2-methyl-4-arylamino-7-alkyl substituted pyrrolo[2,3-d]pyrimidines, processes for their preparation and uses thereof

Abstract

The application belongs to the technical field of biological medicine, and discloses 2-methyl-4-arylamino-7-alkyl substituted pyrrolo[2,3 d ]pyrimidine compounds with a structural formula as shown in formula I. The 2-methyl-4-arylamino-7-alkyl substituted pyrrolo[2,3 d ]pyrimidine compounds can effectively inhibit the in-vitro proliferation of various tumor cells, have a similar mechanism of action as colchicine, can inhibit the aggregation of microtubules, and have potential development and application values.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a 2-methyl-4-arylamino-7-alkyl-substituted pyrrolo[2,3- d Pyrimidine compounds and their preparation methods and uses; more specifically, relating to a 2-methyl-4-arylamino-7-alkyl-substituted pyrrolo[2,3- d Pyrimidine compounds, their preparation methods, pharmaceutical compositions, combination drug compositions and their applications. Background Technology

[0002] Numerous published protein crystal structures demonstrate the crucial role of water molecules in the drug-binding pocket in the binding of compounds to their targets. Replacing water molecules in the binding pocket, or designing interactions between compounds and water molecules, has increasingly led to applications in guiding compound design and modification, enhancing the affinity and selectivity of compounds for targets, and improving pharmacokinetic properties.

[0003] like Figure 1 and Figure 2 As shown, several high-resolution crystal structures of tubulin-colchicine binding sites and their composite crystal structures have been reported. Superposition analysis of the composite crystal structures of tubulin with colchicine (PDB code: 4O2B, resolution: 2.30 Å, https: / / www.rcsb.org / structure / 4O2B), SB-204 (PDB code: 6N47, resolution: 2.60 Å, https: / / www.rcsb.org / structure / 6N47), or compound 1 (PDB code: 6BR1, resolution: 2.30 Å, https: / / www.rcsb.org / structure / 6BR1) revealed that the oxygen atom in the methoxy group at position 2 of the colchicine structure and the 2-chloropyridino[3,2-] in the SB-204 structure... d The nitrogen atom at position 1 of the pyrimidine ring and the 2-chloropyridyl[2,3-] in compound 1 d The nitrogen atom at position 1 of the pyrimidine ring is essentially overlapped, and forms a hydrogen bond network with βCys239 and βVal236 (the amino acids at the same position in 4O2B are encoded as Cys241 and Val238) through water molecules β728, β626 or β675, respectively, suggesting that water molecules at this position may play an important role in the binding of the compound.

[0004] like Figure 3As shown, compound 2 (Journal of Medicinal Chemistry, 2010, 53(22), 8116-8128) is a reported compound that acts on the colchicine binding site of tubulin and exhibits strong proliferative inhibitory activity against various tumor cells. This is achieved by introducing a group that can replace key water molecules into its structure via an alkane chain and... N Structural modification of the -methyl linker may lead to the discovery of novel microtubule aggregation inhibitors with new structural types and modes of action, and potential anticancer applications. Furthermore, given the clinical use of colchicine for anti-inflammatory treatment, the compounds discovered in this invention bind to the same site on microtubules as colchicine, and may also possess potential anti-inflammatory activity. Summary of the Invention

[0005] In view of this, the present invention provides a compound with the structure of Formula I, its preparation method, and its application, based on the "replacement" strategy of water molecules in the microtubule colchicine site binding pocket. This type of compound is a novel 2-methyl-4-arylamino-7-alkyl-substituted pyrrolo[2,3- d This invention relates to a pyrimidine small-molecule microtubule aggregation inhibitor, which, through further experimental confirmation, exhibits significant inhibitory effects on the proliferation of human non-small cell lung cancer A549 cells, human cervical cancer HeLa cells, and human glioma U251 cells. No prior data on its activity have been reported. This invention is significant for enhancing drug specificity and efficacy, reducing toxic side effects, and preventing drug resistance. Furthermore, given the clinical use of colchicine for anti-inflammatory treatment, the compound discovered in this invention binds to the same site on microtubules as colchicine and may also possess potential anti-inflammatory activity.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a class of compounds or pharmaceutically acceptable salts thereof having the structure shown in Formula I: Among them, R 1 For Me or with R 2 Ring formation , , , Equal structure; R 2 It is an amino, hydroxyl, alkylamine, azacyclic alkylamine, alkoxy, alkyloxoamino, or related to R 1 Ring formation , , , structure; R 3Alkyl groups with two or more carbon atoms include 2-hydroxyethyl, 2-aminoethyl, 2-alkoxyethyl, 2-methylaminoethyl, 2-dimethylaminoethyl, 2-cycloalkylaminoethyl, 2-ethoxy-2-oxoethyl, 2-amino-2-oxoethyl, 2-methylamino-2-oxoethyl, 2-cycloalkylamino-2-oxoethyl, 2-hydroxyamino-2-oxoethyl, 3-piperidinylmethyl, 2-dimethylamino-2-oxoethyl, 2-azacycloalkylamino-2-oxoethyl, 2-azacycloalkylaminoethyl, cyanomethyl, 1-cyanoethyl, etc. The cycloalkyl group is C10. 3~6 A saturated alkyl group, optionally containing 0 to 2 N atoms, or simultaneously containing 0 to 1 nitrogen and oxygen atoms, or simultaneously containing 0 to 1 nitrogen and sulfur atoms; when containing nitrogen atoms, it can be C 1~6 The alkyl group is substituted; the cyclic amino group is a 3-6 membered saturated heterocyclic hydrocarbon containing at least one nitrogen atom, optionally containing 0-1 N atom, or simultaneously containing 0-1 nitrogen atom and oxygen atom, or simultaneously containing 0-1 nitrogen atom and sulfur atom, and when containing 2 nitrogen atoms, the other nitrogen atom can be C 1~6 Alkyl substitution.

[0007] Substituent definition As used herein, the term "halogen" refers to fluorine, chlorine, bromine, or iodine. Preferred halogen groups are fluorine, chlorine, or bromine.

[0008] The term "cyano" as used in this article refers to -CN.

[0009] The term "hydroxyl" as used in this article refers to -OH.

[0010] The term "amino" as used in this article refers to -NH2.

[0011] As used herein, the term "alkyl" refers to a saturated straight-chain or branched monovalent hydrocarbon group having 1 to 6 carbon atoms, preferably 1 to 6, 1 to 4, or 1 to 3 carbon atoms. Typical examples of "alkyl" include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, tert-pentyl, neopentyl, hexyl, etc.

[0012] As used herein, the term "cycloalkyl" refers to a saturated cyclic hydrocarbon group having 3 to 12 carbon atoms and having a monocyclic, bicyclic, or multiple fused rings (including fused and bridged ring systems), preferably having 3 to 10, 3 to 8, 5 to 8, 3 to 6, or 5 to 6 carbon atoms. Typical examples of "cycloalkyl" include, but are not limited to, monocyclic structures such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, 1-methylcyclopropyl, 2-methylcyclopentyl, 2-methylcyclooctyl, etc., bicyclic structures such as bicyclic [2.2.1]heptyl, etc., and polycyclic structures such as adamantyl.

[0013] The term "alkoxy" as used in this article refers to the -OR group. 4 , where R 4 Selected from alkyl and cycloalkyl groups as defined herein. Typical examples of "alkoxy" include, but are not limited to, methoxy, ethoxy, propoxy, butoxy, cyclopropyloxy, etc.

[0014] The term "alkylamino" as used in this article refers to the group -NHR 4 , where R 4 Selected from alkyl and cycloalkyl groups as defined herein. Typical examples of "alkylamino" include, but are not limited to, methylamino, ethylamino, propylamino, butylamino, cyclopropylamino, etc.

[0015] The term "azacycloalkylamine" as used in this article refers to a group. Where n = 1~4, R 5 Selected from alkyl and halogen groups as defined herein. Typical examples of “azacycloalkylamine” include, but are not limited to, aziridine, 2-methylaziridine, aziridine, 2-methylaziridine, 3-methylaziridine, pyrrolidine, 2-methylpyrrolidine, 3-methylpyrrolidine, 2,5-dimethylpyrrolidine, 3,3-dimethylpyrrolidine, 3,3-difluoropyrrolidine, piperidine, 2-methylpiperidine, 3-methylpiperidine, 4-methylpiperidine, 2,3-dimethylpiperidine, 2,4-dimethylpiperidine, 2,5-dimethylpiperidine, 2,6-dimethylpiperidine, 4,4-difluoropiperidine, etc.

[0016] The term "dialkylamino" as used in this article refers to the NR group. 6 R 7 , where R 6 and R 7 Each is independently selected from alkyl and cycloalkyl groups as defined herein. Typical examples of "dialkylamino" include, but are not limited to, dimethylamino, diethylamino, dipropylamino, dibutylamino, etc.

[0017] In cases where the compound name used in this article differs from the chemical structural formula, the chemical structural formula shall prevail.

[0018] According to a preferred embodiment of the present invention, R in general formula I 1 For Me or with R 2 Ring formation , , , structure.

[0019] According to a preferred embodiment of the present invention, R in general formula I 2 It is an amino, hydroxyl, alkylamine, azacyclic alkylamine, alkoxy, alkyloxoamino, or related to R 1 Ring formation , , , structure; According to a preferred embodiment of the present invention, R in general formula I 3 Alkyl groups with two or more carbon atoms include 2-hydroxyethyl, 2-aminoethyl, 2-alkoxyethyl, 2-methylaminoethyl, 2-dimethylaminoethyl, 2-cycloalkylaminoethyl, 2-ethoxy-2-oxoethyl, 2-amino-2-oxoethyl, 2-methylamino-2-oxoethyl, 2-cycloalkylamino-2-oxoethyl, 2-hydroxyamino-2-oxoethyl, 3-piperidinylmethyl, 2-dimethylamino-2-oxoethyl, 2-azacycloalkylamino-2-oxoethyl, 2-azacycloalkylaminoethyl, cyanomethyl, 1-cyanoethyl, etc. The cycloalkyl group is C10. 3~6 A saturated alkyl group, optionally containing 0 to 2 N atoms, or simultaneously containing 0 to 1 nitrogen and oxygen atoms, or simultaneously containing 0 to 1 nitrogen and sulfur atoms; when containing nitrogen atoms, it can be C 1~6 The alkyl group is substituted; the cyclic amino group is a 3-6 membered saturated heterocyclic hydrocarbon containing at least one nitrogen atom, optionally containing 0-1 N atom, or simultaneously containing 0-1 nitrogen atom and oxygen atom, or simultaneously containing 0-1 nitrogen atom and sulfur atom, and when containing 2 nitrogen atoms, the other nitrogen atom can be C. 1~6 Alkyl substitution.

[0020] According to a preferred embodiment of the present invention, in general formula I, R 1 With R 2 Ring formation .

[0021] According to a preferred embodiment of the present invention, in general formula I, R 2 With R 1 Ring formation .

[0022] Preferably, the compound of general formula I of the present invention is selected from: ; The corresponding chemical name is: 2-Methyl-4-( N -Methyl-4-methoxyaniline)-7-(2-hydroxyethyl)-7 H -pyrrolo[2,3- d Pyrimidine (compound I1) 2-Methyl-4-( N -Methyl-4-methoxyaniline)-7-(2-ethoxy-2-oxoethyl)-7 H -pyrrolo[2,3- dPyrimidine (compound I2) 2-Methyl-4-( N -Methyl-4-methoxyaniline)-7-(2-amino-2-oxoethyl)-7 H -pyrrolo[2,3- d Pyrimidine (compound I3) 2-Methyl-4-( N -Methyl-4-methoxyaniline)-7-(2-methylamino-2-oxoethyl)-7 H -pyrrolo[2,3- d Pyrimidine (compound I4) 2-Methyl-4-( N -Methyl-4-methoxyaniline)-7-(2-cyclopropylamino-2-oxoethyl)-7 H -pyrrolo[2,3- d Pyrimidine (compound I5) 2-Methyl-4-( N -Methyl-4-methoxyaniline)-7-(2-hydroxyamino-2-oxoethyl)-7 H -pyrrolo[2,3- d Pyrimidine (compound I6) 2-Methyl-4-( N -methyl-4-methoxyaniline)-7-(3-piperidinylmethyl)-7 H -pyrrolo[2,3- d Pyrimidine (compound I7) 2-Methyl-4-( N -Methyl-4-methoxyaniline)-7-(2-dimethylamino-2-oxoethyl)-7 H -pyrrolo[2,3- d Pyrimidine (compound I8) 2-Methyl-4-( N -Methyl-4-methoxyaniline)-7-(2-morpholino-2-oxoethyl)-7 H -pyrrolo[2,3- d Pyrimidine (compound I9) 2-Methyl-4-( N -Methyl-4-methoxyaniline)-7-(2-morpholinylethyl)-7 H -pyrrolo[2,3- d Pyrimidine (compound I10) 2-Methyl-4-( N -methyl-4-methoxyaniline)-7-cyanomethyl-7 H -pyrrolo[2,3- d Pyrimidine (compound I11) 2-Methyl-4-( N -Methyl-4-methoxyaniline)-7-(1-cyanoethyl)-7 H -pyrrolo[2,3- d Pyrimidine (compound I12) 2-Methyl-4-(6-methoxy-3-oxo-3,4-dihydroquinoxaline-2-) H -1-yl)-7-cyanomethyl-7 H -pyrrolo[2,3- d Pyrimidine (compound I13) 2-Methyl-4-(6-methoxy-3-oxo-3,4-dihydroquinoxaline-2-) H -1-yl)-7-(2-ethoxy-2-oxoethyl)-7 H -pyrrolo[2,3- d Pyrimidine (compound I14) 2-Methyl-4-(6-methoxy-3-oxo-3,4-dihydroquinoxaline-2-) H -1-yl)-7-(2-amino-2-oxoethyl)-7 H -pyrrolo[2,3- d Pyrimidine (compound I15).

[0023] A second aspect of the invention provides a 2-methyl-4-arylamino-7-alkyl-substituted pyrrolo[2,3- d The preparation method for pyrimidine compounds is carried out according to the following reaction route: Where R 1 R 2 and R 3 The definition is as described above.

[0024] In the presence of a base or acid, compound 3 reacts with compound of general formula II in a solvent to give compound of general formula III, which then reacts with an alkylating agent to give compounds of general formula I1 to I12. Alternatively, compound 3 can be alkylated to obtain compound IV, which reacts with compound 4 in a solvent in the presence of acid to obtain compound V. After chloroacetyl chloride acylation, compound VI is obtained, and then intramolecular cyclization under alkaline conditions yields compounds I13 to I15 of general formula I.

[0025] More specifically, Step (1) reaction: with 1 to 10 equivalents of base (e.g., sodium hydride, cesium carbonate, sodium carbonate, sodium bicarbonate, triethylamine, pyridine, ... N ,N -Dimethylaminopyridine, N , N The reaction is carried out in the presence of diisopropylethylamine, potassium carbonate, sodium tert-butoxide, cesium carbonate, potassium carbonate, etc., or an acid (such as hydrochloric acid, sulfuric acid, hydrobromic acid, or acetic acid, etc.) in an amount up to 2 equivalents, and if necessary, with the aid of cuprous halide, bis(diphenylphosphinoferrocene) palladium dichloride / 1,1'-bis(diphenylphosphinoferrocene), palladium acetate, X-Phos, etc., in a solvent (such as DMF, acetonitrile, ethanol, tert-butanol, acetone, isopropanol, methanol, THF, toluene, 1,4-dioxane, or DMSO, etc.), at a temperature below 200°C (e.g., temperature controlled by ice / water bath, oil bath, or microwave), for 5 minutes to 24 minutes. After hours, a secondary amine compound is obtained, which is further dissolved in a solvent (such as DMF, acetonitrile, acetone, THF, toluene, 1,4-dioxane, or DMSO), and a base (such as sodium hydride, cesium carbonate, sodium carbonate, sodium bicarbonate, triethylamine, pyridine, potassium carbonate, sodium tert-butoxide, cesium carbonate, potassium carbonate) and an alkylating agent are added. The reaction is carried out at a temperature below 200°C (e.g., using an ice / water bath, oil bath, or microwave temperature control) for 5 minutes to 24 hours to obtain compounds of general formula I, I1 to I12. Alternatively, in 1 to 10 equivalents of alkali (such as sodium hydride, cesium carbonate, sodium carbonate, sodium bicarbonate, triethylamine, pyridine, etc.). N , N -Dimethylaminopyridine, N , N Compound 1 reacts with an alkylating agent under the action of diisopropylethylamine, potassium carbonate, sodium tert-butoxide, cesium carbonate, potassium carbonate, etc., to give compound IV. Compound IV then reacts with 4-methoxy-1,2-phenylenediamine (compound 2) in the presence of an acid (e.g., hydrochloric acid, sulfuric acid, hydrobromic acid, or acetic acid) in a solvent (e.g., DMF, acetonitrile, ethanol, tert-butanol, acetone, isopropanol, methanol, THF, toluene, 1,4-dioxane, or DMSO) at a temperature below 200°C (e.g., temperature controlled by ice / water bath, oil bath, or microwave) for 5 minutes to 24 hours to give compound V. Compound V is then reacted with 1 to 10 equivalents of a base (e.g., sodium hydride, cesium carbonate, sodium carbonate, sodium bicarbonate, triethylamine, pyridine, etc.). N , N -Dimethylaminopyridine, N , NThe acylation reaction of diisopropylethylamine, potassium carbonate, sodium tert-butoxide, cesium carbonate, potassium carbonate, etc., with acylation reagents such as chloroacetyl chloride in solvents (e.g., DMF, acetonitrile, ethanol, tert-butanol, acetone, isopropanol, methanol, THF, toluene, 1,4-dioxane, or DMSO) at temperatures below 200°C (e.g., temperature controlled by ice / water bath, oil bath, or microwave) for 5 minutes to 24 hours directly yields compounds of general formula I, I13 to I15; or the acylated product can be separated and further reacted with 1 to 10 equivalents of base (e.g., sodium hydride, cesium carbonate, sodium carbonate, sodium bicarbonate, triethylamine, pyridine, etc.) in a solvent (e.g., DMF, acetonitrile, ethanol, tert-butanol, acetone, isopropanol, methanol, THF, toluene, 1,4-dioxane, or DMSO) at temperatures below 200°C (e.g., temperature controlled by ice / water bath, oil bath, or microwave) for 5 minutes to 24 hours to directly yield compounds of general formula I, I13 to I15; or the acylated product can be separated and further reacted with 1 to 10 equivalents of base (e.g., sodium hydride, cesium carbonate, sodium carbonate, sodium bicarbonate, triethylamine, pyridine, etc.) in a solvent (e.g., DMF, acetonitrile, ethanol, tert-butanol, acetone, isopropanol, methanol, isopropanol ... N , N -Dimethylaminopyridine, N , N Under the action of diisopropylethylamine, potassium carbonate, sodium tert-butoxide, cesium carbonate, potassium carbonate, etc., at a temperature below 200°C (e.g., temperature controlled by ice / water bath, oil bath or microwave), the reaction proceeds for 5 minutes to 24 hours to obtain compounds of general formula I, I13 to I15.

[0026] A third aspect of the present invention provides a pharmaceutical composition comprising the said compound or a pharmaceutically acceptable salt thereof as a pharmaceutically active component.

[0027] A fourth aspect of the invention provides a combination drug composition comprising the said compound and other pharmaceutically acceptable carriers.

[0028] A fifth aspect of the present invention provides the use of a compound in the preparation of a drug for treating tumors.

[0029] Furthermore, the tumor is any one of hematologic malignancy, ovarian cancer, prostate cancer, testicular cancer, melanoma, pancreatic cancer, lymphoma, breast cancer, stomach cancer, brain cancer, kidney cancer, lung cancer, liver cancer, or colon cancer.

[0030] The compounds of this invention have shown strong inhibitory activity in various cancer cell assays. As described below, the compounds exhibited inhibitory activity comparable to or better than the positive control drug paclitaxel in assays of human non-small cell lung cancer A549 cells, human cervical cancer HeLa cells, and human glioma U251 cells. Therefore, the series of compounds of this invention possess broad-spectrum anticancer activity, and further research on them holds promise for the development of new drugs for treating various tumors or cancers.

[0031] The compounds of this invention can be used either on their own or as pharmaceutically acceptable salts or solvates. Pharmaceutically acceptable salts of compounds of general formula I include salts formed with pharmaceutically acceptable inorganic or organic acids, or with pharmaceutically acceptable inorganic or organic bases. Examples of suitable acid addition salts include salts formed with hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid, perchloric acid, fumaric acid, acetic acid, propionic acid, succinic acid, glycolic acid, formic acid, lactic acid, maleic acid, tartaric acid, citric acid, pyric acid, malonic acid, hydroxymaleic acid, phenylacetic acid, glutamic acid, benzoic acid, salicylic acid, fumaric acid, toluenesulfonic acid, methanesulfonic acid, naphthalene-2-sulfonic acid, benzenesulfonic acid, hydroxynaphthoic acid, hydroiodic acid, malic acid, tannic acid, etc. N , N'- Salts formed from dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, N-methylglucosamine, and procaine. When referring to compounds of this invention, this includes compounds of general formula I and their pharmaceutically acceptable salts or solvates.

[0032] According to the present invention, the pharmaceutical composition comprises a compound of general formula I of the present invention and a conventional pharmaceutical carrier or excipient. The pharmaceutical composition can be administered, for example, orally or parenterally. The pharmaceutical compositions of the present invention can be prepared into various dosage forms according to conventional methods in the art, including but not limited to tablets, capsules, solutions, suspensions, granules, or injections, for administration, for example, orally or parenterally.

[0033] It should also be noted that the dosage and method of administration of the compounds of this invention depend on many factors, including the patient's age, weight, sex, natural health condition, nutritional status, the activity intensity of the compound, the time of administration, metabolic rate, the severity of the disease, and the subjective judgment of the treating physician. The preferred dosage is between 0.01 and 100 mg / kg body weight / day.

[0034] The beneficial effects of this invention are: (1) The 2-methyl-4-arylamino-7-alkyl-substituted pyrrolo[2,3-] discovered in this invention d Pyrimidine microtubule aggregation inhibitors have novel structures and are of great significance for enhancing drug specificity and efficacy, reducing toxic side effects, and preventing drug resistance.

[0035] (2) The 2-methyl-4-arylamino-7-alkyl-substituted pyrrolo[2,3-] discovered in this invention d Pyrimidine compounds are novel microtubule aggregation inhibitors that exhibit strong proliferative inhibitory activity against human non-small cell lung cancer A549 cells, human cervical cancer HeLa cells, and human glioma U251 cells. Their mechanism of action is similar to that of colchicine, which inhibits microtubule polymerization and has good potential for development and application. Attached Figure Description

[0036] Figure 1 The structural formulas of colchicine, SB-204, and compound 1 in the background art are shown below; Figure 2 This is a schematic diagram illustrating the interaction between the microtubule protein molecule and the ligands colchicine, SB-204, or compound 1 in the background art. Figure 3 The structural formula of compound 2 in the background art; Figure 4 This is a schematic diagram of the in vitro microtubule aggregation inhibition test results for compound I11; Figure 5 This is a schematic diagram of the molecular docking of compound I11 with tubulin; in the diagram, amino acid residues are represented by light gray bars; compound I11 is represented by green bars; hydrogen bonds are represented by yellow dashed lines, and the distance between hydrogen bonds is <3 Å. Detailed Implementation

[0037] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0038] Synthetic Method 1: Weigh compound 2 (1 equivalent) and dissolve it in DMF. Add alkali (1-3 equivalents of anhydrous K₂CO₃ or anhydrous Cs₂CO₃), stir at room temperature for 10 minutes, then add the corresponding haloalkane (1-2 equivalents), and continue the reaction under these conditions for 1-48 hours. Pour the reactants into an appropriate amount of ice water and adjust the pH to 7 with dilute hydrochloric acid. Filter or extract, and separate or recrystallize the crude product using a Flash column chromatography column to obtain the target products I1, I2, and I4-I12.

[0039] Synthetic Method 2: At room temperature, 4-chloro-2-methyl-7 H -pyrrolo[2,3- d Pyrimidine (compound 3, 1 equivalent) was dissolved in anhydrous DMF, and alkali (anhydrous K₂CO₃ or anhydrous Cs₂CO₃, 1-3 equivalents) was added. After stirring at room temperature for 10 minutes, the corresponding haloalkane (1-2 equivalents) was added, and the reaction was allowed to proceed for 12-24 hours at room temperature. The extent of the reaction was monitored by TLC. After the reaction was complete, the mixture was poured into ice water, and the pH was adjusted to 7 with 2N hydrochloric acid. The mixture was filtered or extracted, and the crude product was separated by Flash column chromatography or recrystallized to obtain compound IV.

[0040] Synthetic Method 3: Dissolve compound IV (1 equivalent) and 4-methoxy-o-phenylenediamine (compound 4, 1-2 equivalents) in isopropanol, and then add a small amount of... c HCl, reacted in an oil bath at 60-80°C for 4-24 hours or microwaved at 80-150°C for 30 minutes to 3 hours. After the reaction is complete, the reactants are allowed to stand at 4°C to precipitate, which is then filtered. A small amount is collected. i Washing with -PrOH yields compound of general formula V.

[0041] Synthetic Method 4: Dissolve compound V (1 equivalent) in anhydrous DMF, add alkali (anhydrous K₂CO₃, anhydrous Cs₂CO₃, or NaHCO₃, 1-3 equivalents), add chloroacetyl chloride (1-2 equivalents) under ice bath cooling, and continue the reaction at this temperature for 30 min to 2 h. After complete reaction, pour into ice water and adjust the pH to 7 with 2N hydrochloric acid. Filter or extract, and separate or recrystallize the crude product using Flash column chromatography to obtain compound VI.

[0042] Synthetic Method 5: Dissolve compound of general formula VI (0.5 ~ 1 equivalent) in anhydrous DMF, add alkali (anhydrous K2CO3 or anhydrous Cs2CO3, 1 ~ 3 equivalents), react at room temperature for 4 ~ 12 h, after complete reaction, pour into ice water, and adjust pH to 7 with 2N hydrochloric acid. Filter or extract, separate the crude product by Flash column chromatography or recrystallize to obtain target compounds of general formulas I13 ~ I15.

[0043] Synthesis and spectral data of some intermediates: N -(4-Methoxyphenyl)- N 2-Dimethyl-7 H -pyrrolo[2,3- d Pyrimidine-4-amine (compound 2) Compound 3 (6.68 g, 40 mmol) and N 6.99 g (51 mmol) of methyl-4-methoxyaniline was dissolved in 60 mL of isopropanol, and 2 mL of [amount missing] was added. c The reaction was carried out with HCl and heated to reflux in an oil bath for 4 hours. After standing, a solid precipitated out, which was then filtered and alkalized to give 8.47 g of the target compound as a white solid. The yield was 82%; the melting point was 225-227℃; and the HPLC result was 99.38%. 1 H NMR (400 MHz, DMSO- d 6) δ 12.50 (s, 1H, NH), 7.42 (d, J= 8.4 Hz, 2H, Ph-2&6H), 7.13 (d, J = 8.8Hz, 2H, Ph-3&5H), 7.01 (brs, 1H, Ar-6H), 4.60 (brs, 1H, Ar-5H), 3.85(s, 3H,OMe), 3.64(s, 3H, NMe), 2.67(s, 3H, 2-Me).HRMS(ESI): Calcd for C 15 H 17 N4O + [M+H] + : 269.139 7, found: 269.146 5.

[0044] 4-Chloro-2-methyl-7-(cyanomethyl)-7 H -pyrrolo[2,3- d Pyrimidine (compound IVa) Synthetic Method 2. Compound 3 (2.51 g, 15 mmol), bromoacetonitrile (2.16 g, 18 mmol), anhydrous K₂CO₃ (4.15 g, 30 mmol), DMF 30 mL, yielded 2.10 g of the target compound as a white solid, yield: 68%. 1 H NMR (600MHz, d -CDCl3) δ 7.26 (d, J = 3.6 Hz, 1H, Ar-6H), 6.69 (d, J = 3.6 Hz, 1H, Ar-5H),5.20 (s, 2H, CH2), 2.78 (s, 3H, Me). HRMS (ESI) m / s calcd for C9H8ClN4 + [M+H] + 207.043 2, found 207.044 0.

[0045] 2-(4-chloro-2-methyl-7-) H -pyrrolo[2,3- d Acetyl pyrimidin-7-yl (compound IVb) Synthetic Method 2. Compound 3 (531 mg, 3.1 mmol), ethyl bromoacetate (1.01 g, 6.32 mmol), anhydrous K2CO3 (1.2 g, 9 mmol), and DMF 10 mL were used to give 390 mg of the target compound as a white solid, yield: 50%, melting point: 116-118 °C. 1 H NMR (500 MHz, d -CDCl3) δ 7.17 (d, J = 3.5 Hz, 1H, Ar-6H), 6.61 (d, J = 4.0Hz, 1H, Ar-5H), 5.00 (s, 2H, CH2), 4.25 (q, J = 7.0 Hz, 2H, OCH2), 2.74 (s, 3H,2-Me), 1.29 (t, J = 7.5 Hz, 3H, CH3).

[0046] 2-Methyl-4-(2-amino-4-methoxyaniline)-7-cyanomethyl-7 H -pyrrolo[2,3- d Pyrimidine (compound Va) Synthetic method 3. IVa (620 mg, 3 mmol), 4-methoxy-1,2-phenylenediamine (4,415 mg, 3 mmol), yielded 749 mg of the target compound as a white solid, yield: 81%. 1 H NMR (500 MHz, DMSO-) d 6) δ 8.60 (s, 1H,NH), 7.08 (d, J = 3.5 Hz, 1H, Ph-3H), 6.95 (d, J = 8.5 Hz, 1H, Ph-6H), 6.37 (d, J =3.0 Hz, 1H, Ar-6H), 6.17 (dd, J = 8.5, 3.0 Hz, 1H, Ph-5H), 5.30 (s, 2H, CH2), 4.94 (s, 2H, NH2), 3.71 (s, 3H, OMe), 2.41 (s, 3H, 2-Me).HRMS(ESI): Calcd forC 21 H 24 N6O3 + [M+H] +: 309.145 8, found: 309.146 8.

[0047] 2-Methyl-4-(2-amino-4-methoxyaniline)-7-(2-ethoxy-2-oxoethyl)-7 H -pyrrolo[2,3- d Pyrimidine (compound Vb) Synthetic method 3. IVb (304 mg, 1.2 mmol), 4 (138 mg, 1 mmol), yielded 300 mg of the target compound as a white solid, yield: 85%, melting point: 215-217 °C, which was used directly in the next reaction.

[0048] 2-Methyl-4-(2-chloroacetamido-4-methoxyaniline)-7-cyanomethyl-7 H -pyrrolo[2,3- d Pyrimidine (compound VIa) Synthetic Method 4. Va (132 mg, 0.42 mmol), chloroacetyl chloride (42 μL, 0.52 mmol), K2CO3 (138 mg, 1 mmol) were added to give 113 mg of the target compound as a white solid, yield: 70%, melting point: 174-176 °C. 1 H NMR (500MHz, DMSO-) d 6) δ 9.77 (s, 1H, NH), 9.03 (s, 1H, 4-NH), 7.43 (brs, 1H, Ph-3H), 7.31 (d, J =9.0 Hz, 1H, Ph-6H), 7.23 (d, J = 3.5 Hz, 1H, Ar-6H), 6.82 (dd, J = 9.0,3.0 Hz, Ph-5H), 6.38 (brs, 1H, Ar-5H), 5.36 (s, 2H, CH2CN), 4.29 (s, 2H,CH2Cl), 3.78 (s, 3H, OMe), 2.50 (s, 3H, CH3).HRMS(ESI): Calcd for C 18 H 18 ClN6O2 + [M+H] + : 385.117 4, found: 385.118 3.

[0049] 2-Methyl-4-(2-chloroacetamido-4-methoxyaniline)-7-(2-ethoxy-2-oxoethyl)-7 H -pyrrolo[2,3- d Pyrimidine (compound VIb) Synthetic Method 4. Vb (274 mg, 0.7 mmol), chloroacetyl chloride (68 μL, 0.84 mmol) were added to give 173 mg of the target compound as a white solid in 57% yield, which was used directly in the next reaction.

[0050] Example 1: 2-Methyl-4-( N -Methyl-4-methoxyaniline)-7-(2-hydroxyethyl)-7 H -pyrrolo[2,3- d Pyrimidine (compound I1) Synthetic Method 1. 2 (300 mg, 1.12 mmol), 2-bromoethanol (162 mg, 1.3 mmol), Cs₂CO₃ (651 mg, 2 mmol), yielded 227 mg of the target compound as a white solid, yield: 65%, melting point: 90-92 °C, HPLC: 99.38%; 1 H NMR (400 MHz, DMSO-) d 6δ 7.26 (d, J = 8.8 Hz, 2H, Ph-2&6H), 7.03 (d, J = 8.8 Hz, 2H,Ph-3&5H), 6.82 (d, J = 3.2 Hz, 1H, Ar-6H), 4.89 (t, J = 5.2 Hz, 1H, OH), 4.51 (d, J = 3.2 Hz, 1H, Ar-5H), 4.08 (t, J = 6.0 Hz, 2H, OCH2), 3.82 (s, 3H, OMe), 3.62(t, J = 5.6 Hz, 2H, NCH2), 3.45 (s, 3H, N-Me), 2.48 (s, 3H, 2-Me); 13 C NMR (150MHz, Chloroform- d) δ 159.93, 158.82, 156.89, 138.67, 129.36, 123.35, 114.79,101.79, 100.80, 62.76, 55.51, 50.65, 39.20, 25.71;HRMS(ESI): Calcd forC 17 H 21 N4O2 + [M+H] + : 313.165 9, found: 313.167 1.

[0051] Example 2: 2-Methyl-4-( N -Methyl-4-methoxyaniline)-7-(2-ethoxy-2-oxoethyl)-7 H -pyrrolo[2,3- d Pyrimidine (compound I2) Synthetic method 1. 2 (305 mg, 1 mmol), ethyl bromoacetate (222 mg, 2 mmol), Cs₂CO₃ (980 mg, 3 mmol), yielded 340 mg of the target compound as a white solid, yield: 96%, melting point: 147-149 °C, HPLC: 96.15%; 1 HNMR (500 MHz, DMSO- d 6) δ 7.28 (d, J = 8.5 Hz, 2H, Ph-2&6H ), 7.05 (d, J = 8.5 Hz, 2H,Ph-3&5H), 6.81 (d, J = 3.5 Hz, 1H, Ar-6H), 4.89 (s, 2H, NCH2), 4.56 (d, J = 3.5Hz, 1H, Ar-5H), 4.12 (q, J = 7.5 Hz, 2H, OCH2), 3.82 (s, 3H, OMe), 3.46 (s, 3H,NCH3), 2.46 (s, 3H, 2-Me), 1.19 (t, J = 7.0 Hz, 3H, CH3); 13 C NMR (150 MHz, DMSO-) d6) δ 168.68, 159.32, 158.37, 156.14, 151.36, 138.28, 129.40, 123.82, 114.79,100.52, 100.29, 60.92, 55.40, 44.89, 38.87, 25.89, 14.05;HRMS(ESI): Calcd forC 19 H 23 N4O3 + [M+H] + : 355.176 5, found: 355.176 8.

[0052] Example 3: 2-Methyl-4-( N -Methyl-4-methoxyaniline)-7-(2-amino-2-oxoethyl)-7 H -pyrrolo[2,3- d Pyrimidine (compound I3) I₂ (71 mg, 0.2 mmol) was dissolved in 3 mL of ammonia in methanol and reacted at 40 °C in a sealed environment for 24 h to give 49 mg of the target compound as a white solid. Yield: 75%, melting point: 172-174 °C, HPLC: 96.22%. 1 H NMR (500MHz, DMSO-) d 6) δ 7.45 (s, 1H, NH), 7.27 (d, J = 9.0 Hz, 2H, Ph-2&6H), 7.11 (s,1H, NH), 7.04 (d, J = 9.0 Hz, 2H, Ph-3&5H ), 6.76 (d, J = 3.5 Hz, 1H, Ar-6H),4.66 (s, 2H, CH2), 4.54 (d, J = 3.5 Hz, 1H, Ar-5H), 3.82 (s, 3H, OMe), 3.46 (s,3H, NCH3), 2.47 (s, 3H, 2-CH3); 13 C NMR (150 MHz, DMSO-) d6) δ 159.93, 158.52,156.29, 150.66, 138.03, 129.37, 120.04, 118.87, 114.89, 102.10, 100.85,55.44, 38.89, 25.89, 18.73;HRMS(ESI): Calcd for C 17 H 20 N5O2 + [M+H] + : 326.161 2, found: 326.162 2.

[0053] Example 4: 2-Methyl-4-( N -Methyl-4-methoxyaniline)-7-(2-methylamino-2-oxoethyl)-7 H -pyrrolo[2,3- d Pyrimidine (compound I4) Synthetic Method 1. 2 (91 mg, 0.3 mmol), 2-bromo- N -Methylacetamide (91 mg, 0.6 mmol), Cs2CO3 (391 mg, 1.2 mmol), yielded 91 mg of the target compound as a white solid, yield: 89%, melting point: 172-174 °C, HPLC: 97.35%; 1 H NMR (500 MHz, DMSO-) d 6) δ 7.89(d, J = 4.5 Hz, 1H, 7-NH), 7.27(d, J = 8.5Hz, 2H, Ph-2&6H), 7.04(d, J = 8.5 Hz, 2H, Ph-3&5H), 6.77(d, J = 3.5 Hz, 1H, Ar-6H), 4.66(s, 2H, CH2), 4.55(d, J = 3.5 Hz, 1H, Ar-5H), 3.82(s, 3H, OMe), 3.46(s, 3H, NCH3), 2.58(d, J = 4.5 Hz, 3H, NHC H 3), 2.46(s, 3H, 2-CH3); 13 C NMR (150MHz, DMSO-) d6) δ 167.71, 159.07, 158.32, 156.12, 151.33, 138.38, 129.38,124.20, 114.75, 100.59, 99.98, 55.40, 46.04, 38.87, 25.91, 25.57;HRMS(APESI)m / s calcd for C 18 H 22 N5O2 + [M+H] + 340.176 8, found: 340.177 3.

[0054] Example 5: 2-Methyl-4-( N -Methyl-4-methoxyaniline)-7-(2-cyclopropylamino-2-oxoethyl)-7 H -pyrrolo[2,3- d Pyrimidine (compound I5) I2 (70 mg, 0.2 mmol), cyclopropylamine (69 μL, 1 mmol), and DBU (150 μL, 1 mmol) were dissolved in acetonitrile and reacted at 40 °C under a sealed environment for 24 h to give 56 mg of product as a white solid. Yield: 76%, melting point: 169-171 °C, HPLC: 98.25%. 1 H NMR (500 MHz, DMSO-) d 6) δ 8.20 (d, J = 4.0 Hz, 1H, NH), 7.27(d, J = 9.0Hz, 2H, Ph-2&6H), 7.04(d, J = 8.5 Hz, 2H, Ph-3&5H), 6.76(d, J = 3.5 Hz, 1H, Ar-6H), 4.62(s, 2H, CH2), 4.54(d, J = 3.5 Hz, 1H, Ar-5H), 3.82(s, 3H, OMe), 3.46(s, 3H, NMe), 2.64 ~ 2.59(m, 1H, CH), 2.46(s, 3H, 2-Me), 0.63 ~ 0.58(m, 2HCH2), 0.42~0.40(m, 2H, CH2); 13 C NMR (150 MHz, DMSO-) d6) δ 168.72, 159.47, 158.77,151.77, 138.84, 129.84, 124.75, 115.21, 101.00, 100.27, 55.85, 46.32, 26.35,22.75, 6.04;HRMS(ESI): Calcd for C 20 H 24 N5O2 + [M+H] + : 366.192 5, found: 366.192 8.

[0055] Example 6: 2-Methyl-4-( N -Methyl-4-methoxyaniline)-7-(2-hydroxyamino-2-oxoethyl)-7 H -pyrrolo[2,3- d Pyrimidine (compound I6) I2 (106 mg, 0.3 mmol), hydroxylamine hydrochloride (53 mg, 1.5 mmol), and sodium methoxide (1 mL, 5 mmol / mL) were dissolved in 3 mL of methanol and reacted at room temperature for 10 h to give 76 mg of product as a white solid. Yield: 74%, melting point: 165-167 °C, HPLC: 95.63%. 1 H NMR (500 MHz, DMSO-) d 6) δ 10.73 (s, 1H, OH), 8.92 (s, 1H, NH), 7.27 (d, J = 9.0 Hz, 2H, Ph-2&6H), 7.04 (d, J = 9.0 Hz, 2H, Ph-3&5H), 6.78 (d, J =3.5 Hz, 1H, Ar-6H), 4.60 (s, 2H, CH2), 4.55 (d, J = 4.0 Hz, Ar-5H), 3.82 (s,3H, OMe), 3.46 (s, 3H, NMe), 2.47 (s, 3H, 2-Me); 13 C NMR (150 MHz, DMSO-) d6) δ164.36, 159.08, 158.33, 156.13, 138.39, 129.35, 124.12, 114.77, 100.64,99.98, 55.41, 43.94, 39.84, 38.87, 25.90;HRMS(ESI): Calcd for C 17 H 20 N5O3 + [M+H] + :342.156 1, found: 342.156 7.

[0056] Example 7: 2-Methyl-4-( N -methyl-4-methoxyaniline)-7-(3-piperidinylmethyl)-7 H -pyrrolo[2,3- d Pyrimidine (compound I7) Synthetic general method one. 2 (107 mg, 0.4 mmol), 1-tert-butoxycarbonyl-3-(bromomethyl)piperidine (136 mg, 0.48 mmol), Cs₂CO₃ (396 mg, 1.2 mmol), yielded 149 mg of a yellow oil, which is 2-methyl-4-( N -methyl-4-methoxyaniline)-7-( N -tert-butyloxycarbonyl-3-piperidinylmethyl)pyrrolo[2,3- d Pyrimidine. Take 2-methyl-4-( N -methyl-4-methoxyaniline)-7-( N -tert-butyloxycarbonyl-3-piperidinylmethyl)pyrrolo[2,3- d Pyrimidine (47 mg, 0.1 mmol) was dissolved in 5 mL of ethyl acetate, and 0.5 mL of concentrated hydrochloric acid was added. The mixture was stirred overnight at room temperature, and the ethyl acetate was removed under reduced pressure. After alkalization, 36.2 mg of the target compound was obtained as a white solid. Yield: 99%, melting point: 98-100 °C, HPLC: 98.52%. 1 H NMR (500 MHz, DMSO-) d 6) δ 7.27 (d, J = 8.5 Hz, 2H, Ph-2&6H), 7.03 (d, J = 9.0 Hz, 2H, Ph-3&5H),6.78 (d, J = 3.5 Hz, 1H, Ar-6H), 4.52 (d, J= 3.5 Hz, 1H, Ar-5H), 3.88 (d, J = 7.5Hz, 2H, CH2), 3.82 (s, 3H, OMe), 3.45(s, 3H, NMe), 2.74 (d, J = 11.5 Hz, 1H,Pip-2H), 2.56 (dd, J = 12.0, 2.5 Hz, 1H, 7-2H), 2.47(s, 3H, 2-CH3), 2.40~2.34(m, 1H, 7-6H), 2.17~2.12(m, 1H, 7-6H), 1.86~1.79(m, 1H, 7-3H), 1.54~1.47(m,2H, 7-4H, 7-4H ), 1.27~1.18(m, 1H, 7-5H), 1.05~0.96(m, 1H, 7-5H); 13 C NMR (150MHz, DMSO-) d 6) δ 158.94, 158.30, 156.10, 151.21, 138.43, 129.42, 123.46,114.72, 100.57, 99.72, 55.39, 50.00, 47.16, 46.40, 38.85, 37.18, 28.55,26.04, 25.27;HRMS(ESI): Calcd for C 21 H 27 N5O + [M+H] + : 366.228 8, found: 366.2293.

[0057] Example 8: 2-Methyl-4-( N -Methyl-4-methoxyaniline)-7-(2-dimethylamino-2-oxoethyl)-7 H -pyrrolo[2,3- d Pyrimidine (compound I8) Synthetic Method 1. 2 (92 mg, 0.3 mmol), 2-bromo- N , N -Dimethylacetamide (91 mg, 0.6 mmol), K2CO3 (166 mg, 1.2 mmol), yielded 103 mg of the target compound as a white solid, yield: 97%, melting point: 166-168 °C, HPLC: 95.73%;1 H NMR (500 MHz, DMSO-) d 6) δ 7.28 (d, J = 8.5 Hz, 2H, Ph-2&6H), 7.04(d, J = 8.5 Hz, 2H, Ph-3&5H), 6.69 (d, J = 3.5 Hz, 1H, Ar-6H), 4.95 (s, 2H, CH2), 4.54 (d, J = 3.5 Hz, 1H, Ar-5H), 3.82 (s, 3H, OMe), 3.46 (s, 3H, NMe), 3.06 (s,3H, 2-Me), 2.81(s, 3H, CH3), 2.46(s, 3H, CH3); 13 C NMR (150 MHz, DMSO-) d 6) δ168.25, 159.00, 158.31, 156.11, 151.32, 138.39, 129.37, 124.28, 114.75,100.54, 99.81, 55.40, 45.87, 25.89, 22.30, 5.59;HRMS(ESI): Calcd for C 19 H 24 N5O2 + [M+H] + : 354.192 5, found: 354.192 9.

[0058] Example 9: 2-Methyl-4-( N -Methyl-4-methoxyaniline)-7-(2-morpholino-2-oxoethyl)-7 H -pyrrolo[2,3- d Pyrimidine (compound I9) Synthetic Method 1. 2-chloro-1-(morpholin-4-yl)acetone (255 mg, 1.6 mmol), Cs₂CO₃ (623 mg, 2 mmol), yielded 380 mg of the target compound as a white solid, yield: 96%, melting point: 164-166 °C, HPLC: 99.26%; 1 H NMR (600MHz, DMSO-) d 6) δ 7.28(d, J= 9.0 Hz, 2H, Ph-2&6H), 7.04(d, J = 9.0 Hz, 2H, Ph-3&5H), 6.71(d, J = 3.6 Hz, 1H, Ar-6H), 4.98(s, 2H, CH2), 4.54(d, J = 3.0 Hz, 1H, Ar-5H), 3.82(s, 3H, OMe), 3.63(t, J = 4.8 Hz, 4H, OCH2), 3.46(s, 3H, NMe), 3.40(d, J = 5.4 Hz, 4H, NCH2), 2.46(s, 3H, 2-Me). 13 C NMR (150 MHz, DMSO- d 6) δ 166.03, 158.97, 158.32, 156.09, 151.47, 138.40, 129.43, 124.35,114.75, 100.50, 99.87, 65.99, 65.94, 55.39, 44.81, 44.68, 41.77, 38.87,25.90;HRMS(ESI): Calcd for C 21 H 26 N5O3 + [M+H] + : 396.203 9, found: 396.203 0.

[0059] Example 10: 2-Methyl-4-( N -Methyl-4-methoxyaniline)-7-(2-morpholinylethyl)-7 H -pyrrolo[2,3- d Pyrimidine (compound I10) Synthetic Method 1. 2 (273 mg, 1 mmol), 4-(2-chloroethyl)morpholine (177 mg, 1.2 mmol), Cs₂CO₃ (814 mg, 2.5 mmol), yielded 190 mg of the target compound as a white solid, yield: 50%, melting point: 224-226 °C, HPLC: 99.73%; 1 H NMR (600 MHz, d -CDCl3) δ 7.23 (d, J= 8.0 Hz, 2H, Ph-2&6H), 6.97 (d, J =8.0 Hz, 2H, Ph-3&5H), 6.65 (d, J = 3.6 Hz, 1H, Ar-6H), 4.68 (d, J = 3.6 Hz, 1H, Ar-5H), 4.27 (brs, 2H, Ar-7-NCH2), 3.88(s, 3H, OMe), 3.69(brs, 4H, 2×OCH2), 3.58(s, 3H, NMe), 2.65(s, 3H, 2-Me), 2.52 (brs, 4H, 2×NCH2), 2.10 (brs, 2H,CH2).HRMS(ESI): Calcd for C 21 H 27 N5NaO2 + [M+Na] + : 404.205 7, found: 404.174 1.

[0060] Example 11: 2-Methyl-4-( N -methyl-4-methoxyaniline)-7-cyanomethyl-7 H -pyrrolo[2,3- d Pyrimidine (compound I11) Synthetic Method 1. 2 (268 mg, 1 mmol), bromoacetonitrile (137 mg, 1.3 mmol), Cs₂CO₃ (651 mg, 2 mmol) yielded 300 mg of the target compound as a white solid, yield: 98%. Melting point: 148-149 °C, HPLC: 99.68%. 1 H NMR (400 MHz, DMSO-) d 6) δ 7.29 (d, J = 8.4 Hz, 2H, Ph-2&6H), 7.05 (d, J = 8.4 Hz, 2H,Ph-3&5H), 6.93 (d, J = 3.6 Hz, 1H, Ar-6H), 5.27 (s, 2H, CH2), 4.60 (d, J = 3.6Hz, 1H, Ar-5H), 3.82 (s, 3H, OMe), 3.47 (s, 3H, NMe), 2.52 (s, 3H, 2-Me);13 CNMR (150 MHz, DMSO- d 6) δ 160.07, 158.49, 156.27, 151.03, 138.02, 129.36,122.42, 116.44, 114.88, 101.70, 100.75, 55.42, 38.90, 31.75, 25.86;HRMS(ESI):Calcd for C 17 H 18 N5O + [M+H] + : 308.151 6, found: 308.150 6.

[0061] Example 12: 2-Methyl-4-( N -Methyl-4-methoxyaniline)-7-(1-cyanoethyl)-7 H -pyrrolo[2,3- d Pyrimidine (compound I12) Synthetic method 1. 2 (135 mg, 0.5 mmol), 2-bromopropionitrile (95 mg, 0.7 mmol), Cs₂CO₃ (486 mg, 1.5 mmol), yielded 131 mg of the target compound as a white solid, yield: 82%, melting point: 165-167 °C, HPLC: 98.25%; 1 H NMR (600 MHz, DMSO-) d 6) δ 7.30 (d, J = 8.4 Hz, 2H, Ph-2&6H), 7.08 (d, J = 3.6 Hz, 1H, Ar-6H), 7.06 (d, J = 9.0 Hz, 2H, Ph-3&5H), 5.92 (q, J = 7.2 Hz, 1H, CH), 4.60(d, J = 3.6 Hz, 1H, Ar-5H), 3.83 (s, 3H, OMe), 3.47 (s, 3H, NMe), 2.52 (s, 3H,2-Me), 1.74 (d, J = 7.2 Hz, 3H, Me); 13 C NMR (150 MHz, DMSO-) d6) δ 159.92, 158.51,156.27, 150.63, 138.00, 129.38, 120.03, 118.88, 114.88, 102.10, 100.82,55.43, 39.12, 38.89, 25.90, 18.72;HRMS(ESI): Calcd for C 18 H 20 N5O + [M+H] + :322.166 2, found: 322.167 1.

[0062] Example 13: 2-Methyl-4-(6-methoxy-3-oxo-3,4-dihydroquinoxaline-2-) H -1-yl)-7-cyanomethyl-7 H -pyrrolo[2,3- d Pyrimidine (compound I13) Synthetic Method Five. 2-Methyl-4-(2-chloroacetamido-6-methoxyamino)-7-cyanomethylpyrrolo[2,3- d Pyrimidine (VIa, 267 mg, 0.69 mmol), Cs₂CO₃ (425 mg, 1.3 mmol), yielded 180 mg of the target compound as a white solid, yield: 75%, melting point: 255-257 °C, HPLC: 96.%. 1 H NMR (500 MHz, DMSO-) d 6) δ 10.68 (s, 1H,NH), 7.23 (d, J = 3.5 Hz, 1H, Ar-6H), 7.19 (d, J = 8.5 Hz, 1H, Qui-8H), 6.66 (d, J = 3.0 Hz, 1H, Qui-5H), 6.63 (dd, J = 8.5, 3.0 Hz, 1H, Qui-7H), 5.57 (d, J = 3.5Hz, 1H, Ar-5H), 5.37 (s, 2H, CH2CN), 4.66 (s, 2H, CH2), 3.78 (s, 3H, OMe), 2.58 (s, 3H, CH3); 13 C NMR (125 MHz, DMSO- d6) δ 168.38, 160.64, 157.76, 154.43,152.53, 133.83, 124.73, 124.36, 121.18, 116.78, 107.73, 102.27, 102.20,55.87, 49.37, 32.41, 26.10;HRMS(ESI): Calcd for C 18 H 17 N6O2 + [M+H] + : 349.140 8, found: 349.141 5.

[0063] Example 14: 2-Methyl-4-(6-methoxy-3-oxo-3,4-dihydroquinoxaline-2-) H -1-yl)-7-(2-ethoxy-2-oxoethyl)-7 H -pyrrolo[2,3- d Pyrimidine (compound I14) Synthetic Method Five. 2-Methyl-4-(2-chloroacetamido-6-methoxyamino)-7-(2-ethoxy-2-oxoethyl)pyrrolo[2,3- d Pyrimidine (VIb, 82 mg, 0.2 mmol), Cs2CO3 (129 mg, 0.4 mmol), yielded 66 mg of the target compound as a white solid, yield: 84%, melting point: 192-194 °C; 1 H NMR (500 MHz, DMSO-) d 6) δ 10.67 (s,1H, NH), 7.16 (d, J = 8.5 Hz, 1H, Qui-8H), 7.13 (d, J = 3.5 Hz, 1H, Ar-6H), 6.65(t, J = 2.5 Hz, 1H, Qui-5H), 6.63 (d, J = 3.0 Hz, 1H, Qui-7H), 5.51 (d, J = 3.5 Hz,1H, Ar-5H), 5.00 (s, 2H, 4-NH2), 4.65 (s, 2H, NCH2), 4.14 (q, J = 7.0 Hz, 2H,OCH2), 3.78 (s, 3H, OMe), 2.52 (s, 3H, 2-Me), 1.21 (t,J = 7.0 Hz, 3H, CH3); 13 CNMR (125 MHz, DMSO- d 6) δ 168.99, 168.46, 159.91, 157.60, 154.23, 152.93,133.74, 126.20, 124.27, 121.45, 107.68, 102.26, 102.02, 100.79, 61.49, 55.86,49.41, 45.53, 26.10, 14.51;HRMS(ESI): Calcd for C 20 H 21 N5O4 + [M+H] + : 396.166 6found: 396.167 4.

[0064] Example 15: 2-Methyl-4-(6-methoxy-3-oxo-3,4-dihydroquinoxaline-2-) H -1-yl)-7-(2-amino-2-oxoethyl)-7 H -pyrrolo[2,3- d Pyrimidine (compound I15) The synthesis procedure was the same as for compounds I3 and I14. 3 mL of ammonia in methanol yielded 36 mg of the target compound as a white solid, yield: 97%, melting point 232-234 °C. 1 H NMR (500 MHz, DMSO-) d 6) δ 10.66 (s,1H, NH), 7.51 (s, 1H, Qui-8H), 7.21 ~ 7.13 (m, 2H, NH2), 7.08 (d, J = 4.0 Hz, 1H, Ar-6), 6.64 (q, J = 3.0 Hz, 1H, Qui-5H), 6.62 (d, J = 3.0 Hz, 1H, Qui-7H),5.47 (d, J = 3.5 Hz, Ar-5H), 4.75 (s, 2H, Ar-7-NCH2), 4.64 (s, 2H, Qui-2-CH2), 3.77 (s, 3H, OMe), 2.52 (s, 3H, 2-Me); 13C NMR (125 MHz, DMSO- d 6) δ 169.55,168.48, 159.58, 157.52, 154.15, 152.91, 133.67, 126.74, 124.22, 121.59,107.64, 102.22, 102.13, 100.25, 55.86, 49.44, 46.51, 26.11;HRMS(ESI): Calcdfor C 18 H18N6O3 + [M+H] + : 367.151 3, found: 367.152 3.

[0065] Example 16: In vitro tumor cell proliferation inhibition assay of compound I 1. Test method: Human non-small cell lung cancer A549 cells, human cervical cancer HeLa cells, and human glioma U251 cells were given different concentrations of compound I (prepared in Examples 1-15) and incubated in a 37°C, 5% CO2 incubator for 48 h. The inhibition rate of the compound on tumor cells was determined by the CCK-8 assay. Colchicine was used as the positive control. The results are shown in Table 1.

[0066] 2. Experimental Results: Table 1. Inhibitory activity of the compounds of the present invention against the proliferation of A549, HeLa, and U251 tumor cells. Example 17: In vitro microtubule aggregation inhibition experiment of compound I11 1. Test drugs: Compound I11 prepared in Example 11, Compressor (CA-4), and dimethyl sulfoxide (DMSO, control).

[0067] 2. Test methods: Different concentrations of porcine brain tubulin were mixed in a PEM buffer (100 mM PIPES, 1 mM MgCl2, and 1 mM EGTA) containing 1 mM GTP and 5% glycerol. The polymerization process of tubulin was monitored at 37 °C and 340 nm using a SPECTRA MAX 190 spectrophotometer, and the relative absorbance values ​​were calculated.

[0068] 3. Experimental Results: See Figure 4 As shown, by Figure 4 It can be seen that compound I11 can inhibit tubulin aggregation, similar to the mechanism of action of CA-4. The IC50 of compound I11 inhibits tubulin aggregation.50 The value was 6.7 ± 1.1 μM.

[0069] Example 18: Simulated molecular docking of compound I11 with tubulin The composite crystal structure data used for molecular docking were all obtained from the Protein Data Bank (www.rcsb.org), number 6N47. The software used for protein structure preparation and molecular docking was Discovery Studio 3.0 (DS3.0).

[0070] 1. Preprocessing of protein structure 6N47: One α- and β-dimer, along with the ligand and metal ion, were retained from the crystal structure. Water molecules α615 and β675 were retained during docking validation. Only water molecule α615 was retained during I11 docking, and the rest were deleted. The protein structure was prepared using the Prepare Protein module; all other settings were left as default.

[0071] 2. Molecular docking with protein structure 6N47: The prepared 6N47 was used as the target protein for docking. The binding site was a spherical region with a radius of 10 Å centered on the original ligand SB-204. The ligand small molecule was energy minimized using the Full Minimization module, and a Chemistry at Harvard Macromolecular Mechamics (CHARMM) force field was added. Docking was performed using the CDOCKER module with default parameters, including conformational generation, conformational optimization, and simulated annealing. The binding energy was calculated using the Generalized Born with Molecular Volume (GBMV) solvent model.

[0072] 3. First, the docking method was validated using SB-204. The results showed that the optimal conformation was largely consistent with the ligand conformation in the protein structure, with an RMSD value of 0.482 Å and a binding energy of -25.03 kcal / mol, indicating that the docking method was effective. See [link to documentation]. Figure 5 As shown, the docking results of I11 indicate that it also binds well to tubulin, with binding energies of -18.25 kcal / mol. The N atom at the cyano terminus of I11 in its structure basically overlaps with the oxygen atom in the water molecule β626 and forms a hydrogen bond with βC239.

[0073] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0074] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A 2-methyl-4-arylamino-7-alkyl-substituted pyrrolo[2,3- d Pyrimidine compounds, characterized in that, Its structural formula is shown in Formula I: Among them, R 1 For Me or with R 2 Cyclonation is selected from at least one of the following structures: , , , R 2 It is an amino, hydroxyl, alkylamine, azacyclic alkylamine, alkoxy, alkyloxoamino, or related to R 1 Cyclonation is selected from at least one of the following structures: ， ， ， R 3 The alkyl group has two or more carbon atoms, including 2-hydroxyethyl, 2-aminoethyl, 2-alkoxyethyl, 2-methylaminoethyl, 2-dimethylaminoethyl, 2-cycloalkylaminoethyl, 2-ethoxy-2-oxoethyl, 2-amino-2-oxoethyl, 2-methylamino-2-oxoethyl, 2-cycloalkylamino-2-oxoethyl, 2-hydroxyamino-2-oxoethyl, 3-piperidinylmethyl, 2-dimethylamino-2-oxoethyl, 2-azacycloalkylamino-2-oxoethyl, 2-azacycloalkylaminoethyl, cyanomethyl, and 1-cyanoethyl; wherein the cycloalkyl group is C 3~6 A saturated alkyl group, optionally containing 0 to 2 N atoms, or simultaneously containing 0 to 1 nitrogen and oxygen atoms, or simultaneously containing 0 to 1 nitrogen and sulfur atoms; when containing nitrogen atoms, it can be C 1~6 The alkyl group is substituted; the cyclic amino group is a 3-6 membered saturated heterocyclic hydrocarbon containing at least one nitrogen atom, optionally containing 0-1 N atom, or simultaneously containing 0-1 nitrogen atom and oxygen atom, or simultaneously containing 0-1 nitrogen atom and sulfur atom, and when containing 2 nitrogen atoms, the other nitrogen atom can be C 1~6 Alkyl substitution.

2. The 2-methyl-4-arylamino-7-alkyl-substituted pyrrolo[2,3-] according to claim 1 d Pyrimidine compounds, characterized in that: R 1 With R 2 Ring formation .

3. The 2-methyl-4-arylamino-7-alkyl-substituted pyrrolo[2,3-] according to claim 1 d Pyrimidine compounds, characterized in that: R 2 With R 1 Ring formation .

4. The 2-methyl-4-arylamino-7-alkyl-substituted pyrrolo[2,3-] according to claim 1 d Pyrimidine compounds, characterized in that, The compound is any one of the following structural formulas: 。 5. A 2-methyl-4-arylamino-7-alkyl-substituted pyrrolo[2,3- d The method for preparing pyrimidine compounds, applied to the 2-methyl-4-alkylamino-7-alkyl-substituted pyrrolo[2,3-] compounds as described in any one of claims 1 to 4 d Pyrimidine compounds, characterized in that, The preparation method proceeds according to the following reaction route: 。 6. The preparation method according to claim 5, characterized in that, The preparation method includes the following steps: in the presence of a base or acid, compound 3 reacts with compound of general formula II in a solvent to obtain compound of general formula III, and then reacts with an alkylating agent to obtain compound of general formula I 1-12; Alternatively, compound 3 can be alkylated to obtain compound IV, which reacts with compound 4 in a solvent in the presence of acid to obtain compound V. After chloroacetyl chloride acylation, compound VI is obtained, and finally, compound I 13-15 is cyclized under basic conditions.

7. A pharmaceutical composition, characterized in that, The active pharmaceutical ingredient includes the compound of any one of claims 1-4 or a pharmaceutically acceptable salt thereof.

8. A combination drug composition, characterized in that, This includes the compounds described in any one of claims 1-4, as well as other pharmaceutically acceptable carriers.

9. The use of the 2-methyl-4-arylamino-7-alkyl-substituted pyrrolo[2,3-d]pyrimidine compound according to any one of claims 1 to 4 in the preparation of a drug for treating tumors.

10. The application according to claim 9, characterized in that: The tumor is any one of the following: blood cancer, ovarian cancer, prostate cancer, testicular cancer, melanoma, pancreatic cancer, lymphoma, breast cancer, stomach cancer, brain cancer, kidney cancer, lung cancer, liver cancer, or colon cancer.

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