CN111675747B

CN111675747B - Antitumor drug and application

Info

Publication number: CN111675747B
Application number: CN202010750818.0A
Authority: CN
Inventors: 邓洪斌; 刘晓嘉; 冯艳春; 张娜; 宋丹青; 殷明晓; 董靖雯; 曾庆轩
Original assignee: Institute of Medicinal Biotechnology of CAMS
Current assignee: Institute of Medicinal Biotechnology of CAMS
Priority date: 2020-07-30
Filing date: 2020-07-30
Publication date: 2021-04-27
Anticipated expiration: 2040-07-30
Also published as: CN111675747A

Abstract

The invention relates to an anti-tumor medicament and application, in particular to application of a tubeimoside derivative and new application thereof, and particularly relates to application of the tubeimoside derivative shown as a formula I, an optical isomer, a solvate or a pharmaceutically acceptable salt thereof in preparing a medicament for treating tumors, wherein the tubeimoside derivative has a structure shown in the specification, and the compound treats the tumors by reducing PD-L1 on the surface of tumor cells, can enhance the killing activity of immune cells, and has good in-vivo activity and low toxicity.

Description

Antitumor drug and application

Technical Field

The invention belongs to the technical field of medicines, relates to a new application of a tubeimoside B derivative, and particularly relates to the tubeimoside B derivative shown as a compound in a formula I, an optical isomer, a solvate or a pharmaceutically acceptable salt thereof. The invention also relates to the application of the compound in the formula I in preparing the medicine for treating tumors. The bolbostemma glucoside B derivative of the compound of the formula I has a structure shown in the specification, treats tumors by reducing PD-L1 on the surface of tumor cells, can enhance the killing activity of immune cells, and has good in-vivo activity and low toxicity.

Background

Immune checkpoints (Immune checkpoints) refer to some inhibitory signaling molecules present in the body's Immune system to avoid self-tissue damage by modulating the intensity and persistence of the Immune response in peripheral tissues. Immune checkpoint blockers (ICIs) can block the interaction between Immune checkpoints, break the Immune escape mechanism of tumor cells, activate T cells and kill tumor cells, and simultaneously can play an anti-tumor role through ectopic effect. Unlike chemotherapy or targeted therapy, ICIs can induce a sustained immune response in a proportion of patients, and can produce a long-lasting tumor-specific immunological memory even after cessation of therapy. Therefore, immune checkpoint blockade therapy has become a new and effective treatment modality for tumors.

PD-L1 (also named B7-H1), which is an important member of the immunoglobulin superfamily costimulatory molecule, is widely and highly expressed on the surface of various malignant tumors such as melanoma, breast cancer, lung cancer and renal small cell carcinoma cells. The high expression of PD-L1 is closely related to the proliferation and killing activity of T cells. In addition, the interaction of PD-1/PD-L1 can induce antigen-specific T cell apoptosis and promote CD4⁺T cells to Foxp3⁺Regulatory T cells differentiate, leading to the formation of an immunosuppressive tumor microenvironment, allowing tumor cells to escape the body's immune surveillance and killing. Therefore, the PD-1/PD-L1 signaling pathway has become an effective target for immunotherapy of malignant tumors.

The rhizoma bolbostemmae is a traditional Chinese medicine, has long medicinal history, is bitter in taste and cool in nature, and has pharmacological effects of clearing heat and removing toxicity, resisting inflammation and swelling, resisting tumors and the like. The saponin is the main active component in rhizoma Bolbostematis, including Tubeimoside A, Tubeimoside B and Tubeimoside C, wherein Tubeimoside B (TBM-II) is the active component with high yield, good water solubility and stability in rhizoma Bolbostemmatis.

Tubeimoside B was reported as early as 1988 (Orthogalus paniculatus, et al, research on chemical composition of tubeimoside B, C, D, structure of tubeimoside B, C, 04 of 1988), which is white powder with molecular formula C₆₃H₉₈O₃₀，[α]_D6.0(c0.32, pyridine), M_r1334(SIMS：m/z，1335[M+H]⁺). The aglycone is polygalacic acid, and the sugar is glucose, rhamnose, arabinose and xylose with the molar ratio of 1:1:2: 1.1. Method for preparing tubeimoside B¹³The C NMR spectrum showed five sugar terminal carbon signals (. delta.106.4, 104.8, 103.2, 100.8 and 94.3ppm) and the characteristic signals for the 3-methyl-3-hydroxyglutaric acid residue (. delta.171.3, 171.2, 70.2, 47.1, 46.8 and 26.2 ppm). The CAS No.115810-12-3 of tubeimoside B has the following chemical structural formula:

the bolbostemma paniculatum glucoside B contains a unique pentacyclic triterpenoid structure, has obvious inhibition effect on various tumors in vivo and in vitro, but is limited from further clinical application due to side effects such as nausea, vomiting, gastrointestinal reaction and the like.

Therefore, there is still a need in the art for new methods for treating tumors, for example, by structurally modifying tubeimoside b to improve its anti-tumor activity and/or reduce its toxic side effects.

Disclosure of Invention

The invention aims to provide a novel method for treating tumors, for example, by structurally modifying tubeimoside B to improve the antitumor activity and/or reduce the toxic and side effects of the tubeimoside B. It has been surprisingly found that the tubeimoside B derivatives provided by the present invention exhibit one or more unexpected technical effects, and the present invention has been completed based on such findings.

To this end, a first aspect of the invention provides a compound of formula I:

or a pharmaceutically acceptable salt, optical isomer, solvate thereof,

wherein:

r1 is a halogen atom or a halogen atom,

r2 is C_1-6An alkyl group, a carboxyl group,

r3 is phenyl optionally substituted with 1 to 4 groups selected from: hydrogen, halogen, hydroxy, trifluoromethoxy, trifluoromethyl, amino, nitro, C_1-6Alkyl radical, C_1-6An alkoxy group.

A compound according to the first aspect of the invention, wherein the halogen is selected from: fluorine, chlorine, bromine.

A compound according to the first aspect of the present invention, wherein said C_1-6The alkyl group is selected from: methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl.

A compound according to the first aspect of the present invention, wherein said C_1-6Alkoxy is selected from: methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, tert-butoxy.

A compound according to the first aspect of the present invention, wherein R3 is hydrogen or 1 to 3 groups selected from: halogen, hydroxy, trifluoromethoxy, trifluoromethyl, amino, nitro, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, tert-butoxy.

A compound according to the first aspect of the invention, which is a compound selected from the group consisting of:

or a pharmaceutically acceptable salt, optical isomer, solvate thereof.

Further, the second aspect of the present invention provides a process for preparing a compound of formula I, or a pharmaceutically acceptable salt thereof, comprising the steps of:

step (1): dissolving TBM-II in DMF, adding NBS, then adding catalytic amount of AIBN, reacting at room temperature, after the reaction is finished, adding 20ml of water and 30ml of ethyl acetate for treating reaction, collecting a water phase, concentrating and drying to obtain a solid TBM-II-1;

step (2): dissolving TBM-II-1 in DMF, adding imidazole and TIPDSCl under ice bath₂Reacting at room temperature, adding TBSOTf and imidazole into the system after the reaction is finished, reacting at room temperature, treating the reaction with ethyl acetate and water after the reaction is finished, and drying and concentrating the organic phase to obtain a solid TBM-II-2;

and (3): dissolving TBM-II-2 in ACOH: water: stirring at room temperature in a THF mixed solution, treating the reaction solution with ethyl acetate and water after the reaction is finished, concentrating an organic phase, and drying to obtain a solid TBM-II-3;

and (4): dissolving TBM-II-3 in DMF, adding phenylphosphoryl dichloride, tetrazole and DIPEA in an ice bath, adding alanine methyl ester after the reaction is complete, continuing the reaction, monitoring the reaction by TLC, adding TBAF into the system after the reaction is finished, reacting at room temperature, adding water for cataloguing reaction after the reaction is finished, and purifying the residue by a silica gel column to obtain solid TBM-19.

A method according to a second aspect of the invention having the procedure as described in example 1.

Further, in a third aspect, the present invention provides the use of a compound according to any one of the first aspect of the present invention or a compound prepared by the method according to any one of the second aspect of the present invention in the manufacture of a medicament for the treatment of a tumour. For example, the cancer is, but not limited to, lung cancer (e.g., non-small cell lung cancer), pancreatic cancer, ovarian cancer, bladder cancer, breast cancer.

Further, the fourth aspect of the present invention provides an injection solution comprising: TBM-19 compound 10mg, 50mg polyethylene glycol 200, tartaric acid 20mg, and water in appropriate amount to 1 ml.

Further, the injection is prepared by the following method: adding TBM-19 compound, polyethylene glycol and tartaric acid into 80% water, stirring to dissolve, adjusting pH to 6.8 with 1M hydrochloric acid or 1M sodium hydroxide, adding water to full dose, filtering, packaging, and sterilizing to obtain injection.

Any technical feature possessed by any one aspect of the invention or any embodiment of that aspect is equally applicable to any other embodiment or any embodiment of any other aspect, so long as they are not mutually inconsistent, although appropriate modifications to the respective features may be made as necessary when applicable to each other. Various aspects and features of the disclosure are described further below.

All documents cited herein are incorporated by reference in their entirety and to the extent such documents do not conform to the meaning of the present invention, the present invention shall control. Further, the various terms and phrases used herein have the ordinary meaning as is known to those skilled in the art, and even though such terms and phrases are intended to be described or explained in greater detail herein, reference is made to the term and phrase as being inconsistent with the known meaning and meaning as is accorded to such meaning throughout this disclosure.

Paniculate Bolbostemma rhizome (Tubeiimu) is a dried tuber of a plant of the genus Pseudobulbus of the family Cucurbitaceae, is a traditional Chinese medicine and is recorded in Bencao gang mu Shi (supplement to compendium of materia Medica) edited by the Qing Dynasty Zhao Zhi Ming. Its tuber is used to cure mammary abscess, hyperplasia of mammary glands, scrofula and other diseases, and clinically, it has obvious effect of dispelling toxin and eliminating carbuncle and swelling. The tubeimoside B is a monomer effective component separated from traditional Chinese medicine tubeimoside B. The tubeimoside B has good water solubility and strong stability as the tubeimoside A.

It is known that tubeimoside B has obvious inhibition effect on human tumor cells in vitro, and various human malignant tumor cell lines, such as pancreatic cancer (PANC-I), gastric cancer (HCG-27), colon cancer (COLO320DM), cervical cancer (HeLacells), neuroblastoma (GOTO), glioblastoma (A-127), promyelocytic leukemia (HL-60) and low-differentiation nasopharyngeal carcinoma (CNE-2Z) are all sensitive to glucoside A and glucoside B, particularly to cells such as A-127, GOTO, HL-60 and the like, wherein the GOTO and HL-60 cells can also be induced to differentiate. In addition, it is reported that tubeimoside B also has obvious inhibition effect on the growth of transplantable tumor of mice, tubeimoside A and tubeimoside B have obvious inhibition effect on the growth of transplantable tumor Sl80, H22 and ehrlich ascites carcinoma, and when the dosage of tubeimoside A is 4 mg.kg-1.d-1 (im, 19d), the tumor inhibition rate reaches 59.0 percent; the tubeimoside B dose is 4 mg/kg-1/d-1 (ip, 16d), the tumor inhibition rate reaches 71.5 percent, and the effect is higher than that of positive control drugs of fluorouracil and cyclophosphamide. The result of the quantitative structure-activity relationship research shows that the antitumor activity of the tubeimoside B is superior to that of the tubeimoside A. In addition, it is reported that tubeimoside B also has the effect of anti-cancer promotion, and tubeimoside A and tubeimoside B both have obvious anti-cancer effect, but the effect of tubeimoside B is superior to that of tubeimoside A. The local dosage of the glucoside A shows obvious inhibition effect on the mouse ear edema induced by Arachidonic Acid (AA) and 12-O-tetradecylphenol fubo-13-acetate (TPA) at 100 mu g and 50 mu g per ear in dose-effect dependence; the glucoside A has the function of inhibiting TPA from enhancing 3H to be doped into C3H10Tl2 clone 8 cell phospholipid, the effect can be still detected when the concentration of the glucoside A is as low as 5 mu g.mL < -1 >, and the obvious dose-effect dependence relationship is realized; topical application of the glycoside A completely inhibited the development of Dimethylbenzanthracene (DMBA) -stimulated, TPA-promoted mouse dermal papillomas. The addition of glycoside A and glycoside B in the drinking water can reduce the occurrence of skin tumor of mice, but the glycoside C applied through gastrointestinal tract can promote the occurrence of skin tumor of mice. It is known that tubeimoside B or tubeimoside containing tubeimoside B can be used for preparing medicines for resisting angiogenesis, tumor invasion and metastasis. The bolbostemma paniculatum rhizoma et radix calamitis obviously inhibits the proliferation of human umbilical vein endothelial cells ECV-304 and promotes the apoptosis; the tubeimoside B obviously inhibits the angiogenesis of chick embryo chorioallantoic membrane and tumor cells induced by chick embryo chorioallantoic membrane; the tubeimoside B obviously inhibits the generation of tumor tissue micro-vessels; the tubeimoside B obviously inhibits the expression of Vascular Endothelial Growth Factor (VEGF) and basic fibroblast factor (bFGF) of tumor tissues; the tubeimoside B obviously inhibits the experimental transfer of mouse B16 melanoma cells and the spontaneous transfer of Lewis lung cancer cells; the bolbostemoside B induces the expression of transfer promotion genes CD44v6 and ErBb-2 in Lewis lung cancer tissues to be down-regulated, and inhibits the expression of transfer genes nm23-H1 to be up-regulated. The tubeimoside B or tubeimoside containing tubeimoside B can be used for preparing drugs for resisting angiogenesis, tumor invasion and metastasis. The bolbostemma glucoside B can be used for preparing medicinal bait for preventing and treating human and animal viral diseases. The tubeimoside B and tubeimoside inhibit the replication of human and animal viruses, have the effects of preventing and treating human and animal viral diseases, and can be used for preventing and treating various human and animal viral diseases. The tubeimoside B and the saponin show special anti-inflammatory action, can obviously inhibit the growth of human malignant tumor cells and mouse transplantation tumors, prolong the life cycle of tumor-bearing mice, have the effect of inducing differentiation on HL-60 cells, and can resist the promotion action of cancer promoters on the generation of mouse skin tumors.

According to the invention, the derivative obtained by carrying out structural modification on the tubeimoside B has one or more effects.

Drawings

FIG. 1: TBM-19 down-regulates the expression of PD-L1 in tumor cells. After human lung cancer H460 and H157 cells are treated by TBM-19 with different concentrations for 24 hours, western blot detects the change of the protein level of PD-L1 in the cells.

FIG. 2: TBM-19 induced a decrease in the expression of PD-L1 on the surface of tumor cells. (A) After H460 cells were treated for 24H with TBM-19 (5. mu.M), changes in the expression of PD-L1 protein on the cell membrane surface were detected by flow cytometry. (B) After treating H460 cells for 24H for TBM-19 (5. mu.M), the change in binding between H460 cells and PD-1-Fc was observed by fluorescence microscopy.

FIG. 3: TBM-19 enhances the killing of tumor cells by T cells. (A) Schematic diagram of co-culture of T cells and tumor cells. (B) Human T cells were co-cultured with H460 cells treated with DMSO or TBM-19 (5. mu.M), and the change in the mortality of H460 cells was detected by the LDH method. (C) Human T cells were co-cultured with H460 cells treated with DMSO or TBM-19 (5. mu.M), and the change in the mortality of H460 cells was detected by cell impedance assay.

FIG. 4: TBM-19 inhibitorAnd (5) preparing Lewis lung cancer transplantation tumor growth. (A) In vivo Lewis lung carcinoma transplanted tumor mice of TBM-19 are shown in the figure, and five C57 mice are inoculated to each of the control group and the experimental group, and each is inoculated with 2.5 multiplied by 10⁶Tumor cells, administered intraperitoneally beginning on day 3. (B) TBM-19 can effectively inhibit the growth of Lewis transplanted tumor. (C) TBM-19 significantly inhibited the volume of Lewis-transplanted tumors. (D) TBM-19 significantly inhibited the tumor weight of Lewis transplanted tumors. (E) The body weight of the mice changed during the administration of TBM-19.

FIG. 5: TBM-19 increases tumor infiltrating lymphocytes and activates T cells. After tumor tissues of mice are treated by collaenase IV and DNase 1 and digested into single cells, (A) is a method for detecting immune cell proportion and T cell activation after administration by flow cytometry. (B) Detection of immune cells in tumor tissue for flow cytometry (CD 45)^﹢) The fractional situation of (c). (C) Detection of tumor infiltrating T cells (IFN-gamma) for flow cytometry^﹢CD8^﹢CD3^﹢CD45^﹢) The activation condition of (1).

Detailed Description

The present invention will be further described by the following examples, however, the scope of the present invention is not limited to the following examples. It will be understood by those skilled in the art that various changes and modifications may be made to the invention without departing from the spirit and scope of the invention. The present invention has been described generally and/or specifically with respect to materials used in testing and testing methods. Although many materials and methods of operation are known in the art for the purpose of carrying out the invention, the invention is nevertheless described herein in as detail as possible.

Example 1: preparation of TBM-19(R1 ═ bromo, R2 ═ methyl, R3 ═ phenyl)

An exemplary reaction scheme is as follows:

step (1): TBM-II (2g, 1.5mmol) was dissolved in 10mL DMF and NBS (0.3g, 1.65mmol) was added at 0 deg.C followed by catalytic amount of AIBN and reacted at room temperature, monitored by TLC (chloroform: methanol: water ═ 6: 4: 1), and upon completion of the reaction, 20mL of water and 30mL of ethyl acetate were added to treat the reaction, the aqueous phase was collected, concentrated and lyophilized to give 1.8g of TBM-II-1 (MS: M/z 1335[ M: M/z 1335 ] as a yellow solid⁺+Na])。

Step (2): TBM-II-1 (1.6g, 1.13mmol) was dissolved in 25ml DMF and imidazole (0.3g, 4.5mmol), TIPDSCl, was added under ice-bath₂(0.4g and 1.24mmol), reacting at room temperature, monitoring the reaction by TLC, adding TBSOTf (4.5g and 17mmol) and imidazole (1.23g and 18mmol) into the system after the reaction is finished, reacting at room temperature, monitoring the reaction by TLC, treating the reaction with ethyl acetate and water after the reaction is finished, and concentrating the organic phase by drying to obtain 2.5g of similar white solid TBM-II-2;

and (3): TBM-II-2 (2.5g, 0.8mmol) was dissolved in 15ml (ACOH: water: THF 4: 4: 7) solution, stirred at room temperature, monitored by TLC, after the reaction was complete, treated with ethyl acetate and water, and the organic phase was concentrated and dried to give 2.1g of off-white solid TBM-II-3.

And (4): dissolving TBM-II-3 (2g and 0.6mmol) in 10ml DMF, adding phenylphosphoryl dichloride (0.17g and 0.86mmol), tetrazole (18 mg) and DIPEA (0.22g and 0.2mmol) in ice bath, adding alanine methyl ester (0.1g and 0.1mmol) after the reaction is completed, continuing the reaction, monitoring the reaction by TLC, adding TBAF (2g and 7.6mmol) into the system after the reaction is finished, reacting at room temperature, monitoring the reaction by TLC, adding water to freeze-dry the reaction system after the reaction is finished, purifying the residue by a silica gel column to obtain TBM-19 which is a white-like solid (0.7 g and C)₇₃H₁₀₉BrNO₃₃P，MS：m/z 1660[M⁺+Na]))。¹H NMR(500MHz,Pyridiene-D₅/D₂O):δ7.24-7.31(m,5H)，6.09(s,1H),6.01(d,1H),6.00(br,1H),5.48(br,1H),5.17(br,1H),5.14(d,1H),5.08(d,1H),5.01(d,1H),4.82(dd,1H),4.74(m,1H),4.63-4.60(m,2H),4.55(dd,1H),4.50-4.42(m,6H),4.36-4.21(m,8H),4.17-4.00(m,4H),3.93-3.85(m,3H),3.75(m,2H),3.65(s,3H),3.56(m,2H),3.36(d,1H),3.33(m,2H),3.23(d,1H),3.15-3.12(m,5H),3.04(d,1H),2.05(s,3H),1.66(s,3H),1.60-1.57(m,6H)1.54(s,3H),1.53(d,3H),1.45(m,4H)1.34(s,3H),1.28(s,3H),1.25(s,3H),0.95(m,1H)0.93(s,3H),0.87(s,3H)。¹³CNMR(125MHz,Pyridiene-D₅):δ176.3,171.5,171.2,170.8,144.3,134.2,131.1,131.1,128.7,128.7,123.3,106.2,105.6,105.2,102.1,94.5,83.2,79.0,77.9,77.6,77.6,77.3,76.7,75.3,75.0,74.9,73.5,73.3,73.2,72.0,71.7,71.5,70.9,70.7,70.2,68.8,67.9,66.7,66.5,64.6,62.6,62.4,52.3,51.9,49.6,48.4,47.6,47.5,47.1,46.9,44.1,42.7,42.3,41.5,39.7,36.9,34.1,33.3,33.0,32.1,30.7,28.9,26.0,25.7,23.9,23.4,22.8,19.1,18.7,18.4,17.7,17.5,15.4。

[ high performance liquid chromatography A ] or called [ HPLC method A ]:

1) performing high performance liquid chromatography according to VD (supplement VD) in the second part of the 2010 edition of the Chinese pharmacopoeia;

2) chromatographic conditions and system applicability test: a column packed with octyl silane-bonded silica gel (ZORBAX Eclipse XDB-C8, 4.6X 250mm, 5 μm column); using 0.1% phosphoric acid solution (pH value is adjusted to 4.50 +/-0.05 by triethylamine) -acetonitrile (40: 60) as a mobile phase;

the flow rate is 1ml per minute (the retention time of TBM-19 peak is in the range of 8-9 minutes, the detection wavelength is 226nm, and the column temperature is 30 ℃;

taking a proper amount of a test solution, placing the test solution at the temperature of 60 ℃ for 18-20 hours, injecting 10ul of the test solution into a liquid chromatograph, recording a chromatogram, wherein the number of theoretical plates is not less than 3000 calculated according to a TBM-19 peak, an impurity peak (RRT 1.53 impurity in the invention) is required to appear at about 1.53 relative to the retention time of the TBM-19 peak, the separation degree of the impurity peak and the TBM-19 peak is more than 4, and the test solution meets the requirements of a conventional analysis method.

TBM-19 from example 1 was determined to have an HPLC chromatographic purity of 99.26%, an RRT1.53 impurity content of 0.272% as calculated by the principal component control method, less than 0.02% for each of the remaining single impurities, and less than 0.1% for the total of the remaining impurities other than RRT 1.53. This result indicates that TBM-19 obtained in example 1 showed high purity, but that one typical impurity, i.e., RRT1.53, was present. Through preliminary analysis, the RRT1.53 impurity is a bolbostemma paniculatum analogue formed by the cleavage of the phosphoryl group of TBM-19 and shown as the following formula:

example 2: preparation of the pharmaceutical composition of the invention in the form of an injection

As an antitumor drug, it is useful to formulate it in the form of an injection.

This example provides an injection of the following formulation:

TBM-19 Compound: 10mg of the total weight of the mixture,

polyethylene glycol 200: 50mg of the total weight of the powder,

tartaric acid: 20mg of the total weight of the mixture,

water: adding proper amount of the mixture to 1 ml;

the preparation method of the injection comprises the following steps: adding TBM-19 compound, polyethylene glycol and tartaric acid into 80% water, stirring to dissolve, adjusting pH to 6.8 with 1M hydrochloric acid or 1M sodium hydroxide, adding water to full dose, filtering, subpackaging (5 ml/bottle) in divided dose, sealing, packaging, and sterilizing the obtained solution (121 deg.C, 15min) to obtain injection.

Example 2 a: preparation of TBM-19 Compound injection

Referring to the formulation and preparation of example 2, except that no polyethylene glycol was added, an injection solution was obtained.

Example 2 b: preparation of TBM-19 Compound injection

Referring to the formulation and preparation of example 2, except that no tartaric acid was added, an injection solution was obtained.

Example 2 c: preparation of TBM-19 Compound injection

Referring to the formulation and preparation method of example 2, except that polyethylene glycol and tartaric acid were not added, an injection solution was obtained.

Example 3: stability survey

The TBM-19 compound (powder), the injection of example 2a, the injection of example 2b, and the injection of example 2c were left for 5 months at 40 ℃ under sealed conditions, and the contents of RRT1.53 impurities (relative to the TBM-19 compound) at 0 month and 5 months were measured using the "HPLC method a" on these samples.

For each sample, the "percentage increase in high temperature" of the RRT1.53 impurity, i.e., the percentage increase in high temperature (%) of the RRT1.53 impurity content, is calculated by dividing the difference of the 5-month content of the impurity minus the 0-month content of the impurity in the sample by the 0-month content of the impurity multiplied by 100% using the following formula:

the results show that the compounds of example TBM-19 all have an RRT1.53 impurity percentage increase at high temperature of 16.4%.

The percentage of the high temperature increase of the RRT1.53 impurity of the injection of example 2 is 24.2%, the percentage of the high temperature increase of the RRT1.53 impurity of the injection of example 2a is 163.7%, the percentage of the high temperature increase of the RRT1.53 impurity of the injection of example 2b is 211.6%, and the percentage of the high temperature increase of the RRT1.53 impurity of the injection of example 2c is 231.4%.

The above results show that in the preparation of liquid formulations, in particular aqueous liquid formulations, of TBM-19 compounds, the degradation of the active ingredient is effectively inhibited only by the simultaneous use of polyethylene glycol and tartaric acid, the effect of which in the present invention is completely unexpected in the prior art. In addition, the calculation of the content of the active ingredient in the injection of example 2 at month 5 relative to its content at month 0, i.e., the residual content of the active ingredient, gave a result of 99.1%, indicating that the content of the active ingredient in the injection did not significantly change, thereby indicating that the injection composition of the present invention is a particularly stable aqueous liquid pharmaceutical preparation.

Test example 1: expression analysis of cell membrane surface PD-L1

Tumor cells such as H460, MHCC97H, Panc-1, etc. are divided into 5 × 10 cells⁵The concentration of each ml was inoculated into 6-well plates, 1ml per well. Adding the drug to be tested into the drug adding group to ensure that the drug is addedThe final concentration was 10. mu.M, and an equivalent amount of DMSO was added to the control group, and the control group was cultured in a sterile incubator for 24 hours. After the cells were digested with 0.25% trypsin, the cells were harvested by centrifugation at 1500rpm for 5 minutes, washing 2 times with PBS. After resuspending the cells with 200. mu.l PBS, 100. mu.l of the cell suspension was added with 5. mu.l PE-labeled IgG antibody, and the remaining 100. mu.l of the cell suspension was added with 5. mu.l PE-labeled PD-L1 antibody, and incubated at 4 ℃ for 30 minutes. The supernatant was discarded by centrifugation, washed twice with PBS, resuspended in 500. mu.l PBS, filtered through a 300 mesh screen into a flow tube, and tested on the machine.

As a result:

(1) downregulation of PD-L1 expression in tumor cells by TBM-19: human lung cancer H460 and H157 cells were treated with different doses (0.5, 1, 2.5, 5, 7.5. mu.M) of TBM-19 for 24H, and changes in PD-L1 protein expression in tumor cells were detected by Western blot. The result shows that TBM-19 has concentration dependence on the down-regulation effect of the protein level of PD-L1, and the expression level of PD-L1 protein in tumor cells can be obviously reduced when the TBM-19 is used at the concentration of 5-10 mu M. The specific results are shown in FIG. 1, which shows that the TBM-19 can reduce the expression of PD-L1 in tumor cells, and the change of the level of PD-L1 protein in human lung cancer H460 and H157 cells can be detected by western blot after the cells are treated for 24 hours by TBM-19 with different concentrations. In addition, the results of the measurements of TBM01 to TBM18 showed substantial agreement with TBM.

(2) Downregulation of PD-L1 on tumor cell surface by TBM-19: to analyze whether TBM-19 also caused a decrease in PD-L1 on the cell membrane surface, we treated H460 with TBM-19 and examined the change in expression of PD-L1 protein on the cell membrane surface by flow cytometry 24H after treatment. In addition, H460 cells after 24H of TBM-19 pretreatment are interacted with recombinant human PD-1-Fc protein, Fc is labeled by using a fluorescent antibody, and the binding condition of PD-1 and PD-L1 protein is observed under a fluorescent microscope. The flow cytometry experiment results show that TBM-19 can obviously reduce the expression of PD-L1 on the surfaces of H460 and MB231 cell membranes (see FIG. 2A, which shows that the change of the expression of PD-L1 protein on the surfaces of the cell membranes is detected by flow cytometry after H460 cells are treated by TBM-19(5 mu M) for 24 hours). Fluorescence microscopy results also demonstrated that TBM-19 reduced the binding of PD-L1 to PD-1-Fc on the surface of H460 cells (see FIG. 2B, which shows that the change in binding between H460 cells and PD-1-Fc was observed by fluorescence microscopy 24H after TBM-19 (5. mu.M) treatment of H460 cells).

Test example 2: activation assay for in vitro co-cultured cells

Tumor cells were treated at 5X 10⁴The cells were plated at a concentration of 100. mu.l/well in 96-well cell culture plates. Test compounds (in the present invention, DMSO or water is used as a solvent, as not specifically mentioned) were added to the drug-added group at a final concentration of 10. mu.M, and the mixture was placed at 37 ℃ in a 5% CO atmosphere₂The culture was carried out in a sterile incubator for 24 hours. Activating human T cells or NK cells at 2.5X 10⁵/ml、5×10⁵The cells were inoculated in a 96-well plate plated with H460 tumor cells at a concentration of one ml and cultured for 3 hours. Add 20. mu.l of lysis buffer to the maximal LDH release group for 45 min. The 96-well plate was removed, centrifuged at 1500rpm for 5 minutes, 50. mu.l of the supernatant was placed in a new 96-well plate, and 50. mu.l of the substrate was added and reacted for 30min in the dark. Then, 50. mu.l of a stop solution was added to each well, and the absorbance at 492nm was measured. The ratio of the absorbance per well to the absorbance of the maximum LDH release group was statistically calculated. In addition, 100. mu.l of complete medium was added to the E-Plate assay Plate and the background impedance value was determined according to the instructions of a multifunctional real-time unlabeled cell analyzer (RTCA DP, ACEA Biosciences Inc.). Then at 5X 10⁴Inoculating H460 cells into an E-Plate detection Plate at a concentration of/ml, putting the Plate into a detection table for real-time dynamic detection of cell proliferation, adding TBM-19 after 6H of cell culture for continuous treatment for 24H, adding activated T/NK cells for co-culture, and continuously performing real-time dynamic detection of cell proliferation.

As a result:

TBM-19 promotes killing activity of co-cultured T cells against tumor cells: we co-cultured tumor cells treated with TBM-19 with T cells stimulated with IL-2/anti-CD3 (FIG. 3A), and examined the release of LDH from the supernatant to determine the change in killing activity of T cells against tumor cells. LDH experiment results show that after the cells are treated by TBM-19, the killing activity of the T cells to H460 is obviously increased, and the effect is better than that of bolbostemma paniculatum glucoside B (figure 3B, which shows that human T cells are co-cultured with the H460 cells treated by DMSO or TBM-19(5 mu M), and the change of the death rate of the H460 cells is detected by an LDH method). Furthermore, we also examined the killing effect of T cells on H460 cells by Cell impedance method (Cell index assay) using a real-time label-free Cell analyzer. The results indicate that TBM-19 treatment of H460 cells enhanced T cell killing of tumor cells (FIG. 3C, showing co-culture of human T cells with DMSO or TBM-19 (5. mu.M) treated H460 cells and cell impedance assay to detect changes in H460 cell mortality).

Test example 3: inhibition effect of TBM-19 on Lewis lung cancer transplantation tumor mouse tumor

Female C57 BL/6 mice (18-22g, Beijing Wittingle laboratory animal technology Co., Ltd.) with age of 6 weeks were used as experimental subjects, 10 animals per group were inoculated with Lewis lung cancer cells in the underarm. After 72 hours of inoculation, the test groups were administered with 1, 2 and 4mg/kg of normal saline, and the blank control group was administered with 200. mu.l of normal saline once a day for 18 days. After inoculation, the body weight of the mice was measured every two days; tumor volumes were measured every 2 days, beginning on day three of dosing. The mice were sacrificed 19 days after administration, and tumor tissue samples were taken for experiments such as gene chip, immunohistochemistry, and the like; separating tumor infiltrating T cells and NK cells, and detecting activation of the T/NK cells by flow cytometry; taking the heart, liver, spleen, lung and kidney to make pathological sections; blood was taken to determine the change in biochemical index.

As a result:

TBM-19 inhibits the growth of Lewis lung cancer tumor-bearing mice tumors: by using a mouse lung cancer transplantation tumor model, the in vivo anti-tumor effect of TBM-19 is explored. Mice were injected with Lewis lung carcinoma cells into the axilla of C57 BL/6 mice for tumor formation, and TBM-19 (see FIG. 4A, showing in vivo Lewis lung carcinoma transplantation tumor mice experimental pattern of TBM-19, five mice each inoculated with 2.5X 10C 57 on day 3 and administered with 10mg/kg of TBM-19 (see FIG. 4A, for control and experimental groups)⁶Individual tumor cells, beginning intraperitoneal administration on day 3), and end of administration on day 18. The in vivo antitumor effect of TBM-19 was evaluated by measuring the change in body weight and transplanted tumor volume of tumor-bearing mice during administration. The results indicate that TBM-19 significantly slowed the growth of transplanted tumors (see FIG. 4B, showing that TBM-19 effectively inhibited the growth of Lewis transplanted tumors. (C) is the volume of TBM-19 significantly inhibited Lewis transplanted tumors) After the administration, TBM-19 can be observed to have obvious inhibition effect on the tumor weight and the tumor volume of the transplanted tumor (see fig. 4C and 4D, showing that TBM-19 can significantly inhibit the tumor weight of Lewis transplanted tumor). In addition, TBM-19 did not affect the body weight of the mice during the dosing period (see FIG. 4E, which shows the change in body weight of the mice during the TBM-19 dosing period).

Test example 4: tumor infiltrating immune cell assay

Firstly, preparing a tumor tissue single cell suspension: adding a proper amount of prepared dissociation solution (DMEM +400 uni/mL Collagenase IV +100ug/mL DNase 1) into a 6-well plate for later use, killing the mice, separating tumors, weighing, shearing into pieces by sterile ophthalmic scissors, placing the pieces into the plate with the dissociation solution, placing the 6-well plate into a 37 ℃ shaking table for 30-60min, sucking out the dissociated supernatant, filtering by a 70 mu m nylon screen, and then transferring into a centrifuge tube. Centrifuging at 500g for 5min, discarding the supernatant, resuspending with PBS 500uL, centrifuging again, sucking out the supernatant after centrifugation, adding PBS 100uL for resuspension, and obtaining the single cell suspension. A second step of staining the single cells, adding a dead dye reagent and then adding a receptor blocking reagent TruStain fcX to the reagent^TM(anti-mouse CD16/32) Antibody, incubated at 4 ℃ for 15min in a refrigerator. And finally, performing multicolor antibody Staining, taking out the incubated centrifuge tube, centrifuging (350g-500g for 5min), discarding the supernatant after centrifugation, adding 100uL of stabilizing Buffer, shaking or blowing to resuspend the cells, centrifuging again, repeating twice, discarding the supernatant after centrifugation, adding 100uL of stabilizing Buffer for resuspension, then sequentially adding flow antibodies according to the calculated dilution times, placing the centrifuge tube in a refrigerator at 4 ℃ for incubation for 15min, washing, and performing detection on a machine.

As a result:

TBM-19 activates tumor infiltrating T cells: to examine the effect of TBM-19 on tumor infiltrating immune cells, tumor tissues treated with blank control or TBM-19 were selected, digested into single cells by collaenase IV and DNase 1 treatment, and the infiltrating immune cells were isolated and analyzed for the proportion of CD45 positive immune cells and activation of T cells by flow cytometry (see fig. 5A, which shows the method of flow cytometry for detecting immune cell proportion and T cell activation after administration). ResultsIt is shown that TBM-19 can induce CD45 in tumor tissues⁺The number of immune cells increased (see fig. 5B, showing flow cytometry to detect immune cells in tumor tissue (CD 45)^﹢) In the same time) and also activates tumor-infiltrating T cells, expressed as IFN-gamma^﹢CD8^﹢CD3^﹢CD45^﹢The ratio increased (see FIG. 5C, showing flow cytometry to detect tumor infiltrating T cells (IFN-. gamma.))^﹢CD8^﹢CD3^﹢CD45^﹢) Activation of (c).

Claims

1. A compound of formula I:

or a pharmaceutically acceptable salt thereof,

wherein:

r1 is a halogen atom or a halogen atom,

r2 is C_1-6An alkyl group, a carboxyl group,

r3 is phenyl optionally substituted with 1 to 4 groups selected from: halogen, hydroxy, trifluoromethoxy, trifluoromethyl, amino, nitro, C_1-6Alkyl radical, C_1-6An alkoxy group.

2. The compound according to claim 1, wherein the halogen is selected from: fluorine, chlorine, bromine.

3. The compound according to claim 1, wherein said C_1-6The alkyl group is selected from: methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl.

4. The compound according to claim 1, wherein said C_1-6Alkoxy is C_1-4An alkoxy group.

5. The compound according to claim 1, wherein said C_1-6Alkoxy is selected from: methoxy, ethoxy, n-propoxy and isopropylOxy, n-butoxy, isobutoxy, sec-butoxy, tert-butoxy.

6. The compound according to claim 1, which is a compound selected from the following numbers TBM-01 to TBM-19:

numbering R1 R2 R3 TBM-01 Fluorine Methyl radical P-chlorophenyl group TBM-02 Fluorine Methyl radical P-nitrophenyl radical TBM-03 Fluorine Ethyl radical 3-methylphenyl radical TBM-04 Fluorine Propyl radical 4-trifluoromethoxyphenyl group TBM-05 Fluorine Methyl radical 4-hydroxyphenyl group TBM-06 Fluorine Methyl radical Phenyl radical TBM-07 Chlorine Methyl radical Phenyl radical TBM-08 Chlorine Methyl radical P-chlorophenyl group TBM-09 Chlorine Ethyl radical 3-aminophenyl TBM-10 Chlorine Propyl radical 3-methylphenyl radical TBM-11 Chlorine Tert-butyl radical 4-trifluoromethoxyphenyl group TBM-12 Chlorine Methyl radical 4-hydroxyphenyl group TBM-13 Bromine compound Methyl radical P-chlorophenyl group TBM-14 Bromine compound Methyl radical 3-aminophenyl TBM-15 Bromine compound Ethyl radical 3-methylphenyl radical TBM-16 Bromine compound Propyl radical 4-trifluoromethoxyphenyl group TBM-17 Bromine compound Tert-butyl radical 4-hydroxyphenyl group TBM-18 Bromine compound Methyl radical 4-Ethylphenyl TBM-19 Bromine compound Methyl radical Phenyl radical

Or a pharmaceutically acceptable salt thereof.

7. An injection solution, comprising: TBM-19 compound 10mg, 50mg polyethylene glycol 200, tartaric acid 20mg, and water appropriate amount added to 1 ml; the TBM-19 compound is the compound numbered as TBM-19 according to claim 6.

8. The injection according to claim 7, which is prepared as follows: adding TBM-19 compound, polyethylene glycol and tartaric acid into water 80% of the total volume of the preparation solution, stirring to dissolve the materials, adjusting pH of the medicinal liquid to =6.8 with 1M hydrochloric acid or 1M sodium hydroxide, adding water to full dose, filtering the obtained medicinal liquid, subpackaging in divided doses, sealing and packaging, and sterilizing the obtained medicinal liquid to obtain the injection.

9. Use of a compound according to any one of claims 1 to 6 in the manufacture of a medicament for the treatment of a cancer selected from: lung cancer, pancreatic cancer, ovarian cancer, bladder cancer, breast cancer.

10. The use according to claim 9, wherein the lung cancer is non-small cell lung cancer.