阅读说明：本技术 一种羟苯水杨胺羟基化物、制备方法及其应用 (Hydroxyl benzene salicylamine hydroxylate, preparation method and application thereof ) 是由 裘云庆 楼燕 于 2018-06-13 设计创作，主要内容包括：本发明涉及羟苯水杨胺羟基化物,所述羟苯水杨胺羟基化物可通过抑制RR酶而抑制乙肝病毒DNA复制,本发明还介绍了羟苯水杨胺羟基化物的制备方法,有助于解决目前对RR酶具有抑制作用的药物分子仍然比较少见的技术问题。(The invention relates to a hydroxyl benzene salicylamine hydroxylate which can inhibit the replication of hepatitis B virus DNA by inhibiting RR enzyme, and also discloses a preparation method of the hydroxyl benzene salicylamine hydroxylate, which is helpful for solving the technical problem that the existing drug molecules with the inhibiting effect on RR enzyme are still rare.)

1. A hydroxyl benzene salicylamine hydroxylate characterized by the structural formula:

2. the method for preparing hydroxyl benzene salicylamine hydroxylate of claim 1, which is realized by the following steps:

a. dissolving the intermediate and adding a catalyst to form a first mixture; the catalyst is one of palladium carbon, raney nickel, stannic chloride and ferric trichloride, and the intermediate is as follows:

b. the first mixture is hydrogenated to completely react to generate hydroxyl benzene salicylamide hydroxylate, and the reaction temperature is 0-50 ℃;

c. mixing and dissolving the compound A and the compound B to form a second mixture, wherein the molar ratio of the compound A to the compound B to the organic base in the second mixture is 1: 1-3; the organic base is triethylamine, N-diisopropylethylamine, pyridine and 4-dimethylamino pyridine,

the compound A is:

the compound B is:

wherein X is Cl, Br or I;

d. the second mixture is completely reacted to generate an intermediate;

e. cooling the second mixture, wherein the temperature of the second mixture is kept between-15 ℃ and 35 ℃;

f. adding a hydrogen-free organic base to the second mixture, and controlling the temperature of the second mixture to be below 15 ℃.

3. The preparation method according to claim 2, wherein the solvent used in the reaction in step a is dichloromethane, methanol, ethanol, tert-butanol, acetonitrile, tetrahydrofuran, or dioxane.

4. The preparation method according to claim 2, wherein the solvent used in the reaction in step c is a nonpolar solvent, and the nonpolar solvent is at least one of dichloromethane, dichloroethane, tetrahydrofuran, acetonitrile and dioxane.

5. Use of the hydroxyphenyl salicylamide hydroxylate as claimed in claim 1 in the preparation of a medicament for inhibiting ribonucleotide reductase activity.

6. The use of the hydroxyphenyl salicylamide hydroxylate of claim 1 in the preparation of a medicament for inhibiting hepatitis b virus DNA replication.

Technical Field

The invention relates to hydroxyl benzene salicylamine hydroxylate, which has the following structural formula:

the invention also relates to the preparation and application of the hydroxyl benzene salicylamine hydroxylate.

Background

Viral hepatitis B (hepatitis B) is still one of important infectious diseases seriously threatening the health of people in China, and is a main factor causing primary liver cancer in China. The HBsAg carrying rate of people in China is nearly 7.2%, and about 3.5 hundred million people all over the world suffer from HBV infection. The existing research shows that the inhibition of HBV DNA replication can prevent the progress of hepatitis B disease, slow down and even reverse liver fibrosis and cirrhosis and reduce the incidence rate of liver cancer. The nucleotide analogue treatment recommended by the current international anti-HBV treatment guidelines still has more adverse reactions and higher drug resistance rate and virus variation defects. In addition, the existing drugs can not completely eliminate cccDNA (divalently closed circular dna) in the liver cell nucleus of hepatitis b patients, and the continuous existence of cccDNA in patients is a main reason that chronic HBV infection is difficult to cure, and in fact, HBV variation and drug resistance caused by nucleoside (acid) analogues are difficult problems in the global hepatitis b treatment history and are also important obstacles for overcoming hepatitis b.

At present, researches suggest that the drug resistance incidence rate of drugs taking proteins in host cells as targets is obviously reduced compared with drugs directly acting on viruses, and mitochondrial toxicity of nucleoside (acid) analogues can be avoided by taking the proteins in the host cells as drug targets. Therefore, the search of virus-host cell interaction sites as new antiviral targets in the HBV replication cycle, so as to develop a new anti-HBV treatment scheme capable of clearing cccDNA and reducing the incidence of drug resistance, is a key problem to be solved urgently in the treatment of hepatitis B and the prevention and treatment of hepatitis B-related liver cancer at present, and has important clinical and social meanings.

Ribonucleotide Reductase (RR) is a key enzyme in the synthesis and repair of human cellular DNA, and functions to reduce ribonucleotides (NDPs) to deoxyribonucleotides (dNDPs), which are further phosphorylated to dNTPs by kinases to provide starting materials for DNA synthesis. HBV has no gene for encoding RR enzyme, most normal liver cells in the liver of adults are in a static state, and the concentration of dNTPs is very low. HBV (No RR gene) inhibits the combination of hepatocyte regulatory factor x1(RFX1) and the promoter of RRM2 or activates Chk1-E2F1 pathway of DNA damage pathway through HBx protein so as to activate the RR enzyme expression and enzyme activity of host hepatocytes, and the abnormally activated RR enzyme can provide sufficient DNA synthesis raw materials and ensure the replication of HBV and the demand of liver cancer cells on DNA raw materials, so that the RR enzyme is a key factor protein for the occurrence and development of hepatitis B and related liver cancer and is a new target molecule of anti-hepatitis B drugs. The RR enzyme inhibitor can cut off the supply of HBV DNA replication raw materials, inhibit the synthesis of double-stranded relaxed circular molecule rcDNA (strained circular DNA), prevent the replication of a large amount of HBV in liver cells, and is expected to effectively inhibit the formation of HBVcccDNA from the source, thereby relieving the inflammatory injury of liver tissues, preventing the generation and development of hepatitis and converting the hepatitis into liver cancer.

Disclosure of Invention

Although RR enzyme is found to be a key factor protein for the occurrence and development of hepatitis B and related liver cancer at present, drug molecules with an inhibitory effect on RR enzyme are still rare, and the invention provides a drug molecule with an inhibitory effect on RR enzyme, wherein the molecule is hydroxyl-phenyl salicylamide hydroxylate, and the structural formula of the molecule is as follows:

the invention discovers that analysis of the molecule by using molecular docking software eHiTS (Version 12.0, SimBioSys Inc., Canada) of SimBioSys company shows that the eHiTS _ Score value (eHiTS _ Score corresponds to log (Kd) (logarithmic dissociation constant), and the more negative value represents that the binding capacity of the small molecular compound is stronger, such as-6 corresponds to mu M, -9 corresponds to nM, the numerical difference is 1, and the binding capacity is 10 times different) is-3.807.

In addition, the hydroxyl-phenyl salicylamine hydroxylate also has a very strong function of inhibiting the replication of hepatitis B virus DNA, so that the hydroxyl-phenyl salicylamine hydroxylate can be used for preparing a medicament for treating the hepatitis B, and particularly, the hydroxyl-phenyl salicylamine hydroxylate is used for preparing a medicament for preventing and treating the hepatitis (particularly the hepatitis B).

The medicament can be prepared into dosage forms suitable for various administration forms.

The invention also provides a method for preparing hydroxyl benzene salicylamine hydroxylate, which comprises the following steps:

a. a step of dissolving the intermediate and adding a catalyst to form a first mixture;

b. the first mixture is hydrogenated and completely reacted to generate hydroxyl benzene salicylamine hydroxylate;

the catalyst is one of palladium carbon, raney nickel, stannic chloride and ferric trichloride, and the intermediate is as follows:

the reaction yield of the step b can reach more than 80 percent. And (b) dissolving the intermediate in the step a, and adding a catalyst, wherein the solvent selected in the step a can dissolve the intermediate, and is generally dichloromethane, methanol, ethanol, tert-butyl alcohol, acetonitrile, tetrahydrofuran and dioxane, preferably methanol.

Various specifications for the catalyst may be used for the hydrogenation of the intermediate, palladium on carbon being generally preferred for the catalyst. The reaction temperature of the step b is controlled to be 0-50 ℃.

The intermediate can be synthesized by one skilled in the art using various synthetic routes, but in the present invention, the intermediate is generated by reacting compound a:

the compound B is:

wherein X is Cl, Br or I.

For compound a and compound B, the synthesis can be performed by the skilled person in the art by looking up the technical documentation, and the invention is not described further.

The synthesis steps of the intermediate comprise:

c. mixing and dissolving the compound A and the compound B to form a second mixture;

d. and (4) completely reacting the second mixture to generate an intermediate.

The reaction yield of the step d can reach more than 70 percent, and the solvent selected in the step c is a nonpolar solvent, so polar solvents such as alcohols and the like cannot be used. The nonpolar solvent can be at least one of dichloromethane, dichloroethane, tetrahydrofuran, acetonitrile and dioxane, preferably dichloromethane and tetrahydrofuran.

The c step further comprises the step of cooling the second mixture during the mixing of compound a and compound B to form the second mixture to inhibit the formation of by-products. In some embodiments of the invention, the step of cooling the second mixture is capable of maintaining the temperature of the second mixture at-15 ℃ to 35 ℃, preferably-15 ℃ to 15 ℃.

In order to ensure that the compound A and the compound B can smoothly react, the step c further comprises the step of adding a hydrogen-free organic base to the second mixture, and in the step, the second mixture is still cooled, so that the temperature of the second mixture added with the organic base is controlled below 15 ℃, so as to keep the reaction efficiency and simultaneously reduce the generation of byproducts as much as possible. In some embodiments of the invention, the organic base is triethylamine, N-diisopropylethylamine, pyridine, 4-dimethylaminopyridine, preferably triethylamine.

Generally, the molar charge ratio of the compound A, the compound B and the organic base is 1:1-3:1-3, preferably 1:1-2: 1-2.

Theoretically, a person skilled in the art can synthesize the hydroxyphenyl salicylamide hydroxylate by adopting other synthetic routes, but the synthetic method provided by the invention has obvious technical effects:

1. the raw materials are cheap, the cost is low, the purchase is very convenient, the raw materials for synthesizing the compound A and the compound B can be selected as the initial raw materials by technical personnel according to technical documents;

2. the reaction in the step b and the step d is sufficient, and byproducts are not easy to generate, and in fact, the reaction yield in the step b and the step d is over 70 percent;

3. the obtained byproducts have certain water solubility, and the intermediate and the hydroxyl-phenyl salicylamine hydroxylate can be purified by simple water washing and extraction, so that the purification cost is reduced.

Drawings

FIG. 1 is a LC/MS spectrum of the intermediate of example 1;

FIG. 2 is a nuclear magnetic spectrum of hydroxyl-phenyl salicylamine hydroxylate in example 5;

FIG. 3 is a LC/MS spectrum of hydroxyl-phenyl salicylamide hydroxylate of example 5;

FIG. 4 is an HPLC chromatogram of hydroxyl-phenyl salicylamide hydroxylate of example 5;

fig. 5 is a 3D block diagram of the RRM2 according to embodiment 10;

FIG. 6 is a graph showing the effect of each drug on HBV DNA as described in example 11.

Detailed Description

The following describes the implementation of the present invention in further detail with reference to the accompanying drawings.

A. Preparation process of hydroxyphenyl salicylamine hydroxylate

In this section, compound B is

1. For the preparation of intermediates

Example 1

At room temperature, adding dichloromethane (20L) into a reaction kettle, sequentially adding a compound A (5.00kg, 16.37mol, 1.0eq) and a compound B (8.08kg, 32.75mol, 2.0eq) into the reaction kettle, cooling to 0-5 ℃, dropwise adding a dichloromethane solution of 4-dimethylaminopyridine (4.00kg, 32.75mol, 2.0eq), after dropwise adding, keeping the temperature for reaction for 1h, controlling the reaction to be complete, adding water (10L) into the reaction solution, extracting with dichloromethane, directly concentrating to obtain 7.85kg of yellow oily matter, wherein the molar yield is 93%, and the obtained product is directly used in the next step without purification.

EI-MS [ M +1] ═ 516.3, see fig. 1.

Example 2

Adding tetrahydrofuran (400mL) into a reaction kettle at room temperature, sequentially adding a compound A (100.00g, 0.33mol, 1.0eq) and a compound B (162.81g, 0.66mol, 2.0eq) into the reaction kettle, cooling to 0-5 ℃, dropwise adding a dichloromethane solution of triethylamine (66.66g, 0.66mol, 2.0eq), after dropwise adding, keeping the temperature for reaction for 2 hours, controlling the reaction to be complete, adding water (200mL) into the reaction solution, concentrating, extracting with dichloromethane, and concentrating again to obtain 147.91g of yellow oily matter, wherein the molar yield is 87.6%, and the obtained product is directly used in the next step without purification.

Example 3

At room temperature, adding acetonitrile (400mL) into a reaction kettle, sequentially adding a compound A (100.00g, 0.33mol, 1.0eq) and a compound B (162.81g, 0.66mol, 2.0eq) into the reaction kettle, cooling to 0-5 ℃, dropwise adding a dichloromethane solution of N, N-diisopropylethylamine (128.0g, 0.99mol, 3.0eq), after dropwise adding, carrying out heat preservation reaction for 2 hours, controlling the reaction to be complete, adding water (200mL) into a reaction solution, concentrating, extracting with dichloromethane, and concentrating again to obtain 151.77g of yellow oily matter, wherein the molar yield is 89.2%, and the obtained product is directly used in the next step without purification.

Example 4

At room temperature, adding dichloromethane (1200mL) into a reaction kettle, sequentially adding a compound A (100.00g, 0.33mol, 1.0eq) and a compound B (162.81g, 0.66mol, 3.0eq) into the reaction kettle, cooling to 0-5 ℃, dropwise adding a dichloromethane solution of 4-dimethylaminopyridine (120.95g, 0.99mol, 3.0eq), after dropwise adding, keeping the temperature for reaction for 2 hours, controlling the reaction to be complete, adding water (200mL) into the reaction liquid, extracting with dichloromethane, and concentrating again to obtain 148.35g of yellow oily matter, wherein the molar yield is 87.2%, and the obtained product is directly used in the next step without purification.

2. Preparation of hydroxyl benzene salicylamine hydroxylate

Example 5

Methanol (100L) is added into a reaction kettle at room temperature, then the intermediate obtained in the examples 1-4 (7.85kg, 15.22mol, 1.0eq) and palladium carbon (785g, 10%) are sequentially added, hydrogen is filled into the reaction kettle, the pressure of 0.2MPa is maintained, the reaction is carried out for 10 hours at room temperature, after the reaction is finished, the reaction product is filtered, the filtrate is concentrated to be dry, and 3.20kg of pink solid is obtained, and the molar yield is 85.6%.

The nuclear magnetic spectrum is shown in FIG. 2.

EI-MS [ M +1] ═ 246.3, see fig. 3.

HPLC of hydroxyl phenyl salicylamine hydroxylate is shown in FIG. 4.

Example 6

Tetrahydrofuran (800mL) was added to the reaction vessel at room temperature, followed by the sequential addition of the intermediates (100g, 0.19mol, 1.0eq) obtained in examples 1-4, raney nickel (5g, 5%), followed by hydrogen, maintaining a pressure of 0.2MPa, reaction at 50 ℃ for 24h, after completion of the reaction, filtration, and concentration of the filtrate to dryness to obtain 38.67g of a pink solid with a molar yield of 81.3%.

Example 7

Methanol (1L) is added into a reaction kettle at room temperature, then the intermediate (100g, 0.19mol, 1.0eq) obtained in the example 1-4 and palladium carbon (10g, 10%) are sequentially added, hydrogen is filled in, the pressure of 0.2MPa is kept, the reaction is carried out for 5h at room temperature, after the reaction is finished, the filtration is carried out, the filtrate is concentrated to be dry, 39.14g of pink solid is obtained, and the molar yield is 82.3%.

Example 8

At room temperature, tert-butanol (300mL) was added to the reaction kettle, followed by the sequential addition of the intermediate obtained in examples 1-4 (100g, 0.19mol, 1.0eq), palladium on carbon (1g, 1%), hydrogen was charged, the pressure was maintained at 0.2MPa, the reaction was carried out at room temperature for 10h, after completion of the reaction, filtration was carried out, and the filtrate was concentrated to dryness to obtain 36.48g of a pink solid with a molar yield of 78.3%.

Example 9

Methanol (1L) is added into a reaction kettle at room temperature, then the intermediate (100g, 0.19mol, 1.0eq) obtained in the example 1-4 and palladium carbon (10g, 10%) are sequentially added, hydrogen is filled in, the pressure of 0.2MPa is kept, the reaction is carried out for 5h at room temperature, after the reaction is finished, the filtration is carried out, the filtrate is concentrated to be dry, 39.14g of pink solid is obtained, and the molar yield is 82.3%.

B. The function of hydroxyl-benzene salicylamide hydroxylate in inhibiting RR enzyme and hepatitis B virus DNA replication

1. Function of hydroxyl benzene salicylamine hydroxylate in inhibiting RR enzyme

Example 10

Molecular docking calculation of salicylidene (Osalmid) and derivatives thereof

(1) The magnitude of the binding capacity of 4 compounds to the target RRM2 was predicted by the molecular docking software eHiTS of SimBioSys corporation (Version 12.0, SimBioSys inc.

The 4 compounds are:

(2) molecular docking method

Preparation of small molecules: the 2D small molecules were converted to 3D structures using OpenBabel 2.3.0 for molecular docking studies.

Target and active pocket definitions: the crystal structure of RRM2 was obtained from the PDB library (PDB code:3OLJ), and the definition of its active pocket was described in Xia Liu et al, Biochemical Pharmacology, i.e., the region where F244, D271, R330 and E334 are located, is defined as the active site.

The molecular docking method comprises the following steps: molecular docking studies were performed using eHiTS software from Wiley. eHiTS is a precise and fast flexible molecular docking software. The ehITS can automatically preprocess the target; and the butted small molecules only need to be in a 3D structure and do not need to be in the lowest-energy conformation. The accuracy parameter during docking, accuracracy, is set to 6, i.e., the highest accuracy molecular docking is performed. Oxybenzene salicylamine and its derivatives were separately docked to this site to evaluate their binding capacity to RRM 2.

(3) Scoring the binding Capacity of the Compounds

The docking scores of the 4 small molecules are as follows, and the sequencing is carried out according to the strength of the binding capacity:

Name	eHiTS-Score
		OH-1.mol	-3.807
Osalmid.mol	-2.885
		OH-2.mol	-2.424
HU.mol	-2.289

note: the eHiTS software evaluates the amount of binding ability of small molecule compounds to targets by eHiTS _ Score. eHiTS _ Score corresponds to log (kd) (log of dissociation constant), more negative values indicate stronger binding of small molecule compounds, e.g., -6 for μ M, -9 for nM; the difference in the values was 1, and the binding capacity was 10-fold.

2. Function of hydroxyl benzene salicylamine hydroxylate in inhibiting hepatitis B virus DNA replication

Example 11

Experiment on anti-HBV activity of hydroxyl benzene salicylamine hydroxylate in vitro.

1. Experimental Material

(1) Cell lines: HepG 2.2.15 cells (stably transfected HBV gene, HBV genome replication and expression can be stably carried out, and the cells are preserved in the laboratory.

(2) The main reagents are as follows: hydroxyphenyl salicylamide hydroxylate (OH-1, Hangzhou nuclide chemical technologies, Ltd.), hydroxyphenyl salicylamide (Osalmid, Beijing Bailingwei science and technology, Ltd.), lamivudine (3-TC, Beijing Bailingwei science and technology, Ltd.), hydroxyurea (HU, Beijing Bailingwei science and technology, Ltd.), and a hepatitis B virus nucleic acid quantitative determination kit (Hunan Shengxiang biotechnology, Ltd.)

(3) The main apparatus is as follows: carbon dioxide incubator (Thermo Forma corporation, USA), real-time fluorescence quantitative PCR instrument (ABI corporation, USA 7500Fast)

2. The experimental method comprises the following steps:

(1) cell culture:

HepG 2.2.15 and A64 cells were cultured in DMEM high-sugar medium (supplemented with 10% fetal bovine serum, 1% streptomycin, 400. mu.g/ml G418, 2mmol/L glutamine) and placed in a cell culture incubator at 37 ℃ with 5% CO₂And (5) culturing under an environment. When the cells grow to 70-80%, the cells are digested and passaged by pancreatin containing EDTA.

(2) Determining the inhibitory effect of the drug on HBV DNA in the supernatant of HepG 2.2.15 cell culture:

compounding hydroxyl benzene salicylamide hydroxylate into HepG 2.2.15 cells with DMEM medium without G418⁴each/mL of the cells was densely plated in 24-well plates, 500. mu.L of the cell suspension was added to each well, and CO was added₂After 24h of incubation at 37 ℃, the cells adhere to the wall and grow well, the culture solution is aspirated, and 500 mu L of culture medium containing medicine is added into each hole, wherein each concentration is 3 multiple holes. Collecting culture medium supernatant on the 4 th, 6 th and 8 th days of culture to detect HBV DNA content. Adding 5 mul of sample releasing agent into 5 mul of cell culture supernatant, fully mixing uniformly, incubating at room temperature for 10min, adding 38 mul of reaction solution, 2 mul of enzyme mixed solution and 0.2 mul of internal standard, and amplifying according to the following conditions: after pre-denaturation at 50 ℃ for 2min and 94 ℃ for 5min, the cycle is repeated for 40 cycles at 94 ℃ for 15s and 57 ℃ for 30s and signals are collected. And (3) automatically calculating the quantitative result of the HBV DNA by a ratio instrument of the Ct value of the standard product.

3. Statistical analysis:

the experimental data were processed with SPSS 13.0 software and the results were as followsIt is shown that the comparison of the mean of the multiple samples was analyzed using One-way ANOVA, and that p < 0.05 was statistically significantly different.

4. The experimental results are as follows:

compared with a blank group control group, the hydroxyl benzene salicylamine hydroxylate has obvious inhibitory effect (P is less than 0.01) on HBV copy number at the dose of 10 mu M on 4 th, 6 th and 8 th days after drug addition, and the inhibitory effect is enhanced along with the prolonging of the acting time. The inhibition effect is superior to that of the prototype hydroxyphenyl salicylamine and lower than that of lamivudine.

Table 1 effect of each drug on HepG 2.2.15 cell culture supernatant HBV DNA (n-3,)

p < 0.01, P < 0.001, P < 0.0001 compared with control group

The above description is only an embodiment of the present invention, and is not intended to limit the design of the present invention, and all equivalent changes made according to the design key of the present invention fall within the protection scope of the present invention.