CN106822129B

CN106822129B - Application of the aloperine derivative in the drug of preparation treatment tumour

Info

Publication number: CN106822129B
Application number: CN201710136438.6A
Authority: CN
Inventors: 宋丹青; 邓洪斌; 张欣; 张靖溥; 窦玥莹; 唐胜; 汪燕翔; 李迎红; 张娜
Original assignee: Institute of Medicinal Biotechnology of CAMS
Current assignee: Institute of Medicinal Biotechnology of CAMS
Priority date: 2017-03-08
Filing date: 2017-03-08
Publication date: 2019-09-03
Anticipated expiration: 2037-03-08
Also published as: CN106822129A

Abstract

The present invention relates to a kind of new applications of aloperine derivative, in particular to aloperine derivative, its optical isomer shown in formula I, solvate or pharmaceutically acceptable salt are preparing the application in the drug for treating tumour, wherein, the structure of aloperine derivative is as shown in specification, such compound treats tumour by reducing the PD-L1 of tumor cell surface, the killing activity of immunocyte can be enhanced simultaneously, and there is good activity in vivo and toxicity is low.

Description

Application of aloperine derivative in preparing medicine for treating tumor

Technical Field

The invention belongs to the field of medical application, and particularly relates to application of aloperine derivatives in preparation of a medicine for treating tumors.

Background

The occurrence of tumor has close correlation with the immune function of the body. Usually, the immune system of the host will produce some killing effect on tumor cells, and this action mechanism is mainly that tumor cells containing specific antigen are phagocytized by dendritic cells and presented to T cells via the dendritic cells, and then the activated T cells recognize the tumor cells and release lymphokines, or directly kill the tumor cells. While some malignant cells may escape the killing effect of immune cells in different ways. The approach for realizing immune escape mainly comprises the following steps: (1) tumor self-modification and metabolism; (2) changes in the tumor microenvironment. Therefore, studying the tumor microenvironment is a new exploratory approach for tumor therapy. Tumor immunotherapy targeting the tumor microenvironment is becoming a progressive research hotspot.

It is well known that as an important member of the immune system, T cell activation requires the synergy of dual signals, namely an antigen presentation signal (antigenic peptide-MHC/TCR) and a costimulatory signal provided by B7/CD 28. The costimulatory molecules can participate in the immune response of the body by providing positive or negative regulatory signals to lymphocytes, and programmed death molecule 1(PD-1), and its ligands (PD-L1 and PD-L2) belong to the B7/CD28 family. The research shows that PD-L1 is in a high expression state in various tumors, such as melanoma, breast cancer, lung cancer, renal small cell carcinoma and the like. Activated T cells can also enhance the killing effect by secreting cytokines such as IFN-gamma, and the expression of programmed death factor 1 ligand (PD-L1) on the surface of tumor cells is obviously up-regulated under the stimulation of IFN-gamma. Many studies show that PD-1/PD-L1 acts as a pair of immunosuppressive co-stimulators, and the activation of its signaling pathway can inhibit T cell mediated immune response and even induce T cell apoptosis, thereby leading to the formation of immunosuppressive tumor microenvironment and leading tumor cells to escape from immune surveillance and killing of the body. While blocking this signaling pathway may enhance endogenous anti-tumor immune effects. Therefore, the PD-1/PD-L1 signal channel is expected to become a new target point for tumor immunotherapy.

At present, the anti-PD-L1 antibody and the anti-PD-1 antibody have good activities on tumors such as melanoma, lung cancer, kidney cancer, bladder cancer and the like in clinical tests. The monoclonal antibodies have the advantages of high targeting property, high safety, small side effect, strong blocking effect, no drug resistance and the like when being used as drugs for treating tumors. However, it has the corresponding limitations, such as short half-life, high production cost, etc., and for some solid tumors, the monoclonal antibody has a large molecular weight and cannot enter into the tumor cell space, so the range of action is limited to the edge of the tumor.

Aloperine is an alkaloid separated from sophora alopecuroides, and the structural formula of the aloperine is as follows:

research shows that the aloperine has various physiological activities including arrhythmia resistance, negative muscle strength resistance, antibiosis, antivirus, anti-inflammation, smooth muscle relaxation, immunosuppression, anticancer, free radical elimination, toxic and side effects, insecticidal action and the like.

In addition, the applicant designs and synthesizes a series of aloperine derivatives by taking aloperine as a mother nucleus, and the structural general formula of the aloperine derivatives is as follows:

wherein,

the X group being-CH₂-, -C (O) -or-S (O)_a-, wherein a is 1 or 2;

the Y radical being C_1-10An alkyl group, or a 3-15 membered cycloalkyl, aryl or heterocyclyl group formed by atoms selected from C, N, O and S; optionally, wherein said C_1-10The alkyl, 3-15 membered cycloalkyl, aryl or heterocyclyl is each independently substituted by one or more groups selected from halogen, amino, cyano, nitro, carboxy, C_1-6Alkyl radical, C_1-6Alkoxy, halo C_1-6Alkyl, halo C_1-6Alkoxy radical, C_1-6Alkoxycarbonyl, C_1-6Alkyl acyl radical, C_1-6Alkylamine acyl, C_1-6Alkylamido radical, C_1-6Alkylsulfonyl and 8-15 membered benzoheterocyclyl.

The aloperine derivative has excellent antiviral activity. However, other uses of the aloperine derivatives have not been reported in the prior art.

Disclosure of Invention

The invention provides a new application of aloperine derivative, namely the application of aloperine derivative in preparing medicaments for treating tumors.

The specific technical scheme of the invention is as follows:

the invention provides a new application of aloperine derivative, namely the application of aloperine derivative shown in formula I, optical isomer, solvate or pharmaceutically acceptable salt thereof in preparing a medicament for treating tumor,

wherein R is substituted by one or more groups selected from halogen, trifluoromethoxy, trifluoromethyl, amino, nitro, cyano, carboxyl, C_1-4Alkyl radical, C_1-4Alkoxy radical, C_1-4Alkoxycarbonyl, C_1-4Alkylamine acyl, C_1-6Alkylamido or C_1-6Alkylsulfonyl-substituted phenyl, imidazolyl, benzyl, pyridyl, thienyl, thiazolyl, piperazinyl or benzofuranyl.

In a further refinement, R is substituted with one or more substituents selected from the group consisting of halogen, trifluoromethoxy, trifluoromethyl, amino, nitro, cyano, carboxy, C_1-4Alkyl radical, C_1-4Alkoxy-substituted phenyl or imidazolyl.

In a further improvement, R is phenyl or imidazolyl substituted with one selected from trifluoromethoxy, methyl.

In a further improvement, R is methyl-substituted imidazolyl.

In a further improvement, R is phenyl substituted by trifluoromethoxy.

In a further improvement, the aloperine derivative shown in the formula I is 12-N- (1-methyl-1H-imidazole-4-sulfonyl) aloperine (NKD-49), and the structural formula is as follows:

in a further improvement, the aloperine derivative shown in the formula I is 12-N- (4-trifluoromethoxybenzenesulfonyl) aloperine (NKD-50), and the structural formula is as follows:

in a further improvement, the aloperine derivative shown in the formula I, an optical isomer, a solvate or a pharmaceutically acceptable salt thereof is applied to the preparation of a medicament for reducing the expression of the total protein PD-L1 in tumor cells.

In a further improvement, the tumor is selected from one of melanoma, breast cancer, lung adenocarcinoma, lung cancer and renal small cell carcinoma.

The term C as used in the present invention_1-4Alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, and the like, and the alkyl groups may be substituted or unsubstituted, and when substituted, include trifluoromethyl, trifluoroethyl, hydroxymethyl, hydroxyethyl, aminomethyl, aminoethyl, and the like.

The term C as used herein_1-4Alkoxy groups include methoxy, ethoxy, propoxy, isopropoxy, and tert-butoxy, and the like.

The term C as used in the present invention_1-4The alkoxycarbonyl group includes methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, t-butoxycarbonyl, etc.

The term C as used in the present invention_1-4The alkanoyl group includes acetyl, propionyl, butyryl and the like.

The term C as used in the present invention_1-4The alkylaminoalkyl group includes methyl aminoacyl, ethyl aminoacyl, N-dimethyl aminoacyl, N-diethyl aminoacyl, etc.

The term C as used in the present invention_1-4The alkylamido group includes formylamino group, acetylamino group and the like.

The term C as used in the present invention_1-4Alkylsulfonyl includes methylsulfonyl, ethylsulfonyl and the like.

The term "isomer" as used herein includes all possible isomeric (e.g., enantiomeric, diastereomeric, geometric, conformational, epimeric, and rotational isomers, etc.) forms of the aloperine derivatives of formula I of the present invention. For example, the respective R and S configurations of the asymmetric centers, (Z) and (E) double bond isomers, and (Z) and (E) conformational isomers are within the scope of the present invention.

The aloperine derivative shown in the formula I can form a solvate, such as a hydrate, an alcoholate and the like. In general, the solvate forms with pharmaceutically acceptable solvents such as water, ethanol, and the like are comparable to the non-solvate forms.

The aloperine derivatives of formula I of the present invention may also be prodrugs or forms which release the active ingredient after metabolic changes in the body. The selection and preparation of suitable prodrug derivatives is well known to those skilled in the art and is not intended to be limiting.

The compound shown in the formula I or the pharmaceutically acceptable salt thereof can also exist in crystals, and the invention comprises any crystal form of the aloperine derivative shown in the formula I or the pharmaceutically acceptable salt thereof.

The aloperine derivative shown in the formula I, the optical isomer, the solvate or the pharmaceutically acceptable salt thereof can be administered by the following routes: parenteral, topical, intravenous, oral, subcutaneous, intraarterial, intradermal, transdermal, rectal, intracranial, intraperitoneal, intranasal, intramuscular routes, or as inhalants.

The aloperine derivative shown in the formula I, the optical isomer, the solvate or the pharmaceutically acceptable salt thereof can be administered in the form of a pharmaceutical preparation, and the pharmaceutical composition comprises the aloperine derivative shown in the formula I, the optical isomer, the solvate or the pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier or auxiliary material.

The aloperine derivative shown in the formula I, the optical isomer, the solvate or the pharmaceutically acceptable salt thereof can be prepared into various suitable dosage forms according to the administration route.

When administered orally, the compounds of the present invention may be formulated in any orally acceptable dosage form, including but not limited to tablets, capsules, aqueous solutions or suspensions. Among these, carriers for tablets generally include lactose and corn starch, and additionally, lubricating agents such as magnesium stearate may be added. Diluents used in capsule formulations generally include lactose and dried corn starch. Aqueous suspension formulations are generally prepared by mixing the active ingredient with suitable emulsifying and suspending agents. Optionally, some sweetener, aromatic or colorant may be added into the above oral preparation.

When applied topically to the skin, the compounds of the present invention may be formulated in a suitable ointment, lotion, or cream formulation wherein the active ingredient is suspended or dissolved in one or more carriers. Carriers that may be used in ointment formulations include, but are not limited to: mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyethylene oxide, polypropylene oxide, emulsifying wax and water; carriers that can be used in lotions or creams include, but are not limited to: mineral oil, sorbitan monostearate, tween 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.

The aloperine derivative shown in the formula I, the optical isomer, the solvate or the pharmaceutically acceptable salt thereof can also be used in the form of sterile injection preparations, including sterile injection water or oil suspension or sterile injection solution, and also can be in a freeze-dried form. Among the carriers and solvents that may be employed are water, ringer's solution and isotonic sodium chloride solution. In addition, the sterilized fixed oil may also be employed as a solvent or suspending medium, such as a monoglyceride or diglyceride.

The pharmaceutical preparation of the present invention includes any preparation which can be pharmaceutically practiced, for example, oral preparations, parenteral preparations and the like.

The invention has the beneficial effects that: the invention provides a new application of aloperine derivatives, the compounds treat tumors by reducing PD-L1 on the surface of tumor cells, can enhance the killing activity of immunocytes, and have good in-vivo activity and low toxicity.

Drawings

FIG. 1a shows the results of expression of total protein PD-L1 in H157 cells after different times of administration of NKD-49 by immunoblot analysis; wherein GAPDH is a reference;

FIG. 1b shows the result of expression of total protein PD-L1 in H157 cells after immunoblot analysis given NKD-50 at various times; wherein GAPDH is a reference;

FIG. 2a is a graph showing the results of immunoblot analysis of the expression of total PD-L1 protein in H157 cells given different concentrations of NKD-49; wherein GAPDH is a reference;

FIG. 2b is a graph showing the results of immunoblot analysis of the expression of total PD-L1 protein in H157 cells given different concentrations of NKD-50; wherein GAPDH is a reference;

FIG. 3 is a graph showing the expression results of total PD-L1 protein in NKD-49 and NKD-50 treated H157 cells, PD-L1⁺% indicates the proportion of PD-L1 positive cells;

FIG. 4a is a schematic diagram of co-culture of tumor cells and CIK cells;

FIG. 4b is a graph of the ratio of uptake per well to the maximal LDH release group for CIK cells co-cultured with NKD-49-pretreated A549 cells;

FIG. 4c is a graph of absorbance per well versus absorbance of the maximal LDH release group co-cultured with CIK cells and A549 cells pretreated with NKD-50;

FIG. 5 is a graph showing the survival rate of NKD-49 and NKD-50 treated A549 cells;

FIG. 6 shows the anti-tumor activity in mice, wherein a is the change in tumor weight after NKD-49 treatment, (top) blank control, (middle 1) NKD-49 administered at a dose of 20 mg/kg; (middle 2) NKD-49, administered at a dose of 50 mg/kg; (below) NKD-49, administered at a dose of 100 mg/kg; b is tumor weight change after NKD-50 treatment, (upper) blank control, (middle) NKD-50, and the administration dose is 50 mg/kg; (below) NKD-50 at a dose of 200 mg/kg; NKD-49 treatment post-mouse weight change, (upper) blank control, (middle 1) NKD-49 administered at a dose of 20 mg/kg; (middle 2) NKD-49, administered at a dose of 50 mg/kg; (below) NKD-49, administered at a dose of 100 mg/kg; change in body weight of mice treated with NKD-50; (upper) blank control, (middle) NKD-50, administered at 50 mg/kg; (below) NKD-50 at a dose of 200 mg/kg; FIG. 7 is a graph showing the change in tumor weight of NKD-49 and NKD-50 treated mice, wherein A represents a blank control and B represents NKD-49 administered at a dose of 20 mg/kg; c represents NKD-49, and the administration dosage is 50 mg/kg; d represents NKD-49 and the dosage is 100 mg/kg.

Detailed Description

Test example 1 NKD-49 and NKD-50 time-effect relationship

1.1 test methods

H157 cells were digested with 0.25% trypsin to make a cell suspension at 5X 10⁵The culture medium was inoculated into 6-well cell culture plates at a concentration of 1ml per well, and the cells were incubated at 37 ℃ with 5% CO₂Culturing for 24 hours in an aseptic culture box; then randomly divided into three groups, namely a control group, an NKD-49 group and an NKD-50 group.

NKD-49 and NKD-50 were added to the drug-adding wells of NKD-49 and NKD-50 groups, respectively, to a final concentration of 20. mu.M, and the control group was added with DMSO of the same volume, and the mixture was left at 37 ℃ with 5% CO₂Culturing for 3h, 6h, 9h, 12h and 24h in the sterile incubator.

3. After the culture, the cells were harvested, the cell culture medium was aspirated, the plates were washed with 1ml PBS 2 times, 50. mu.l of lysis buffer was added to each well of the six-well plate, the cells were scraped off with a spatula, lysed on ice for 30min, and centrifuged at 12000rpm for 15min at 4 ℃.

4. After centrifugation, the supernatant was transferred to a new EP tube, and 2. mu.l of the supernatant was added to 600. mu.l of Bradford, and left to stand for 10min to determine the protein concentration.

5. 50 μ g of total protein was taken from each sample into a new EP tube, with a 4: 1, adding 5 xSDS Loading, mixing uniformly, and incubating at 100 ℃ for 10 min.

6. After electrophoresis of the samples on 10% and 15% SDS-PAGE gels, the proteins were transferred to PVDF membranes, blocked with 5% skim milk at room temperature for 1h, and incubated overnight at 4 ℃.

7. After the primary antibody incubation was completed, the primary antibody was recovered, washed with TBS-T for 30min (10 min each), and the secondary antibody was incubated, and after the secondary antibody incubation was completed, washed with TBS-T for 30min (10 min each), and exposed in an imager, and the results of the detection are shown in FIG. 1a and FIG. 1 b.

1.2 test results

As can be seen from FIG. 1a, NKD-49 decreased the expression of total protein PD-L1 in H157 cells after 12H of drug administration, and significantly decreased the expression of total protein PD-L1 after 24H of drug administration; from FIG. 1b, it can be seen that NKD-50 began to reduce the expression of total protein PD-L1 in H157 cells after 3H of drug administration, and the expression of total protein PD-L1 was significantly reduced after 12H of drug administration.

1.3 conclusion of the test

Both NKD-49 and NKD-50 were able to reduce the expression of total PD-L1 protein in cells and were time-dependent.

Test example 2 dose-Effect relationship between NKD-49 and NKD-50

2.1 test methods

H157 cells were digested with 0.25% trypsin to make a cell suspension at 5X 10⁵The culture medium was inoculated into 6-well cell culture plates at a concentration of 1ml per well, and the cells were incubated at 37 ℃ with 5% CO₂Cultured in a sterile incubator for 24 hours, then randomly divided into three groups, which are respectively used as controlsGroup, NKD-49 group and NKD-50 group.

NKD-49 and NKD-50 groups were added to the wells to a final concentration of 5. mu.M, 10. mu.M, 15. mu.M, 20. mu.M, respectively, to the NKD-49 and NKD-50 groups, to the control group, an equal volume of DMSO was added thereto, and the resulting mixture was set at 37 ℃ and 5% CO₂Culturing for 24h in the sterile incubator.

5. 50 μ g of total protein was taken from each sample into a new EP tube, with a 4: 1 plus 5 x SDS Loading. After mixing, incubation was carried out at 100 ℃ for 10 min.

7. After the primary antibody incubation was completed, the primary antibody was recovered, washed with TBS-T for 30min (10 min each), the secondary antibody was incubated, and after the secondary antibody incubation was completed, washed with TBS-T for 30min (10 min each), and exposed in an imager, and the results of the detection are shown in FIG. 2a and FIG. 2 b.

2.2 test results

As can be seen from FIG. 2a, after H157 cells are dosed for 24H, the NKD-49 dosing dose is 10 μ M, and the expression of PD-L1 total protein can be remarkably reduced; as can be seen from FIG. 2b, the NKD-50 dose was 15. mu.M after H157 cells were dosed for 24H, which decreased the expression of PD-L1 total protein.

2.3 conclusion of the test

Both NKD-49 and NKD-50 were able to reduce the expression of total PD-L1 protein in cells and were dose-dependent.

Test example 3NKD-49 and NKD-50 decrease PD-L1 in tumor cells

3.1 test methods

NKD-49 and NKD-50 were added to the drug wells of NKD-49 and NKD-50 groups, respectively, to give final concentrations of 15. mu.M, 20. mu.M, and 25. mu.M, respectively, and the control group was incubated in a sterile incubator for 24 hours with the addition of an equal amount of DMSO.

3. After the culture, the cells were harvested, digested with 0.25% trypsin, washed 2 times with PBS, centrifuged at 1500rpm for 5min, and harvested.

4. The cells were resuspended in 200. mu.l PBS, then 100. mu.l was removed and 5. mu.l PE-labeled IgG antibody was added to the remaining 100. mu.l cell suspension, and 5. mu.l PE-labeled PD-L1 antibody was added and incubated at 4 ℃ for 30 min.

5. 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, with the test results shown in FIG. 3.

3.2 test results

As can be seen in FIG. 3, NKD-49 and NKD-50 significantly reduced the expression of total PD-L1 protein in H157 cells at 20. mu.M concentration compared to the control group.

3.3 conclusion of the test

NKD-49 and NKD-50 can significantly reduce the expression of total PD-L1 protein in cells.

Test examples 4 NKD-49 and NKD-50 enhance the killing activity of immune cells

4.1 test methods

A549 cells are digested by pancreatin to prepare a cell suspension, and the cell suspension is prepared by 5 multiplied by 10⁴The cells were inoculated in 96-well cell culture plates at a concentration of 100. mu.l/well, and a blank control group, a normal cell group, a maximum LDH release group, an IFN-. gamma.group and an addition group were placed in 3 duplicate wells at 37 ℃ with 5% CO₂Culturing for 24h in the sterile incubator.

Adding NKD-49 or NKD-50 with final concentration of 20 μ M into the medicated group of A549 cells, treating for 1 hr, adding IFN- γ, standing at 37 deg.C and 5% CO₂And culturing for 24h in the sterile incubator.

3. The medium of A549 was replaced with 100. mu.l of complete medium of CIK cells, after which the CIK cells were cultured at 5X 10⁵The cells were seeded in 96-well plates 100. mu.l/well plated with A549 cells at 37 ℃ in 5% CO₂Culturing for 3h in the sterile incubator.

4. Add 20. mu.l of lysis buffer to the maximal LDH release group and place at 37 ℃ with 5% CO₂The sterile incubator is cracked for 45 min.

5. 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.

6. Then, 50. mu.l of a stop solution was added to each well, and the absorbance at 492nm was measured.

7. The ratio of the absorbance per well to the absorbance of the maximum LDH release group was statistically calculated and the results are shown in fig. 4 a-4 c.

4.2 test results

As can be seen from FIGS. 4a, 4b and 4c, in the presence of IFN-gamma, the killing activity of the CIK cells on the A549 cells can be remarkably improved after the A549 cells and the CIK cells are co-cultured at a ratio of 1:10 and the A549 cells are pretreated by NKD-49 or NKD-50.

4.3 conclusion of the test

NKD-49 or NKD-50 can enhance the killing activity of immune cells.

Test example 5 NKD-49 and NKD-50 cytotoxicity in vitro

5.1 test methods

A549 cells are digested by pancreatin to prepare a cell suspension, and the cell suspension is prepared by 5 multiplied by 10⁴Inoculating to 96-well cell culture plate with concentration of 100 μ l per well, setting blank control group, normal cell group and drug adding group in 3 multiple wells, standing at 37 deg.C and 5% CO₂Culturing for 24h in the sterile incubator.

2.NKD-49 or NKD-50 was added to the drug-added group to give final concentrations of 2. mu.M, 5. mu.M, 10. mu.M, 20. mu.M, 100. mu.M, 200. mu.M, and 500. mu.M, respectively, and the mixture was cultured in a sterile incubator for 48 hours using the medium as a blank.

3. 4 hours before the end of the culture, 20. mu.l of 5mg/ml MTT solution was added to each well, and after the end of the culture, the supernatant was aspirated by a 2ml syringe, 100. mu.l of dimethyl sulfoxide was added to each well, and after shaking for 10min, the light absorption was measured at 570nm, and the results are shown in FIG. 5.

5.2 test results

As can be seen from FIG. 5, NKD-49 and NKD-50 reduced the survival rate of A549 cells, and the survival rate of A549 cells was reduced by about 40% when the concentration of NKD-49 or NKD-50 reached 500. mu.M.

5.3 conclusion of the test

NKD-49 and NKD-50 have small cytotoxicity to tumor cells.

Test examples 6NKD-49 and NKD-50 in vivo Activity

6.1 test methods

1. 40C 57BL/6 mice (female, 6 weeks old, 18-22g, Beijing Wintotonghua laboratory animal technology Co., Ltd.) were selected, murine lung carcinoma (Lewis lung carcinoma) cells were inoculated into the right axilla of the mice, and randomly divided into experiment 1 group, experiment 2 group and blank control group.

2. After 72h of inoculation, the injection is orally taken and gastric lavage is carried out, NKD-49 and NKD-50 are respectively administered to the experiment group 1 and the experiment group 2, the administration dose is respectively 20mg/kg, 50mg/kg and 100mg/kg, 200 mu l of normal saline is administered to the blank control group, and the administration is carried out once a day for 10 days.

3. 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.

4. Two days after the administration, the mice were treated, tumor tissues were photographed and weighed, and the results of the measurements were shown in FIG. 6.

6.2 test results

As can be seen in FIG. 7, the tumor volume in NKD-49 and NKD-50 treated mice was significantly reduced compared to the control group; as can be seen from the graph a in FIG. 6, the tumor inhibition rate in mice is 57% when NKD-49 is administered at a predose of 20mg/kg, and the tumor inhibition rate is 56% when NKD-49 is administered at a dose of 50 mg/kg; when the administration dosage of NKD-49 is 100mg/kg, the inhibition rate is 65.8%; as can be seen from the b-diagram, the dose of NKD-50 administered was 50mg/kg, and the inhibition rate was 43%; the administration dosage of NKD-50 is 200mg/kg, and the inhibition rate is 57%; as can be seen from the graphs c and d, the body weight of mice was not significantly changed by the administration of NKD-49 and NKD-50 at different doses.

6.3 conclusion of the test

NKD-49 and NKD-50 can inhibit the growth of tumors in mice, and have the curative effect of treating tumors without toxic or side effect.

Claims

1. An application of aloperine derivative or pharmaceutically acceptable salt shown in formula I in preparing medicine for treating lung cancer,

wherein R is 1-methyl substituted imidazolyl or 4-trifluoromethoxyphenyl.

2. The use as claimed in claim 1, wherein the aloperine derivative of formula i is 12-N- (1-methyl-1H-imidazole-4-sulfonyl) aloperine.

3. The use as claimed in claim 1, wherein the aloperine derivative of formula i is 12-N- (4-trifluoromethoxybenzenesulfonyl) aloperine.

4. The use according to claim 1, wherein the aloperine derivative of formula i or a pharmaceutically acceptable salt thereof is used for the manufacture of a medicament for reducing the expression of total PD-L1 protein in lung cancer cells.