Pyridopyrrolo compounds, methods of making and using the same

Abstract

The present application relates to the technical field of compound preparation, in particular to pyridopyrrolo compounds, a preparation method thereof and application thereof. The pyridopyrrolo compound is selected from the compounds shown in the following structural formula: wherein R 1 and R 2 are independently selected from substituted or unsubstituted C6-C23 aryl, X is N, and Y is C. The pyridopyrrolo compound has good inhibitory effect on LSD1 / GSK3β, has good therapeutic effect on tumors such as breast cancer, cervical cancer, colon cancer and glioma, and can be applied to the treatment of tumors.

Description

Technical Field

[0001] This invention relates to the field of compound preparation technology, and more specifically, to pyridopyrrole compounds, their preparation methods, and their applications. Background Technology

[0002] LSD1, short for lysine-specific demethylase 1 (also known as KDM1A), is the first histone demethylase discovered in humans. It is a key molecule in epigenetic regulation, widely involved in multiple biological processes such as cell development and tumorigenesis. For example, LSD1 is a crucial epigenetic enzyme for maintaining the stemness of leukemia stem cells and remodeling the tumor immune microenvironment, making it a popular therapeutic target for various solid tumors and hematological malignancies. However, existing LSD1 inhibitors suffer from insufficient selectivity, limited efficacy as monotherapy, and significant issues with drug resistance.

[0003] GSK3β (glycogen synthase kinase-3β) is a highly conserved serine / threonine kinase. Its overactivation is closely related to the progression of various diseases, including diabetes, Alzheimer's disease, cancer, and periodontal disease. Therefore, inhibiting GSK3β activity has become a key strategy for disease treatment. GSK3β inhibitors are a class of drug molecules that target glycogen synthase kinase-3β and are currently a popular therapeutic direction in the pharmaceutical field for various diseases. The following are the core research progress and application directions. However, GSK3β inhibitors also have drawbacks, including insufficient selectivity, high risk of off-target side effects, poor metabolic stability, easily elucidated structure, and insufficient blood-brain barrier function.

[0004] Furthermore, there are currently no compounds in the technology that can simultaneously inhibit GSK3β and LSD1. To simultaneously inhibit GSK3β and LSD1, it is necessary to combine compounds that separately inhibit GSK3β and LSD1. However, the effect of combinations of compounds with different mechanisms and effects is uncertain. Therefore, there is an urgent need for a compound that can simultaneously inhibit GSK3β and LSD1 and thus treat tumors.

[0005] In view of this, the present invention is proposed. Summary of the Invention

[0006] The purpose of this invention is to provide pyridopyrrole compounds, their preparation methods, and their applications. The pyridopyrrole compounds provided in the embodiments of this invention have good inhibitory effects on LSD1 / GSK3β and also show good therapeutic effects on tumors such as breast cancer, cervical cancer, colon cancer, and glioma, and can be applied to the treatment of tumors.

[0007] This invention is implemented as follows: In a first aspect, the present invention provides a pyridopyrrole compound selected from compounds shown in the following structural formulas: , where R 1 and R 2 Each is independently selected from substituted or unsubstituted C6-C23 aryl groups, where X is N and Y is C.

[0008] Secondly, the present invention provides a method for preparing the pyridopyrrole compounds described in the foregoing embodiments, wherein the synthesis is carried out according to the following synthetic route: .

[0009] Thirdly, the present invention provides the use of the pyridopyrrole compounds described in the foregoing embodiments in the preparation of antitumor drugs.

[0010] The present invention has the following beneficial effects: The embodiments of the present invention provide a new pyrrolopyridine compound with good inhibitory effect on LSD1 / GSK3β, and also has good therapeutic effect on tumors such as breast cancer, cervical cancer, colon cancer and glioma, and can be applied to the treatment of tumors. Attached Figure Description

[0011] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0012] Figure 1 This is a graph showing the effect of compound T7 on the migration of MCF7 cells in an embodiment of the present invention. Figure 2 This is a bar chart showing the effect of compound T7 on the migration of MCF7 cells in an embodiment of the present invention. Figure 3 This is a graph showing the effect of the compounds in the embodiments of the present invention on apoptosis in MCF7 cells. Detailed Implementation

[0013] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0014] In a first aspect, embodiments of the present invention provide a pyridopyrrole compound selected from compounds shown in the following structural formulas: , where R 1 and R 2Each is independently selected from substituted or unsubstituted C6-C23 aryl groups, for example, R 1 and R 2 Each is independently a substituted or unsubstituted phenyl group; X is N and Y is C.

[0015] Specifically, R 1 The substituted phenyl group can be any one of C1-C5 unsubstituted alkyl-substituted phenyl, hydroxy-substituted phenyl, halogen-substituted phenyl, C1-C5 unsubstituted alkoxy-substituted phenyl, C3-C8 heterocyclic alkyl-substituted phenyl and C3-C8 heterocyclic alkyl fused phenyl, the heteroatom is at least one of O, S and N, and the number of heteroatoms is ≥1, for example, the number of heteroatoms is 1, 2, etc.

[0016] Furthermore, R 1 The substitution of a substituted phenyl group can be monosubstituted, such as meta-methyl substituted phenyl, para-methyl substituted phenyl, methoxy substituted phenyl, and hydroxy substituted phenyl, or the above monosubstituted. The substitution can also be disubstituted, trisubstituted, or polysubstituted, such as 3-fluoro-5-methyl substituted phenyl, 3-hydroxy-4-methyl substituted phenyl, 3,4-2-fluoro substituted phenyl, 3-fluoro-5-methyl substituted phenyl, etc.

[0017] Furthermore, R 2 It is any one of phenyl, C1-C5 unsubstituted alkyl-substituted phenyl, halogen-substituted phenyl, C1-C5 unsubstituted alkoxy-substituted phenyl and cyano-substituted phenyl.

[0018] Furthermore, R 2 The substituted phenyl group is a monosubstituted phenyl group, such as meta-methyl-substituted phenyl, para-methyl-substituted phenyl, ortho-methyl-substituted phenyl, ethyl-substituted phenyl, n-propyl-substituted phenyl, and halogen-substituted phenyl can also be monosubstituted as described above. The substitution can also be disubstituted, trisubstituted, or polysubstituted, such as 3-fluoro-5-methyl-substituted phenyl, 3-hydroxy-4-methyl-substituted phenyl, 3,4-2-fluoro-substituted phenyl, 3-fluoro-5-methyl-substituted phenyl, etc. A monosubstituted phenyl group is preferred; a para-substituted phenyl group is more preferred.

[0019] It should be noted that the above-mentioned C1-C5 unsubstituted alkyl groups include alkyl groups such as methyl, ethyl, n-propyl, and isopropyl.

[0020] C1-C5 unsubstituted alkoxy groups include methoxy, ethoxy, propoxy, and other alkoxy groups.

[0021] Halogens include fluorine, chlorine, and bromine.

[0022] Furthermore, the pyridopyrrole compounds are selected from any one of the compounds shown in the following structural formulas: .

[0023] It should be noted that the symbols listed below the compounds in the above structural formulas are the corresponding compound labels.

[0024] Secondly, embodiments of the present invention provide a method for preparing pyridopyrrole compounds, which are synthesized according to the following synthetic route: The specific process is as follows: Step i includes: performing an amide condensation reaction by mixing M1 with an amine under the conditions of a condensing agent; wherein, M1 is... ; Step ii involves: mixing compound M2 with an organoboronic acid reagent under palladium catalysis to carry out a Suzuki-Miyaura reaction; wherein, M2 is... .

[0025] Further, using 5-bromo-1H-pyrrolo[2,3-b]pyridine-3-carboxylic acid (M1) as a raw material, it was dissolved in N,N-dimethylformamide (DMF), then N-methylimidazolium (NMI) was added, followed by the corresponding amine, and then N,N,N,N-tetramethylchloroformamidine hexafluorophosphate (TCFH) was added to react with it to obtain the target amide product intermediate (M2).

[0026] Subsequently, through a Suzuki-Miyaura reaction with organoboronic acid catalyzed by palladium, M2 formed a C-C bond with the organoboronic acid to obtain the target compound M3. Finally, the reaction yielded the final products T1~T25.

[0027] Each reaction step requires post-processing to obtain compounds with higher purity. Post-processing methods include pH adjustment, rotary evaporation, drying, and extraction, which will not be described in detail in the embodiments of this invention.

[0028] The pyridopyrrole compounds provided in this invention have good inhibitory effects on LSD1 and GSK3β. Therefore, these pyridopyrrole compounds can be used to prepare LSD1 / GSK3β inhibitors.

[0029] Furthermore, the pyridine-pyrrole compounds provided in the embodiments of the present invention have good therapeutic effects on tumors such as breast cancer, cervical cancer, colon cancer and glioma. Therefore, they can be used to prepare drugs for treating anti-tumor diseases.

[0030] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0031] Example 1 This invention provides a method for preparing a pyridopyrrole compound (denoted as T1), following the synthetic route described below:

[0032] The specific process is as follows: 4 g (approximately 16.59 mmol, 1 equivalent) of 5-bromo-1H-pyrrolo[2,3-b]pyridine-3-carboxylic acid was placed in a 100 mL reaction flask, and 40 mL of N,N-dimethylformamide (DMF) was added. The mixture was stirred at room temperature with a magnetic stirrer until fully dissolved. Then, 4 equivalents of N-methylimidazolium (NMI) and 1.3 equivalents of various substituted anilines were added sequentially. The system was stirred at room temperature for nearly 30 minutes until the solution became clear and transparent. Subsequently, 1.2 equivalents of N,N,N,N-tetramethylchloroformamidine hexafluorophosphate (TCFH) were added, and the entire system was heated to 100 °C and reacted for 3 hours. Thin-layer chromatography (TLC) was used as the developing solvent, with petroleum ether (PE):ethyl acetate (EA) = 1:1, and the reaction was observed under ultraviolet light. After the reaction is complete, slowly add saturated sodium bicarbonate (NaHCO3) solution to the reaction flask. At this time, a large number of bubbles are generated. Place the reaction flask on a magnetic stirrer and stir at room temperature until the bubbles disappear. Then let it stand to separate into layers. A large amount of precipitate is generated in the lower layer. After the precipitate stops precipitating, vacuum filter it and dry it to obtain the intermediate. Then place it in a refrigerator at 4°C for later use.

[0033] One equivalent of the above intermediate was added to a 100 mL two-necked round-bottom flask, followed by a mixed solvent of N,N-dimethylformamide (DMF):ethanol (EtOH):water = 2:1:1. The mixture was then purged three times with nitrogen (N2) to ensure a nitrogen-protected environment. Subsequently, 2.5 equivalents of potassium carbonate (K2CO3), 0.05 equivalents of tetrakis(triphenylphosphine)palladium, and 2 equivalents of the corresponding organoboronic acid were added. The flask was placed in a constant-temperature oil bath and heated to 100°C for 12 hours. TLC monitoring showed that the reaction was complete, with dichloromethane (DCM):anhydrous methanol (MeOH) = 20:1 as the developing solvent. The solution was dark brown. The reaction was stopped, and the mixture was cooled to room temperature. Saturated sodium bicarbonate (NaHCO3) was added to adjust the solution to neutral. The solution was then transferred to a separatory funnel, and the product was extracted three times with 300 mL of ethyl acetate (EA), followed by washing three times with saturated brine. The organic layer was collected and dried with an appropriate amount of anhydrous sodium sulfate (Na2SO4). The product was then concentrated under reduced pressure using a rotary evaporator and separated and purified by silica gel column chromatography to obtain the final product, which was then stored in a -20°C refrigerator for later use.

[0034] Examples 2-25 The pyridopyrrole compounds corresponding to Examples 2-25 (labeled T2-T25) were synthesized using the same method as in Example 1. The synthesis methods were basically the same, except that the reactants and reaction conditions were modified accordingly. The reaction conditions were also within the scope of the embodiments of this invention and will not be described in detail here.

[0035] The characterization data of the pyridopyrrole compounds prepared in Examples 1-25 are as follows: T1: N-(3-methoxy-4-methylphenyl)-5-(p-methoxyphenyl)-1H-pyrrolo[2,3-b]pyridine-3-carboxamide Purity > 95%. 1 H NMR (400 MHz, DMSO-d6) δ 12.32 (s, 1H), 9.77 (s, 1H), 8.68 (s, 1H), 8.59 (s, 1H), 8.45 (s, 1H), 7.62 (d, J = 8.2 Hz, 2H), 7.49 (s,1H), 7.32 (d, J = 8.3 Hz, 2H), 7.25 (d, J = 7.7 Hz, 1H), 7.07 (d, J = 8.1 Hz, 1H), 3.80 (s, 3H), 2.37 (s, 3H), 2.11 (s, 3H).

[0036] HRMS(ESI)m / z: (M+H) + calcd for C 23 H 21 N3O2: 372.1707; found: 372.1714.

[0037] T2: N,5-Dibenzoyl-1H-pyrrolo[2,3-b]pyridine-3-carboxamide Purity > 95%. 1 H NMR (400 MHz, DMSO-d6) δ 12.30 (s, 1H), 9.77 (s, 1H),8.68 (s, 1H), 8.59 (s, 1H), 8.44 (s, 1H), 7.63 (dd, J = 15.3, 6.6 Hz, 4H),7.32 (d, J = 6.7 Hz, 2H), 7.15 (d, J = 7.0 Hz, 2H), 2.37 (s, 3H), 2.28 (s, 3H).

[0038] HRMS(ESI)m / z: (M+H) + calcd for C 22 H 19 N3O: 342.1601; found: 342.1599.

[0039] T3: 5-(4-fluorophenyl)-N-(3-methoxy-4-methylphenyl)-1H-pyrrolo[2,3-b]pyridine-3-carboxamide Purity > 95%. 1 H NMR (400 MHz, DMSO-d6) δ 12.36 (s, 1H), 9.79 (s, 1H), 8.69 (s, 1H), 8.59 (s, 1H), 8.47 (s, 1H), 7.79 – 7.74 (m, 2H), 7.50 (s, 1H), 7.33 (t, J = 8.8 Hz, 2H), 7.25 (d, J = 7.8 Hz, 1H), 7.07 (d, J = 7.9 Hz, 1H), 3.80 (s, 3H), 2.11 (s, 3H).

[0040] HRMS(ESI)m / z: (M+H) + calcd for C 22 H 18 FN3O2: 376.1456; found: 376.1462.

[0041] T4: 5-(4-fluorophenyl)-N-(2-hydroxyphenyl)-1H-pyrrolo[2,3-b]pyridine-3-carboxamide Purity > 95%. 1 H NMR (400 MHz, DMSO-d6) δ 12.44 (s, 1H), 9.85 (s, 1H),9.34 (s, 1H), 8.63 (d, J = 19.1 Hz, 2H), 8.49 (s, 1H), 7.80 – 7.74 (m, 2H),7.70 (d, J = 7.9 Hz, 1H), 7.33 (t, J = 8.9 Hz, 2H), 7.02 (t, J = 7.6 Hz, 1H), 6.93 (d, J = 7.8 Hz, 1H), 6.84 (t, J = 7.6 Hz, 1H).

[0042] HRMS(ESI)m / z: (M+H) + calcd for C 20 H 14 FN3O2: 348.1143; found: 348.1146.

[0043] T5: N-(3-fluorophenyl)-5-(4-methoxyphenyl)-1H-pyrrolo[2,3-b]pyridine-3-carboxamide Purity >95%. 1H NMR (400 MHz, DMSO-d6) δ 12.30 (s, 1H), 9.77 (s, 1H), 8.65 (s, 1H), 8.56 (s, 1H), 8.45 (d, J = 3.1 Hz, 1H), 7.66 (d, J = 8.7 Hz, 3H), 7.56 (d, J = 7.7 Hz, 1H), 7.22 (t, J = 7.8 Hz, 1H), 7.08 (d, J = 8.7 Hz, 2H), 6.88 (d, J = 7.0 Hz, 1H), 3.82 (s, 3H).

[0044] HRMS(ESI)m / z: (M+H)+ calcd for C 21 H 16 FN3O2: 362.1299; found: 362.1305.

[0045] T6: N-(3-hydroxy-4-methylphenyl)-5-(4-methoxyphenyl)-1H-pyrrolo[2,3-b]pyridine-3-carboxamide Purity > 95%. 1 H NMR (400 MHz, DMSO-d6) δ 12.27 (s, 1H), 9.64 (d, J =15.8 Hz, 1H), 9.27 (s, 1H), 8.65 (s, 1H), 8.56 (s, 1H), 8.43 (s, 1H), 7.65(d, J = 7.0 Hz, 2H), 7.44 (s, 1H), 7.10 – 6.98 (m, 4H), 3.83 (d, J = 1.8 Hz,3H), 2.09 (s, 3H).

[0046] HRMS(ESI)m / z: (M+H) + calcd for C 22 H 19 N3O3: 374.1499; found: 374.1506.

[0047] T7: 5-(4-methoxyphenyl)-N-(p-methoxyphenyl)-1H-pyrrolo[2,3-b]pyridine-3-carboxamide Purity > 95%. 1H NMR (400 MHz, DMSO-d6) δ 12.29 (s, 1H), 9.77 (s, 1H), 8.65 (t, J = 2.1 Hz, 1H), 8.56 (t, J = 2.1 Hz, 1H), 8.43 (s, 1H), 7.66 (d, J= 6.5 Hz, 4H), 7.15 (d, J = 7.0 Hz, 2H), 7.08 (dd, J = 8.6, 1.9 Hz, 2H), 3.82 (d, J = 2.0 Hz, 3H), 2.28 (s, 3H).

[0048] HRMS(ESI)m / z: (M+H) + calcd for C 22 H 19 N3O2: 358.1550; found: 358.1560.

[0049] T8: 5-(4-fluorophenyl)-N-(4-(piperidin-1-yl)phenyl)-1H-pyrrolo[2,3-b]pyridine-3-carboxamide Purity > 95%. 1 H NMR (400 MHz, DMSO-d6) δ 12.30 (s, 1H), 9.66 (s, 1H),8.67 (d, J = 2.3 Hz, 1H), 8.58 (d, J = 2.3 Hz, 1H), 8.41 (d, J = 2.6 Hz, 1H),7.78 – 7.74 (m, 2H), 7.58 (d, J = 9.0 Hz, 2H), 7.36 – 7.31 (m, 2H), 6.91 (d,J = 9.0 Hz, 2H), 3.08 (t, J = 5.5 Hz, 4H), 1.57 (dd, J = 39.1, 4.9 Hz, 6H).

[0050] HRMS(ESI)m / z: (M+H) + calcd for C 25 H 23 FN4O: 415.1929; found: 415.1920.

[0051] T9: 5-(4-Chlorophenyl)-N-(2,3-Dihydrobenzo[b][1,4]dioxacyclopentan-6-yl)-1H-pyrrolo[2,3-b]pyridine-3-carboxamide Purity > 95%. 1 H NMR (400 MHz, DMSO-d6) δ 9.70 (s, 1H), 8.68 (s, 1H), 8.61 (s, 1H), 8.42 (s, 1H), 7.76 (d, J = 8.2 Hz, 2H), 7.56 (d, J = 8.4 Hz, 2H), 7.39 (s, 1H), 7.17 (d, J = 8.3 Hz, 1H), 6.82 (d, J = 8.8 Hz, 1H), 5.75 (s, 1H), 4.23 (d, J = 5.4 Hz, 4H).

[0052] HRMS(ESI)m / z: (M+H) + calcd for C 22 H 16 ClN3O3:406.0953; found: 406.0952.

[0053] T10: 5-(4-chlorophenyl)-N-(2-hydroxyphenyl)-1H-pyrrolo[2,3-b]pyridine-3-carboxamide Purity > 95%. 1 H NMR (400 MHz, DMSO-d6) δ 9.34 (s, 1H), 8.67 (s, 1H), 8.62 (s, 1H), 8.48 (s, 1H), 7.73 (dd, J = 28.4, 8.1 Hz, 4H), 7.55 (d, J = 8.6Hz, 2H), 7.00 (d, J = 7.5 Hz, 1H), 6.92 (d, J = 8.1 Hz, 1H), 6.84 (d, J = 7.6Hz, 1H), 5.74 (s, 1H).

[0054] HRMS(ESI)m / z:(M+H) + calcd for C 20 H 14 ClN3O2: 364.0847; found: 364.0850.

[0055] T11: N-(2,3-dihydrobenzo[b][1,4]dioxacyclopenta[d]phenyl-6-yl)-5-(5-fluoro-2-methoxyphenyl)-1H-pyrrolo[2,3-b]pyridine-3-carboxamide Purity > 95%. 1H NMR (400 MHz, DMSO-d6) δ 9.68 (s, 1H), 8.56 (s, 1H), 8.39 (s, 2H), 7.37 (s, 1H), 7.27 – 7.13 (m, 5H), 6.82 (s, 1H), 4.23 (d, J =6.0 Hz, 4H), 3.77 (s, 3H).

[0056] HRMS(ESI)m / z:(M+H) + calcd for C 23 H 18 FN3O4: 420.1354; found: 420.1361.

[0057] T12: 5-(3,5-dichlorophenyl)-N-(3-hydroxy-4-methylphenyl)-1H-pyrrolo[2,3-b]pyridine-3-carboxamide Purity > 95%. 1 H NMR (400 MHz, DMSO-d 6) δ 9.72 (d, J = 9.5 Hz, 1H), 9.27 (s, 1H), 8.70 (d, J = 14.8 Hz, 1H), 8.47 (s, 1H), 7.81 (s, 1H), 7.68 (d, J =12.1 Hz, 2H), 7.60 (d, J = 7.2 Hz, 2H), 7.40 (d, J = 15.5 Hz, 1H), 6.99 (d, J = 16.0 Hz, 2H), 2.09 (d, J = 6.1 Hz, 3H).

[0058] HRMS(ESI)m / z:(M+H) + calcd for C 21 H 15 Cl2N3O2: 412.0614; found: 412.0618.

[0059] T13: 5-(3-Chlorophenyl)-N-(3-Fluoro-4-methylphenyl)-1H-pyrrolo[2,3-b]pyridine-3-carboxamide Purity > 95%. 1H NMR (400 MHz, DMSO-d6) δ 12.45 (s, 1H), 9.98 (s, 1H), 8.72 (s, 1H), 8.66 (s, 1H), 8.49 (s, 1H), 7.82 – 7.71 (m, 3H), 7.57 – 7.43(m, 3H), 7.25 (s, 1H), 2.22 (s, 3H).

[0060] HRMS(ESI)m / z:(M+H) + calcd for C 21 H 15 ClFN3O: 380.0960; found: 380.0966. T14: 5-(3-chloro-4-hydroxyphenyl)-N-(2,5-dimethoxyphenyl)-1H-pyrrolo[2,3-b]pyridine-3-carboxamide Purity > 95%. 1 H NMR (400 MHz, DMSO-d6) δ 12.39 (s, 1H), 10.33 (s, 1H), 8.99 (s, 1H), 8.56 (s, 2H), 8.43 (s, 1H), 7.69 (s, 2H), 7.52 (d, J = 8.4 Hz,2H),, 6.69 (s, 2H), 3.84 (s, 3H), 3.73 (s, 3H).

[0061] HRMS(ESI)m / z:(M+H) + calcd for C 22 H 18 ClN3O4: 424.1059; found: 424.1066.

[0062] T15: 5-(5-fluoro-2-methoxyphenyl)-N-(3-hydroxy-4-methylphenyl)-1H-pyrrolo[2,3-b]pyridine-3-carboxamide Purity > 95%. 1 H NMR (400 MHz, DMSO-d6) δ 12.29 (s, 1H), 9.65 (s, 1H), 9.27 (s, 1H), 8.58 (s, 1H), 8.41 (d, J = 16.4 Hz, 2H), 7.42 (s, 2H), 7.24 (s,1H), 6.98 (s, 3H), 3.77 (s, 3H), 2.07 (s, 3H).

[0063] HRMS(ESI)m / z: (M+H) + calcd for C 22 H 18 FN3O3: 392.1405; found: 392.1411.

[0064] T16: 5-(3,4-difluorophenyl)-N-(2-hydroxyphenyl)-1H-pyrrolo[2,3-b]pyridine-3-carboxamide Purity > 95%. 1 H NMR (400 MHz, DMSO-d6) δ 12.44 (s, 1H), 9.83 (s, 1H), 9.32 (s, 1H), 8.63 (d, J = 14.5 Hz, 2H), 8.47 (s, 1H), 7.66 (s, 1H), 7.56 (s,2H), 7.00 (s, 1H), 6.91 (d, J = 8.4 Hz, 1H), 6.82 (s, 2H).

[0065] HRMS(ESI)m / z: (M+H) + calcd for C 20 H 13 F2N3O2: 366.1049; found: 366.1042.

[0066] T17: 5-(3-fluoro-5-methylphenyl)-N-(3-fluorophenyl)-1H-pyrrolo[2,3-b]pyridine-3-carboxamide Purity > 95%. 1 H NMR (400 MHz, DMSO-d6) δ 12.45 (s, 1H), 10.05 (s, 1H), 8.70 (s, 1H), 8.64 (s, 1H), 8.49 (s, 1H), 7.82 (s, 1H), 7.51 (s, 2H), 7.42 –7.37 (m, 2H), 7.05 (s, 1H), 6.89 (s, 1H), 2.43 (s, 3H).

[0067] HRMS(ESI)m / z: (M+H) + calcd for C 21 H 15 F2N3O: 364.1256; found: 364.1261.

[0068] T18: N-(4-fluorophenyl)-5-(3-methoxy-4-methylphenyl)-1H-pyrrolo[2,3-b]pyridine-3-carboxamide Purity > 95%. 1 H NMR (400 MHz, DMSO-d6) δ 12.36 (s, 1H), 9.93 (s, 1H), 8.65 (d, J = 18.7 Hz, 2H), 8.44 (s, 1H), 7.78 (s, 2H), 7.29 – 7.16 (m, 5H), 3.91 (s, 3H), 2.21 (s, 3H).

[0069] HRMS(ESI)m / z: (M+H) + calcd for C 22 H 18 FN3O2: 376.1456; found: 376.1461.

[0070] T19: 5-(2,4-dimethylphenyl)-N-(2-hydroxyphenyl)-1H-pyrrolo[2,3-b]pyridine-3-carboxamide Purity > 95%. 1 H NMR (400 MHz, DMSO-d6) δ 12.39 (s, 1H), 9.83 (s, 1H),9.25 (s, 1H), 8.44 (s, 1H), 8.33 (s, 1H), 8.24 (s, 1H), 7.68 (d, J = 7.7 Hz,1H), 7.15 (d, J = 9.0 Hz, 2H), 7.08 (d, J = 7.3 Hz, 1H), 6.96 (d, J = 7.3 Hz,1H), 6.88 (d, J = 7.8 Hz, 1H), 6.80 (t, J = 7.5 Hz, 1H), 2.31 (s, 3H), 2.21(s, 3H).

[0071] HRMS(ESI)m / z: (M+H) + calcd for C 22 H 19 N3O2: 358.1550; found: 358.1555.

[0072] T20: 5-(4-cyanophenyl)-N-(p-methoxyphenyl)-1H-pyrrolo[2,3-b]pyridine-3-carboxamide Purity > 95%. 1 H NMR (400 MHz, DMSO-d6) δ 12.45 (s, 1H), 9.81 (s, 1H), 8.79 (d, J = 2.4 Hz, 1H), 8.70 (d, J = 2.3 Hz, 1H), 8.50 (d, J = 3.2 Hz, 1H), 7.97 (s, 3H), 7.63 (d, J = 15.8 Hz, 1H), 7.55 (d, J = 8.3 Hz, 1H), 7.22 (t, J= 7.8 Hz, 1H), 6.89 (d, J = 7.3 Hz, 1H), 3.51 (s, 1H), 2.32 (s, 3H).

[0073] HRMS(ESI)m / z: (M+H) + calcd for C 22 H 16 N4O: 353.1397; found: 353.1398.

[0074] T21: 5-(4-cyanophenyl)-N-(4-(piperidin-1-yl)phenyl)-1H-pyrrolo[2,3-b]pyridine-3-carboxamide Purity > 95%. 1 H NMR (400 MHz, DMSO-d6) δ 12.40 (s, 1H), 9.69 (s, 1H), 8.78 (d, J = 2.2 Hz, 1H), 8.69 (d, J = 2.3 Hz, 1H), 8.44 (s, 1H), 8.32 (d, J= 28.9 Hz, 1H), 7.96 (s, 3H), 7.57 (t, J = 8.7 Hz, 2H), 6.92 (d, J = 9.2 Hz,2H), 3.07 (d, J = 4.9 Hz, 4H), 1.63 (s, 4H), 1.52 (d, J = 4.3 Hz, 2H).

[0075] HRMS(ESI)m / z: (M+H) + calcd for C 26 H 23 N5O: 444.1795; found: 444.1786.

[0076] T22: 5-(4-cyanophenyl)-N-(3-hydroxy-4-methylphenyl)-1H-pyrrolo[2,3-b]pyridine-3-carboxamide Purity > 95%. 1 H NMR (400 MHz, DMSO-d6) δ 12.40 (s, 1H), 9.69 (s, 1H), 9.25 (s, 1H), 8.75 (d, J = 2.3 Hz, 1H), 8.67 (d, J = 2.3 Hz, 1H), 8.45 (s,1H), 7.93 (s, 4H), 7.40 (s, 1H), 7.02 – 6.94 (m, 2H), 2.06 (s, 3H).

[0077] HRMS(ESI)m / z: (M+H) + calcd for C 22 H 16 N4O2: 369.1346; found: 369.1348.

[0078] T23: 5-(4-cyanophenyl)-N-(2,3-dihydrobenzo[b][1,4]dioxacyclopentan-6-yl)-1H-pyrrolo[2,3-b]pyridine-3-carboxamide Purity > 95%. 1 H NMR (400 MHz, DMSO-d6) δ 12.44 (s, 1H), 9.74 (s, 1H), 8.77 (s, 1H), 8.70 (s, 1H), 8.45 (s, 1H), 7.97 (s, 4H), 7.40 (s, 1H), 7.18(d, J = 9.8 Hz, 1H), 6.83 (d, J = 8.9 Hz, 1H), 4.24 (d, J = 6.2 Hz, 4H).

[0079] HRMS(ESI)m / z: (M+H) + calcd for C 23 H 16 N4O3: 419.1115; found: 419.1119.

[0080] T24: 5-(4-cyanophenyl)-N-(4-fluorophenyl)-1H-pyrrolo[2,3-b]pyridine-3-carboxamide Purity > 95%. 1H NMR (400 MHz, DMSO-d6) δ 12.49 (s, 1H), 9.96 (s, 1H),8.78 (s, 1H), 8.72 (s, 1H), 8.49 (s, 1H), 7.98 (s, 4H), 7.80 (t, J = 6.3 Hz,2H), 7.20 (t, J = 9.2 Hz, 2H).

[0081] HRMS(ESI)m / z: (M+H) + calcd for C 21 H 13 FN4O: 357.1146; found: 357.1152.

[0082] T25: 5-(4-cyanophenyl)-N-(3-fluorophenyl)-1H-pyrrolo[2,3-b]pyridine-3-carboxamide Purity > 95%. 1 H NMR (400 MHz, DMSO-d6) δ 12.52 (s, 1H), 10.08 (s, 1H), 8.78 (s, 1H), 8.72 (s, 1H), 8.52 (s, 1H), 7.97 (s, 4H), 7.80 (d, J = 11.5 Hz,1H), 7.52 (d, J = 7.5 Hz, 1H), 7.38 (d, J = 7.8 Hz, 1H), 6.89 (s, 1H).

[0083] HRMS(ESI)m / z: (M+H) + calcd for C 21 H 13 FN4O: 357.1146; found: 357.1145.

[0084] Experimental Example 1 The in vitro antitumor activity of the pyridopyrrole compounds (T1-T25) provided in the embodiments of the present invention was detected using the Cell Counting Kit 8 (CCK8) method.

[0085] The cells used were: MCF7 (breast cancer cells), HeLa (cervical cancer cells), SW480 (colon cancer cells), and U87 (human glioma cells).

[0086] Specifically, seed 4000 cells / well in a 96-well plate, with a liquid volume of 100 μL per well. Do not seed the edge of the plate; instead, add 100 μL of PBS buffer to the wells at the edge to prevent edge effects from affecting the experiment. After seeding, transfer the plate to an incubator and incubate for 24 hours before administering the drug.

[0087] Five concentrations of pyridopyrrole compounds and positive control drugs were set up: 10 μM, 5 μM, 2.5 μM, 1.25 μM, and 0.625 μM. 100 μL of the corresponding drug group was added to each well of the cell plate (three wells per drug group). 100 μL of the corresponding complete culture medium was added to three wells of the blank control group (three replicates). A blank control group was also included. The administered compounds were then incubated for 72 h.

[0088] Against a relatively dark background, add 10 μL of CCK8 solution to each well of the cell plate after 72 hours of culture (avoid adding air bubbles as much as possible to prevent them from affecting the experimental results). Then, place the cell plate in an incubator and culture for 1-4 hours. Finally, place the cell plate in a microplate reader to detect the OD value.

[0089] The formula for calculating cell viability is as follows: Cell viability (%) = [OD experimental group - OD blank control group] / [OD positive control group - OD blank control group] × 100% The results are shown in Tables 1-4.

[0090] Table 1 Inhibition rate

[0091] Table 2 Inhibition rate

[0092] Table 3 Results of in vitro antiproliferative activity

[0093] Table 4 Results of in vitro antiproliferative activity

[0094] Therefore, it can be seen that the pyridine-pyrrole compounds provided in the embodiments of the present invention can inhibit breast cancer cells, cervical cancer cells, colon cancer cells and human glioma cells.

[0095] Experiment Example 2 GSK3β kinase inhibitory activity study The specific process is as follows: 1. Dilute the compound with DMSO to 50× of the final reaction concentration, and transfer 100 μL of the mother liquor into a 96-well plate.

[0096] 2. In the same 96-well plate, add 100 μL of DMSO as a compound-free control and an enzyme-free control, and label the plate as the original plate.

[0097] 3. Preparation of intermediate plate: Transfer 10 μL of the compound stock solution from the original plate to a new 96-well plate as an intermediate plate. Add 90 μL of 1×kinase buffer to each well of the intermediate plate and shake for 10 minutes.

[0098] 4. Prepare a 2.5-fold enzyme solution: Add GSK3β kinase to 1× kinase buffer.

[0099] 5. Prepare a 2.5-fold peptide solution: Add the FAM-labeled peptide and ATP to a 1× kinase buffer.

[0100] 6. Add 5 μL of the test compound solution to each well of the 384-well detection plate, then add 10 μL of 2.5× enzyme solution to each well, and incubate at room temperature for 10 minutes.

[0101] 7. Add 10 μL of 2.5× peptide solution to each well of the 384-well detection plate, incubate at 28°C for 1 hour, then add 25 μL of stop solution to stop the reaction.

[0102] 8. Data was collected using Caliper. The inhibition rate of the enzyme (%Inh) = (max-conversion) / (max-min)*100.

[0103] The results are shown in Table 5-6.

[0104] Table 5. Inhibition rate of pyridine-pyrrole compounds on GSK3β

[0105] Table 6. Inhibition rate of pyridine-pyrrole compounds on GSK3β

[0106] As shown in the table above, the pyridine-pyrrole compounds provided in the embodiments of the present invention have a good inhibitory effect on GSK3β.

[0107] Experimental Example 3 LSD1 enzyme inhibitory activity study The specific process is as follows: 1. Preparation of reaction system: 1x modified Tris buffer (pH=7.5); serial dilution of sample solution, with a final DMSO concentration of 1%; enzyme solution: add LSD1 to 1x enzyme buffer solution; substrate peptide solution: add H3K4me2 to 1x enzyme buffer solution.

[0108] 2. Using the Echo sound-controlled pipetting system, transfer 5 μL of the test compound solution or blank control solution to a 384-well cell plate, and then pre-incubate with the enzyme (final concentration 5 nM) at room temperature for 15 minutes.

[0109] 3. Add 5 μL of substrate peptide solution (final concentration 100 nM) to the reaction mixture and incubate at room temperature for 60 min to start the reaction.

[0110] 4. Add 15 μL of acceptor and donor solution to terminate the reaction, incubate at room temperature for 60 minutes in the dark.

[0111] 5. Read data using EnVision®'s Alpha mode.

[0112] 6. Data Processing: Inhibition rate (%Inh) = (Max - signal) / (Max - Min) * 100, where Max contains histone demethylase protein solution and dimethyl sulfoxide solution, and Min contains only buffer solution and dimethyl sulfoxide solution; Substitute %Inh and compound concentration into the following formula in GraphPad Prism 5 software to obtain the IC50 value: Y = Bottom + (Top - Bottom) / (1 + 10^((LogIC50 - X) * Hill Slope)), where Y is %Inh and X is the compound concentration.

[0113] The results are shown in Table 7.

[0114] Table 7 Inhibition rate of LSD1

[0115] As shown in the table above, the pyridine-pyrrole compound provided in one embodiment of the present invention has a good inhibitory effect on LSD1.

[0116] Experiment Example 4 Research on the detection of cell migration The specific process is as follows: Cell growth was observed in real time using an inverted microscope. MCF7 cells with excellent growth and regular morphology were selected and seeded into 6-well cell culture plates, with the cell density controlled at approximately 4 × 10⁶ cells per well. 3Add 2 mL of complete culture medium to each well, gently agitate the culture plate to ensure cells are evenly distributed at the bottom of the wells, and then incubate the culture dish in a constant temperature cell culture incubator for 24 hours. Once the cells have completely adhered and densely covered the bottom of the wells in a monolayer, gently aspirate the existing culture medium from the wells using a pipette. Add 1 mL of PBS buffer to each well and slowly rinse twice to remove residual culture medium and suspended cells. Then replace with fresh complete culture medium. Using a 1 mL sterile pipette tip, draw a straight, uniform cell scratch perpendicular to the bottom of the well at a uniform speed. Aspirate the culture medium again and rinse the detached suspended cells with PBS buffer. The experimental groups were set as follows: wells with drug-containing culture medium were designated as the drug treatment group, and wells with only an equal volume of blank complete culture medium were designated as the blank control group. 2 mL of the corresponding culture medium was added to each well in each group. After treatment, the culture plate was placed under an inverted fluorescence microscope to observe the initial state and photograph the changes. The morphological changes of cell scratch healing were observed at two time points: 0 hours and 24 hours of culture, and images were taken simultaneously to record the data.

[0117] See results Figure 1 and Figure 2 ,in Figure 1 The figure shows the effect of compound T7 on the migration of MCF7 cells; Figure 2 This is a bar chart showing the effect of compound T7 on the migration of MCF7 cells. Based on... Figure 1 and Figure 2 It is known that the pyridine-pyrrole compounds provided in the embodiments of the present invention can inhibit the migration of MCF7 cells and exhibit a certain concentration dependence.

[0118] Experimental Example 5 Study on the detection of apoptosis The specific process is as follows: MCF7 cells in good condition were seeded into 6-well cell culture plates following the steps in Example 4. 2 mL of culture medium was added, and the plates were gently shaken until the cells were homogeneous. The plates were then incubated in a cell culture incubator for 24 hours. When the cells reached 80% confluence, the culture medium was aspirated, and each well was washed with 1 mL of PBS. The wells containing the drug-treated medium served as the treatment group, while the wells with fresh culture medium served as the control group. In this experiment, the drug-treated group was divided into three concentrations: high, medium, and low, with drug concentrations of 0.5 μM, 0.25 μM, and 0.125 μM, respectively. The 6-well plates were incubated for 24 hours, followed by staining.

[0119] Collect the cell culture medium from each well into a centrifuge tube for later use. Add the PBS used for washing to the corresponding centrifuge tube to prevent loss of apoptotic cells. Add 600 μL of trypsin to each well for digestion, and stop digestion with the culture medium in the corresponding centrifuge tube. After centrifugation, wash the cells twice with PBS, resuspend the cells in Binding Buffer diluted with deionized water, and adjust the cell concentration to 1 × 10⁻⁶ cells / well.6 Cells / mL. Take 100 μL of cell suspension and place it in a flow cytometry tube for later use. Four control groups were set up for the experiment, including a blank group, a PI-only group, and an Annexin V-only group. The FITC group and the double-staining group were used for flow cytometry voltage and compensation regulation. 5 μL of Annexin V was first added to the flow cytometer tube. After mixing with FITC, the mixture was incubated at room temperature in the dark for 5 min. Then, 5 μL of PI and 200 μL of PBS were added, and the mixture was immediately analyzed by flow cytometry. Statistical analysis of the experimental data was performed using GraphPad Prism 5 software. The t-test was used to compare the drug-treated group and the control group. P < 0.05 was considered statistically significant (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

[0120] See results Figure 3 , Figure 3 To investigate the effect of compound T7 on apoptosis in MCF7 cells, based on... Figure 3 It is known that the pyridine-pyrrole compounds provided in the embodiments of the present invention can promote apoptosis of MCF7 cells at a certain concentration, and this effect is concentration-dependent, suggesting that the compounds may induce apoptosis by activating the apoptosis pathway and gradually increasing the concentration.

[0121] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A pyridopyrrole compound, characterized in that, It is selected from compounds with the following structural formulas: , where R 1 and R 2 Each is independently selected from substituted or unsubstituted C6-C23 aryl groups, where X is N and Y is C.

2. The pyridopyrrole compound according to claim 1, characterized in that, R 1 and R 2 Each can be a substituted or unsubstituted phenyl group, independently.

3. The pyridopyrrole compound according to claim 1 or 2, characterized in that, R 1 It is any one of C1-C5 unsubstituted alkyl-substituted phenyl, hydroxy-substituted phenyl, halogen-substituted phenyl, C1-C5 unsubstituted alkoxy-substituted phenyl, C3-C8 heterocyclic alkyl-substituted phenyl and C3-C8 heterocyclic alkyl fused phenyl, the heteroatom is at least one of O, S and N, and the number of heteroatoms is ≥1; Preferably, R 1 The substituted phenyl group is a monosubstituted phenyl or a disubstituted phenyl; preferably a monosubstituted phenyl; more preferably a meta-substituted phenyl. Preferably, R 1 The substituents of the substituted phenyl group are halogens and C1-C5 unsubstituted alkyl groups.

4. The pyridopyrrole compound according to claim 1 or 2, characterized in that, R 2 It is any one of C1-C5 unsubstituted alkyl-substituted phenyl, halogen-substituted phenyl, C1-C5 unsubstituted alkoxy-substituted phenyl and cyano-substituted phenyl; Preferably, R 2 The substituted phenyl group is a monosubstituted phenyl or a disubstituted phenyl; preferably a monosubstituted phenyl; more preferably a para-substituted phenyl. Preferably, R 2 The substituents of the substituted phenyl group are any one of halogen, C1-C5 unsubstituted alkyl, and C1-C5 unsubstituted alkoxy groups.

5. The pyridopyrrole compound according to claim 1 or 2, characterized in that, The pyridopyrrole compounds are selected from any one of the compounds shown in the following structural formulas: 。 6. A method for preparing the pyridopyrrole compound according to claim 1, characterized in that, Perform the synthesis according to the following synthesis path: 。 7. The preparation method according to claim 6, characterized in that, include: M1 is mixed with an amine under the condition of a condensing agent to carry out an amide condensation reaction; wherein, M1 is... ; M2 was mixed with an organoboronic acid reagent under palladium catalysis to carry out the Suzuki reaction, wherein M2 was... .

8. The use of a pyridopyrrole compound according to claim 1 in the preparation of an antitumor medicament.

9. The application according to claim 8, characterized in that, The tumors include breast cancer, cervical cancer, colon cancer, and glioma.

10. The application according to claim 8, characterized in that, The drug is an inhibitor of LSD1 / GSK3β.

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