Process for the preparation of dihydropyrrolone derivatives and use thereof

Abstract

The application belongs to the technical field of medicines, and particularly discloses a preparation method and application of a dihydropyrrolone derivative. The application provides the dihydropyrrolone derivative with high tumor immunotherapy activity, and the structural formula is shown in the following formula (I). The dihydropyrrolone derivative provided by the application has good immunosuppressive activity on melanoma, lung cancer and breast cancer cells, and can provide a potential lead compound for development of a new immunological drug.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of medicine, and particularly relates to a preparation method of a dihydropyrrolone derivative and application thereof. BACKGROUND

[0002] Tumor immunotherapy is one of the focuses of medical research in recent years. With the deepening of immunology research and the progress of science and technology, the position of immunotherapy in clinical application is rising, which provides more choices for the clinical treatment of cancer. At present, the main ways of immunotherapy are tumor vaccine, cell therapy and immune checkpoint inhibitors. Among them, immune checkpoint inhibitors such as PD-1 / PD-L1 and CTLA-4 have achieved great success in the treatment of multiple cancers and have been approved for use in second-line or first-line treatment, which is of great significance to improve tumor cure rate, prolong survival time and improve the quality of life of patients. However, due to factors such as tumor immune escape, lack of T cell infiltration and inhibitory tumor microenvironment, the proportion of patients who can truly obtain long-term and sustained response from immune checkpoint inhibitor treatment is still very low. Although the combination of other treatment methods or other existing immune checkpoint inhibitors can improve the response rate, the combination will also be accompanied by serious toxicity. In addition, many patients may develop acquired drug resistance after initial response. Therefore, it is urgent to find new targets and combination methods to solve these problems.

[0003] The mechanism of action of immune checkpoint inhibitors is that in the tumor microenvironment, tumor cells highly express PD-L1, which binds to PD-1 on the surface of T cells, resulting in inhibition of T cell function and inability to effectively kill tumor cells. PD-1 / PD-L1 inhibitors block this binding to relieve the inhibition of T cells, allowing T cells to restore their ability to kill tumor cells, thereby achieving the effect of treating tumors. However, the effectiveness of this therapy depends not only on whether T cell function can be restored, but also on the sensitivity of tumor cells to immune killing. Tumor cells often use countermeasures to achieve immune escape, which greatly hinders the effectiveness of immunotherapy, so it is far from enough to treat tumors by only improving T cell function to achieve the desired effect.

[0004] Tumor immune escape refers to the phenomenon that tumor cells evade recognition and attack by the body's immune system through various mechanisms, thereby surviving and proliferating in the body. The main mechanisms of tumor immune escape include antigen loss, high expression of immune checkpoints, initiation of anti-apoptotic cascade, resistance to ferroptosis, down-regulation of antigen presentation, release of factors promoting immune tolerance, etc. In addition, the tumor microenvironment also plays an immunosuppressive role, allowing tumor cells to escape immune surveillance. Immune escape is an important reason for resistance to immunotherapy.

[0005] Abnormal expression of MHC-I molecules on the surface of tumor cells is one of the most important reasons for tumor immune escape. The major histocompatibility complex class I (MHC-I or human leukocyte antigen HLA) antigen presentation (AP) pathway is essential for the activation and proliferation of CD8+ T cells. Tumor cells often evade anti-tumor immunity by down-regulating MHC-I. Previous studies have confirmed that MHC-I expression is positively correlated with the prognosis of patients with various cancer types, while down-regulation of MHC-I is associated with disease progression and poor prognosis in various cancers. And since the therapeutic effect of immune checkpoint inhibitors depends on the recognition of tumor cell surface cytoplasmic antigens presented by MHC-I, the reduction of MHC-I expression is also closely related to the resistance of immune checkpoint inhibitor therapy. At present, the research on improving MHC-I expression is relatively limited, and some studies have found that CDK4 and CDK6 inhibitors or EZH2 inhibitor GSK126 treatment for 7 days can improve MHC-I expression, which indicates that it is feasible to regulate the expression of MHC-I by drugs or treatment methods. Therefore, exploring more drugs or treatment methods that can effectively improve the expression and function of MHC-I has important significance for improving the response rate of existing immunotherapy.

[0006] Therefore, the design and development of new compounds with high tumor immunocompetent activity have a very broad application prospect in future cancer treatment. SUMMARY

[0007] In view of the deficiencies of the prior art, the purpose of the present application is to provide a preparation method of dihydropyrrolone derivatives and their immunotherapy applications.

[0008] To achieve the above-mentioned purpose, the technical scheme adopted by the present application is as follows: the application of dihydropyrrolone derivatives in the preparation of tumor immunotherapy drugs.

[0009] The application of dihydropyrrolone derivatives in the preparation of immune checkpoint inhibitors.

[0010] The application of dihydropyrrolone derivatives in improving the antigen presentation capacity of tumor cell surface.

[0011] The application of dihydropyrrolone derivatives in up-regulating the expression of MHC-I molecules on the surface of tumor cells.

[0012] The application of dihydropyrrolone derivatives in the preparation of drugs for inducing high expression of MHC-I molecules on the surface of tumor cells and / or improving the tumor killing ability of T cells (in tumor cells).

[0013] The application of dihydropyrrolone derivatives in improving the tumor microenvironment and increasing the infiltration of CD4+ T cells and / or CD8+ T cells.

[0014] In the application described, the concentration of the dihydropyrrolidone derivative is 0.1~20 μM, preferably 10 μM.

[0015] Furthermore, the tumors include solid tumors, such as melanoma, lung cancer, and breast cancer.

[0016] Among them, MHC-I molecules refer to major histocompatibility complex type I.

[0017] The structural formula of the dihydropyrrolidone derivative is as follows ( As shown in the image:

[0018]

[0019] Mode( In ), R is C 1-28 Aliphatic hydrocarbon groups, aromatic hydrocarbon groups, heterocyclic groups and their derivative groups.

[0020] Furthermore, the formula ( The structural formulas of the compounds are shown in compounds 1, 2, and 3 below:

[0021]

[0022] Where R1 is C 1-9 Alkyl, C 6-10 Alicyclic hydrocarbon groups and their monohalogenated or polyhalogenated groups, C 6-10 Cycloolefin groups and their monohalogenated or polyhalogenated groups, C 8-10 The halogen atom is any one of benzocycloalkyl or five- to six-membered heterocyclic groups, wherein the halogen atom is at least one of fluorine, chlorine, bromine, or iodine; R2 is hydrogen or C. 1-9 Alkyl, C 2-9 alkenyl, C 2-9 Any one of the alkynyl groups; R3, R4, R5, R6, and R7 may be the same or different, and are respectively any one of hydrogen, fluorine, chlorine, bromine, iodine, carboxyl, hydroxyl, methoxy, ethoxy, methyl, ethyl, propyl, isopropyl, trifluoromethyl, nitrile, acetyl, sulfonyl, p-toluenesulfonate, benzenesulfonate, and trifluoromethanesulfonate.

[0023] Preferred formula ( The compound is selected from any one of the following compounds 1a~1h, 2a~2b, 3a~3b:

[0024]

[0025] More preferably, formula ( The compound is selected from any one of compound 1b, compound 2b (Talaroconvolutin A), and compound 3a.

[0026] Compared with the prior art, the present application has the beneficial effects of:

[0027] The dihydropyrrolone derivative provided by the present application has good immunosuppressive activity on melanoma, lung cancer and breast cancer cells. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 The hydrogen spectrum (600MHz, DMSO) of compound 1a synthesized in Example 1 is as follows:

[0029] Figure 2 The carbon spectrum (151MHz, DMSO) of compound 1a synthesized in Example 1 is as follows:

[0030] Figure 3 The hydrogen spectrum (600MHz, DMSO) of compound 1b synthesized in Example 1 is as follows:

[0031] Figure 4 The carbon spectrum (151MHz, DMSO) of compound 1b synthesized in Example 1 is as follows:

[0032] Figure 5 The hydrogen spectrum (600MHz, DMSO) of compound 1c synthesized in Example 1 is as follows:

[0033] Figure 6 The carbon spectrum (151MHz, DMSO) of compound 1c synthesized in Example 1 is as follows:

[0034] Figure 7 The hydrogen spectrum (600MHz, DMSO) of compound 1d synthesized in Example 1 is as follows:

[0035] Figure 8 The carbon spectrum (151MHz, DMSO) of compound 1d synthesized in Example 1 is as follows:

[0036] Figure 9 The hydrogen spectrum (600MHz, DMSO) of compound 1e synthesized in Example 1 is as follows:

[0037] Figure 10 The carbon spectrum (151MHz, DMSO) of compound 1e synthesized in Example 1 is as follows:

[0038] Figure 11 The hydrogen spectrum (600MHz, DMSO) of compound 1f synthesized in Example 1 is as follows:

[0039] Figure 12 The carbon spectrum (151MHz, DMSO) of compound 1f synthesized in Example 1 is as follows:

[0040] Figure 13 The hydrogen spectrum (600 MHz, DMSO) of compound 1g synthesized in Example 1;

[0041] Figure 14 The carbon spectrum (151 MHz, DMSO) of compound 1g synthesized in Example 1;

[0042] Figure 15 The hydrogen spectrum (600 MHz, DMSO) of compound 1h synthesized in Example 1;

[0043] Figure 16 The carbon spectrum (151 MHz, DMSO) of compound 1h synthesized in Example 1;

[0044] Figure 17 The hydrogen spectrum (600 MHz, DMSO) of compound 2a synthesized in Example 1;

[0045] Figure 18 The carbon spectrum (151 MHz, DMSO) of compound 2a synthesized in Example 1;

[0046] Figure 19 The hydrogen spectrum (600 MHz, DMSO) of compound 3a synthesized in Example 1;

[0047] Figure 20 The carbon spectrum (151 MHz, DMSO) of compound 3a synthesized in Example 1;

[0048] Figure 21 The hydrogen spectrum (600 MHz, DMSO) of compound 3b synthesized in Example 1;

[0049] Figure 22 The carbon spectrum (151 MHz, DMSO) of compound 3b synthesized in Example 1;

[0050] Figure 23A The HLA-A / B / C flow cytometry results of compound 1b, 2b, 3a on A549 cells in Example 2;

[0051] Figure 23B The HLA-A / B / C flow cytometry results of compound 1b, 2b, 3a on BT-474 cells in Example 2;

[0052] Figure 23C The H-2K flow cytometry results of compound 1b, 2b, 3a on B16 cells in Example 2 b

[0053] Figure 24 The apoptosis flow cytometry results of OT-I T cells on B16 cells in the killing function test in Example 2; ​

[0054] Figure 25A CCK8 results of compound 1b, 2b, 3a on A549 cell viability in Example 2;

[0055] Figure 25B CCK8 results of compound 1b, 2b, 3a on BT-474 cell viability in Example 2;

[0056] Figure 25C CCK8 results of compound 1b, 2b, 3a on B16 cell viability in Example 2;

[0057] Figure 26 Tumor growth curve of compound 1b on B16 in Example 2;

[0058] Figure 27 Statistical chart of T cell infiltration in B16 tumor of compound 1b in Example 2. DETAILED DESCRIPTION

[0059] The application will be further described in detail below with reference to the examples.

[0060] Those skilled in the art will understand that the following examples are only for illustration of the present application and should not be regarded as limiting the scope of the present application. If no specific technique or condition is specified in the examples, the technique or condition described in the literature in the art or according to the product manual is used. If no manufacturer of the material or equipment is specified, it is a conventional product that can be obtained by purchase.

[0061] Example 1

[0062] Synthesis of dihydropyrrolone derivatives

[0063]

[0064] Note: -C7H in 1b 15 Specifically, n-heptyl.

[0065] Compound 4 was prepared according to the method reported in the literature (Chinese Journal of Chemistry 2024, 42, 1509-1514.) and patent application (Synthetic process of natural product Talaroconvolutin A with anticancer activity, application number: CN202311866192.X).

[0066] Compound 4 (0.5 mmol) was dissolved in 6 mL of tetrahydrofuran under nitrogen, cooled to -78 °C, and a solution of LiHMDS (0.6 mmol) in tetrahydrofuran was added dropwise slowly. After 45 min, a solution of aldehyde (1.0 mmol) in tetrahydrofuran (most of the aldehydes used were commercially available. Only the aldehyde used to prepare compound 2a needed to be synthesized by following the procedure used to synthesize the aldehyde in the synthesis of Talaroconvolutin A.) and boron trifluoride etherate (1.0 mmol) were added dropwise in sequence. The reaction was allowed to proceed for 3 h, quenched by the addition of saturated solution of sodium bicarbonate, extracted with ethyl acetate three times, and the organic phase was washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate. Concentration under reduced pressure and purification by silica gel column chromatography afforded the compound as an oil. The compound was dissolved in 4 mL of dichloromethane, cooled to 0 °C, and pyridine (3.6 mmol), water (1.98 mmol), and Dess-Martin periodinane (1.8 mmol) were added in sequence. The reaction was allowed to proceed for 1 h at room temperature. The reaction was quenched with a mixture of saturated sodium bicarbonate and 10% sodium thiosulfate solution (1:1, v / v), and the aqueous phase was extracted with dichloromethane three times. The organic phase was washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by silica gel column chromatography to afford the compound as a yellow oil. The compound was dissolved in 4 mL of methanol, and p-toluenesulfonic acid monohydrate (0.11 mmol) was added. The reaction was allowed to proceed for 6 h at 50 °C. The solid was allowed to precipitate upon cooling, and the target product 1a~1h, 2a~2b, 3a~3b was obtained by filtration and washing with methanol.

[0067] The yield, high resolution mass spectrometry data, nuclear magnetic hydrogen spectrum, and carbon spectrum data of compounds 1a~1h, 2a~2b, 3a~3b are as follows, respectively.

[0068]

[0069] Yield: 27.4%

[0070] High resolution mass spectrometry data: HRMS calcd for C 13 H 11 NNaO3 + [M+Na + ] 252.0631 found252.0630.

[0071] Nuclear magnetic data: 1 H NMR (600 MHz, DMSO) δ 10.50 (brs, 1H), 10.11 (s, 1H),7.85 (d, J = 1.97 Hz, 1H), 7.54 (d, J = 8.70 Hz, 2H), 6.82 (d, J = 8.70 Hz,2H), 6.48 (s, 1H), 2.45 (s, 3H).

[0072] 13 C NMR (151 MHz, DMSO) δ 193.13, 169.64, 159.01, 145.62, 132.42,132.35, 129.05, 125.11, 121.26, 116.09, 29.00.

[0073]

[0074] Yield: 29.4%

[0075] High resolution mass spectrum data: HRMS calcd for C 19 H 23 NNaO3 + [M+Na + ] 336.1570 found336.1568.

[0076] NMR data: 1 H NMR (600 MHz, DMSO) δ 10.48 (brs, 1H), 10.10 (brs, 1H),7.83 (d, J = 2.4 Hz, 1H), 7.53 (d, J = 8.4 Hz, 1H), 6.81 (d, J = 8.4 Hz, 1H),6.46 (s, 1H), 2.87 (t, J = 7.2 Hz, 2H), 1.53 (m, 2H), 1.26 (m, 6H), 0.85 (t,J = 7.2 Hz, 3H). 13 C NMR (151 MHz, DMSO) δ 195.71, 169.60, 158.97, 145.40,132.41, 132.37, 129.01, 125.12, 121.04, 116.07, 40.74, 31.21, 28.64, 28.62,23.34, 22.11, 13.99.

[0077]

[0078] Yield: 27.7%

[0079] High resolution mass spectrum data: HRMS calcd for C 18 H 19 NNaO3 + [M+Na + ] 320.1257 found320.1255.

[0080] NMR data: 1 H NMR (600 MHz, DMSO) δ 10.47 (s, 1H), 10.11(s, 1H), 7.85(d, J = 1.8 Hz, 1H), 7.54 (d, J = 8.4 Hz, 2H), 6.82 (d, J = 8.4 Hz, 2H), 6.46(s, 1H), 3.31 (m, 1H), 1.71 (m, 5H), 1.23 (m, 5H). 13 C NMR (151 MHz, DMSO) δ198.7, 169.5, 159.0, 146.1, 132.5, 132.4, 128.5, 125.1, 121.0, 116.1, 46.9,28.1, 25.6, 25.3.

[0081]

[0082] Yield: 36.6%

[0083] High resolution mass spectrum data: HRMS calcd for C 18 H 17 F2NNaO3 + [M+Na + ] 356.1069 found356.1067. NMR data: 1 H NMR (600 MHz, DMSO) δ 10.55 (s, 1H), 10.14(s, 1H), 7.92(d, J = 1.8 Hz, 1H), 7.56 (d, J = 9.0 Hz, 2H), 6.83 (d, J = 9.0 Hz, 2H), 6.51(s, 1H), 3.46 (m, 1H), 2.07 (m, 2H), 1.90 (m, 4H), 1.53 (m, 2H). 13 C NMR (151MHz, DMSO) δ 197.28, 169.41, 159.14, 146.53, 132.53, 132.44, 128.03, 125.05,123.84, 121.63, 116.12, 44.29, 32.31, 24.46.

[0084]

[0085] Yield: 10.5%

[0086] High resolution mass spectral data: HRMS calcd for C 22 H 23 NNaO3 + [M+Na + ] 372.1570 found372.1569.

[0087] NMR data: 1 H NMR (600 MHz, DMSO) δ 10.43 (s, 1H), 9.99 (s, 1H), 7.54(d, J = 1.8 Hz, 1H), 7.50 (d, J = 9.0 Hz, 2H), 6.81 (d, J = 9.0 Hz, 1H), 6.32(s, 1H), 2.01 (m, 3H), 1.87 (m, 6H), 1.69 (m, 6H). 13 C NMR (151 MHz, DMSO) δ204.16, 169.99, 158.86, 141.96, 133.11, 132.26, 131.40, 125.66, 118.70,116.40, 46.45, 37.31, 36.41, 27.80.

[0088]

[0089] Yield: 22.6%

[0090] High resolution mass spectral data: HRMS calcd for C 22 H 19 NNaO3 + [M+Na + ] 368.1257 found368.1258.

[0091] NMR data: 1H NMR (600 MHz, DMSO) δ 10.60 (s, 1H), 10.16(s, 1H), 7.96(d, J = 1.8 Hz, 1H), 7.58 (d, J = 9.0 Hz, 2H), 7.11 (m, 2H), 7.06 (m, 1H),6.92 (d, J = 7.8 Hz, 1H), 6.84 (d, J = 9.0 Hz, 2H), 6.53 (s, 1H), 4.98 (t, J= 6.0 Hz, 1H), 2.74 (m, 2H), 2.02 (m, 1H), 1.89 (m, 1H), 1.71 (m, 2H). 13 C NMR(151 MHz, DMSO) δ 198.04, 170.01, 159.58, 147.14, 138.00, 135.06, 132.98,132.86, 130.04, 129.40, 128.97, 126.64, 125.96, 125.52, 122.09, 116.57,48.20, 29.15, 26.52, 20.31.

[0092]

[0093] Yield: 16.2%

[0094] High resolution mass spectral data: HRMS calcd for C 21 H 21 NNaO3 + [M+Na + ] 358.1414 found358.1413.

[0095] NMR data: 1 H NMR (600 MHz, DMSO) δ 10.40 (s, 1H), 7.51 (d, J = 8.4 Hz,2H), 7.47 (s, 1H), 6.87 (m, 1H), 6.81 (d, J = 8.4 Hz, 2H), 6.31 (s, 1H), 4.75(m, 2H), 3.17 (s, 1H), 2.42 (m, 2H), 2.17 (m, 3H), 1.85 (m, 1H), 1.74 (s,3H), 1.43 (m, 1H). 13C NMR (151 MHz, DMSO) δ 190.91, 169.99, 158.94, 148.89, 144.92, 142.97, 138.72, 133.01, 132.26, 130.56, 125.63, 118.77, 116.42, 109.91, 49.07, 31.44, 26.79, 23.52, 21.01.

[0096]

[0097] Yield: 4.7%

[0098] High resolution mass spectrum data: HRMS calcd for C 19 H 17 NNaO3 + [M+Na + ] 330.1101 found330.1100.

[0099] NMR data: 1 H NMR (600 MHz, DMSO) δ 10.48 (s, 1H), 10.10(brs, 1H), 7.88(d, J = 1.8 Hz, 1H), 7.55 (d, J = 8.4 Hz, 2H), 6.82 (d, J = 8.4 Hz, 2H), 6.48(s, 1H), 6.21 (m, 2H), 3.25 (m, 1H), 2.91 (m, 2H), 1.85 (m, 1H), 1.30 (m,2H), 1.22 (m, 1H). 13 C NMR (151 MHz, DMSO) δ 197.80, 169.93, 159.45, 146.32, 138.40, 136.65, 132.87, 132.83, 129.25, 125.56, 121.47, 116.53, 48.61, 45.95, 45.89, 41.90, 29.40.

[0100]

[0101] Yield: 23.3%

[0102] High resolution mass spectrum data: HRMS calcd for C 25 H 29 NNaO3 + [M+Na +] 414.2040 found 414.2039.

[0103] NMR data: 1 H NMR (600 MHz, CDCl3) δ 9.13 (s, 1H), 7.68 (d, J = 1.8 Hz,1H), 7.42 (d, J = 8.4 Hz, 2H), 6.93 (d, J = 8.4 Hz, 2H), 6.38 (s, 1H), 5.22(s, 1H), 3.88 (m, 1H), 2.27 (dd, J = 10.2, 17.4 Hz, 1H), 2.10 (dd, J = 6.0,17.4 Hz, 1H), 1.64 (s, 3H), 1.59 (m, 3H), 1.40 (m, 2H), 1.25 (m, 1H), 0.97(s, 3H), 0.87 (m, 2H), 0.81 (d, J = 6.0 Hz, 3H). 13 C NMR (151 MHz, CDCl3) δ201.75, 170.21, 158.12, 144.60, 134.01, 132.70, 131.91, 130.46, 129.40,126.29, 122.46, 116.78, 48.14, 45.92, 44.62, 35.72, 35.61, 34.20, 27.42,24.79, 23.15, 22.75, 20.50.

[0104]

[0105] Data related to compound 2b can be found in the literature (Chinese Journal of Chemistry 2024, 42, 1509-1514.) and the patent application (a synthetic process for a natural product with anticancer activity, Talaroconvolutin A, application number: CN202311866192.X).

[0106]

[0107] Yield: 31.6%

[0108] High-resolution mass spectrometry data: HRMS calcd for C 20 H 15 NNaO3 + [M+Na +] 340.0944 found 340.0942.

[0109] NMR data: 1 H NMR (600 MHz, DMSO) δ 10.60 (s, 1H), 10.17 (s, 1H), 8.04 (d, J = 2.4 Hz, 1H), 7.95 (d, J = 15.6 Hz, 2H), 7.75 (m, 2H), 7.70 (d, J = 15.6 Hz, 2H), 7.59 (d, J = 8.4 Hz, 2H), 7.48 (m, 3H), 6.85 (d, J = 8.4 Hz, 2H), 6.54 (s, 1H). 13 C NMR (151 MHz, DMSO) δ 184.25, 170.26, 159.60, 147.01, 143.09, 135.11, 133.11, 133.02, 131.19, 129.74, 129.56, 129.05, 125.56, 124.66, 122.08, 116.57.

[0110]

[0111] Yield: 13.3%

[0112] High resolution mass spectrum data: HRMS calcd for C 21 H 17 NNaO5 + [M+Na + ] 386.0999 found 386.0998.

[0113] 1 H NMR (600 MHz, DMSO) δ 10.53 (s, 1H), 9.79 (brs, 1H), 7.97 (d, J = 1.8 Hz, 1H), 7.71 (d, J = 15.6 Hz, 1H), 7.63 (d, J = 15.6 Hz, 1H), 7.57 (d, J = 9.0 Hz, 2H), 7.31 (d, J = 2.4 Hz, 1H), 7.23 (m, 1H), 6.85 (m, 3H), 6.50 (s, 1H), 3.84 (s, 3H). 13C NMR (151 MHz, DMSO) δ 184.23, 170.28, 159.44, 150.24, 148.37, 146.33, 144.13, 133.11, 132.87, 130.17, 126.65, 125.62, 123.51, 121.56, 121.42, 116.54, 116.32, 112.67, 56.14.

[0114] Example 2

[0115] (1) Detection of MHC class I molecules on tumor cell surface

[0116] The mouse melanoma cells B16, human lung cancer cells A549, and human breast cancer cells BT-474 in logarithmic growth phase were digested with 0.25% trypsin, and the cell suspensions were adjusted to a concentration of 3 x 10^5 / mL using high-sugar DMEM medium. 1 mL was inoculated into each well of a 12-well plate, and the plate was placed in a 37°C, 5% CO2 constant-temperature carbon dioxide incubator for incubation. After 4 h, the original culture medium was discarded, the experimental group was replaced with high-sugar DMEM medium containing 10 μM of compound 1b, 2b, or 3a, and the negative control (NT) group was replaced with ordinary high-sugar DMEM medium. The plate was placed in a 37°C, 5% CO2 constant-temperature carbon dioxide incubator for incubation for 24 h.

[0117] After the incubation, the cells in the experimental group and the negative control (NT) group were digested with 0.25% trypsin for 2 min, resuspended in a flow tube with ordinary high-sugar DMEM medium, and centrifuged at 1000 rpm for 5 min. The supernatant was discarded. 100 μL of 1 μg / mL H-2K b -PE (B16) or HLA-A / B / C-PE (A549, BT-474) was added to each tube. Incubation was performed on ice for 30 min, and the cells were collected and detected by flow cytometry. MHC-I fold increase = MFI 实验组 / MFI 阴性对照组 .

[0118] The flow cytometry results are shown in Figure 23A 、 Figure 23B 、 Figure 23C, the results show that the NT group has low expression of MHC-I, and the experimental groups treated with compounds 1b, 2b and 3a have high expression of MHC-I in B16, A549 and BT-474 tumor cells. Among them, in A549 cells, the expression of MHC-I after treatment with compound 1b is 1.6 times that of the NT group, the expression of MHC-I after treatment with compound 2b is 5.3 times that of the NT group, and the expression of MHC-I after treatment with compound 3a is 1.3 times that of the NT group; in BT474 cells, the expression of MHC-I after treatment with compound 1b is 2.3 times that of the NT group, the expression of MHC-I after treatment with compound 2b is 7.7 times that of the NT group, and the expression of MHC-I after treatment with compound 3a is 1.8 times that of the NT group; in B16 cells, the expression of MHC-I after treatment with compound 1b is 7.7 times that of the NT group, the expression of MHC-I after treatment with compound 2b is 5.0 times that of the NT group, and the expression of MHC-I after treatment with compound 3a is 1.8 times that of the NT group.

[0119] Further, according to the foregoing method, the tumor cell surface MHC-I molecules of compounds 1a, 1c, 1d, 1e, 1f, 1g, 1h, 2a, 3b in B16, A549, BT-474 tumor cells were detected respectively. The effects of compounds 1a-1h, 2a-2b, 3a-3b on the expression of MHC-I in BT-474, B16 and A549 cells are shown in Table 1. The experimental groups treated with these compounds have high expression of MHC-I in BT-474 cells.

[0120] Table 1 Effects of compounds 1a-1h, 2a-2b, 3a-3b on the expression of MHC-I in BT-474, B16 and A549 cells

[0121]

[0122] (2) Detection of the killing function of OT-I T cells on tumor cells

[0123] The mouse melanoma cell B16 was transfected with OVA antigen plasmid to obtain the mouse melanoma cell B16-OVA stably expressing OVA antigen.

[0124] Take the logarithmic growth phase of mouse melanoma cells B16-OVA, after 0.25% trypsin digestion, using high glucose DMEM medium to adjust the cell suspension concentration to 3x10^5 / mL, take 1 mL inoculated in 12 well plate, the culture plate was placed in 37℃, 5% CO2 constant temperature carbon dioxide incubator. After 4h, the original culture medium was discarded, the experimental group was replaced with high glucose DMEM medium containing 10μM compound 1b, the negative control (NT) group was replaced with ordinary high glucose DMEM medium, and the culture was continued for 24h. After the end of the culture, the experimental group and the negative control (NT) group cells were digested with 0.25% trypsin for 2min, resuspended with ordinary high glucose DMEM medium and centrifuged at 1000rpm for 5min, and the supernatant was discarded. The cell suspension was adjusted to 6x10^5 / mL with high glucose DMEM medium, 0.5mL was taken per well, and the experimental group and the negative control (NT) group cells were inoculated in 12 well plates, and the culture plates were placed in 37℃, 5% CO2 constant temperature carbon dioxide incubator.

[0125] Take the spleen of OT-I mice, grind the tissue with a glass slide with a frosted edge and resuspend with PBS, centrifuge at 1500rpm for 5min, discard the supernatant, add 10mL red cell lysis solution, stand at room temperature for 10min, then centrifuge at 1500rpm for 5min, discard the supernatant. The cell pellet was resuspended with 10mL T cell complete medium (RPMI-1640 medium, add 0.05mM final concentration of β-mercaptoethanol, 20mM HEPES solution, 2mM L-glutamine, 1mM sodium pyruvate), and filtered through a 70μm screen. After cell counting, the cell suspension was adjusted to 3x10^6 / mL with T cell complete medium containing 1μg / mL OVA peptide and inoculated in 12 well plates, and the culture plates were placed in 37℃, 5% CO2 constant temperature carbon dioxide incubator.

[0126] After the end of the culture, resuspend with T cell complete medium and centrifuge at 1500rpm for 5min, discard the supernatant. Adjust the cell suspension concentration to 6x10^5 / mL with T cell complete medium, take 0.5mL per well, and inoculate in 12 well plates containing B16-OVA cells of the experimental group and the negative control (NT) group, respectively, and place the culture plates in 37℃, 5% CO2 constant temperature carbon dioxide incubator for 16h.

[0127] After co-culture, use the apoptosis kit to stain, and use flow cytometry to detect.

[0128] The flow cytometry results are shown in Figure 24, the horizontal axis represents Annexin V-FITC, and the vertical axis represents PI. The results show that the addition of 10 μM compound 1b has little effect on cell apoptosis, while the addition of OT-Ⅰ cell co-culture group significantly increases cell apoptosis, and the addition of 10 μM compound 1b further increases cell apoptosis caused by OT-Ⅰ cell co-culture.

[0129] Summary: The above results show that compound 1b can increase the expression of MHC-Ⅰ of tumor cells, thereby promoting the recognition ability of immune cells to tumors, and can more effectively eliminate tumors.

[0130] (3) Tumor cell viability test

[0131] Take the logarithmic growth period of mouse melanoma cells B16, human lung cancer cells A549, and human breast cancer cells BT-474, and adjust the cell suspension concentration to 3×10^5 / mL after 0.25% trypsin digestion. Take 100 μL and inoculate in a 96-well plate. Place the culture plate in a 37°C, 5% CO2 constant temperature carbon dioxide incubator and incubate. After 4h, discard the original culture medium and replace it with 100 μL of high-sugar DMEM medium containing 0 μM, 1 μM, 2 μM, 5 μM, 10 μM, 20 μM, 30 μM, 40 μM, and 50 μM compound 1b, 2b, and 3a. Place the culture plate in a 37°C, 5% CO2 constant temperature carbon dioxide incubator and incubate for 24h. Perform CCK8 detection.

[0132] CCK8 results are shown in Figure 25A , Figure 25B , Figure 25C The results show that compound 1b at a concentration of 10 μM or less has a small inhibitory effect on the activity of mouse melanoma cells B16, human lung cancer cells A549, and human breast cancer cells BT-474. Compound 2b and 3a can significantly inhibit the activity of some tumor cells at a concentration of 10 μM or more.

[0133] From the above cell experiment results, it can be seen that compounds 1b, 2b, and 3a can be effective inducers of tumor cell surface MHC-Ⅰ, making tumor cells more sensitive to T cell killing in co-culture experiments, but have little direct cytotoxic effect on tumor cells, and have certain safety.

[0134] (4) B16 tumor-bearing experiment

[0135] The mouse subcutaneous tumor experiment was divided into two groups: a control group and a compound 1b administration group. The logarithmic growth period mouse melanoma cells B16 were taken, digested with 0.25% trypsin, resuspended with high-sugar DMEM medium, washed twice with PBS after centrifugation, resuspended with PBS and adjusted to a concentration of 5x10^6 / mL, and 100 μL was injected into the inguinal subcutaneous position of C57 mice. From the seventh day, the compound 1b administration group was injected intraperitoneally with a compound 1b solution according to a total drug amount of 10 mg / kg (3.2 mM), with a drug volume of 200 μL (0.2 mg of drug was dissolved in 20 μL of DMSO, then 80 μL of PEG300 was added, mixed uniformly to be clear, then 10 μL of Tween 80 was added, mixed uniformly to be clear, then 90 μL of PBS was added, mixed uniformly to be clear), and the administration was performed once every 2 days; the control group was administered with PBS intraperitoneally according to 200 μL, and the administration was performed once every 2 days. The tumor size was monitored every 2 days, the tumor growth curve was drawn, the mice were sacrificed on the 20th day after inoculation of B16 tumor cells, the mouse tumor tissue was taken, chopped and ground, then treated with tumor tissue digestion solution to obtain a single cell suspension and subjected to flow staining, and the proportion of different immune cells in the tumor tissue was analyzed.

[0136] The tumor growth line graph is shown in Figure 26 , and the T cell infiltration is shown in Figure 27 The results show that the tumor growth in the compound treatment group is inhibited in the mouse body, and the proportion of CD4+ T cells and CD8+ T cells in CD45+ cells in the tumor infiltration is increased. It is indicated that the compound 1b treatment can improve the tumor microenvironment and increase the infiltration of CD4+ T cells and CD8+ T cells.

[0137] The above shows and describes the basic principles, main features and advantages of the present application. It should be understood by those skilled in the art that the present application is not limited by the above examples, and the above examples and descriptions in the specification are only to illustrate the principles of the present application, and various changes and improvements can be made without departing from the spirit and scope of the present application, and these changes and improvements all fall within the scope of the claimed present application. The scope of protection of the present application is defined by the appended claims and their equivalents.

Claims

1. The application of dihydropyrrolidone derivatives in the preparation of immunotherapy drugs for tumor treatment, characterized in that, The tumor is melanoma, and the structural formula of the dihydropyrrolidone derivative is selected from any one of the following compounds 1a-1h, 2a-2b, and 3a-3b:

2. The application of dihydropyrrolidone derivatives in the preparation of immunotherapy drugs for tumor treatment, characterized in that, The tumor is breast cancer, and the structural formula of the dihydropyrrolidone derivative is selected from any one of the following compounds 1a-1h, 2a-2b, and 3a-3b:

3. The application of dihydropyrrolidone derivatives in the preparation of immunotherapy drugs for tumor treatment, characterized in that, The tumor is lung cancer, and the structural formula of the dihydropyrrolidone derivative is selected from any one of the following compounds 1b and 3a:

4. The application according to any one of claims 1-3, characterized in that, The tumor immunotherapy drug mentioned is an immune checkpoint inhibitor.

5. The application according to any one of claims 1-3, characterized in that, The aforementioned tumor immunotherapy drug can upregulate the expression of MHC-I molecules on the surface of tumor cells, and / or enhance the tumor-killing ability of T cells, improve the tumor microenvironment, and / or increase the infiltration of CD4+ T cells and / or CD8+ T cells.

6. The application according to any one of claims 1-3, characterized in that, The concentration of the dihydropyrrolidone derivative is 0.1–20 μM.

7. The application according to claim 6, characterized in that, The concentration of the dihydropyrrolidone derivative is 10 μM.

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