CN113637045A - Protopanaxadiol derivative and preparation method and application thereof - Google Patents

Abstract

The invention discloses a protopanoxadiol derivative and a preparation method thereof, and application of the protopanoxadiol derivative as a medicament for treating cancers, and treatment application of the protopanoxadiol derivative and a PD-L1/PD-L2 antibody combination in combination, wherein the treatment effect on the anti-tumor treatment of the lung cancer can be improved, and meanwhile, side effects are reduced. By the combined medication, cancer can be effectively treated.

Description

Protopanaxadiol derivative and preparation method and application thereof

Technical Field

The invention relates to a protopanoxadiol derivative, a preparation method and application thereof, and application of the protopanoxadiol derivative and a PD-L1/PD-L2 antibody in preparation of a medicament for treating cancer.

Background

Programmed death receptor-1 (PD-1) is a transmembrane receptor on T cells and was first obtained using subtractive methods in apoptotic T cell hybridomas. It is named because it is involved in apoptosis. PD-1 can interact with ligands PD-L1 and PD-L2 to inhibit T cell proliferation. PD-L1 and PD-L2 are mainly expressed on antigen presenting cells (such as DC cells and macrophages). Although the monoclonal antibody of PD-L1 and PD-1 has achieved unusual performance in clinical application, the response rate of cancer patients to the monoclonal antibody of PD-L1 and PD-1 is only about 20-30% so far. The search for a drug combination for improving the clinical response rate of the monoclonal antibody of PD-L1 and PD-1 becomes important in the research of the current cancer drugs.

Ginseng is the most important 'qi-tonifying medicine' in traditional Chinese medicines, and the main active ingredient of the medicine is ginsenoside. Ginsenosides are classified into diol saponins and triol saponins according to differences in aglycone structures. Oral administration of panaxadiol saponins mainly comprises blood components and main active metabolite Rh 2; compound K and protopanaxadiol have many reports on the anticancer effect of protopanaxadiol, and Chinese patent CN1418633A discloses an anticancer auxiliary drug containing 20(S) -protopanaxadiol as effective component and its application. Chinese patent CN102366417A discloses the possibility of protopanoxadiol as anticancer drug, and according to the patent data, the 50% growth inhibitory concentration IC50 value of protopanoxadiol on different cell strains is between 3.34-918 μ M. It can be seen from the prior art that the structure of protopanaxadiol and the derivative thereof is further researched on the basis of the protopanaxadiol and the derivative thereof, so that a more effective anticancer product is developed, and the method is the key point for researching and developing related products at the present stage.

Disclosure of Invention

Aiming at the key points of the research and development of related PD-1, protopanaxadiol and derivatives thereof at the present stage, the invention further researches the structure of the protopanaxadiol and discovers a more effective anticancer compound, namely a novel derivative of the protopanaxadiol.

The protopanoxadiol derivative has the general formulas of formula I and II:

in the formula I, R₁H or Val; r₂H or Val, wherein Val represents valine.

The preparation method of the protopanoxadiol derivative comprises the following steps:

taking protopanoxadiol, DMAP, EDCI and Boc protected valine according to an equivalent ratio of 1: 3: 4: 4, the solvent is pyridine, the mixture is stirred for 10 hours at the temperature of 40 ℃, then the reaction liquid is concentrated to remove part of the pyridine, ethyl acetate is added into the reaction liquid, then the reaction liquid is washed twice by 10% hydrochloric acid, twice by saturated sodium carbonate solution and once by saturated salt water solution, an organic layer is dried and then concentrated, and the Boc-protected protopanaxadiol valine ester is obtained.

b, mixing the product obtained in the previous step and methanesulfonic acid according to an equivalent ratio of 1: 1.8, reacting, wherein the solvent is a mixed solvent of toluene and isopropanol, stirring for 8 hours at 50 ℃, then adding ethyl acetate, washing with saturated sodium bicarbonate solution for three times, washing with saturated salt solution for one time, drying an organic layer, concentrating to obtain viscous light yellow liquid, performing silica gel column chromatography, and eluting with ethyl acetate/petroleum ether (16-50%) to obtain 3-Val-PPD and 3-Val-PPD protopanoxadiol valine derivatives dehydrated at the C-20 position.

And C, dissolving the purified 3-Val-PPD and the 3-Val-PPD protopanoxadiol valine derivative dehydrated at the C-20 position into ethyl acetate, adding an HCl ethyl acetate solution, precipitating, and performing suction filtration to obtain the protopanaxadiol valine derivative hydrochloride.

The invention discloses a protopanoxadiol derivative which can improve the tumor inhibition effect of a monoclonal antibody PD-1/PDL-1.

The invention also discovers that the protopanoxadiol derivative has obvious tumor inhibition effect in mice, and the tumor inhibition effect is obviously increased when the protopanoxadiol derivative is combined with PD1/PDL 1.

The invention uses the combination of the protopanoxadiol derivative and PD1/PDL1 and uses an animal non-small cell lung cancer model to verify the anticancer effect of the protopanoxadiol derivative. The dosage of protopanaxadiol derivative is 50mg/kg, and the dosage of PDL1/PDL2 is 10 mg/kg.

The invention provides a preparation method of protopanoxadiol derivatives and application thereof in preparing anti-cancer drugs.

Drawings

FIG. 1 is a nuclear magnetic resonance hydrogen spectrum of 3-Val-PPD (solvent: CD)₃Cl₃)。

FIG. 2 is a mass spectrum (ESI-MS) of 3-Val-PPD.

FIG. 3 is a mass spectrum (ESI-MS) of C20-dehydrated 3-Val-PPD.

FIG. 4 is a mass spectrum (ESI-MS) of 3, 12-Val-PPD.

FIG. 5 is anatomical tumor size and weight after sacrifice

FIG. 6 is a lifetime of groups

Detailed Description

The following examples are intended to illustrate the invention, but not to limit the substance of the invention.

Example 1 preparation of Boc protected Valinic acid protopanoxadiol ester

1.89g of Boc-protected valine was dissolved in 40mL of Py, and 1.66g of EDCI was added thereto, followed by activation at room temperature for half an hour, followed by addition of 0.80g of DMAP and 1g of protopanoxadiol in this order, and after completion of the addition, the reaction was carried out by heating to 40 ℃. After 10 hours of reaction, the reaction mixture was concentrated under reduced pressure to remove part of the pyridine, 50mL of EA was added to the remaining viscous substance, and then washed with 50mL of 10% diluted HCl for 2 times, 50mL of saturated Na₂CO₃The solution was washed 2 times, once with 40mL of saturated brine, and the organic layer was dried over anhydrous sodium sulfate. Suction filtration and concentration under reduced pressure gave 1.8g of a yellow solid.

Example 2 preparation of Val-valinate Protopanaxadiol

Dissolving 0.8g of the solid obtained in the previous step in a mixed solvent of 6mL of toluene and 4.5mL of isopropanol, replacing argon for protection, cooling to-10 ℃, dropwise adding 0.2mL of methanesulfonic acid, and reacting at 50 ℃ for eight hoursThereafter, 10mL of EA was added, followed by washing three times with 10mL of a saturated sodium carbonate solution, once with 10mL of a saturated saline solution, and the organic layer was dried over anhydrous sodium sulfate. Vacuum filtering, concentrating under reduced pressure to obtain crude product 0.6g, performing silica gel column chromatography, eluting with ethyl acetate/petroleum ether (16% -50%) to obtain 3-Val-PPD protopanaxadiol valine derivative 0.3g with yield of 44%, and adding C₂₀0.35g of 3-Val-PPD protopanaxadiol valine derivative which is dehydrated at the position. The yield thereof was found to be 50%.

EXAMPLE 3 preparation of Valinic acid Protopanaxadiol hydrochloride

0.3g of purified 3-Val-PPD and C₂₀Dissolving 0.35g 3-Val-PPD protopanaxadiol valine derivative in ethyl acetate, adding HCl ethyl acetate solution, precipitating, and vacuum filtering to obtain corresponding protopanaxadiol valine derivative hydrochloride 0.3g and C₂₀0.35g of a valine derivative of protopanaxadiol dehydrated at the position.

The hydrogen spectrum and mass spectrum data of 3-Val-PPD are as follows:

¹H-NMR(600MHz,CDCl₃):δ5.17(t,J＝7.2Hz,1H),4.53(dd,J₁＝11.6Hz,J₂＝4.9Hz,1H),3.60(td,J₁＝10.4Hz,J₂＝5.2Hz,1H),3.33-3.26(m,1H),2.17(s,1H),2.14-1.99(m,4H),1.91-1.80(m,2H),1.70(s,3H),1.64(s,3H),1.58-1.38(m,7H),1.33-1.24(m,6H),1.20(s,3H),1.16-1.06(m,3H),1.03-0.98(m,6H),0.92-0.86(m,15H).ESI-MS(m/z):560.5[M+H]⁺.

C₂₀-dehydrated 3-Val-PPD：ESI-MS(m/z):542.4[M+H]⁺.

3,12-diVal-PPD：ESI-MS(m/z):659.6[M+H]⁺.

effect example 1 anticancer Effect of protopanoxadiol and its derivatives

1.1 materials

1.1.1 cell culture

Lewis murine lung adenocarcinoma cells (LLC) were purchased from the Cell Culture center of the Chinese Academy of medicine Sciences (CAMS; the republic of China, Beijing). 90% DMEM medium (Dulbecco's modified Eagle's medium, Gibco BRL, USA), 10% FBS fetal bovine serum (total bone serum, Gibco BRL, USA), in the presence of 5% CO2, 95% O2 constant temperature and humidity 37 deg.C incubator. The culture medium is replaced once every 2 to 3 days or whether to passage is judged according to the cell density.

1.1.2 Experimental animals

Healthy SPF-grade female C57BL/6 mice, 50 mice, 6 weeks old, were purchased from the Institute of laboratory animal Science (CAMS) of the national people's republic, Beijing. The animals were fed sterile water and Beijing KeAoXieli rodent chow (qualified) free (ad libitum). All animals were acclimated for one week prior to starting the experiment.

1.2 methods

1.2.1 tumor cell inoculation

C57BL/6 mice were acclimatized for 1 week and LLC cells were abundant before being used for inoculation and molding. 100ul of 5X 10^5 LLC cells per ml were injected subcutaneously into the right axilla of mice, the day of inoculation being day 0.

1.2.2 Experimental groups and dosing

The successfully inoculated 50C 57BL/6 mice were randomly divided into 5 groups of 10 mice each, numbered by toe clipping. After grouping, each group was fed normally every day. Experimental animals were grouped as follows:

(1) model group (group 4): 10mg IgG antibody was dissolved in 10ml buffer, i.e., 1000ug/ml, and each mouse was injected intraperitoneally with 0.2ml (200ug), and treated on days 1,3,6,8, and 11 with 0.2ml IgG solution.

(2) PD-L1/PD-L2 antibody group (group 3): 50mg of PD-L1 antibody (murine PD-L1 antibody, bisoxcell) was dissolved in 50ml buffer (1000 ug/ml), 0.2ml (200ug) was intraperitoneally injected into each mouse, and 0.2ml of PD-L1/PD-L2 solution was intraperitoneally injected on days 1,3,6,8, and 11.

(3) Protopanoxadiol derivative (protopanaxadiol valine ester hydrochloride) single drug group (group 2): the treatment is carried out on the 1 st to 12 th days by intraperitoneal injection of 200ul every day.

(4) PD-L1/PD-L2+ protopanaxadiol derivative (protopanaxadiol valine ester hydrochloride) combined administration group (group 1): mice were dosed simultaneously with the two groups of doses as described above.

1.2.3 tumor size and weight assessment

From the day of administration, the mice were weighed once a day, and the tumor size of the test animals was measured by measuring the major and minor diameters of the tumor with a vernier caliper, and calculated by the following formula.

Tumor size long diameter short diameter 2

1.2.4 sacrifice after completion of dosing and dissect the tumor for weighing statistics.

1.3 data processing

1.3.1 dissect tumor size after sacrifice, statistics for tumor weight raw data are as follows:

FIG. 5 shows the tumor size and weight of mice.

1.3.2 groups of lifetimes are shown in FIG. 6.

1.4 results of the experiment

1.4.1 compared with the model group, the protopanaxadiol derivative (10 mg/kg/mouse) has obvious inhibition effect on non-small cell lung adenocarcinoma tumors of C57BL/6J mice.

1.4.2 compared with the model group, the combined administration group of the protopanoxadiol derivative (10 mg/kg/mouse) and PD-L1/PD-L2 has more significance on the size of the mouse tumor, and the significant inhibition effect of the tumor of the combined group is confirmed.

From the above findings, when the PD-L1/PD-L2 antibody was administered, by using the protopanoxadiol derivative in combination, a significant inhibitory effect of C57BL/6J mouse tumor was confirmed by a synergistic effect of the two.

Claims (9)

1. Derivatives of protopanoxadiol having the general formulae i and ii:

in the formula R₁H or Val; r₂H or Val, wherein Val represents valine.

2. A compound according to claim 1, R₁Is Val; r₂Is Val.

3. A process for the preparation of a compound according to claim 2, characterized in that: mixing and stirring protopanoxadiol, DMAP, EDCI and Boc protected valine, then concentrating a reaction solution, adding ethyl acetate into the reaction solution, carrying out acid washing, alkali washing and concentration, reacting an obtained product with methanesulfonic acid in a mixed solvent, then adding ethyl acetate, carrying out alkali washing, drying, concentrating, carrying out silica gel column chromatography, and eluting ethyl acetate/petroleum ether to obtain the compound.

4. A pharmaceutical combination of the protopanaxadiol derivative of claim 1 and a PD-L1/PD-L2 antibody for the treatment of cancer, comprising administering to the subject: a) a therapeutically effective amount of at least one inhibitor of an immune checkpoint, b) a therapeutically effective amount of the protopanaxadiol derivative of claim 1; wherein the disease, disorder or condition is lung cancer or other various benign or malignant tumors that preferably express PD-L1 and/or PD-L2.

5. A composition according to claim 4, wherein said protopanaxadiol derivative is a valine ester of protopanaxadiol.

6. The composition according to claim 5, wherein the protopanoxadiol derivative is contained in an amount of 50 mg/unit.

7. The composition of claim 6, wherein the protopanaxadiol derivative and the PD-L1/PD-L2 antibody are administered in a ratio of 5: 1.

8. A pharmaceutical composition comprising

a) A therapeutically effective amount of an immune antibody according to any one of claims 1-7 and a pharmaceutically acceptable diluent, carrier or excipient; b) a therapeutically effective amount of a protopanaxadiol derivative drug according to any one of claims 1-7, and a pharmaceutically acceptable diluent, carrier or derivative.

9. The pharmaceutical composition of claim 8, wherein the excipient is an isotonic injection solution.

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