Indole amide derivatives and their mechanism of inhibiting tumor growth through autophagy apoptosis

Abstract

The present application belongs to the field of medicine synthesis, and provides an indole amide derivative as shown in formula I, and pharmaceutically acceptable salts, hydrates, solvates or prodrugs of the compound, a preparation method thereof and the use of the compound as a tumor treatment, especially as an LSD1 inhibitor.

Description

Technical Field

[0001] This invention belongs to the field of drug synthesis and relates to indoleamide derivatives, as well as pharmaceutically acceptable salts, hydrates, solvates or prodrugs of said compounds, their preparation methods and their use in treating tumors, particularly as LSD1 inhibitors, specifically involving the study of indoleamide derivatives and their mechanism of inhibiting tumor growth through autophagy and apoptosis. Background Technology

[0002] Cancer is one of the major diseases threatening human health, primarily caused by the relentless division and metastasis of cancer cells. Cancer severely impacts human health, imposing a tremendous burden on affected families and society. According to a World Health Organization report, the number of new cancer cases worldwide (an average of over 14 million per year) and the number of cancer-related deaths (approximately 8.8 million deaths annually, accounting for one-sixth of all deaths) have been increasing year by year over the past decade. Cancer treatment remains a global challenge. Traditional chemotherapy methods are prone to causing severe toxic side effects, reducing patients' quality of life. Currently, the development of targeted therapies offers relatively better safety and tolerability, and has become a major direction in the development of anti-tumor drugs.

[0003] Histone lysine-specific demethylase 1 (LSD1) is the first discovered flavin adenine dinucleotide-dependent demethylase, belonging to the amine oxidase family. With the participation of the cofactor flavin adenine dinucleotide (FAD), LSD1 specifically removes monomethylation and dimethylation of H3K4 and H3K9 through the oxidation reaction that produces formaldehyde. Located in the cell nucleus, it acts as a histone methylation eraser. Numerous studies have shown that LSD1 and its downstream target proteins are involved in a wide range of biological functions, including embryonic development and tumor growth and metastasis. Due to its high expression in various malignant tumors, inactivating or downregulating LSD1 expression can be an effective method for inhibiting tumor cells. Researching and developing highly effective and low-toxicity LSD1 inhibitors has become a new approach for the prevention and treatment of tumors. Studies have shown that LSD1 is closely related to autophagy and is considered a negative regulator of autophagy. LSD1 can inhibit autophagy through autophagy-related pathways such as the SESN2-mTOR and Akt / mTOR pathways. It can also inhibit autophagy by affecting the synthesis of the key autophagy complex ATG5-ATG12-ATG16 and disrupting p62 protein stability. Furthermore, in tumor cells such as ovarian cancer, LSD1 has been shown to negatively regulate autophagy; decreased LSD1 activity or low expression can promote mTOR-mediated autophagy. However, the regulation of autophagy via LSD1 as a switch is subject to co-regulation by different pathways and autophagy-related genes and proteins, and the relationships and interactions between these pathways and their impact on LSD1 are not yet fully understood.

[0004] To obtain highly active LSD1 inhibitors with novel scaffolds, we used LSD1 as the target protein and combined it with previously reported LSD1 inhibitors. We then modified its structure by using scaffold transitions to design and synthesize a class of indoleamide derivatives with outstanding LSD1 inhibitory activity. Summary of the Invention

[0005] This invention addresses the shortcomings of existing technologies by providing an indoleamide derivative, its geometric isomer, or a pharmaceutically acceptable salt, hydrate, solvate, or prodrug thereof, and its application as an LSD1 inhibitor. Specifically, it relates to the study of indoleamide derivatives and their mechanism of inhibiting tumor growth through autophagy and apoptosis.

[0006] To achieve the above objectives, the general structural formula of the indoleamide derivatives provided by the present invention is as follows: , in, R1 is selected from hydrogen, C1-C6 alkyl, halogen-substituted C1-C6 alkyl, or C1-C6 alkoxy; R2 is selected from C1-C6 aliphatic amines, 5-7 member alicyclic amines, and N-alkyl substituted 5-7 member alicyclic amines; Preferably, R1 is selected from C1-C3 alkyl groups or halogen-substituted C1-C3 alkyl groups; Preferably, R2 is selected from tetrahydropyrrole, piperidine, N-methylpiperidine, and C2-C4 fatty amines.

[0007] The structure of the indoleamide derivative is as follows: .

[0008] Furthermore, according to some common methods in the field to which this invention pertains, some compounds of general formula (I) in this invention have basic groups and can react with acids to form pharmaceutically acceptable salts. Pharmaceutically acceptable addition salts include addition salts of inorganic and organic acids, with salts that add to the following acids being particularly preferred: hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, benzenesulfonic acid, naphthalenedisulfonic acid, acetic acid, propionic acid, lactic acid, trifluoroacetic acid, maleic acid, citric acid, fumaric acid, oxalic acid, tartaric acid, benzoic acid, etc. Hydrochloric acid is most preferred.

[0009] In this invention, "hydrate" refers to an associative compound formed when the solvent molecules are water.

[0010] The term "solvent" in this invention refers to an association formed by one or more solvent molecules with the compound of this invention. Solvents that form solvates include, but are not limited to, water, isopropanol, ethanol, methanol, dimethyl sulfoxide, ethyl acetate, acetic acid, and aminoethanol.

[0011] The present invention also includes prodrugs of the derivatives of the present invention. The prodrugs of the derivatives of the present invention are derivatives of general formula (I), which may have weak or no activity on their own, but are converted into the corresponding biologically active form under physiological conditions (e.g., through metabolism, solvation or other means) after administration.

[0012] Compounds of general formula (I) can exist in both unsolvable and solvable forms containing pharmaceutically acceptable solvents (such as water, ethanol, etc.). Compounds of general formula (I) may contain asymmetric or chiral centers and therefore can exist in different stereoisomers. All stereoisomers of the present invention, including but not limited to diastereomers, enantiomers, and transisomers, as well as mixtures thereof (such as racemic mixtures), are included within the scope of the present invention.

[0013] The compound represented by general formula (I) can exist in different tautomer forms, all of which are included within the scope of this invention. The terms "tautomer" or "tautomer form" refer to structural isomers with different energies that interconvert via low energy barriers.

[0014] In this invention, "alkyl" refers to a straight-chain or branched alkyl group, wherein the C1-C6 group refers to the part having 1-6 carbon atoms, that is, the group contains 1, 2, 3, 4, 5 or 6 carbon atoms.

[0015] In this invention, "alkoxy" refers to alkyl ether alkyl, such as methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, and tert-butoxy.

[0016] In this invention, "halogen" refers to fluorine, chlorine, bromine, or iodine.

[0017] This invention can contain derivatives of general formula (I), and pharmaceutically acceptable salts, hydrates, solvates, or prodrugs as active ingredients, mixed with pharmaceutically acceptable carriers or excipients to prepare compositions, and formulated into clinically acceptable dosage forms. The aforementioned pharmaceutically acceptable excipients refer to any diluent, adjuvant, and / or carrier that can be used in the pharmaceutical field. The derivatives of this invention can be used in combination with other active ingredients, provided they do not produce other adverse effects, such as allergic reactions.

[0018] The derivatives of this invention containing general formula (I) can be synthesized by methods well known in the field of chemistry, especially according to the description of this invention. The methods for preparing the derivatives of this invention can all be carried out according to route 1, specifically as follows: Intermediate 2 was produced by reacting 4H-thiophene[3,2-b]pyrrole-5-carboxylic acid ethyl ester with a haloalkane under alkaline conditions, followed by hydrolysis to obtain key intermediate 3. Intermediate 5 was obtained by coupling p-hydroxybenzaldehyde with a hydroxy heterocycle, followed by reductive halogenation to obtain key intermediate 6.

[0019] Synthesis of the target compound: Following the synthetic method of route 2, 7-aminoindole was used as the starting material. It was first protected with Boc to obtain intermediate 8, which then underwent a substitution reaction with the key intermediate 6 under alkaline conditions to obtain intermediate 9. After deprotection with Boc in hydrochloric acid and methanol, intermediate 10 was obtained. Finally, intermediate 10 and intermediate 3 were condensed under HATU to obtain the target compound.

[0020] The synthetic route is as follows.

[0021] .

[0022] Route 1 Reagents and conditions: (a) NaH, DMF, rt; (b) NaOH, MeOH, rt; (c) corresponding alcohols, PPh3, DIAD, THF, −20°C; (d) i) NaBH4, MeOH, 0°C; ii) I2, PPh3, DCM.

[0023] .

[0024] Route 2 Reagents and conditions: (a) Boc2O, THF, 0°C; (b) NaH, DMF, rt; (c) 2N HCl / MeOH, rt; (d) HATU, DIPEA, DMF, rt.

[0025] The anti-tumor drugs described in this invention specifically target breast cancer, lung cancer, stomach cancer, and prostate cancer.

[0026] The invention has significant technical advantages.

[0027] Based on literature review, the inventors designed and synthesized a series of indoleamide derivatives using computer-aided drug design techniques. These compounds exhibit significant LSD1 inhibitory activity, possess a novel scaffold, and can significantly inhibit tumor cell growth. Experimental results show that the indoleamide derivatives synthesized in our laboratory possess strong antitumor activity and can be used to prepare antitumor drugs, demonstrating promise for clinical development. Attached Figure Description

[0028] Figure 1Example 1: Inhibition of breast cancer MCF-7 (Figure A), prostate cancer PC3 (Figure B), and lung cancer A549 (Figure C) cell viability assays. *P<0.05; **P<0.01; ***P<0.001.

[0029] Figure 2 Example 1: Determination of the inhibitory activity of combined death inhibitors on lung cancer A549. The small molecule inhibitors included 3-MA (autophagy inhibitor), Ferrostatin (ferroptosis inhibitor), Z-VAD-FMk (apoptosis inhibitor), and NAC (antioxidant inhibitor). *P<0.05; **P<0.01; ***P<0.001.

[0030] Figure 3 Example 1: After A549 cells were treated, flow cytometry was used to test the apoptosis of A549 cells.

[0031] Figure 4 Western blot was used to detect changes in marker apoptosis proteins. The first lane was the blank control group, the second lane was the test cells with 0.78125 μM drug, the third lane was the test cells with 1.5625 μM drug, and the fourth lane was the test cells with 3.125 μM drug. Detailed Implementation

[0032] The following examples are intended to illustrate, but not limit, the scope of the invention. The proton NMR spectra of the compounds were determined using a Bruker ARX-400, and the mass spectra were determined using an Agilent 1100 LC / MS. The raw materials are generally available from commercial sources or prepared using methods well known to those skilled in the art, or prepared according to the methods described in this invention. Unless otherwise specified, all reagents used are of analytical or chemical purity.

[0033] Synthesis of Example 1.

[0034] .

[0035] Step 1: Synthesis of ethyl 4-methyl-4H-thiopheno[3,2-b]pyrrole-5-carboxylate.

[0036] Sodium hydride (0.74 g, 30.73 mmol) was added to a DMF solution of ethyl 4H-thiophene[3,2-b]pyrrole-5-carboxylate (2.00 g, 10.24 mmol), and the mixture was stirred at room temperature for 30 min. Then, methyl iodoform (4.36 g, 30.73 mmol) was added, and the reaction was continued at room temperature for 6 h. After the reaction was complete as detected by TLC, the reaction solution was poured into 100 mL of water, and a solid precipitated. The solid was filtered and dried to give 1.64 g of a pale yellow solid, yield 76.5%. ESI-MS m / z: 210.2 [M+H]+ .

[0037] Step 2: Synthesis of 4-methyl-4H-thieno[3,2-b]pyrrole-5-carboxylic acid.

[0038] Ethyl 4-methyl-4H-thiophene[3,2-b]pyrrole-5-carboxylate (1.0 g, 4.78 mmol) was dissolved in 20 mL of methanol, and then 5 mL of 2N sodium hydroxide was added and stirred at room temperature for 6 h. The reaction was confirmed by TLC. The methanol was removed under reduced pressure, and the pH was adjusted to 5-6 with 1N hydrochloric acid, resulting in the precipitation of a white solid. The solid was filtered to give 0.79 g of product, with a yield of 91.2%.

[0039] Step 3: Preparation of N-methyl-4-piperidine-4-oxybenzaldehyde.

[0040] p-Hydroxybenzaldehyde (2.0 g, 16.38 mmol), N-methyl-4-hydroxypiperidine (1.9 g, 16.38 mmol), and triphenylphosphine (6.4 g, 24.57 mmol) were dissolved in anhydrous tetrahydrofuran. The mixture was cooled to -20 °C, and DIAD (4.97 g, 24.57 mmol) was slowly added dropwise. The mixture was gradually brought to room temperature and reacted for 12 h. The reaction was monitored by TLC until complete. The solvent was evaporated under reduced pressure, and the mixture was extracted with 40 mL of water and 30 mL of ethyl acetate. The organic layer was washed with saturated brine and dried overnight in Na₂SO₄. The drying agent was filtered off, and the solvent was evaporated under reduced pressure. The residue was purified by silica gel column chromatography to give 3.05 g of a white solid, with a yield of 84.93%. ESI-MS m / z: 220.3 [M+H] + .

[0041] Step 44: Synthesis of (1-methylpiperidin-4-oxy)phenylmethanol.

[0042] N-methyl-4-piperidin-4-oxybenzaldehyde (3.0 g, 13.68 mmol) was dissolved in methanol. After cooling to 0 °C, sodium borohydride (0.78 g, 20.52 mmol) was added in portions, and the mixture was gradually heated to room temperature. After 2 h, the reaction was detected by TLC to indicate completion. The reaction was quenched by adding saturated ammonium chloride, and the solvent was evaporated under reduced pressure. Then, 40 mL of water and 30 mL of ethyl acetate were added for extraction. The organic layer was washed with saturated brine and dried overnight with Na2SO4. The drying agent was filtered off, and the solvent was evaporated under reduced pressure. The residue was directly used for the next reaction.

[0043] Step 54: Preparation of (4-(iodomethyl)phenoxy)-1-methylpiperidine.

[0044] Iodine (3.47 g, 13.68 mmol) and triphenylphosphine (3.59 g, 13.68 mmol) were dissolved in dichloromethane. After stirring at room temperature for 1 h, the above-mentioned 4-(1-methylpiperidin-4-oxy)phenylmethanol was added, and the reaction was continued at room temperature for 6 h. The reaction was detected by TLC to indicate completion. Then, 50 mL of water was added, and the mixture was extracted with dichloromethane. The organic layer was washed with saturated brine and dried overnight with Na2SO4. The drying agent was filtered off, the solvent was removed by vacuum distillation, and the residue was purified by silica gel column chromatography to give 2.15 g of a pale yellow solid, with a yield of 47.45%. ESI-MS m / z: 332.2 [M+H] + .

[0045] Step 6: Preparation of N-Boc-7-aminoindole.

[0046] 7-Aminoindole (2.00 g, 15.13 mmol) was dissolved in tetrahydrofuran, and the mixture was cooled to 0 °C in an ice bath. Then, a tetrahydrofuran solution of di-tert-butyl dicarbonate (3.30 g, 15.13 mmol) was slowly added dropwise. After the addition was complete, the reaction was continued at 0 °C for 2 h. The reaction was detected by TLC to indicate completion. The solvent was removed by vacuum distillation, and the residue was purified by silica gel column chromatography to give 3.02 g of a white solid, yield: 85.92%.

[0047] Step 7: Preparation of N-Boc-1-(4-(1-methylpiperidin-4-oxy)benzyl)-1H-7-aminoindole.

[0048] N-Boc-7-aminoindole (2.00 g, 8.61 mmol) was dissolved in DMF, and sodium hydride (0.69 g, 17.22 mmol) was added. The mixture was stirred at room temperature for 10 min, and then 4-(4-(iodomethyl)phenoxy)-1-methylpiperidine (2.45 g, 8.61 mmol) was added. The reaction was continued for 2 h, and the reaction was detected by TLC to be complete. The reaction solution was poured into water, and a solid precipitated. The solid was filtered to obtain 3.04 g of the target product, with a yield of 84.15%.

[0049] Step 81: Preparation of (4-(1-methylpiperidin-4-oxy)benzyl)-1H-7-aminoindole.

[0050] N-Boc-1-(4-(1-methylpiperidin-4-oxy)benzyl)-1H-7-aminoindole (3.00 g, 6.89 mmol) was dissolved in methanol, and 5 mL of 3N hydrochloric acid-methanol solution was added. The reaction was carried out at room temperature for 2 h. The reaction was detected by TLC to indicate completion. The solvent was removed under reduced pressure, and the pH was adjusted to 9-10 with water. Then, 30 mL of ethyl acetate was added for extraction, and the organic layer was washed with saturated brine and dried over Na2SO4 overnight. The drying agent was filtered off, and the solvent was removed under reduced pressure to give 2.01 g of white solid, yield 88.0%.

[0051] Step 9: Synthesis of the target compound.

[0052] 4-Methyl-4H-thieno[3,2-b]pyrrole-5-carboxylic acid (0.54 g, 2.98 mmol) was dissolved in DMF. Then, HATU (1.36 g, 3.58 mmol), DIEA (0.77 g, 5.96 mmol), and 1-(4-(1-methylpiperidin-4-oxy)benzyl)-1H-7-aminoindole (1.00 g, 2.98 mmol) were added sequentially to the solution, and the mixture was heated to 70°C. After 8 hours, the reaction was detected by TLC to indicate completion. The reaction solution was poured into water, and a solid precipitated. The crude product was filtered and purified by silica gel column chromatography to obtain 0.86 g of a white powdery solid, with a yield of 57.8%.

[0053] 1 H NMR (400 MHz, DMSO-d6) δ 9.22 (s, 1H), 7.78 (d, J = 8.0 Hz, 1H), 7.45(d, J = 5.4 Hz, 1H), 7.44 (d, J = 7.8 Hz, 1H), 7.30(d, J = 8.0 Hz, 2H),7.14 (d, J = 5.4 Hz, 1H), 6.86 (s, 1H), 6.74(d, J = 8.0 Hz, 2H), 6.38-6.30(m, 3H),4.65 (s, 2H), 4.18 (m, 1H), 3.93 (s, 3H), 2.64–2.56 (m, 2H),2.18 (s,3H), 2.15 -2.10 (m, 2H), 1.90-1.81 (m, 2H), 1.62-1.53 ​​(m, 2H). ESI-MS m / z:499.4[M+H] + .

[0054] Following the method of Example 1, using 4H-thiophene[3,2-b]pyrrole-5-carboxylic acid ethyl ester, p-hydroxybenzaldehyde, and 7-aminoindole as raw materials, Examples 2-8 were obtained sequentially according to the synthesis method of Example 1.

[0055] Example 2.

[0056] .

[0057] 1H NMR (400 MHz, DMSO-d6) δ 9.23 (s, 1H), 7.79 (d, J = 8.2 Hz, 1H), 7.44(d, J = 5.4 Hz, 1H), 7.45 (d, J = 7.8 Hz, 1H), 7.31(d, J = 8.0 Hz, 2H),7.15 (d, J = 5.4 Hz, 1H), 6.85 (s, 1H), 6.75(d, J = 8.1 Hz, 2H), 6.38-6.30(m, 3H), 4.65 (s, 2H), 4.18-4.15 (m, 1H), 3.93 (s, 3H), 2.64–2.55(m, 2H),2.55 -2.50 (m, 2H), 1.90-1.81 (m, 2H), 1.62-1.53 ​​(m, 2H). ESI-MS m / z: 485.2[M+H] + .

[0058] Example 3.

[0059] .

[0060] 1 H NMR (400 MHz, DMSO-d6) δ 9.21 (s, 1H), 7.76 (d, J = 7.8 Hz, 1H), 7.45(d, J = 5.3 Hz, 1H), 7.42 (d, J = 7.9 Hz, 2H), 7.31(d, J = 8.0 Hz, 2H),7.14 (d, J = 5.4 Hz, 1H), 6.86 (s, 1H), 6.74(d, J = 8.0 Hz, 2H), 6.38-6.30(m, 3H), 4.65 (s, 2H), 3.93 (s, 3H), 3.88-3.78 (m, 1H), 2.99 (s, 2H),2.81-2.71 (m, 2H), 2.04-1.95 (m, 2H).ESI-MS m / z: 471.1[M+H] + .

[0061] Example 4.

[0062] .

[0063] 1H NMR (400 MHz, DMSO-d6) δ 9.22 (s, 1H), 7.80 (d, J = 8.0 Hz, 1H),7.42(d, J = 5.0 Hz, 1H), 7.42 (d, J = 7.6 Hz, 1H), 7.32(d, J = 8.1 Hz, 2H),7.14 (d, J = 5.4 Hz, 1H), 6.85 (s, 1H), 6.75(d, J = 8.1 Hz, 2H), 6.38-6.30(m, 3H), 4.64-4.57(m, 4H), 4.18-4.14 (m, 1H), 2.64–2.55 (m, 2H),2.55 -2.49(m, 2H), 1.90-1.82 (m, 2H), 1.62-1.55 (m, 2H),1.22 (t, J = 14.2 Hz, 3H). ESI-MS m / z: 499.2[M+H] + . .

[0064] Example 5.

[0065] .

[0066] 1 H NMR (400 MHz, DMSO-d6) δ 9.20 (s, 1H), 7.82 (d, J = 8.2 Hz, 1H),7.45(d, J = 5.4 Hz, 1H), 7.44 (d, J = 7.8 Hz, 1H), 7.31(d, J = 8.0 Hz, 2H),7.15 (d, J = 5.4 Hz, 1H), 6.85 (s, 1H), 6.75(d, J = 8.1 Hz, 2H), 6.38-6.30(m, 3H), 5.22-5.18 (m, 1H), 4.64 (s, 2H), 4.18-4.14 (m, 1H), 2.63–2.56(m,2H), 2.56 -2.51 (m, 2H), 1.91-1.82 (m, 2H), 1.60-1.54 (m, 2H), 1.32 (t, J = 5.8Hz, 6H). ESI-MS m / z: 513.2[M+H] + . .

[0067] Example 6.

[0068] .

[0069] 1 H NMR (400 MHz, DMSO-d6) δ 9.24 (s, 1H), 7.78 (d, J = 8.2 Hz, 1H), 7.43 (d, J = 5.0 Hz, 1H), 7.43 (d, J = 7.8 Hz, 1H), 7.36-7.31 (m, 3H), 7.15(d,J = 5.4 Hz, 1H), 6.85 (s, 1H), 6.74 (d, J = 8.0 Hz, 2H), 6.38-6.31 (m, 3H), 4.66 (s, 2H), 4.18-4.15 (m, 1H), 2.64–2.55 (m, 2H), 2.57 -2.51 (m, 2H), 1.88-1.80 (m, 2H), 1.61-1.52 (m, 2H). ESI-MS m / z:521.4[M+H] + .

[0070] Example 7.

[0071] .

[0072] 1 H NMR (400 MHz, DMSO-d6) δ 9.26 (s, 1H), 7.76 (d, J = 8.0 Hz, 1H), 7.45 (d, J = 5.4 Hz, 1H), 7.46 (d, J = 7.7 Hz, 2H), 7.32 (d, J = 8.0 Hz, 2H),7.12(d, J = 5.2 Hz, 1H), 6.88 (s, 1H), 6.76 (d, J = 7.8 Hz, 2H), 6.38-6.30(m, 3H), 4.65 (s, 2H),4.06-4.01 (m, 2H), 4.18 (m, 1H), 3.93 (s, 3H), 2.68-2.61 (m, 2H), 1.98-1.91 (m, 2H). ESI-MS m / z: 459.2[M+H] + .

[0073] Example 8.

[0074] .

[0075] 1H NMR (400 MHz, DMSO-d6) δ 9.21 (s, 1H), 7.79 (d, J = 8.0 Hz, 1H), 7.43 (d, J = 5.4 Hz, 1H), 7.40 (d, J = 7.8 Hz, 2H), 7.31 (d, J = 8.0 Hz,2H),7.15 (d, J = 5.4 Hz, 1H), 6.87 (s, 1H), 6.74 (d, J = 8.0 Hz, 2H), 6.38-6.30(m, 3H), 4.64 (s, 2H), 4.31–4.26 (m, 2H) 4.18-4.12 (m, 1H), 3.94 (s, 3H),3.34–3.30 (m, 2H). ESI-MS m / z: 445.3[M+H] + .

[0076] Pharmacological activity testing.

[0077] I. LSD1 inhibitory activity test.

[0078] Weigh 1-2 mg of sample (Examples 1-8) into a 1.5 mL EP tube, add DMSO to prepare a 10 mM solution, and store at 4 °C for later use. Dilute with PBS according to the required concentration during experiments (dilution concentrations: 3.125 nM, 6.25 nM, 12.5 nM, 25 nM, 50 nM, 100 nM, 200 nM, 400 nM). Incubate the sample with LSD1 protein at room temperature, add the LSD1 reaction substrate H3K4me2 and incubate. Finally, add the fluorescent dye Amplex and horseradish peroxidase HRP and incubate at room temperature. Detect fluorescence values ​​using a microplate reader with excitation light at 530 nm and emission light at 590 nm. The activity test results are shown in Table 1.

[0079] Table 1. Results of in vitro LSD1 inhibitory activity assay.

[0080] II. Cell proliferation inhibition assay (MTT assay).

[0081] Cells were added to 96-well plates at a concentration of approximately 5000 per well. After incubation for 72 hours, fresh culture medium containing different concentrations of the compound (Examples 1-8) (diluted to 1.56 μM, 3.125 μM, 6.25 μM, 12.5 μM, 25 μM, and 50 μM) was added to each well. The plates were incubated for another 24 hours. The culture medium was then removed, and a solution of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazol bromide (MTT) was added to each well. The plates were then incubated at 37°C for another 4 hours. Afterward, the culture medium was removed, and 150 μL of DMSO was added to each well. The plates were then shaken on a plate shaker to dissolve any remaining formazan crystals. The absorbance was measured at 490 nm using a microplate reader. The MTT cell proliferation inhibition rate is shown in Table 2.

[0082] Table 2. MTT cell proliferation inhibition rate.

[0083] Next, to further verify that the compounds designed in this invention have significant antitumor activity, Example 1 was used as the test drug. The inhibitory activity of Example 1 on three types of cells is shown in [reference needed]. Figure 1 .

[0084] III. The inhibitory activity of the combination of death inhibitors in Example 1 against lung cancer A549 was determined using the MTT assay. The small molecule inhibitors mentioned were 3-MA (autophagy inhibitor), Ferrostatin (ferroptosis inhibitor), Z-VAD-FMk (apoptosis inhibitor), and NAC (antioxidant inhibitor). See [link to MTT assay]. Figure 2 .

[0085] A549 cells were administered at a rate of 5 × 10⁻⁶. 3 / wells were seeded into 96-well plates and incubated at 37°C for 24 h. Culture media containing different small molecule inhibitors were prepared (3-MA 1 mM; NAC 5 mM; Ferrostatin 1 μM; Z-VAD-FMk 50 μM). The sample was diluted to 3.125 μM with this culture medium and then administered to the cells. Each concentration was used in 6 replicates, and the cells were incubated for another 24 h.

[0086] The results showed that after A549 cells were treated with the apoptosis inhibitor, autophagy inhibitor and antioxidant inhibitor in Example 1, the cell death rate showed a decreasing trend compared with Example 1. Therefore, it is speculated that the signaling pathway of tumor cells inhibited by Example 1 may be related to the apoptosis pathway and autophagy induced by oxidative stress.

[0087] Furthermore, this invention employs flow cytometry for detection, with A549 cells at a concentration of 1×10⁻⁶. 7The cells were seeded into 6-well plates and allowed to adhere to the plates overnight with a confluence of over 80%. After adding the A549 cells treated in Example 1, the cells were cultured for another 24 hours. The culture medium was discarded, and the cells were digested and collected in centrifuge tubes. The cells were washed three times with PBS, and then 300 μL of 1× Binding Buffer, 3 μL (2.5 μg / mL) of Annexin-V-FITC, and 3 μL (50 μg / mL) of PI (propidium iodide) were added respectively. After incubation at room temperature in the dark for 15 minutes, apoptosis was detected in A549 cells treated with the solutions from Example 1 (0.78125 μM, 1.5625 μM, 3.125 μM) using flow cytometry. (See [link to relevant documentation]). Figure 3 The results showed that, at the inhibitory concentration, significant apoptosis occurred in the cells.

[0088] Furthermore, this invention employs Western blotting to further verify the expression of relevant proteins. The test cells used in this invention are A549 cells, and the drug-treated groups are those described in Example 1 (0.78125 μM, 1.5625 μM, 3.125 μM). After 24 hours of drug treatment, protein samples were collected according to conventional techniques in the art. Cells were lysed, and after BCA quantification, conventional Western blotting techniques were used (10%-12% separating gel, 5% stacking gel, electrophoresis conditions: 80V, 1-2 h; 120V, 1-2 h; transfer conditions: 200mA constant current transfer for 2 h; primary antibody incubation at 4℃ overnight; secondary antibody incubation at room temperature for 1.5-2 h). The results are shown in [Figure number missing]. Figure 4 The results showed that the expression of the anti-apoptotic protein Bcl-2 was significantly reduced in the drug-treated group, while the expression of the pro-apoptotic protein Bax was slightly increased; Beclin-1 protein was significantly increased. This indicates that apoptosis and autophagy in the drug-treated group mutually promoted each other, jointly inhibiting tumor cell growth. However, the mechanism by which indole compounds trigger Beclin-1-dependent autophagy in tumor cells remains unclear and requires further investigation.

[0089] In summary, based on the experimental results of this invention, indole compounds, as inhibitors of LSD1, directly inhibit LSD1 enzyme activity, thereby increasing the expression level of the basal protein Beclin-1 at the protein level. This promotes the continuous advancement of the autophagy cascade involving Beclin-1, ultimately achieving upregulation of autophagy levels and activating autophagy in tumor cells. Furthermore, the indole compounds of this invention also induce apoptosis. Through the combined effects of autophagy and apoptosis, precise killing of tumor cells is achieved, providing a clear molecular basis and translational pathway for the development of novel LSD1-targeted anti-tumor drugs.

Claims

1. An indoleamide derivative and its geometric isomers, or a pharmaceutically acceptable salt, hydrate, solvate, or prodrug thereof, characterized in that, The structural formula of the derivative is as follows: ， R1 is selected from hydrogen, C1-C6 alkyl, halogen-substituted C1-C6 alkyl, or C1-C6 alkoxy; R2 is selected from C1-C6 aliphatic amines, 5-7 cyclic aliphatic amines, and N-alkyl-substituted 5-7 cyclic aliphatic amines.

2. The indoleamide derivative and its geometric isomers as described in claim 1, or their pharmaceutically acceptable salts, hydrates, solvates, or prodrugs, characterized in that, R1 is selected from C1-C3 alkyl groups and C1-C3 alkyl groups substituted with halogens.

3. The indoleamide derivatives and their geometric isomers, or their pharmaceutically acceptable salts, hydrates, solvates, or prodrugs as described in claim 1, characterized in that, R2 is selected from tetrahydropyrrole, piperidine, N-methylpiperidine, and C2-C4 fatty amines.

4. The indoleamide derivatives and their geometric isomers, or their pharmaceutically acceptable salts, hydrates, solvates, or prodrugs as described in any one of claims 1-3, characterized in that, The derivatives are selected from: 。 5. The use of the indoleamide derivatives as described in any one of claims 1-4 in the preparation of antitumor drugs.

6. The indoleamide derivative according to any one of claims 5 is used to prepare an antitumor drug, characterized in that, The anti-tumor treatments specifically target breast cancer, lung cancer, stomach cancer, and prostate cancer.

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