was changed to sugar-free DMEM (which had been placed in an anaerobic chamber for 20min to remove O therefrom)2Washed three times) and put into an anaerobic tank (95% N)2+5％CO2) Incubated at 37 ℃ for 2 h.
R: after 2 hours, the cells were removed from the anaerobic chamber and the cell culture medium was changed to NBM/B27 medium-low glutamine version. At 37 ℃ with 5% CO2And (5) incubating for 24h in a cell incubator.
Cell viability was determined with CCK8 kit: add 10. mu. LCCK8 solution per wellAfter being liquefied, the temperature is controlled at 37 ℃ and the CO content is 5 percent2And (4) incubating in a cell incubator for 2h, and measuring the absorbance of each hole at 450 nm.
The experimental data were analyzed by data analysis software supplied by Graphpad Prism, where single factor analysis was used. Table 1 was obtained.
TABLE 1 Compound protective Effect on fetal rat primary cortical neuron cells modeled by OGD/R
Figure BDA0002765401380000261
Figure BDA0002765401380000271
Note: +: 0% -20%, +: 20% -40%, +++: more than 40 percent
t-test, # # #: p < 0.001;
t-test, compared to the modeling group,.: p <0.001, x: p < 0.05.
As can be seen from Table 1, in this experiment, the significant difference exists between the control group and the model group, which indicates that the OGD/R molding is successful and the cell survival rate is reduced after the OGD/R treatment; the building block group and the tested compound group have significant difference, which shows that the positive medicine NBP and the compound of the invention have protective effect on nerve cells damaged by OGD/R, wherein the protective effect under the same concentration of V-7 and V-12 is obviously superior to that of the positive medicine NBP.
Example 41: the invention discloses the properties of the compounds in the pharmacokinetics of oral drugs in rats
Experiment design:
SD rats 3 (male) were tested according to the following table.
Figure BDA0002765401380000272
Collecting samples: each animal was subjected to orbital extraction of 0.10mL of blood, anticoagulated with EDTAK2 at 15min, 30min, 1h, 2h, 4h, 6h, and 8h after administration of the test substance. Blood samples were collected on ice and plasma was centrifuged within 30 minutes (centrifugation conditions: 5000 rpm, 10 minutes, 4 ℃). The samples were stored at-80 ℃ before analysis.
LC-MS/MS conditions:
the liquid phase method comprises the following steps: a chromatographic column: ACQUITY BEH C182.1x50mm 1.7 μm
Mobile phase A: 0.1% formic acid water; mobile phase B: acetonitrile; flow rate: 0.35mL/min
Flow phase ratio varies with time:
Figure BDA0002765401380000281
sample introduction amount: 10 μ L
The mass spectrometry method comprises the following steps: capillary voltage: 3.5kV, desolvation gas temperature: 500 ℃, desolventizing gas flow: 1000L/Hr, cone orifice gas flow: 50L/Hr.
Drawing a drug concentration standard curve: diluting V-7 and NBP stock solutions with 50% methanol water to obtain standard working solutions containing compounds with concentrations of 40, 100, 200, 400, 1000, 2000, 4000, 10000 and 20000ng/mL and quality control working solutions of 120, 1200 and 12000 ng/mL; 47.5 μ L of blank matrix was taken and added with 2.50 μ L of standard curve working solution and quality control working solution, respectively, to prepare quality control samples containing 2.00, 5.00, 10.00, 20.00, 50.00, 100.00, 200.00, 500.00, 1000.00ng/mL of standard curve V-7 or NBP and 6.00, 60.00, and 600.00ng/mL of concentration, respectively, 200 μ L of acetonitrile (containing loratadine 1ng/mL) was added, after vortex oscillation for 3min, 20000rcf was centrifuged at 4 ℃ for 10min, and the supernatant was taken for LC-MS/MS analysis.
And (3) blood concentration measurement: plasma samples were taken at 50. mu.L, 200. mu.L acetonitrile (containing loratadine at 1ng/mL) was added, vortexed and shaken for 3min, centrifuged at 20000rcf at 4 ℃ for 10min, and the supernatant was analyzed by LC-MS/MS. And calculating the blood concentration and the drug half-life through a standard curve and drawing. Table 2 and Table 3 were obtained together with FIG. 1, FIG. 2 and FIG. 3.
TABLE 2 plasma NBP concentration (ng/mL) (IG10mg/kg)
Figure BDA0002765401380000282
Figure BDA0002765401380000291
Note: BLQ: below the detection limit; NA: not counting
TABLE 3 plasma concentration of V-7 (ng/mL) (IG10mg/kg)
Figure BDA0002765401380000292
As can be seen from table 2 and table 3 and fig. 1, fig. 2 and fig. 3, compound V-7 of the present invention rapidly reached a higher concentration in SD rat plasma upon oral administration, and reached a peak around 1 hour. After reaching the peak value, the half-life period is 7.71 hours, and compared with the shorter half-life period of butylphthalide, the method has obvious advantage and can still maintain higher blood concentration after 8 hours. The experimental result shows that the compound disclosed by the invention can rapidly reach higher blood concentration and longer drug half-life in vivo, and can become an ideal oral drug candidate molecule.
In conclusion, the tetrazole derivative has wide application prospects in preventing and resisting cardiovascular and cerebrovascular diseases and improving cardiovascular and cerebrovascular circulatory disorders or resisting thrombus.

Claims (10)

1. A tetrazole derivative having a structure represented by general formula (I):
Figure FDA0002765401370000011
or an optical isomer thereof;
or a pharmaceutically acceptable salt or solvate thereof;
wherein:
R1is H, C1-C3An alkyl chain or haloalkyl chain of (a);
R2is H, amino, nitro, hydroxyl, ether bond, methyl, ester group, carbonyl group, -CF3，-OCF3N-butyl, isopropyl, a peptide bond, one or more Cl, one or more F, one or more Br, or a combination of at least two of one or more Cl, one or more F, one or more Br;
R3independently selected from
Figure FDA0002765401370000012
Wherein R is4Is H or-C (═ O) C1-C3An alkyl group;
a is benzene ring or six-membered heterocycle containing at least one N atom or five-membered heterocycle containing at least one N atom;
R1when H and A are benzene rings, R2Is not H, F, Cl or Br.
2. The tetrazole derivative according to claim 1, having a structure represented by general formula (II):
Figure FDA0002765401370000013
or an optical isomer thereof;
or a pharmaceutically acceptable salt or solvate thereof;
wherein:
R1is H, C1-C3An alkyl chain or haloalkyl chain of (a);
R2is H, amino, nitro, trifluoromethyl, one or more Cl, one or more F, one or more Br, or a combination formed from at least two of one or more Cl, one or more F, one or more Br;
R3independently selected from
Figure FDA0002765401370000021
Wherein R is4Is H or-C (═ O) C1-C3An alkyl group;
x is C or N.
3. The tetrazole derivative according to claim 1, wherein the tetrazole derivative comprises one or more of the following compounds:
1- (4-bromo-2- (1-methyl-1H-tetrazol-5-yl) phenyl) pentan-1-ol
1- (6-bromo-2- (1H-tetrazol-5-yl) pyridin-3-yl) pentan-1-ol
1- (6-bromo-2- (1-methyl-1H-tetrazol-5-yl) pyridin-3-yl) pentan-1-ol
1- (4-chloro-2- (1-methyl-1H-tetrazol-5-yl) phenyl) pentan-1-ol
1- (6-chloro-2- (1H-tetrazol-5-yl) pyridin-3-yl) pentan-1-ol
1- (6-chloro-2- (1-methyl-1H-tetrazol-5-yl) pyridin-3-yl) pentan-1-ol
1- (4-fluoro-2- (1-methyl-1H-tetrazol-5-yl) phenyl) pentan-1-ol
1- (6-fluoro-2- (1H-tetrazol-5-yl) pyridin-3-yl) pentan-1-ol
1- (6-fluoro-2- (1-methyl-1H-tetrazol-5-yl) pyridin-3-yl) pentan-1-ol
1- (2- (1-methyl-1H-tetrazol-5-yl) phenyl) pent-1-ol
1- (2- (1H-tetrazol-5-yl) pyridin-3-yl) pent-1-ol
1- (2- (1-methyl-1H-tetrazol-5-yl) pyridin-3-yl) pentan-1-ol
1- (4-amino-2- (1H-tetrazol-5-yl) phenyl) pentan-1-ol
1- (4-amino-2- (1-methyl-1H-tetrazol-5-yl) phenyl) pentan-1-ol
1- (4-nitro-2- (1H-tetrazol-5-yl) phenyl) pentan-1-ol
1- (4-Nitro-2- (1-methyl-1H-tetrazol-5-yl) phenyl) pentan-1-ol
1- (2- (1H-tetrazol-5-yl) -4- (trifluoromethyl) phenyl) pentan-1-ol
1- (2- (1-methyl-1H-tetrazol-5-yl) -4- (trifluoromethyl) phenyl) pentan-1-ol
Or other optical isomers of the above compounds; or a pharmaceutically acceptable salt or solvate of the above compound; or an optical isomer thereof.
4. The tetrazole derivative according to claim 1, wherein the pharmaceutically acceptable salt is a salt with one or more of the following bases: sodium hydroxide, potassium hydroxide, lithium hydroxide, sodium methoxide, potassium methoxide, sodium ethoxide, potassium ethoxide, triethylamine and tert-butylamine.
5. The tetrazole derivative according to claim 1, wherein the tetrazole derivative comprises:
1- (6-bromo-2- (1H-tetrazol-5-yl) pyridin-3-yl) pentan-1-ol sodium salt
Potassium 1- (6-bromo-2- (1H-tetrazol-5-yl) pyridin-3-yl) pentan-1-ol salt
Lithium 1- (6-bromo-2- (1H-tetrazol-5-yl) pyridin-3-yl) pentan-1-ol salt
1- (6-bromo-2- (1H-tetrazol-5-yl) pyridin-3-yl) pent-1-ol triethylamine salt
1- (6-bromo-2- (1H-tetrazol-5-yl) pyridin-3-yl) pent-1-ol tert-butylamine salt
Sodium salt of 1- (2- (1H-tetrazol-5-yl) -4- (trifluoromethyl) phenyl) pentan-1-ol
Potassium 1- (2- (1H-tetrazol-5-yl) -4- (trifluoromethyl) phenyl) pent-1-ol salt
Lithium 1- (2- (1H-tetrazol-5-yl) -4- (trifluoromethyl) phenyl) pent-1-olate
1- (2- (1H-tetrazol-5-yl) -4- (trifluoromethyl) phenyl) pent-1-ol triethylamine salt
1- (2- (1H-tetrazol-5-yl) -4- (trifluoromethyl) phenyl) pent-1-ol tert-butylamine salt
Sodium salt of 1- (4-amino-2- (1H-tetrazol-5-yl) phenyl) pentan-1-ol
Potassium salt of 1- (4-amino-2- (1H-tetrazol-5-yl) phenyl) pent-1-ol
Lithium 1- (4-amino-2- (1H-tetrazol-5-yl) phenyl) pent-1-ol salts
1- (4-amino-2- (1H-tetrazol-5-yl) phenyl) pent-1-ol triethylamine salt
1- (4-amino-2- (1H-tetrazol-5-yl) phenyl) pent-1-ol tert-butylamine salt
Or other optical isomers of the above compounds; or a pharmaceutically acceptable solvate of the above compound.
6. A preparation method of the tetrazole derivative as claimed in any one of claims 1-5, wherein the compound (1) reacts with an acyl protection reagent to obtain a compound (2), the compound (2) reacts with sodium azide, then hydrochloric acid is added to remove a protecting group and eliminate residual sodium azide at the same time to obtain a compound (3), and the compound (3) reacts with a Grignard reagent to obtain a compound shown in a formula (I); optionally, the compound shown in the formula (I) is further reacted with an alkylating agent to obtain a tetrazolylalkyl substitution compound shown in the formula (I'):
Figure FDA0002765401370000041
7. the process for producing a tetrazole derivative according to claim 6, wherein the acyl protecting agent is ethylene glycol and the deprotecting agent is hydrochloric acid.
8. The method for preparing a tetrazole derivative according to claim 6, wherein the temperature of the reaction of the compound (1) with the acyl protective agent is 110-130 ℃; the reaction temperature of the compound (2) and sodium azide is 90-110 ℃.
9. A pharmaceutical composition comprising at least one active ingredient which is a tetrazole derivative according to any one of claims 1 to 8, and one or more pharmaceutically acceptable carriers or excipients.
10. Use of the tetrazole derivative of any one of claims 1-8 in preparation of drugs for preventing and resisting cardiovascular and cerebrovascular diseases, improving cardiovascular and cerebrovascular circulatory disorders or resisting thrombosis.
CN202011231596.8A 2020-11-06 2020-11-06 Tetrazole derivative, preparation method thereof, pharmaceutical composition containing tetrazole derivative and application of pharmaceutical composition Pending CN112457265A (en)

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