阅读说明：本技术 含有胍基的取代嘧啶衍生物、其药物组合物及其制备方法和应用 (Substituted pyrimidine derivative containing guanidyl, pharmaceutical composition thereof, and preparation method and application thereof ) 是由 王伟 江涛 王金鹏 徐萃婧 于 2019-10-31 设计创作，主要内容包括：本发明提出一种含有胍基的取代嘧啶衍生物、其药物组合物及其制备方法和应用,属于有机合成技术领域。该技术方案提供了一种具有由通式I所示的含有胍基的取代嘧啶衍生物：<Image he="376" wi="700" file="DDA0002255890770000011.GIF" imgContent="drawing" imgFormat="GIF" orientation="portrait" inline="no"></Image>其中,取代基R为<Image he="143" wi="289" file="DDA0002255890770000012.GIF" imgContent="drawing" imgFormat="GIF" orientation="portrait" inline="no"></Image>R<Sub>1</Sub>、R<Sub>2</Sub>、R<Sub>3</Sub>和R<Sub>4</Sub>分别独立选自-H或-OCH<Sub>3</Sub>,n为2-6的正整数。本发明为新型抗HSV药物的理性设计提供了新的结构和思路,并为开发基于新靶标的抗单纯疱疹病毒药物提供了重要的理论参考。(The invention provides a substituted pyrimidine derivative containing guanidyl, a pharmaceutical composition thereof, and a preparation method and application thereof, and belongs to the technical field of organic synthesis. The technical scheme provides a substituted pyrimidine derivative containing guanidyl as shown in a general formula I: wherein the substituent R is R 1 、R 2 、R 3 And R 4 Each independently selected from-H or-OCH 3 And n is a positive integer of 2-6. The invention provides a new structure and thought for the rational design of the novel anti-HSV medicamentAnd provides important theoretical reference for developing anti-herpes simplex virus medicines based on new targets.)

1. Substituted pyrimidine derivatives having a guanidino group represented by the general formula I:

wherein the substituent R is

2. The guanidino-containing substituted pyrimidine derivative according to claim 1, wherein the derivative is selected from one of the following compounds:

a1: 2- (4, 6-bis (4-methoxystyryl) pyrimidin-2-oxy) ethylguanidinium trifluoroacetate;

a2: 3- (4, 6-bis (4-methoxystyryl) pyrimidin-2-oxy) propylguanidinium trifluoroacetate;

a3: 4- (4, 6-bis (4-methoxystyryl) pyrimidin-2-oxy) butylguanidinium trifluoroacetate;

a4: 5- (4, 6-bis (4-methoxystyryl) pyrimidin-2-oxy) pentylguanidine trifluoroacetate;

a5: 6- (4, 6-bis (4-methoxystyryl) pyrimidin-2-oxy) hexylguanidinium trifluoroacetate;

b1: 2- (4, 6-bis (3,4, 5-trimethoxystyryl) pyrimidin-2-oxy) ethylguanidinium trifluoroacetate;

b2: 3- (4, 6-bis (3,4, 5-trimethoxystyryl) pyrimidin-2-oxy) propylguanidine trifluoroacetate;

b3: 4- (4, 6-bis (3,4, 5-trimethoxystyryl) pyrimidin-2-oxy) butylguanidine trifluoroacetate;

b4: 5- (4, 6-bis (3,4, 5-trimethoxystyryl) pyrimidin-2-oxy) pentylguanidine trifluoroacetate;

b5: 6- (4, 6-bis (3,4, 5-trimethoxystyryl) pyrimidin-2-oxy) hexylguanidine trifluoroacetate;

c1: 2- (4, 6-bis (3, 4-dimethoxystyryl) pyrimidin-2-oxy) ethylguanidinium trifluoroacetate;

c2: 3- (4, 6-bis (3, 4-dimethoxystyryl) pyrimidin-2-oxy) propylguanidinium trifluoroacetate;

c3: 4- (4, 6-bis (3, 4-dimethoxystyryl) pyrimidin-2-oxy) butylguanidine trifluoroacetate;

c4: 5- (4, 6-bis (3, 4-dimethoxystyryl) pyrimidin-2-oxy) pentylguanidine trifluoroacetate;

c5: 6- (4, 6-bis (3, 4-dimethoxystyryl) pyrimidin-2-oxy) hexylguanidinium trifluoroacetate;

d1: 2- (4, 6-bis (3-methoxystyryl) pyrimidin-2-oxy) ethylguanidinium trifluoroacetate;

d2: 3- (4, 6-bis (3-methoxystyryl) pyrimidin-2-oxy) propylguanidinium trifluoroacetate;

d3: 4- (4, 6-bis (3-methoxystyryl) pyrimidin-2-oxy) butylguanidinium trifluoroacetate;

d4: 5- (4, 6-bis (3-methoxystyryl) pyrimidin-2-oxy) pentylguanidine trifluoroacetate;

d5: 6- (4, 6-bis (3-methoxystyryl) pyrimidin-2-oxy) hexylguanidinium trifluoroacetate;

e1: 2- (4, 6-bis (2-methoxystyryl) pyrimidin-2-oxy) ethylguanidinium trifluoroacetate;

e2: 3- (4, 6-bis (2-methoxystyryl) pyrimidin-2-oxy) propylguanidinium trifluoroacetate;

e3: 4- (4, 6-bis (2-methoxystyryl) pyrimidin-2-oxy) butylguanidinium trifluoroacetate;

e4: 5- (4, 6-bis (2-methoxystyryl) pyrimidin-2-oxy) pentylguanidine trifluoroacetate;

e5: 6- (4, 6-bis (2-methoxystyryl) pyrimidin-2-oxy) hexylguanidinium trifluoroacetate.

3. The method for preparing a substituted pyrimidine derivative containing a guanidino group according to claim 1, comprising the steps of:

dissolving 2-hydroxy-4, 6-di (methoxyl substituted styryl) pyrimidine and potassium carbonate in a molar ratio of 1 (2-2.5) in DMF, stirring at room temperature for 1-1.5h, adding bromine substituted tert-butyl dimethyl silyl ether, stirring at 70-80 ℃ for reaction for 12-16h, and removing tert-butyl dimethyl silyl by using TBAF/THF solution to obtain a4, 6-di (methoxyl substituted styryl) pyrimidine compound with 2-hydroxy substituted by alkyl alcohol;

stirring the obtained 4, 6-bis (methoxy-substituted styryl) pyrimidine compound of which the hydrogen of the 2-position hydroxyl is substituted by alkyl alcohol, N' -bis (tert-butoxycarbonyl) guanidine in a molar ratio of 1 (1.5-2) and triphenylphosphine in a molar ratio of 1 (1.5-2) in anhydrous tetrahydrofuran, dropwise adding an anhydrous tetrahydrofuran solution of 20-60% DIAD in a molar ratio of 1 (1-1.5) under ice bath, and transferring to room temperature for reaction for 3-5 h; then adding acid to remove Boc group at 0-5 ℃, and purifying to obtain substituted pyrimidine derivative containing guanidyl.

4. The production method according to claim 3, wherein the 2-hydroxy-4, 6-bis (methoxy-substituted styryl) pyrimidine is selected from any one of 2-hydroxy-4, 6-bis (3-methoxystyryl) pyrimidine, 2-hydroxy-4, 6-bis (3, 4-dimethoxystyryl) pyrimidine, 2-hydroxy-4, 6-bis (4-methoxystyryl) pyrimidine, 2-hydroxy-4, 6-bis (2-methoxystyryl) pyrimidine and 2-hydroxy-4, 6-bis (3,4, 5-trimethoxystyryl) pyrimidine;

the bromine-substituted tert-butyl dimethyl silicon ether is any one selected from 3-bromopropyl tert-butyl dimethyl silicon ether, 2-bromoethyl tert-butyl dimethyl silicon ether, 4-bromobutyl tert-butyl dimethyl silicon ether, 5-bromoamyl tert-butyl dimethyl silicon ether and 6-bromohexyl tert-butyl dimethyl silicon ether.

5. The method of claim 3, wherein the acid added for removing Boc group is selected from any one of hydrochloric acid, sulfuric acid, organic carboxylic acid, organic sulfonic acid and trifluoroacetic acid.

6. A pharmaceutical composition for inhibiting infection and replication of HSV-1 and HSV-2, comprising a therapeutically effective amount of one or more guanidino-containing substituted pyrimidine derivatives of claim 1 or 2, together with at least one pharmaceutically acceptable adjuvant.

7. Use of a substituted pyrimidine derivative containing a guanidino group according to claim 1 or 2 for the manufacture of a medicament for the treatment or prevention of inhibition of HSV-1 and HSV-2 viral infection and replication.

8. Use according to claim 7, characterized in that the substituted pyrimidine derivative containing a guanidino group is used for the preparation of a medicament for the treatment or prophylaxis of herpes simplex virus.

Technical Field

The invention relates to a substituted pyrimidine derivative containing guanidyl, a pharmaceutical composition containing the derivative and a preparation method of the derivative.

Background

The guanidyl has wide biological activity and has important functions of resisting tumor, resisting virus, reducing blood sugar, killing bacteria and the like in the development of medicaments for treating various diseases. The Y-shaped forked structure of the guanidinium cationic group enables the guanidinium cationic group to form strong non-covalent interaction with anions by means of hydrogen bond interaction or charge pairing, can be used for identifying molecules in cells, and has good binding capacity on intracellular anionic polysaccharide.

The pyrimidine stilbene is a D-pi-A-pi-D structure with good fluorescence property, and the compound has various biological activities such as tumor resistance, Alzheimer disease resistance and the like, has important application in fluorescence imaging, and can be used for activity imaging of living cell protein and organelle to detect and treat diseases.

The synthesis methods of the pyrimidine derivatives are many, but the synthesis methods of the pyrimidine guanidine compounds are few, if the structure modification of the pyrimidine stilbene compounds can be carried out by utilizing the guanidine groups, the stilbene pyrimidine derivative compounds containing the guanidine groups can be synthesized simply and efficiently, and the stilbene pyrimidine derivative compounds can be expected to have wide market prospects.

Disclosure of Invention

The invention provides a substituted pyrimidine derivative containing guanidyl, a pharmaceutical composition thereof, a preparation method and an application thereof, provides a new structure and thought for rational design of a novel anti-HSV drug, and provides an important theoretical reference for developing an anti-herpes simplex virus drug based on a new target.

In order to achieve the above object, the present invention provides a substituted pyrimidine derivative having a guanidino group represented by the general formula I:

wherein the substituent R is

R₁、R₂、R₃And R₄Each independently selected from-H or-OCH₃And n is a positive integer of 2-6.

Preferably, the derivative is selected from one of the following compounds:

a1: 2- (4, 6-bis (4-methoxystyryl) pyrimidin-2-oxy) ethylguanidinium trifluoroacetate;

a2: 3- (4, 6-bis (4-methoxystyryl) pyrimidin-2-oxy) propylguanidinium trifluoroacetate;

a3: 4- (4, 6-bis (4-methoxystyryl) pyrimidin-2-oxy) butylguanidinium trifluoroacetate;

a4: 5- (4, 6-bis (4-methoxystyryl) pyrimidin-2-oxy) pentylguanidine trifluoroacetate;

a5: 6- (4, 6-bis (4-methoxystyryl) pyrimidin-2-oxy) hexylguanidinium trifluoroacetate;

b1: 2- (4, 6-bis (3,4, 5-trimethoxystyryl) pyrimidin-2-oxy) ethylguanidinium trifluoroacetate;

b2: 3- (4, 6-bis (3,4, 5-trimethoxystyryl) pyrimidin-2-oxy) propylguanidine trifluoroacetate;

b3: 4- (4, 6-bis (3,4, 5-trimethoxystyryl) pyrimidin-2-oxy) butylguanidine trifluoroacetate;

b4: 5- (4, 6-bis (3,4, 5-trimethoxystyryl) pyrimidin-2-oxy) pentylguanidine trifluoroacetate;

b5: 6- (4, 6-bis (3,4, 5-trimethoxystyryl) pyrimidin-2-oxy) hexylguanidine trifluoroacetate;

c1: 2- (4, 6-bis (3, 4-dimethoxystyryl) pyrimidin-2-oxy) ethylguanidinium trifluoroacetate;

c2: 3- (4, 6-bis (3, 4-dimethoxystyryl) pyrimidin-2-oxy) propylguanidinium trifluoroacetate;

c3: 4- (4, 6-bis (3, 4-dimethoxystyryl) pyrimidin-2-oxy) butylguanidine trifluoroacetate;

c4: 5- (4, 6-bis (3, 4-dimethoxystyryl) pyrimidin-2-oxy) pentylguanidine trifluoroacetate;

c5: 6- (4, 6-bis (3, 4-dimethoxystyryl) pyrimidin-2-oxy) hexylguanidinium trifluoroacetate;

d1: 2- (4, 6-bis (3-methoxystyryl) pyrimidin-2-oxy) ethylguanidinium trifluoroacetate;

d2: 3- (4, 6-bis (3-methoxystyryl) pyrimidin-2-oxy) propylguanidinium trifluoroacetate;

d3: 4- (4, 6-bis (3-methoxystyryl) pyrimidin-2-oxy) butylguanidinium trifluoroacetate;

d4: 5- (4, 6-bis (3-methoxystyryl) pyrimidin-2-oxy) pentylguanidine trifluoroacetate;

d5: 6- (4, 6-bis (3-methoxystyryl) pyrimidin-2-oxy) hexylguanidinium trifluoroacetate;

e1: 2- (4, 6-bis (2-methoxystyryl) pyrimidin-2-oxy) ethylguanidinium trifluoroacetate;

e2: 3- (4, 6-bis (2-methoxystyryl) pyrimidin-2-oxy) propylguanidinium trifluoroacetate;

e3: 4- (4, 6-bis (2-methoxystyryl) pyrimidin-2-oxy) butylguanidinium trifluoroacetate;

e4: 5- (4, 6-bis (2-methoxystyryl) pyrimidin-2-oxy) pentylguanidine trifluoroacetate;

e5: 6- (4, 6-bis (2-methoxystyryl) pyrimidin-2-oxy) hexylguanidinium trifluoroacetate.

The invention also provides a preparation method of the substituted pyrimidine derivative containing guanidine group according to the technical scheme, which comprises the following steps:

dissolving 2-hydroxy-4, 6-di (methoxy substituted styryl) pyrimidine and potassium carbonate in DMF in a molar ratio of 1 (2-2.5), stirring at room temperature for 1-1.5h, adding bromine substituted tert-butyl dimethyl silyl ether, stirring at 70-80 ℃ for reaction for 12-16h, and removing tert-butyl dimethyl silyl by using TBAF/THF solution to obtain a compound of which the hydrogen of 2-hydroxy is substituted by alkyl alcohol;

stirring the obtained 4, 6-bis (methoxy-substituted styryl) pyrimidine compound of which the hydrogen of the 2-position hydroxyl is substituted by alkyl alcohol, N' -bis (tert-butoxycarbonyl) guanidine in a molar ratio of 1 (1.5-2) and triphenylphosphine in a molar ratio of 1 (1.5-2) in anhydrous tetrahydrofuran, dropwise adding an anhydrous tetrahydrofuran solution of 20-60% DIAD in a molar ratio of 1 (1-1.5) under ice bath, and transferring to room temperature for reaction for 3-5 h; then adding acid to remove Boc group at 0-5 ℃, and purifying to obtain substituted pyrimidine derivative containing guanidyl.

In the above technical scheme, the preparation methods of N, N' -bis (tert-butoxycarbonyl) guanidine, bromo-substituted tert-butyldimethylsilyl ether, and 2-hydroxy-4, 6-bis (methoxy-substituted styryl) pyrimidine are conventional methods, and are not limited to the above preparation methods, and can be prepared by other synthetic routes, but the above synthetic routes are preferred, and are simple and easy to operate.

Preferably, the 2-hydroxy-4, 6-bis (methoxy-substituted styryl) pyrimidine is selected from any one of 2-hydroxy-4, 6-bis (3-methoxystyryl) pyrimidine, 2-hydroxy-4, 6-bis (3, 4-dimethoxystyryl) pyrimidine, 2-hydroxy-4, 6-bis (4-methoxystyryl) pyrimidine, 2-hydroxy-4, 6-bis (2-methoxystyryl) pyrimidine and 2-hydroxy-4, 6-bis (3,4, 5-trimethoxystyryl) pyrimidine;

the bromine-substituted tert-butyl dimethyl silicon ether is any one selected from 3-bromopropyl tert-butyl dimethyl silicon ether, 2-bromoethyl tert-butyl dimethyl silicon ether, 4-bromobutyl tert-butyl dimethyl silicon ether, 5-bromoamyl tert-butyl dimethyl silicon ether and 6-bromohexyl tert-butyl dimethyl silicon ether.

Preferably, the acid added for removing Boc group is selected from any one of hydrochloric acid, sulfuric acid, organic carboxylic acid, organic sulfonic acid and trifluoroacetic acid. In a preferred embodiment, the acid added to remove Boc groups is trifluoroacetic acid.

The invention also provides a pharmaceutical composition for inhibiting HSV-1 and HSV-2 virus infection and replication, which comprises a therapeutically effective amount of one or more substituted pyrimidine derivatives containing guanidino and at least one pharmaceutically acceptable adjuvant.

The invention also provides application of the substituted pyrimidine derivative containing guanidino in preparing a medicament for treating or preventing HSV-1 and HSV-2 virus infection and replication.

Preferably, the use of a substituted pyrimidine derivative containing a guanidino group for the manufacture of a medicament for the treatment or prophylaxis of anti-herpes simplex virus.

Compared with the prior art, the invention has the advantages and positive effects that:

1. the invention provides a substituted pyrimidine derivative containing guanidino and a preparation method thereof, and the substituted pyrimidine derivative containing guanidino is taken as a research object of anti-herpes simplex virus, so that the in vitro action mechanism and the structure-activity relationship thereof are comprehensively explained, the huge influence of guanidino substitution on the anti-HSV activity of the pyrimidine derivative is proved, and a new structure and thought are provided for the rational design of a novel anti-HSV drug;

2. the invention discloses the envelope glycoprotein gB and cell PI3K/Akt/mTOR signal pathways of the guanidyl-containing substituted pyrimidine derivatives with the action targets on the surface of the virus for the first time, which have obvious difference with the action mechanism of nucleoside drugs for inhibiting DNA polymerase in clinical application at present, and can provide important theoretical reference for developing anti-herpes simplex virus drugs based on new targets.

Drawings

FIG. 1 shows an example of the present invention containing substituted pyrimidine compounds, wherein when n is 2-6, R is

R₁、R₃、R₄Is H, R₂Is OCH₃Reaction scheme of (1);

FIG. 2 shows an example of the compound of the present invention containing substituted pyrimidine, wherein when n is 2-6, R is

R₁、R₂、R₃Is OCH₃，R₄Reaction scheme for H;

FIG. 3 shows an example of a compound of the present invention containing a substituted pyrimidine, wherein when n is 2-6, R is

R₁、R₂Is OCH₃，R₃、R₄Reaction scheme for H;

FIG. 4 shows an example of a compound of the present invention containing a substituted pyrimidine, wherein when n is 2-6, R is

R₁、R₂Is H, R₃、R₄Is OCH₃Reaction scheme of (1);

FIG. 5 shows an example of a compound of the present invention containing a substituted pyrimidine, wherein when n is 2-6, R isR₁、R₂、R₃Is H, R₄Is OCH₃Reaction scheme of (1);

FIG. 6 is the plaque reduction test result of Compound C1 in an example of the present invention;

FIG. 7 is a graph of the inhibitory effect of various concentrations of C1 on viral protein expression in examples of the present invention;

FIG. 8 shows the results of immunofluorescence detection of anti-HSV-1 with Compound C1 of the present invention under different modes of action;

FIG. 9 shows the results of immunofluorescence assay of anti-HSV-2 with compound C1 of the present invention under different modes of action;

FIG. 10 is the results of anti-HSV effect of compound C1 of the present invention at various time periods after adsorption;

FIG. 11 shows the results of CPE-inhibition experiments with various concentrations of A0, A1, and C1 in accordance with the present invention;

FIG. 12 shows the plaque assay results of compounds A0, A1 and C1 for HSV inhibition in accordance with the present invention;

FIG. 13 shows the confocal results of fluorescence probe labeling of organelles in an embodiment of the invention;

FIG. 14 shows the confocal results of co-localization of Compound C1 with organelles in the examples of the present invention;

FIG. 15 is an enlarged electronic view of co-localization of Compound C1 with organelles in an example of the present invention;

FIG. 16 shows the results of cellular co-localization of Compound A0 with viral gB protein in examples of the present invention;

FIG. 17 shows the results of cellular co-localization of Compound C1 with viral gB protein in examples of the present invention;

FIG. 18 is a graph of the inhibitory effect of various concentrations of C1 on HSV-1 induced membranous fusion in accordance with an embodiment of the present invention;

FIG. 19 is a graph of the inhibitory effect of various concentrations of C1 on HSV-2 induced membranous fusion in accordance with an embodiment of the present invention;

FIG. 20 is a graph of the inhibitory effect of compounds A0, A1 and C1 of the examples on virus-induced membrane fusion;

FIG. 21 is the effect of compound C1 of the present example on the adsorption process of HSV-1 in HeLa cells;

FIG. 22 is the effect of Compound C1 of the present invention on the HSV-1 entry process in HeLa cells;

FIG. 23 is a graph of the effect of Compound C1 of the present example on the adsorption process of HSV-1 in Vero cells;

FIG. 24 is a graph of the effect of Compound C1 of the present example on the HSV-1 entry process in Vero cells;

FIG. 25 shows the binding relationship between magnetic beads and viral proteins in an embodiment of the present invention;

FIG. 26 shows the binding relationship between magnetic beads and viral proteins and a sample according to an embodiment of the present invention;

FIG. 27 is a graph showing the effect of compound C1 on the PI3K/Akt/mTOR signaling pathway in an example of the present invention;

FIG. 28 is a graph showing the change in body weight of a mouse according to an embodiment of the present invention;

FIG. 29 is a survival curve of a mouse in an example of the present invention;

FIG. 30 shows the change in viral mRNA levels in lung tissue of mice in accordance with an embodiment of the present invention;

FIG. 31 shows the change in viral mRNA levels in spinal cord tissue of mice in accordance with an embodiment of the present invention;

FIG. 32 is a pathological section of mouse brain tissue according to an embodiment of the present invention;

FIG. 33 is a pathological section of mouse lung tissue in an example of the invention.

Detailed Description

In order to clearly and fully introduce the substituted pyrimidine derivatives containing guanidino, pharmaceutical compositions thereof, and methods for preparing and using the same provided in the embodiments of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.