WO2020083353A1

WO2020083353A1 - 3-caffeoylquinic acid derivative, preparation method therefor and use thereof

Info

Publication number: WO2020083353A1
Application number: PCT/CN2019/113160
Authority: WO
Inventors: 张洁; 张亮; 姬勋; 张飞; 黄望
Original assignee: 四川九章生物科技有限公司
Priority date: 2018-10-25
Filing date: 2019-10-25
Publication date: 2020-04-30
Also published as: CN111100006A; CN111100006B

Abstract

The present invention provides a 3-caffeoylquinic acid derivative, a preparation method therefor and use thereof. The 3-caffeoylquinic acid derivative is as shown in formula I. In addition, the 3-caffeoylquinic acid derivative also has the effects of antisepsis and anti-inflammation and antioxidation. The 3-caffeoylquinic acid derivative can be used for preparing anti-tumor and antiviral drugs, particularly drugs for treating the specific tumors and viruses, and can also be used for preparing antibacterial, anti-inflammatory and antioxidant drugs.

Description

3-caffeoylquinic acid derivative, and preparation method and use thereof

Technical method

The invention belongs to the field of chemical medicine, and specifically relates to a 3-caffeoylquinic acid derivative and a preparation method and application thereof.

Background technique

3-Caffeoylquinic acid, also known as chlorogenic acid (Chlorogenic acid, CGA), is a phenolic acid produced by caffeic acid and quinic acid, which is a plant's aerobic respiration A phenylpropanoid compound produced by the shikimate pathway in the structure is shown below.

3-Caffeoylquinic acid is widely present in plants, with higher contents in honeysuckle and eucommia. Because of its wide range of biological activities, it has anti-cancer, antibacterial, anti-viral, hepatoprotective, gallbladder lowering, blood pressure, blood lipid, scavenging free radicals, and exciting central nervous system functions. For example, 3-caffeoylquinic acid as a free radical scavenger and antioxidant has been proved by a large number of tests; studies have shown that 3-caffeoylquinic acid can prevent and inhibit the occurrence of gastric cancer and colon cancer; 3-caffeoylquinic acid Niacin can also inhibit the growth of Escherichia coli, Shigella sonnei, Staphylococcus aureus, Bacillus subtilis, Micrococcus luteus and Legionella.

Although 3-caffeoylquinic acid has so many pharmacological activities, its efficacy needs to be further improved, and there are no reports of improving its activity.

Summary of the invention

The object of the present invention is to provide a 3-caffeoylquinic acid derivative and its preparation method and use.

The present invention provides a compound represented by formula I:

The invention also provides a method for preparing the aforementioned compound, which includes the following steps:

(1) Dissolve 3-caffeoylquinic acid and D-mannitol in an organic solvent, and react under an acidic system to obtain a reaction solution;

(2) The compound reaction solution is prepared by precipitation, adsorption analysis, washing, crystallization and recrystallization, and freeze-drying.

further,

In step (1), the molar ratio of 3-caffeoylquinic acid to D-mannitol is (1: 1) to (5: 1);

And / or, in step (1), the acidic system is a p-toluenesulfonic acid, hydrochloric acid or sulfuric acid system;

And / or, in step (1), the reaction temperature is 20-60 ° C., and the reaction time is 2-24 h;

And / or, in step (2), the chromatography column used for the adsorption analysis is a non-polar or weakly polar macroporous adsorption resin chromatography column;

And / or, in step (2), the washing is washing with an organic solvent.

further,

In step (1), the molar ratio of 3-caffeoylquinic acid to D-mannitol is (1: 1) to (3: 1);

And / or, in step (1), the acidic system is a p-toluenesulfonic acid, hydrochloric acid or concentrated sulfuric acid system;

And / or, in step (1), the reaction temperature is 20-60 ° C., and the reaction time is 24 h;

And / or, in step (1), the organic solvent is N, N-dimethylformamide or absolute ethanol;

And / or, in step (2), the model of the macroporous adsorption resin chromatography column is HPD-100, HPD-450 or LS-46;

And / or, in step (2), the chromatography column is eluted with 5% to 80% ethanol after the chromatography column is completed, the eluent is collected, concentrated to dryness, and dissolved in water with saturation;

And / or, in step (2), the organic solvent used for the washing is ethyl acetate;

And / or, in step (2), the crystallization is refrigerated crystallization;

And / or, in step (2), the recrystallization is recrystallization with water.

The invention also provides the use of the aforementioned compounds in the preparation of anti-tumor drugs.

Further, the tumor is a borderline tumor and / or a malignant tumor;

Preferably, the borderline tumor is a borderline tumor of the ovary; and / or, the malignant tumor is lung cancer, liver cancer, kidney cancer, breast cancer, cervical cancer, and prostate cancer.

The present invention also provides the use of the aforementioned compound in the preparation of antiviral drugs; preferably, the virus is an immunodeficiency virus; more preferably, the immunodeficiency virus is an AIDS virus.

The invention also provides the application of the aforementioned compound in the preparation of antibacterial and anti-inflammatory drugs.

The invention also provides the application of the aforementioned compound in the preparation of anti-oxidation medicine.

The invention also provides a medicine, which is a preparation prepared by taking the aforementioned compound as an active ingredient, together with commonly used pharmaceutical excipients or auxiliary ingredients.

In the present invention, the room temperature is 25 ± 5 ° C, and overnight is 12 ± 2h.

The 3-caffeoylquinic acid derivatives of the present invention have good antitumor and antiviral effects, especially for ovarian borderline tumors in borderline tumors, lung cancer, liver cancer, kidney cancer, breast cancer, cervical in malignant tumor Cancer, prostate cancer, and HIV in immunodeficiency virus have a good inhibitory effect. In addition, the 3-caffeoylquinic acid derivatives of the present invention also have antibacterial, anti-inflammatory and anti-oxidant effects. The 3-caffeoylquinic acid derivatives of the present invention can be used to prepare anti-tumor and anti-virus, especially the aforementioned specific tumor and virus drugs, and can also be used to prepare anti-bacterial anti-inflammatory and anti-oxidation drugs.

Obviously, according to the above content of the present invention, in accordance with the ordinary technical knowledge and conventional means in the art, other various forms of modification, replacement or alteration can be made without departing from the above basic technical idea of the present invention.

The above content of the present invention will be further described in detail below through specific implementations in the form of examples. However, it should not be understood that the scope of the above subject matter of the present invention is limited to the following examples. All technologies implemented based on the above contents of the present invention belong to the scope of the present invention.

BRIEF DESCRIPTION

Figure 1 is the nuclear magnetic resonance spectrum of the 3-caffeoylquinic acid derivative of the present invention; A: ¹ H-NMR spectrum, B: ¹ H- ¹ H COSY spectrum, C: heavy water exchange hydrogen spectrum, D: ¹³ C-NMR Map, E: ¹³ C-DEPT map, F: ¹ H- ¹³ C HSQC map, G: ¹ H- ¹³ C HMBC map.

Figure 2 shows the inhibition rate of Lewis lung cancer in mice in each experimental group.

Figure 3 is the inhibition rate of each experimental group on mouse breast cancer MET-6.

Figure 4 is a photomicrograph (× 100) of each experimental group's inhibitory effect on HIV-1; a is a normal cell control group, b is a virus control group, c is a CT cytotoxicity control group, and d is a drug efficacy group (5mg / ml), e is AZT positive control group.

detailed description

Example 1. Preparation of 3-caffeoylquinic acid derivatives of the present invention

Take 200 g of 3-caffeoylquinic acid and 100 g of D-mannitol, dissolve in N, N-dimethylformamide, add 50 mL of p-toluenesulfonic acid, and stir at room temperature for 24 hours to obtain a reaction solution.

Take the reaction solution, dilute it with water to 1 to 10 times, then apply the HPD-100 macroporous adsorption resin chromatography column. After loading the column, wash with water until the 3-caffeoylquinic acid and D-mannose are not detected in the washing solution. The sugar alcohol was eluted with 80% ethanol. The 80% ethanol eluate was collected and concentrated to dryness. Take the dried product, dissolve it in water, wash it with ethyl acetate, refrigerate and crystallize it, filter it, take the crystallized product, recrystallize it with water, and freeze-dry it to obtain 180g, the content is 98.2%.

The nuclear magnetic resonance spectrum of the 3-caffeoylquinic acid derivative is shown in FIG. ^{The 1} H-NMR spectrum (FIG. 1A) clearly shows that the 3-caffeoylquinic acid derivative is a compound in which 3-caffeoylquinic acid and D-mannitol are reacted to form an ester. Due to the symmetry of the molecular structure of free D-mannitol, the chemical shift of C-17 and C-22 to H is basically the same. After D-mannitol and 3-caffeoylquinic acid form an ester, the group connected to C-17 changes from a hydroxyl group to an ester group. The H on C-17 is affected by the ester group, and the chemical shift moves to a lower field. Significantly increased; because C-22 is still connected to the hydroxyl group, the H chemical shift on C-22 is not greatly affected, and the H chemical shift on the compound C-17 for all compounds of D-mannitol and 3-caffeoylquinic acid ester δ (4.19, 3.99) will be significantly higher than the H chemical shift δ (3.60, 3.37) on C-22. ¹ H- ¹³ C HMBC (Figure 1G) also clearly shows that the derivative is a compound in which 3-caffeoylquinic acid and D-mannitol react to form an ester, and δ173.35 is the C-in 3-caffeoylquinic acid 1 Chemical shift, when 3-caffeoylquinic acid and D-mannitol are reacted to form an ester, the H on C-1 and C-17 can form a triple bond J _CH coupling, which is shown in ¹ H- ¹³ C HMBC Strong correlation.

The C spectrum of the derivative shows that there are 21 kinds of C in the molecule, and the DEPT spectrum (Figure 1E) shows that there are 4 secondary carbons, 11 tertiary carbons, and 6 quaternary carbons in the molecule, while the molecular formula has 22 Cs, indicating that there are 1 pair Tertiary carbons with the same chemical shift are analyzed as two tertiary carbons, C-4 and C-18.

The ¹ H-NMR, ¹ H- ¹ HCOSY and heavy water exchange hydrogen spectrum measurement data are listed in Table 1, combined with ¹³ C-NMR, ¹³ C-DEPT-135, ¹³ C-DEPT-90, ¹ H- ¹³ C HSQC , ¹ H- ¹³ C HMBC data, to determine the attribution of each H.

The ¹³ C-NMR spectrum data are listed in the order of C chemical shift from large to small in Table 2. DEPT spectrum data is used to determine the type of C, HSQC spectrum is used to determine the attribution of directly connected CH, and HMBC spectrum is used to target C Attribution of H on atoms with similar atoms, HSQC and HMBC data and analysis are also listed in Table 2.

Table 1 The attribution of measured data of ¹ -H-NMR, ¹ H-1H COSY and hydrogen exchange spectrum of 3-caffeoylquinic acid derivatives of the present invention

Table 2 ¹³ C-NMR, ¹³ C-DEPT, ¹ H- ¹³ C HSQC, ¹ H- ¹³ C HMBC measurement data of the 3-caffeoylquinic acid derivatives of the present invention

After testing, the structural formula of 3-caffeoylquinic acid derivatives is as follows: its molecular formula is C ₂₂ H ₃₀ O ₁₄ and its molecular weight is 518.47.

Example 2. Preparation of 3-caffeoylquinic acid derivatives of the present invention

Take 500 g of 3-caffeoylquinic acid and 100 g of D-mannitol, dissolve it in absolute ethanol by heating, add 100 mL of concentrated sulfuric acid, and stir at 40 ° C for 24 hours to obtain a reaction solution.

Take the reaction solution, place it below 0 ° C to allow sufficient precipitation, filter, and collect the filtrate; after the filtrate is concentrated and recovered into ethanol, it will be loaded onto a macroporous adsorption resin chromatography column of model HPD-450. After the column is completed, it is washed with water until it is not detected in the washing solution. 3-Caffeoylquinic acid and D-mannitol, eluted with 20% ethanol, collect the 20% ethanol eluent and concentrate to dryness. Take the dried product, dissolve it in water, wash it with ethyl acetate, refrigerate and crystallize it, filter it, take the crystallized product, recrystallize it with water, and freeze-dry it to obtain 120g, the content is 98.6%.

The 3-caffeoylquinic acid derivative has been tested and has the same structure as in Example 1.

Example 3. Preparation of the 3-caffeoylquinic acid derivative of the present invention

Take 300 g of 3-caffeoylquinic acid and 100 g of D-mannitol, dissolve in absolute ethanol, add 150 ml of hydrochloric acid, and stir at 60 ° C for 24 hours to obtain a reaction solution.

Take the reaction solution, place it below 0 ° C to allow sufficient precipitation, filter, and collect the filtrate; after the filtrate is concentrated and recovered into ethanol, a large pore adsorption resin chromatography column of model LS-46 is used. After the column is completed, it is washed with water until it is not detected in the washing solution. 3-Caffeoylquinic acid and D-mannitol, eluted with 5% ethanol, collect the 5% ethanol eluate and concentrate to dryness. Take the dried product, dissolve it in water, wash it with ethyl acetate, refrigerate and crystallize it, filter it, take the crystallized product, recrystallize it with water, and freeze-dry it to obtain 140g, with a content of 98.3%.

Example 4. The prescription of the 3-caffeoylquinic acid derivative oral pharmaceutical preparation of the present invention

1. Prescription 1

1000g of 3-caffeoylquinic acid derivatives.

Preparation method: Aseptically weigh out 3-caffeoylquinic acid derivatives and aseptically dispense into powder.

2. Prescription 2

3-caffeoylquinic acid derivative 1000g, filler 500g, binder 5g.

Preparation method: Weigh 3-caffeoylquinic acid derivative, filler and binder according to the prescription, granulate, whole granulate and divide into granules.

3. Prescription 3

3-caffeoylquinic acid derivative 1000g, filler 500g, binder 5g, lubricant 3g.

Preparation method: Weigh 3-caffeoylquinic acid derivatives, fillers, binders according to the prescription, granulate, adjust granules, add lubricant, and press to obtain tablets.

The above filler is one or more of mannitol, lactose, starch, microcrystalline cellulose, dextrin; binder is sodium carboxymethyl cellulose, PVP; lubricant is magnesium stearate, talc, Micronized silica gel.

Example 5. The prescription of the 3-caffeoylquinic acid derivative injection preparation of the present invention

1. Prescription 1

1000g of 3-caffeoylquinic acid derivatives.

Preparation method (1): Aseptically weigh the 3-caffeoylquinic acid derivative according to the prescription and aseptically divide into powder injections.

Preparation method (2): Weigh the 3-caffeoylquinic acid derivative according to the prescription, dissolve it in water for injection, filter and sterilize, freeze-dry, and fill to obtain a lyophilized powder injection.

2. Prescription 2

3-caffeoylquinic acid derivative 1000g, scaffold 2667g, antioxidant 67g.

Preparation method: Weigh 3-caffeoylquinic acid derivative, scaffold, and antioxidant according to the prescription, dissolve it in water for injection, filter sterilize, fill, freeze-dry, and obtain a freeze-dried powder injection.

The above scaffolds are mannitol, lactose, glucose; antioxidants are sodium bisulfite, vitamins, glutathione, folic acid.

The following specific examples demonstrate the beneficial effects of the present invention.

Test Example 1. Animal test of the 3-caffeoylquinic acid derivative of the present invention for treating lung cancer

1. Test materials

1.1 Test animal

C57BL / 6 mice, male, SPF grade, 120, each 18-20g. Source: Nanjing Junke Biological Engineering Co., Ltd.

1.2 Tested cell line

Mouse Lewis lung cancer tumor strains were subcultured by the Pharmacology Department of the Institute of Materia Medica, Chinese Academy of Medical Sciences.

1.3 Test drug

Positive drug 1: Docetaxel injection, manufacturer: Jiangsu Oxicon Pharmaceutical Co., Ltd.

Positive drug 2: Chlorogenic acid, provided by Sichuan Jiuzhang Biological Technology Co., Ltd.

Test drug: 3-caffeoylquinic acid derivatives prepared in the examples of the present application.

1.4 The dosage and administration method of each drug

Positive drug 1 (Docetaxel injection): The experimental dose is 5 mg / kg, which is administered intraperitoneally, once a day for 5 consecutive days.

Positive drug 2 (chlorogenic acid): the experimental dose is 20 mg / kg, which is administered intraperitoneally, once a day for 10 consecutive days.

Test drug (3-caffeoylquinic acid derivative): The experimental doses were 5mg / kg, 10mg / kg, 20mg / kg, 40mg / kg, intraperitoneal injection, once a day, continuous administration of 10 day.

1.5 Preparation of drugs

Positive drug 1: Each bottle of docetaxel injection is dissolved with 1.5ml of special solvent, divided into 4 tubes, each 500μl (including docetaxel 5mg), stored at -20 ℃. Take a branch dissolved in 20ml of physiological saline and administer 0.2ml intraperitoneally per 10g of mouse body weight. Use before delivery every day.

Positive drug 2: Take 30mg of chlorogenic acid and dissolve it in 30ml of saline. According to the weight of mice, every 10g of intraperitoneal injection of 0.2ml administration. Use before delivery every day.

Test drug 1 (40 mg / kg dose group): Take 60 mg 3-caffeoylquinic acid derivative and dissolve in 30 ml of physiological saline to obtain a solution of test drug 1.

Test drug 2 (20 mg / kg dose group): Take 15 ml of test drug 1 solution, add 15 ml of physiological saline and double it to obtain test drug 2 solution.

Test drug 3 (10 mg / kg dose group): Take 15 ml of test drug 2 solution, add 15 ml of physiological saline and double it to obtain test drug 3 solution.

Test drug 4 (5 mg / kg dose group): Take 10 ml of test drug 3 solution and dilute it by adding 10 ml of physiological saline to obtain test drug 4 solution.

The above-prepared test drugs 1 to 4 are administered intraperitoneally by 0.2 mL per 10 g of mouse body weight. Use before delivery every day.

2. Test method

2.1 Route of administration

Intraperitoneal injection (ip).

2.2 Method of administration

Mouse Lewis lung cancer model, C57BL / 6 mice, male, 18-20g each. During the experiment, the Lewis lung cancer tumor strains of well-grown mice were shredded, ground, and diluted with sterile saline at a volume ratio of 1: 3 to make a tumor cell suspension, and each mouse was inoculated with 0.2 ml of tumor fluid. . The animals were randomly divided into groups the next day after inoculation, weighed, and started to administer the drug.

The experimental animals are divided into 8 groups with 15 animals in each group:

Negative control group: the mice are not given any medicine after inoculated with lung cancer tumor strains.

Solvent control group: mice are inoculated with lung cancer tumor strains, and they are administered intraperitoneally with 0.2 ml of normal saline per 10 g of mice, once a day.

Positive control group:

(1) Docetaxel 5mg / kg administration group: After inoculation of lung cancer tumors, mice were injected intraperitoneally with 0.2ml per 10g of mice, once a day for 5 consecutive days, and the administration was stopped from the 6th day.

(2) Chlorogenic acid 20mg / kg administration group: After inoculation of lung cancer tumors, mice were injected intraperitoneally with 0.2ml per 10g of mice, once a day for 10 consecutive days, and the administration was stopped from the 11th day.

Test drug group: including four dose groups of 3-caffeoylquinic acid derivatives 5mg / kg, 10mg / kg, 20mg / kg, 40mg / kg, all were injected intraperitoneally with 0.2ml per 10g mice, once a day , 10 consecutive days of administration, and stop administration on the 11th day.

2.3 Evaluation of anti-tumor effect

The animals in each test group were sacrificed the next day after stopping the drug, weighed, exfoliated and weighed. The tumor inhibition rate (%) was calculated based on the tumor weight. Body weight and tumor re-use mean ± standard deviation

Show and perform t test between each administration group and negative control group, and between each administration group and positive control group docetaxel group and chlorogenic acid group.

3. Test results

The test results are shown in Table 3 and Figure 2. Intraperitoneal injection of 3-caffeoylquinic acid derivatives has a significant inhibitory effect on the growth of Lewis lung cancer, and has a better dose-effect relationship. 3-caffeoylquinic acid derivatives Compared with the docetaxel group and the chlorogenic acid group at the dose of 20 mg / kg, the tumor inhibition rate was enhanced (Table 3), with significant differences. It can be seen from the weight of the mice that the chlorogenic acid group and the 3-caffeoylquinic acid derivative group at a dose of 10-40 mg / kg significantly increased the body weight of the mice, which was significantly different from the negative control group. It shows that chlorogenic acid and 3-caffeoylquinic acid derivatives have no obvious toxicity at the doses used (Table 3).

Table 3 The inhibitory effect of each test group on the growth of mouse Lewis lung cancer (

n = 15)

Note: * P <0.01, compared with the negative control group. ^# P <0.05, compared with the docetaxel group; ⁺ P <0.05, compared with the chlorogenic acid group.

4. Test conclusion

(1) Intraperitoneal injection of 3-caffeoylquinic acid derivatives showed a significant inhibitory effect on the growth of transplanted Lewis lung cancer in mice, with an effective dose group of 10-40 mg / kg. Peak value; at 20mg / kg and 40mg / kg, there are significant differences compared with the docetaxel group and the chlorogenic acid group, indicating that the 3-caffeoylquinic acid derivative has better inhibitory effect on lung cancer than docetaxel He race and chlorogenic acid.

(2) 3-Caffeoylquinic acid derivatives No obvious toxic and side effects were observed at the doses used.

Test Example 2. Animal test of the 3-caffeoylquinic acid derivative of the present invention in the treatment of breast cancer

1. Test materials

1.1 Test animal

BALB / c mice, male, SPF grade, 120, each 18-20g. Source: Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.

1.2 Tested cell line

The mouse breast cancer MET-6 tumor strain was subcultured and maintained by the Pharmacology Department of the Institute of Materia Medica, Chinese Academy of Medical Sciences.

1.3 Test drug

Positive drug 1: Doxorubicin hydrochloride (doxorubicin) for injection, manufacturer: Shenzhen Wanle Pharmaceutical Co., Ltd.

1.4 Drug dosage and administration method

Positive drug 1 (doxorubicin): the experimental dose is 3 mg / kg, which is administered intraperitoneally, every other day, for a total of 7 times.

Positive drug 2 (chlorogenic acid): The experimental dose is 20 mg / kg, which is administered intraperitoneally, once a day for 13 consecutive days.

Test drug (3-caffeoylquinic acid derivative): The experimental doses were 5 mg / kg, 10 mg / kg, 20 mg / kg, 40 mg / kg, which were administered by intraperitoneal injection, once a day, continuous administration of 13 day.

1.5 Preparation of drugs

Positive drug 1: Weigh 15 mg of doxorubicin powder for injection (including 3 mg of doxorubicin) and dissolve it in 20 ml of physiological saline at a concentration of 0.15 mg / ml. According to the weight of mice, every 10g of intraperitoneal injection of 0.2ml administration. Use before dispensing.

2. Test method

2.1 Route of administration

Intraperitoneal injection (ip).

2.2 Method of administration

The mouse breast cancer MET-6 model, the selected animal is BALB / c mice, male, each 18-20g. During the experiment, the well-grown mouse breast cancer MET-6 tumor strain was taken, shredded, ground, diluted with sterile saline at a volume ratio of 1: 3, and then made into a tumor cell suspension, and each mouse was inoculated with 0.2 ml tumor fluid. The animals were randomly divided into groups the next day after inoculation, weighed, and started to administer the drug.

The volume of doxorubicin injection was 0.2 ml per 10 g of mice, and doxorubicin was given every other day for 7 times. The volume of chlorogenic acid administered was 0.2 ml per 10 g of mice intraperitoneally, once a day for 13 consecutive days. Each dose of 3-caffeoylquinic acid derivative was administered by intraperitoneal injection of 0.2 ml per 10 g of mice once a day for 13 consecutive days.

Negative control group: After the mice were inoculated with breast cancer tumors, no medicine was given.

Solvent control group: mice were inoculated with 0.2ml of normal saline per 10g of mice after inoculation with breast cancer tumors, once a day.

Positive control group:

(1) Doxorubicin injection administration group: After inoculation of breast cancer tumors, mice were injected intraperitoneally with 0.2 ml per 10 g of mice. Doxorubicin was administered every other day for a total of 7 times, and then the drug was stopped.

(2) Chlorogenic acid 20mg / kg administration group: After inoculation of breast cancer tumors, mice were injected intraperitoneally with 0.2ml per 10g of mice, once a day for 13 consecutive days, and then stopped.

Test drug group: including four dose groups of 3-caffeoylquinic acid derivatives 5mg / kg, 10mg / kg, 20mg / kg, 40mg / kg, all were injected intraperitoneally with 0.2ml per 10g mice, once a day , For 13 consecutive days, and then stop.

2.3 Evaluation of anti-tumor effect

Show and perform t test between each administration group and negative control group, and between each medication group and positive control group doxorubicin group and chlorogenic acid group.

3. Test results

The test results are shown in Table 4 and Figure 3. Intraperitoneal injection of 3-caffeoylquinic acid derivatives has a significant inhibitory effect on the growth of mouse breast cancer MET-6, and has a better dose-effect relationship, 3-caffeoyl Compared with the positive control adriamycin group and the chlorogenic acid group, the quinic acid derivative at the dose of 20 mg / kg had an enhanced tumor suppression rate (Table 4), with significant differences. It can be seen from the weight of the mice that the chlorogenic acid group and the 3-caffeoylquinic acid derivative group at a dose of 10-40 mg / kg significantly increased the body weight of the mice, which was significantly different from the negative control group. It showed that chlorogenic acid and 3-caffeoylquinic acid derivatives had no obvious toxicity at the doses used (Table 4).

Table 4 Inhibitory effect of each test group on the growth of mouse breast cancer MET-6 (

n = 15)

Note: * P <0.01, compared with the negative control group. ^# P <0.05, compared with the doxorubicin group; ⁺ P <0.05, compared with the chlorogenic acid group.

4. Test conclusion

(1) Intraperitoneal injection of 3-caffeoylquinic acid derivatives showed a significant inhibitory effect on the growth of MET-6 in breast cancer in mice, with an effective dose group of 10-40 mg / kg, and an inhibitory effect on tumors at 20 mg / kg Peak; at 20 mg / kg, there is a significant difference compared with the doxorubicin group and the chlorogenic acid group, indicating that the 3-caffeoylquinic acid derivative has better inhibitory effect on breast cancer than doxorubicin and chlorogenic at this dose acid.

Test Example 3. The in vitro antiviral test of the 3-caffeoylquinic acid derivative of the present invention for AIDS treatment

1. Test materials

1.1 Test cells

MT-4 cells were provided by the Institute of Virology, Chinese Academy of Preventive Medicine, and subcultured with RPMI 1640 medium.

1.2 Tested virus strain

The HIV-1 IIIB virus strain was provided by the Institute of Virology, Chinese Academy of Preventive Medicine.

1.3 Test drug

Positive drug: Zidovudine (AZT), provided by Shanghai Dissino Chemical Pharmaceutical Co., Ltd.

The above two drugs are prepared by serum-free RPMI 1640 medium and then sterilized by 0.22μm microporous filter membrane for use.

1.4 Other reagents and materials

RPMI 1640: available from GIBCO, powder, each small package (1L amount) was added to 900mL of deionized water was added 2g NaHCO _3, volume to lL, stirring and blending, microporous membrane filter sterilized 0.22μm, sub Install and store at 4 ℃ for future use. When ready to use, add 1% 2mM glutamine, 1% 100U / mL penicillin and 100μg / mL streptomycin and 10% fetal bovine serum, and adjust the PH value to 7.2 ～ 7.3.

Fetal bovine serum: product of GIBCO company.

PBS (Phosphate Buffer Solution): NaCl 8.0g, KCl 0.2g, Na ₂ HPO ₄ · 12H ₂ O 3.58g, KH ₂ PO ₃ 0.24g, dilute to 1L with deionized water, and autoclave at 121 ° C for 30min.

MTT: light yellow powder, product of Amresco. Weigh 100mg MTT and dissolve it in 20mL PBS. After fully dissolved, filter and sterilize, dispense and store at 20 ℃.

1.5 Drug concentration and preparation:

Positive drug: Zidovudine (AZT) experiment concentration is 0.7mg / ml, prepared with serum-free RPMI 1640 medium, and then sterilized by 0.22μm microporous filter membrane, put in 4 ℃ refrigerator for use.

Test drug: The concentration of 3-caffeoylquinic acid derivative used in the experiment is 0.1mg / ml, 0.5mg / ml, 1mg / ml, 5mg / ml, 10mg / ml, and it is formulated with serum-free RPMI 1640 medium. Concentration, and then filter and sterilize through 0.22μm microporous membrane, put in 4 ℃ refrigerator for use.

2. In vitro antiviral test

2.1 Cytology experiment

2.1.1 Cell culture methods

Under sterile conditions, repeatedly pipette MT-4 cell solution 5 times with a pipette to evenly disperse the cells. Place the cells in a centrifuge tube and centrifuge at 1000r / min for 5-10 minutes at low speed. Discard the supernatant. The cell pellet is completely cultured with a small amount of RPMI 1640 Resuspend, take the suspension for cell counting, divide the suspension into cell culture flasks, dilute with RPMI 1640 complete medium to make the final concentration of cells in the culture flask 2 × 10 ⁵ ～ 4 × 10 ⁵ / mL, Incubate at 37 ° C in a 5% CO ₂ incubator, usually change the fluid after 3 days.

2.1.2 Cell freezing and recovery methods

Cryopreservation method: take MT-4 cells in logarithmic growth phase, centrifuge at 1000r / min at low speed for 5-10min, discard supernatant, resuspend cell pellet in cryopreservation solution, count cells, and still dilute with cryopreservation solution to make cells The density is between 1 × 10 ⁶ ～ 5 × 10 ⁶ / mL, divide the cell liquid into the cryopreservation tube, and mark the cell name and the date of cryopreservation. Place it in the + 4 ℃ refrigerator for more than 2h, -20 ℃ refrigerator for more than 24h, and store it in the -70 ℃ refrigerator or put it into the -196 ℃ liquid nitrogen tank for long-term storage.

Resuscitation method: Remove the cryopreservation tube from liquid nitrogen, quickly put it in warm water at 37-38 ° C, and shake it quickly to melt it (about 1 minute). Once the cells melt, the cells should not be stored in the cryopreservation solution at room temperature Dilute with RPMI 1640 complete culture solution at about 25 ° C to 4 times the original volume within 5 minutes, centrifuge at 1000r / min for 5-10min, remove the supernatant, add fresh RPMI 1640 complete culture solution to the cell pellet, mix well, set 37 Incubate in a 5% CO ₂ incubator at ℃.

2.2 Virus culture method

Take MT-4 cells in logarithmic growth phase, centrifuge at 1000r / min at low speed for 5-10min, remove the supernatant, and resuspend the cell pellet with RPMI 1640 complete medium to make the concentration between l × 10 ⁶ and 2 × 10 ⁶ / mL. Add HIV-1ⅢB virus, incubate at 37 ℃, 5% CO ₂ incubator for 2h, then add RPMI 1640 complete medium to make the cell virus concentration in 2 × 10 ⁵ ～ 3 × 10 ⁵ / mL, set at 37 ℃, 5 Incubate in a% CO ₂ incubator, change the fluid day by day and observe the state of the cells to see if syncytia is produced. Generally, after 6 days, the cell lesion reaches 70% or more. Centrifuge at 3500r / min for 10min at 4 ℃, fill the supernatant separately, and store in -196 ℃ liquid nitrogen tank.

2.3 Determination of half of his tissue cell culture infection dose (TCID50)

Take the logarithmic phase of MT-4 cells and inoculate in 96-well plate, 100 μl per well, the cell concentration is 2.5 × 10 ⁴ cells / well, and dilute the virus stock solution 10 times with the culture solution (on ice), 37 ℃, 5% CO ₂ incubator. Change the fluid once on the third day, and observe the cytopathy (CPE) on the sixth day. Cytopathology is + for 25% or less, ++ for 26% to 50%, ++ for 51% to 75%, and ++++ for 76% to 100%. The Reed-Muench method was used to calculate the half of the virus tissue infection dose (TCID50).

2.4 In vitro antiviral test

2.4.1 Observation of cytopathy (CPE method)

The experiment is divided into 13 groups:

Normal cell control group (CC group): simple normal growth of MT-4 cells;

Virus control group (VC group): HIV-1ⅢB virus and MT-4 cell culture;

Positive drug control group (AZT group): Zidovudine at 0.7 mg / ml was cultured with virus-inoculated cells;

Administration group (TT group): the 3-caffeoylquinic acid derivative of the present invention, the experimental concentration is 0.1mg / ml, 0.5mg / ml, 1mg / ml, 5mg / ml, 10mg / ml, and the virus inoculated Cell culture;

Drug cytotoxicity control group (CT group): different concentrations (0.1 mg / ml, 0.5 mg / ml, 1 mg / ml, 5 mg / ml, 10 mg / ml) 3-caffeoylquinic acid derivatives and normal growth MT-4 The cells are cultured.

The specific experimental methods are as follows:

The 96-well plates were streaked into groups, each small component was 7 wells, 100μl RPMI 1640 complete medium was added to each well, and 100μl (2.5 × 10 ⁴ cells / well) MT-4 cells were added to normal cell control group and drug cells respectively To the corresponding wells of the toxicity control group, add 100 μl of the pre-warmed cell virus suspension to the corresponding wells of the virus control group and each administration group, and then add different drugs to the positive control group, each administration group In the wells of each drug cytotoxicity control group, place a 5% CO ₂ incubator at 37 ° C to continue culturing for 3 days. Change the medicine once after the third day, and take photos on the sixth day.

2.4.2 MTT staining method

Add 100 μl of adjusted concentration of MT4 cells (25,000 cells / well) to each well of the 96-well plate, and then add 100 μl of the drug solution prepared by the filtered RPMI 1640 medium to each well, and set the concentration of each drug to 3 Three control wells were used as cell controls at the same time. They were mixed gently and incubated in a 37 ° C, 5% CO ₂ incubator. On the third day, draw 100μl of supernatant from each well and discard it (note that the cells below cannot be aspirated), add 10μl of prepared MTT solution to each well, mix gently, and incubate in a 37 ° C, 5% CO ₂ incubator for 4h ; Add 150μl acidified isopropanol to each well and dissolve the crystals by pipetting; determine the absorbance OD value of each well (OD _570/630 ): calculate the inhibition rate of the drug on normal MT-4 cells at each concentration according to the formula.

3. Test results

3.1 Micrograph results

The microscopic results are shown in Figure 4 of the specification. It can be seen from the figure that the MT-4 cells in the CC group are in good growth state, and the cells grow in clusters and pieces, and the number of cells is significantly more than that in the VC group. The growth state of the cells in the VC group was poor, a large number of cells died, and the number of cells decreased significantly. The cells in the positive drug AZT group grew well, similar to the cells in the CC group. The cells in the drug group at the concentration of 5mg / ml had no obvious toxic and side effects, and the cells also grew in clusters and pieces. After adding the virus, the cell growth was significantly better than the virus control group, which was similar to the AZT group, indicating that there was a certain resistance Virus effect.

3.2 Related parameters

Table 5 Inhibitory effect of each test group on HIV-1 ⅢB virus strain

As can be seen from the above table, the cell survival rate of 3-caffeoylquinic acid derivatives is relatively high, which is significantly higher than that of the virus control, and the highest rate of inhibition of the virus at a concentration of 5 mg / ml is 89.37%, indicating that the concentration The antiviral effect is good, which provides new ideas for the treatment of HIV in the future.

4. Test conclusion

(1) The cells of the 3-caffeoylquinic acid derivative drug group grew well, the cell survival rate was higher than that of the virus control group, it had a certain antiviral effect, and had no obvious toxic and side effects on normal cells.

(2) 3-Caffeoylquinic acid derivatives have obvious inhibitory effect on HIV-1ⅢB virus strain, and the inhibitory effect is obvious at 5mg / ml in this system.

In summary, the 3-caffeoylquinic acid derivatives of the present invention have good anti-tumor and anti-viral effects, especially for ovarian borderline tumors in borderline tumors, lung cancer, liver cancer, kidney cancer, breast in malignant tumors Cancer, cervical cancer, prostate cancer, and HIV in immunodeficiency virus have a good inhibitory effect. In addition, the 3-caffeoylquinic acid derivatives of the present invention also have antibacterial, anti-inflammatory and anti-oxidant effects. The 3-caffeoylquinic acid derivatives of the present invention can be used to prepare anti-tumor and anti-virus, especially the aforementioned specific tumor and virus drugs, and can also be used to prepare anti-bacterial anti-inflammatory and anti-oxidation drugs.

Claims

A compound of formula I:
A method for preparing a compound according to claim 1, characterized in that it includes the following steps:

(1) Dissolve 3-caffeoylquinic acid and D-mannitol in an organic solvent, and react under an acidic system to obtain a reaction solution;

(2) The compound reaction solution is prepared by precipitation, adsorption analysis, washing, crystallization and recrystallization, and freeze-drying.
The preparation method according to claim 2, characterized in that:

In step (1), the molar ratio of 3-caffeoylquinic acid to D-mannitol is (1: 1) to (5: 1);

And / or, in step (1), the acidic system is a p-toluenesulfonic acid, hydrochloric acid or sulfuric acid system;

And / or, in step (1), the reaction temperature is 20-60 ° C., and the reaction time is 2-24 h;

And / or, in step (2), the chromatography column used for the adsorption analysis is a non-polar or weakly polar macroporous adsorption resin chromatography column;

And / or, in step (2), the washing is washing with an organic solvent.
The preparation method according to claim 3, characterized in that:

In step (1), the molar ratio of 3-caffeoylquinic acid to D-mannitol is (1: 1) to (3: 1);

And / or, in step (1), the acidic system is a p-toluenesulfonic acid, hydrochloric acid or concentrated sulfuric acid system;

And / or, in step (1), the reaction temperature is 20-60 ° C., and the reaction time is 24 h;

And / or, in step (1), the organic solvent is N, N-dimethylformamide or absolute ethanol;

And / or, in step (2), the model of the macroporous adsorption resin chromatography column is HPD-100, HPD-450 or LS-46;

And / or, in step (2), the chromatography column is eluted with 5% to 80% ethanol after the chromatography column is completed, the eluent is collected, concentrated to dryness, and dissolved in water with saturation;

And / or, in step (2), the organic solvent used for the washing is ethyl acetate;

And / or, in step (2), the crystallization is refrigerated crystallization;

And / or, in step (2), the recrystallization is recrystallization with water.
Use of the compound of claim 1 in the preparation of anti-tumor drugs.
The use according to claim 5, characterized in that the tumor is a borderline tumor and / or a malignant tumor;

Preferably, the borderline tumor is a borderline tumor of the ovary; and / or, the malignant tumor is lung cancer, liver cancer, kidney cancer, breast cancer, cervical cancer, and prostate cancer.
The use of the compound according to claim 1 in the preparation of antiviral drugs; preferably, the virus is an immunodeficiency virus; more preferably, the immunodeficiency virus is AIDS virus.
Use of the compound according to claim 1 in the preparation of antibacterial and anti-inflammatory drugs.
Use of the compound according to claim 1 in the preparation of anti-oxidation drugs.
A medicine, characterized in that it is a preparation prepared by using the compound as claimed in claim 1 as an active ingredient, together with commonly used pharmaceutical excipients or auxiliary ingredients.