Preparation method and application of dihydropyrrolone derivative

Abstract

The invention belongs to the technical field of medicines, and particularly discloses a preparation method and application of dihydropyrrolidone derivatives. The invention provides a dihydropyrrolone derivative with efficient tumor immunotherapy activity. The structural formula of the dihydropyrrolone derivative is as shown in the following formula (# imgabs 0 #): # imgabs 1 #. The dihydropyrrolone derivative provided by the invention has good immunosuppressive activity on melanoma, lung cancer and breast cancer cells, and can provide a potential lead compound for developing novel immune drugs.

Description

Technical Field

[0001] The invention belongs to the technical field of medicines, and particularly relates to a preparation method and application of dihydropyrrolidone derivatives. Background Art

[0002] Tumor immunotherapy has been a focus of medical research in recent years. With the deepening of immunological research and advances in science and technology, immunotherapy is gaining increasing prominence in clinical applications, providing more options for cancer treatment. Currently, the main immunotherapy approaches include tumor vaccines, cell therapy, and immune checkpoint inhibitors. Immune checkpoint inhibitors, such as PD-1 / PD-L1 and CTLA-4, have demonstrated significant success in treating multiple cancer types and are approved for second-line or first-line treatment. They are crucial for improving cure rates, prolonging survival, and enhancing patients' quality of life. However, due to factors such as tumor immune escape, lack of T cell infiltration, and a suppressive tumor microenvironment, the proportion of patients who achieve long-term, durable responses from immune checkpoint inhibitors remains low. While combining these approaches with other treatment modalities or existing immune checkpoint inhibitors can improve response rates, these combinations can also be associated with significant toxicities. Furthermore, many patients may develop acquired resistance after an initial response. Therefore, there is an urgent need to identify new targets and combination therapies to address these issues.

[0003] The mechanism of action of immune checkpoint inhibitors is that within the tumor microenvironment, tumor cells overexpress PD-L1, which binds to PD-1 on the surface of T cells, inhibiting T cell function and preventing them from effectively killing tumor cells. PD-1 / PD-L1 inhibitors block this binding, releasing the inhibition on T cells and enabling them to regain their ability to kill tumor cells, thereby achieving a therapeutic effect. However, the effectiveness of this therapy depends not only on restoring T cell function but also on the sensitivity of the tumor cells themselves to immune killing. Tumor cells often employ countermeasures to escape the immune system, significantly hindering the effectiveness of immunotherapy. Therefore, simply improving T cell function to treat tumors is far from sufficient to achieve the desired effect.

[0004] Tumor immune escape refers to the phenomenon in which tumor cells evade recognition and attack by the immune system through various mechanisms, allowing them to survive and proliferate in the body. The main mechanisms of tumor immune escape include antigen loss, overexpression of immune checkpoints, activation of anti-apoptotic cascades, resistance to ferroptosis, downregulation of antigen presentation, and the release of factors that promote immune tolerance. Furthermore, the tumor microenvironment also exerts an immunosuppressive effect, allowing tumor cells to evade immune surveillance. Immune escape is a key cause of resistance to immunotherapy.

[0005] Abnormal expression of MHC-I molecules on the surface of tumor cells is one of the most important causes of tumor immune evasion. The major histocompatibility complex class I (MHC-I or human leukocyte antigen HLA) antigen presentation (AP) pathway is crucial for the activation and proliferation of CD8+ T cells. Tumor cells often evade antitumor immunity by downregulating MHC-I. Previous studies have demonstrated a positive correlation between MHC-I expression and patient prognosis in various cancer types, while MHC-I downregulation is associated with disease progression and poor prognosis in many cancers. Furthermore, because the therapeutic efficacy of immune checkpoint inhibitors relies on cytotoxic T cells recognizing cytoplasmic antigens presented by MHC-I on tumor cells, decreased MHC-I expression is closely associated with resistance to immune checkpoint inhibitor therapy. Currently, research on increasing MHC-I expression is relatively limited. However, studies have shown that treatment with CDK4 and CDK6 inhibitors or the EZH2 inhibitor GSK126 for 7 days can increase MHC-I expression, suggesting that modulating MHC-I expression through pharmacological or therapeutic approaches is feasible. Therefore, exploring more drugs or treatments that can effectively improve MHC-I expression and function is of great significance for improving the response rate of existing immunotherapy.

[0006] Therefore, the design and development of new compounds with highly effective tumor immune activity has very broad application prospects in future cancer treatment. Summary of the Invention

[0007] In view of the shortcomings of the prior art, the present invention aims to provide a method for preparing dihydropyrrolidone derivatives and their immunotherapy application.

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

[0009] Application of dihydropyrrolidone derivatives in the preparation of immune checkpoint inhibitors.

[0010] Application of dihydropyrrolidone derivatives in improving the antigen presentation ability of tumor cells.

[0011] Application of dihydropyrrolidone derivatives in upregulating the expression of MHC-I molecules on the surface of tumor cells.

[0012] The use of dihydropyrrolidone derivatives in the preparation of drugs that induce high expression of MHC-I molecules on the surface of tumor cells and / or enhance the tumor-killing ability of T cells (in tumor cells).

[0013] Application of dihydropyrrolidone derivatives in improving tumor microenvironment and increasing infiltration of CD4+ T cells and / or CD8+ T cells.

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

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

[0016] Here, MHC-I molecule refers to major histocompatibility complex class I.

[0017] The structural formula of the dihydropyrrolidone derivatives is as follows: ) as shown:

[0018]

[0019] Mode( ), 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 Cycloalkene and its monohalogenated or polyhalogenated groups, C 8-10 Any one of benzocycloalkyl and five- to six-membered heterocyclic groups, the halogenated atom is at least one of fluorine, chlorine, bromine and iodine; R2 is hydrogen, 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 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] Preferably, the formula ( ) The compound is selected from any one of the following compounds 1a~1h, 2a~2b, 3a~3b:

[0024]

[0025] More preferably, the 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 invention has the following beneficial effects:

[0027] The dihydropyrrolidone derivatives provided by the present invention have good immunosuppressive activity against melanoma, lung cancer and breast cancer cells. Therefore, the present invention can provide potential lead compounds for the development of new immune drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 1a is the hydrogen spectrum (600 MHz, DMSO) of compound 1a synthesized in Example 1;

[0029] Figure 2 is the carbon spectrum (151 MHz, DMSO) of compound 1a synthesized in Example 1;

[0030] Figure 3 1b is the hydrogen spectrum (600 MHz, DMSO) of compound 1b synthesized in Example 1;

[0031] Figure 4 is the carbon spectrum (151 MHz, DMSO) of compound 1b synthesized in Example 1;

[0032] Figure 5 1 is the hydrogen spectrum (600 MHz, DMSO) of compound 1c synthesized in Example 1;

[0033] Figure 6 is the carbon spectrum (151 MHz, DMSO) of compound 1c synthesized in Example 1;

[0034] Figure 7 1d is the hydrogen spectrum (600 MHz, DMSO) of compound 1d synthesized in Example 1;

[0035] Figure 8 is the carbon spectrum (151 MHz, DMSO) of compound 1d synthesized in Example 1;

[0036] Figure 9 1 is the hydrogen spectrum (600 MHz, DMSO) of compound 1e synthesized in Example 1;

[0037] Figure 10 is the carbon spectrum (151 MHz, DMSO) of compound 1e synthesized in Example 1;

[0038] Figure 11 1f is the hydrogen spectrum (600 MHz, DMSO) of compound 1f synthesized in Example 1;

[0039] Figure 12 is the carbon spectrum (151 MHz, DMSO) of compound 1f synthesized in Example 1;

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

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

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

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

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

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

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

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

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

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

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

[0051] Figure 23B This is the flow cytometry result of compounds 1b, 2b, and 3a on BT-474 cells HLA-A / B / C in Example 2;

[0052] Figure 23C The effects of compounds 1b, 2b, and 3a on B16 cell H-2K b Flow cytometry result graph;

[0053] Figure 24 This is a graph showing the apoptosis flow cytometry results of the detection of the killing function of OT-Ⅰ T cells on B16 cells in Example 2;

[0054] Figure 25A This is a graph showing the CCK8 results of compounds 1b, 2b, and 3a on A549 cell viability in Example 2;

[0055] Figure 25B This is a graph showing the CCK8 results of compounds 1b, 2b, and 3a on BT-474 cell viability in Example 2;

[0056] Figure 25C This is a graph showing the CCK8 results of compounds 1b, 2b, and 3a on B16 cell viability in Example 2;

[0057] Figure 26 This is the discounted rate graph of compound 1b on B16 tumor growth in Example 2;

[0058] Figure 27 This is a statistical graph showing the effect of compound 1b in Example 2 on T cell infiltration in B16 tumors. DETAILED DESCRIPTION

[0059] The present invention is described in further detail below with reference to the embodiments.

[0060] Those skilled in the art will understand that the following examples are intended to illustrate the present invention only and should not be construed as limiting the scope of the present invention. Where specific techniques or conditions are not specified in the examples, the techniques or conditions described in the literature in the art or in the product specifications were used. Materials or equipment used without manufacturer identification are commercially available conventional products.

[0061] Example 1

[0062] Synthesis of dihydropyrrolidone derivatives

[0063]

[0064] Note: 1b - C7H 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 the patent application (A synthesis process of the natural product Talaroconvolutin A with anticancer activity, application number: CN202311866192.X).

[0066] Under nitrogen, compound 4 (0.5 mmol) was dissolved in 6 mL of tetrahydrofuran and cooled to -78°C. A solution of LiHMDS (0.6 mmol) in tetrahydrofuran was slowly added dropwise. After a 45-min reaction, a solution of an aldehyde (1.0 mmol) in tetrahydrofuran (most of the aldehydes used were commercially available. Only the aldehyde used in the preparation of compound 2a required reference to the synthesis of aldehydes in the synthesis of Talaroconvolutin A) and boron trifluoride etherate (1.0 mmol) were added dropwise. The reaction was continued for 3 hours. The mixture was quenched by the addition of a saturated sodium bicarbonate solution, extracted three times with ethyl acetate, washed with a saturated sodium chloride solution, and dried over anhydrous sodium sulfate. The mixture was concentrated under reduced pressure and purified by silica gel column chromatography to obtain an oily compound. The oil was dissolved in 4 mL of dichloromethane and cooled to zero degrees Celsius. Pyridine (3.6 mmol), water (1.98 mmol), and Dess-Martin periodinane (1.8 mmol) were added sequentially, and the reaction was continued at room temperature for 1 hour. The reaction was quenched with a mixed solution (saturated sodium bicarbonate and 10% sodium thiosulfate, 1:1 volume ratio). The aqueous phase was extracted three times with dichloromethane, and the organic phase was washed with saturated sodium chloride, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by silica gel column chromatography to afford a yellow oily compound. This oil was dissolved in 4 mL of methanol, and p-toluenesulfonic acid monohydrate (0.11 mmol) was added. The mixture was heated to 50°C for 6 hours. The precipitated solid was cooled, filtered, and washed with methanol to afford the desired products 1a-1h, 2a-2b, and 3a-3b.

[0067] The yields, high-resolution mass spectrometry data, H NMR spectra, and C NMR spectra of compounds 1a-1h, 2a-2b, and 3a-3b are as follows:

[0068]

[0069] Yield: 27.4%

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

[0071] NMR 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 spectrometry data: HRMS calculation 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 spectrometry data: HRMS calculation 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 spectrometry data: HRMS calculation 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 spectrometry data: HRMS calculation 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 spectrometry data: HRMS calculation 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 spectrometry data: HRMS calculation 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 spectrometry data: HRMS calculation 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 spectrometry data: HRMS calculation for C 25 H 29 NNaO3 + [M+Na +] 414.2040 found414.2039.

[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] The relevant data of compound 2b can be found in the literature (Chinese Journal of Chemistry 2024, 42, 1509-1514.) and the patent application (A synthesis process of the natural product Talaroconvolutin A with anticancer activity, application number: CN202311866192.X).

[0106]

[0107] Yield: 31.6%

[0108] High-resolution mass spectrometry data: HRMS calculation for C 20 H 15 NNaO3 + [M+Na +] 340.0944 found340.0942.

[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 spectrometry data: HRMS calculation for C 21 H 17 NNaO5 + [M+Na + ] 386.0999 found386.0998.

[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 the surface of tumor cells

[0116] Mouse melanoma B16 cells, human lung cancer A549 cells, and human breast cancer BT-474 cells in the logarithmic growth phase were digested with 0.25% trypsin and then suspended in high-glucose DMEM medium to a concentration of 3 × 10^5 cells / mL. 1 mL was plated per well in a 12-well plate and incubated in a 37°C, 5% CO2 incubator. After 4 hours, the original medium was discarded and replaced with high-glucose DMEM medium containing compounds 1b, 2b, and 3a at a concentration of 10 μM for the experimental group and standard high-glucose DMEM medium for the negative control (NT) group. The plates were then incubated in a 37°C, 5% CO2 incubator for 24 hours.

[0117] After the culture, the cells of the experimental group and the negative control (NT) group were digested with 0.25% trypsin for 2 minutes, resuspended in ordinary high-glucose DMEM medium, aspirated into the flow tube, and centrifuged at 1000 rpm for 5 minutes, and the supernatant was discarded. 100 μL of 1 μg / mL H-2K was added to each tube. b -PE (B16) or HLA-A / B / C-PE (A549, BT-474). Incubate on ice for 30 minutes, collect cells and analyze by flow cytometry. MHC-Ⅰ enhancement factor = MFI 实验组 / MFI 阴性对照组 .

[0118] The flow cytometry results are shown in Figure 23A 、 Figure 23B 、 Figure 23CThe results showed that the NT group expressed low MHC-Ⅰ, while the experimental groups treated with compounds 1b, 2b, and 3a all expressed high MHC-Ⅰ in the three tumor cells B16, A549, and BT-474. In A549 cells, the MHC-Ⅰ level increased by 1.6 times after treatment with compound 1b, by 5.3 times after treatment with compound 2b, and by 1.3 times after treatment with compound 3a, compared with that in the NT group; in BT474 cells, the MHC-Ⅰ level increased by 2.3 times after treatment with compound 1b, by 7.7 times after treatment with compound 2b, and by 1.8 times after treatment with compound 3a, compared with that in the NT group; in B16 cells, the MHC-Ⅰ level increased by 7.7 times after treatment with compound 1b, by 5.0 times after treatment with compound 2b, and by 1.8 times after treatment with compound 3a, compared with that in the NT group.

[0119] Furthermore, compounds 1a, 1c, 1d, 1e, 1f, 1g, 1h, 2a, and 3b were tested for MHC class I expression on the surface of B16, A549, and BT-474 tumor cells, respectively, using the aforementioned method. The effects of compounds 1a-1h, 2a-2b, and 3a-3b on MHC class I expression in BT-474, B16, and A549 cells are shown in Table 1. All experimental groups treated with these compounds showed high MHC class I expression in BT-474 cells.

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

[0121]

[0122] (2) Detection of OT-Ⅰ T cell cytotoxicity against tumor cells

[0123] Mouse melanoma cells B16 were transfected with OVA antigen plasmid to obtain mouse melanoma cells B16-OVA stably expressing OVA antigen.

[0124] Logarithmically growing mouse melanoma B16-OVA cells were digested with 0.25% trypsin and then suspended in high-glucose DMEM medium to a concentration of 3 × 10^5 cells / mL. 1 mL of the suspension was seeded into a 12-well plate and incubated in a 37°C, 5% CO2 incubator. After 4 hours, the culture medium was discarded and replaced with high-glucose DMEM medium containing 10 μM compound 1b for the experimental group and with standard high-glucose DMEM medium for the negative control (NT) group. Culture was continued for 24 hours. Following the incubation period, cells in both the experimental and negative control (NT) groups were digested with 0.25% trypsin for 2 minutes, resuspended in standard high-glucose DMEM medium, and centrifuged at 1000 rpm for 5 minutes. The supernatant was discarded. The cell suspension concentration was adjusted to 6 × 10^5 cells / mL using high-glucose DMEM medium, 0.5 mL was taken from each well, and the cells in the experimental group and negative control (NT) group were inoculated into 12-well plates. The culture plates were placed in a constant temperature carbon dioxide incubator at 37°C and 5% CO2 for static culture.

[0125] Spleens from OT-I mice were minced using the frosted edge of a glass slide and resuspended in PBS. The tissue was centrifuged at 1500 rpm for 5 minutes, the supernatant discarded, and 10 mL of red blood cell lysis buffer was added and allowed to stand at room temperature for 10 minutes. The pellet was then centrifuged at 1500 rpm for 5 minutes, and the supernatant discarded. The cell pellet was resuspended in 10 mL of complete T cell culture medium (RPMI-1640 medium supplemented with a final concentration of 0.05 mM β-mercaptoethanol, 20 mM HEPES, 2 mM L-glutamine, and 1 mM sodium pyruvate) and filtered through a 70 μm mesh. After counting the cells, the cell suspension was adjusted to a concentration of 3 × 10^6 cells / mL using complete T cell culture medium supplemented with 1 μg / mL OVA peptide. The cells were seeded into 12-well plates and incubated in a 37°C, 5% CO2 incubator for 4 hours.

[0126] After the incubation period, cells were resuspended in complete T cell culture medium and centrifuged at 1500 rpm for 5 minutes, and the supernatant was discarded. The cell suspension concentration was adjusted to 6 × 10^5 cells / mL using complete T cell culture medium. 0.5 mL was plated into each well of a 12-well plate containing experimental and negative control (NT) B16-OVA cells. The plates were then placed in a 37°C, 5% CO2 incubator for 16 hours.

[0127] After co-culture, cells were stained with an apoptosis kit and detected by flow cytometry.

[0128] The flow cytometry results are shown in Figure 24The horizontal axis represents Annexin V-FITC, and the vertical axis represents PI. The results showed that the addition of 10 μM compound 1b had little effect on cell apoptosis, while the apoptosis ratio of the OT-Ⅰ cell co-culture group was significantly increased. The addition of 10 μM compound 1b further increased the 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 in tumor cells, thereby promoting the ability of immune cells to recognize tumors and effectively eliminate tumors.

[0130] (3) Cell viability detection of tumor cells

[0131] Mouse melanoma B16 cells, human lung cancer A549 cells, and human breast cancer BT-474 cells in the logarithmic growth phase were digested with 0.25% trypsin and then suspended in high-glucose DMEM medium to a concentration of 3 × 10^5 cells / mL. 100 μL of the suspension was seeded into a 96-well plate and the plate was incubated at 37°C in a 5% CO2 incubator. After 4 hours, the medium was discarded and replaced with 100 μL of high-glucose DMEM medium containing compounds 1b, 2b, and 3a at concentrations of 0 μM, 1 μM, 2 μM, 5 μM, 10 μM, 20 μM, 30 μM, 40 μM, and 50 μM. The plate was then incubated at 37°C in a 5% CO2 incubator for 24 hours. CCK8 assay was performed.

[0132] CCK8 results are shown in Figure 25A 、 Figure 25B 、 Figure 25C The results showed that compound 1b had a low inhibitory rate on the activity of mouse melanoma cells B16, human lung cancer cells A549, and human breast cancer cells BT-474 at concentrations below 10 μM. Compounds 2b and 3a significantly inhibited the activity of some tumor cells at concentrations of 10 μM and above.

[0133] The above cell experiment results show that compounds 1b, 2b, and 3a can serve as effective inducers of MHC-I on the surface of tumor cells, making tumor cells more sensitive to T cell killing in co-culture experiments, but have less direct cytotoxic effects on tumor cells and have a certain safety.

[0134] (4) B16 tumor-bearing experiment

[0135] The subcutaneous tumor formation experiment in mice was conducted in two groups: a control group and a compound 1b-treated group. Logarithmically growing mouse melanoma B16 cells were digested with 0.25% trypsin and resuspended in high-glucose DMEM medium. After centrifugation, the cells were washed twice with PBS and resuspended in PBS at a concentration of 5×10^6 cells / mL. 100 μL of the suspension was injected subcutaneously into the groin of C57 mice. Starting on day 7, the compound 1b-treated group received intraperitoneal injections of 200 μL of a 10 mg / kg (3.2 mM) compound 1b solution (0.2 mg dissolved in 20 μL DMSO, 80 μL PEG 300 added, mixed until clear, 10 μL Tween 80 added, mixed until clear, and 90 μL PBS added, mixed until clear). Dosing was repeated every two days. The control group received intraperitoneal injections of 200 μL of PBS every two days. The tumor size was monitored every 2 days, and a tumor growth curve was drawn. The mice were killed on the 20th day after inoculation with B16 tumor cells. The tumor tissues were collected, chopped and ground, and then processed into single-cell suspensions using tumor tissue digestion fluid and flow cytometry staining to analyze the proportions of different immune cells in the tumor tissues.

[0136] Tumor growth line graph Figure 26 , T cell infiltration Figure 27 The results showed that tumor growth in mice treated with the compound was inhibited, and the proportion of tumor-infiltrating CD4+ T cells and CD8+ T cells in CD45+ cells increased. This indicates that compound 1b treatment can improve the tumor microenvironment and increase the infiltration of CD4+ T cells and CD8+ T cells.

[0137] The basic principles, main features, and advantages of the present invention are shown and described above. Those skilled in the art should understand that the present invention is not limited to the foregoing embodiments. The foregoing embodiments and descriptions are merely illustrative of the principles of the present invention. Various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention. Such changes and modifications are intended to fall within the scope of the present invention. The scope of protection claimed in the present invention is defined by the appended claims and their equivalents.

Claims

1. The use of dihydropyrrolidone derivatives in the preparation of tumor immunotherapy drugs, characterized in that: The structural formula of the dihydropyrrolidone derivatives is as follows: ) as shown: ， Mode( ), R is C 1-28 Aliphatic hydrocarbon groups, aromatic hydrocarbon groups, heterocyclic groups and their derivative groups.

2. The use of dihydropyrrolidone derivatives in the preparation of immune checkpoint inhibitors, characterized in that: The structural formula of the dihydropyrrolidone derivative is shown in claim 1.

3. The use of dihydropyrrolidone derivatives in improving the antigen presentation ability of tumor cells, characterized in that: The structural formula of the dihydropyrrolidone derivative is shown in claim 1.

4. Use of dihydropyrrolidone derivatives in upregulating the expression of MHC-I molecules on the surface of tumor cells, characterized in that: The structural formula of the dihydropyrrolidone derivative is shown in claim 1.

5. Use of dihydropyrrolidone derivatives in the preparation of drugs for inducing high expression of MHC-I molecules on the surface of tumor cells and / or enhancing the tumor killing ability of T cells, characterized in that: The structural formula of the dihydropyrrolidone derivative is shown in claim 1.

6. Use of dihydropyrrolidone derivatives in improving tumor microenvironment and increasing infiltration of CD4+ T cells and / or CD8+ T cells, characterized in that: The structural formula of the dihydropyrrolidone derivative is shown in claim 1.

7. The use according to any one of claims 1 to 6, characterized in that: Mode( ) The structural formula of the compound is shown as follows: Compounds 1, 2, and 3: ； Where R1 is C 1-9 Alkyl, C 6-10 Alicyclic hydrocarbon groups and their monohalogenated or polyhalogenated groups, C 6-10 Cycloalkene and its monohalogenated or polyhalogenated groups, C 8-10 Any one of benzocycloalkyl and five- to six-membered heterocyclic groups, the halogenated atom is at least one of fluorine, chlorine, bromine and iodine; R2 is hydrogen, 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 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.

8. The use according to any one of claims 1 to 6, characterized in that: Mode( ) The compound is selected from any one of the following compounds 1a~1h, 2a~2b, 3a~3b: 。 9. The use according to any one of claims 1 to 6, characterized in that: Such tumors include solid tumors such as melanoma, lung cancer, and breast cancer.

10. The use according to any one of claims 1 to 6, characterized in that: The concentration of the dihydropyrrolidone derivative is 0.1-20 μM, preferably 10 μM.

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