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CN115124534B - Non-nucleotide PRMT5 small molecule inhibitor, preparation method and application - Google Patents

Non-nucleotide PRMT5 small molecule inhibitor, preparation method and application Download PDF

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CN115124534B
CN115124534B CN202111397709.6A CN202111397709A CN115124534B CN 115124534 B CN115124534 B CN 115124534B CN 202111397709 A CN202111397709 A CN 202111397709A CN 115124534 B CN115124534 B CN 115124534B
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phenyl
prmt5
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CN115124534A (en
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王元相
黄洵
荣德钦
耿美玉
杨红
施琼宇
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Shanghai Institute of Materia Medica of CAS
Sun Yat Sen University
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Sun Yat Sen University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D473/00—Heterocyclic compounds containing purine ring systems
    • C07D473/26—Heterocyclic compounds containing purine ring systems with an oxygen, sulphur, or nitrogen atom directly attached in position 2 or 6, but not in both
    • C07D473/32—Nitrogen atom
    • C07D473/34—Nitrogen atom attached in position 6, e.g. adenine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D473/00—Heterocyclic compounds containing purine ring systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
    • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
    • C07D487/04—Ortho-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D519/00—Heterocyclic compounds containing more than one system of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring system not provided for in groups C07D453/00 or C07D455/00

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Abstract

The invention discloses a non-nucleotide PRMT5 small molecule inhibitor, a preparation method and application thereof. The non-nucleotide PRMT5 small molecule inhibitor has a structure shown in a formula (I):the non-nucleotide PRMT5 small molecule inhibitor provided by the invention has the advantages of high selectivity, strong drug effect and good patentability, and can be used for treating tumors, cancers and the like.

Description

Non-nucleotide PRMT5 small molecule inhibitor, preparation method and application
Technical Field
The invention relates to the fields of pharmaceutical chemistry and pharmacotherapeutics, in particular to a non-nucleotide PRMT5 small molecule inhibitor, a preparation method and application thereof.
Background
Arginine methylation is a post-translational modification that is widely found in mammalian cells and regulates a variety of biological processes including transcription, cell signaling, mRNA translation, DNA damage, receptor trafficking, protein stability, and post-transcriptional miRNAs regulation. Protein arginine methyltransferases (protein arginine methyltransferase, PRMTs) accomplish this modification by catalyzing the transfer of methyl groups from S-adenosylmethionine (SAM) to the guanidine nitrogen atom of the arginine residue, releasing 1 equivalent of S-adenosyl-L-homocysteine (SAH). Depending on the methylation product, it can be classified into type I, type II and type III: PRMT type I catalyzes the formation of monomethyl arginine and asymmetric dimethyl arginine; PRMT type II catalyzes the formation of monomethyl arginine and symmetrical dimethyl arginine; PRMT type III catalyzes the formation of monomethyl arginine. PRMT5 belongs to type II.
PRMT5 is both an important epigenetic enzyme and an important oncogene that mediates tumorigenesis and development. For example, PRMT5 can inhibit transcription of several oncogenes, including tumor suppressor gene 7 (ST 7), metastasis suppressor gene (NM 23), retinoblastoma (Rb) family, and apoptosis 4 (PDCD 4). PRMT5 can interact with a variety of genes, regulating cell growth, such as P53, E2F-1, IL-2, cyclin E1, NF-KB, TRAIL, MITF, SC/PRDM 4, CDK4 complex, and E-cadherin (E-cadherin), among others. Recent studies (j.med. Chem.2018,61, 9429-9441.) have further shown that PRMT5 is overexpressed in a variety of cancers, including glioblastoma, leukemia/lymphoma, prostate cancer, melanoma, and colorectal cancer, and is associated with poor prognosis. In summary, targeting therapy specific to PRMT5 is a novel therapeutic strategy, and research and development of novel small molecule inhibitors LYL-283, EPZ015666, JNJ64619178, GSK3326595, PF-06939999, PRT-543 and the like of PRMT5 also prove the feasibility of research and development of drugs aiming at PRMT5 targets. However, most PRMT5 small molecule inhibitors are SAM analogues, so that the problems of poor membrane permeability, unstable metabolism, low oral bioavailability and the like exist in the pharmaceutical evaluation. Therefore, the design and synthesis of the non-nucleotide PRMT5 small molecule inhibitor with a novel drug framework have important research significance and medical value.
Disclosure of Invention
The invention aims to overcome the defects or shortages of poor membrane permeability, unstable metabolism, low oral bioavailability and the like of the traditional PRMT5 small molecule inhibitor in the patent drug property evaluation, and provide a non-nucleotide PRMT5 small molecule inhibitor. The non-nucleotide PRMT5 small molecule inhibitor provided by the invention has the advantages of high selectivity, strong drug effect and good patentability, and can be used for treating tumors, cancers and the like.
Another object of the present invention is to provide a method for preparing the above non-nucleotide PRMT5 small molecule inhibitor.
Another object of the present invention is to provide the use of the above non-nucleotide PRMT5 small molecule inhibitor or a pharmaceutically acceptable salt or solvate thereof in the preparation of a medicament for preventing and/or treating tumor or cancer.
It is another object of the present invention to provide a medicament.
In order to achieve the above object of the present invention, the present invention provides the following technical solutions:
a non-nucleotide PRMT5 small molecule inhibitor having a structure according to formula (i):
wherein:
x is selected from N or CH; y is selected from N or CH; n is n 1 Is an integer of 1 to 3; n is n 2 Is an integer of 1 to 3;
R 1 selected from hydrogen, halogen, cyano, hydroxy, amino, substituted or unsubstituted C 1～8 Alkyl, substituted or unsubstituted C 1～8 Alkoxy, substituted or unsubstituted C 3～8 Cycloalkyl, substituted or unsubstituted C 3～8 Cycloalkoxy, substituted or unsubstituted C 1～8 Alkylamino groupSubstituted or unsubstituted C 3～8 Cycloalkylamino, substituted or unsubstituted aryl or heteroaryl and their benzo derivatives, 3-to 8-membered heterocyclyl, -COR containing 1-2 heteroatoms selected from N, O or S a 、-CO 2 R a 、-CONR a R b 、-NR a C(O)R b 、-NR a SO 2 R b 、-SR a 、-SOR a 、-SO 2 R a 、-SO 2 NR a R b 、-OC(O)R a or-OC (O) NR a R b ；
R a 、R b Independently hydrogen, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-6 Cycloalkyl, aryl or heteroaryl;
R 2 、R 4 independently selected from hydrogen, halogen, cyano, nitro, amino, hydroxy, trifluoromethyl, trifluoromethoxy, substituted or unsubstituted C 1-6 Alkyl, substituted or unsubstituted C 1-6 Alkoxy, substituted or unsubstituted C 1-6 Alkylamino, substituted or unsubstituted C 3-8 Cycloalkyl, substituted or unsubstituted C 3-8 Is C, substituted or unsubstituted 3-8 Substituted or unsubstituted aryl or heteroaryl and its benzo derivatives, substituted or unsubstituted 3-to 8-membered heterocyclyl containing 1-4 heteroatoms selected from N and O, -COR c 、-CO 2 R c 、-CONR c R d 、-NR c C(O)R d 、-NR c SO 2 R d 、-SR c 、-SOR c 、-SO 2 R c 、-SO 2 NR c R d 、-OC(O)R c or-OC (O) NR c R d ；
R c And R is d Independently hydrogen, C 1-6 Alkyl, C 3-6 Cycloalkyl, aryl or heteroaryl;
R 3 independently selected from hydrogen, deuterium, fluorine, substituted or unsubstituted C 1-6 Alkyl, substituted or unsubstituted C 3-6 Cycloalkyl groups.
The inventionThrough repeated researches, the inventor constructs a series of small molecule inhibitors with drug-like dominant frameworks through design and transformation of the compounds, and the drug-like dominant frameworks are formed by the following steps of R 1 Introducing various suitable groups R 4 Various proper groups are introduced, so that the inhibition activity of the compound is improved; at R 2 Introducing various suitable groups, R 3 The metabolic stability of the compound is improved by introducing various proper groups, and the obtained non-nucleotide PRMT5 small molecule inhibitor has better inhibition effect on arginine methyltransferase PRMT5, has the advantages of high selectivity, strong drug effect and good pharmacy, and can be used for treating tumors, cancers and the like.
Preferably, said R 1 Is amino, substituted or unsubstituted C 3～8 Cycloalkoxy, substituted or unsubstituted C 1～8 Alkylamino, substituted or unsubstituted C 3～8 Cycloalkylamino, substituted or unsubstituted aryl or heteroaryl and its benzo derivative, substituted or unsubstituted 3-to 8-membered heterocyclyl containing 1-4 heteroatoms selected from N and O.
Preferably, R 2 Is H, halogen, substituted or unsubstituted C 1-6 Alkyl, substituted or unsubstituted C 1-6 An alkoxy group.
Preferably, R 3 Is hydrogen, deuterium, substituted or unsubstituted C 1-6 An alkyl group.
Preferably, R 4 Is hydrogen, C 1-6 Alkyl, halogen, nitro, cyano, trifluoromethyl or trifluoromethoxy.
Preferably, R 1 、R 2 、R 4 、R a 、R b 、R c 、R d Wherein said aryl or heteroaryl is independently selected from the group consisting of: furyl, thienyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, benzofuranyl, benzothienyl, benzoxazolyl, benzothiazolyl, phenyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, quinolinyl or naphthyl.
Preferably, said R 1 Wherein the 3-to 8-membered heterocyclic group containing 1-2 hetero atoms selected from N, O or S is morpholinyl, thiomorpholinylPiperazinyl, pyrrolidinyl, imidazolidinyl, tetrahydrofuranyl, tetrahydropyrrolyl, piperidinyl, pyranyl, pyrazolyl, N-methylpyrazolyl, N-methylpiperazinyl, N-ethylpiperazinyl, aminopiperidinyl, homopiperazinyl, N-methylpiperazinyl, tetrahydropyridinyl, dimethylpiperazine, piperazinonyl or N-methylsulfonylpiperazinyl.
Preferably, said R 2 Or R is 4 The 3-to 8-membered heterocyclic group containing 1-4 hetero atoms selected from N and O is tetrahydropyrrolyl or piperidinyl.
Preferably, the substitution means that at least 1 site is substituted with the following substituents: halogen, cyano, nitro, amino, hydroxy, carboxyl, C 1～4 Alkyl, C 1～4 Haloalkyl, C 1～4 Alkoxy or C 1-4 An alkylamino group.
Preferably, the non-nucleotide PRMT5 small molecule inhibitor has the structure shown in the numbers 1-56:
the preparation method of the non-nucleotide PRMT5 small molecule inhibitor comprises the following steps:
when X is N and Y is CH, S1: the method comprises the steps of mixing a substance shown in a formula (1) and a substance shown in a formula (2) in a solvent, heating and reacting to generate an intermediate shown in a formula (3), and then carrying out substitution reaction on the intermediate shown in the formula (3) and corresponding amine under alkaline conditions to obtain a compound shown in a formula (I), or carrying out SUZUKI coupling reaction on the intermediate shown in the formula (3) and corresponding pinacol borate to obtain the compound shown in the formula (I);
the reaction process is as follows:
preferably, the solvent in S1 is one or more of acetone, 1, 2-dichloroethane, tetrahydrofuran, N-dimethylformamide, dichloromethane, methanol, dioxane or water.
Preferably, the reaction temperature of the ring closure reaction in S1 is 60-90 ℃; the reaction temperature of the substitution reaction is 80-110 ℃; the reaction temperature of the SUZUKI coupling reaction is 80-110 ℃.
Preferably, in S1, the alkaline condition is regulated by adding a base, wherein the base is one or more of N, N-diisopropylethylamine, potassium carbonate, lithium diisopropylamide or sodium hydroxide.
Preferably, the molar ratio of the intermediate shown in formula (3) and the corresponding pinacol borate in the SUZUKI coupling reaction of S1 is 1 (1.1-1.5).
Preferably, in the substitution reaction of S1, the molar ratio of the intermediate represented by formula (3) to the corresponding amine is 1 (1.2 to 2.0).
Preferably, a catalyst is also added in the SUZUKI coupling reaction of S1, the catalyst is a palladium catalyst, and the dosage of the catalyst is 5-10% of the molar quantity of the intermediate shown in the formula (3).
Preferably, the corresponding amine in S1 is a compound which can react with an intermediate of formula (3) to give a compound having the formula R 1 Substances of the same residue, for example ammonia, morpholine, thiomorpholine, piperazine, N-methylpiperazine, N-ethylpiperazine, aminopiperidine, homopiperazine, N-methylpiperazine, dimethylpiperazine, piperazinone, N-methylsulfonylpiperazine, 4-methyl-4-hydroxypiperidine, 1-cyclopropylpiperazine, 1- (3-oxetanyl) piperazine, 2-oxa-7-aza-spiro [3,5 ]]Nonane, N' -trimethylethylenediamine, and the like.
Preferably, the corresponding pinacol borate as depicted in S1 is reacted with an intermediate of formula (3) to give a product which is reacted with R 1 Substances of the same residues, such as pinacol borate: 1-methylpyrazole-4-boronic acid pinacol ester, 4-pyrazoleboronic acid pinacol ester, N-t-butyl Oxycarbonyl-1, 2,5, 6-tetrahydropyridine-4-boronic acid pinacol ester and the like.
When X is CH, Y is CH or N, S2: the method comprises the steps of (1) mixing a substance shown in a formula (4) and a substance shown in a formula (5) in a solvent, carrying out SUZUKI coupling reaction to obtain an intermediate shown in a formula (6), and carrying out substitution reaction on the intermediate shown in the formula (6) and corresponding amine under alkaline conditions to obtain a compound shown in a formula (I);
the reaction process is as follows:
preferably, the solvent in S2 is one or more of acetone, 1, 2-dichloroethane, tetrahydrofuran, N-dimethylformamide, dichloromethane, methanol, dioxane or water.
Preferably, the reaction temperature of the substitution reaction in S2 is 80-110 ℃; the reaction temperature of the SUZUKI coupling reaction is 80-110 ℃.
Preferably, a base is added in the S2 to regulate and control alkaline conditions, wherein the base is one or more of N, N-diisopropylethylamine, potassium carbonate, lithium diisopropylamide or sodium hydroxide.
Preferably, the molar ratio of the substances represented by formula (4) and formula (5) in the SUZUKI coupling reaction of S2 is 1 (1.1 to 1.5).
Preferably, in the substitution reaction of S2, the molar ratio of the intermediate represented by formula (6) to the corresponding amine is 1 (1.2 to 2.0).
Preferably, a catalyst is also added in the SUZUKI coupling reaction of S2, the catalyst is a palladium catalyst, and the dosage of the catalyst is 5-10% of the molar weight of the reaction raw materials.
Preferably, the corresponding amine in S2 is a compound which can react with an intermediate of formula (6) to give a compound which reacts with R 1 Substances of the same residue, e.g. ammonia, morpholine, thiomorpholine, piperazine, N-methylpiperazine, N-ethylpiperazine,Aminopiperidine, homopiperazine, N-methyl homopiperazine, dimethylpiperazine, piperazinone, N-methylsulfonylpiperazine, 4-methyl-4-hydroxypiperidine, 1-cyclopropylpiperazine, 1- (3-oxetanyl) piperazine, 2-oxa-7-aza-spiro [3,5 ]]Nonane, N' -trimethylethylenediamine, and the like.
Or when X is CH, Y is CH or N, S3: dissolving a substance shown in a formula (7) in a solvent, sequentially obtaining a substance shown in a formula (8), a substance shown in a formula (9), a substance shown in a formula (10) and an intermediate shown in a formula (11) through sulfonylation, halogen exchange, bromination and deprotection, then carrying out a first SUZUKI coupling reaction on the intermediate shown in the formula (11) and the corresponding pinacol borate to obtain an intermediate shown in a formula (12), and carrying out a second SUZUKI coupling reaction on the intermediate shown in the formula (12) and the substance shown in the formula (5) to obtain a compound shown in a formula (I);
the reaction process is as follows:
preferably, the solvent in S3 is one or more of acetone, 1, 2-dichloroethane, tetrahydrofuran, N-dimethylformamide, dichloromethane, methanol, dioxane or water.
Preferably, a base is added in the S3 to regulate and control alkaline conditions, wherein the base is one or more selected from N, N-diisopropylethylamine, potassium carbonate, lithium diisopropylamide or sodium hydroxide.
Preferably, the reaction temperature of the sulfonylation reaction in S3 is 0-25 ℃, the reaction temperature of the deprotection reaction is 0-25 ℃, the reaction temperature of the halogen exchange reaction is 50-70 ℃, the reaction temperature of the bromination reaction is-78-40 ℃, and the reaction temperature of the SUZUKI coupling reaction in the first step is 80-110 ℃; the reaction temperature of the SUZUKI coupling reaction in the second step is 80-110 ℃.
Preferably, the molar ratio of the intermediate shown in formula (11) to the pinacol borate in the first step of SUZUKI coupling reaction of S3 is 1 (1.1-1.5).
Preferably, a catalyst is also added in the first step SUZUKI coupling reaction of S3, the catalyst is a palladium catalyst, and the catalyst dosage is 5-10% of the molar weight of the reaction raw material (formula (11)).
Preferably, the second step SUZUKI coupling reaction of S3 has a molar ratio of the substance represented by formula (12) to the substance represented by formula (5) of 1 (1.1-1.5)
Preferably, a catalyst is also added in the second step SUZUKI coupling reaction of S3, the catalyst is a palladium catalyst, and the catalyst dosage is 5-10% of the molar weight of the reaction raw material (formula (12)).
The corresponding pinacol borate in S3 can react with an intermediate shown in the formula (11) to obtain the product with R 1 Substances of the same residues, such as pinacol borate: 1-methylpyrazole-4-boronic acid pinacol ester, 4-pyrazoleboronic acid pinacol ester, N-t-butoxycarbonyl-1, 2,5, 6-tetrahydropyridine-4-boronic acid pinacol ester, and the like.
The use of the above-mentioned non-nucleotide PRMT5 small molecule inhibitors or pharmaceutically acceptable salts or solvates thereof in the preparation of a medicament for the prevention and/or treatment of a tumor or cancer is also within the scope of the present invention.
The non-nucleotide PRMT5 small molecule inhibitors may comprise a basic functional group and are thus capable of forming pharmaceutically acceptable salts (acid addition salts) by treatment with a suitable acid.
Preferably, the acid is a pharmaceutically acceptable inorganic acid and a pharmaceutically acceptable organic acid.
Specifically, representative pharmaceutically acceptable acid addition salts include hydrochloride, hydrobromide, nitrate, methyl nitrate, sulfate, bisulfate, sulfamate, phosphate, acetate, glycolate, phenylacetate, propionate, butyrate, isobutyrate, valerate, maleate, hydroxymaleate, acrylate, fumarate, malate, tartrate, citrate, salicylate, p-aminosalicylate, glycolate, lactate, heptanoate, phthalate, oxalate, succinate, benzoate, o-acetoxybenzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, mandelate, tannic acid, formate, stearate, ascorbate, palmitate, oleate, pyruvate, pamoate, malonate, laurate, glutarate, glutamate, propionate laurate (estolate), methanesulfonate, ethanesulfonate, 2-hydroxyethanesulfonate, benzenesulfonate, p-toluenesulfonate (toluenesulfonate), naphthalene-2-sulfonate, and the like.
Preferably, the tumor or cancer is lung cancer, bone cancer, stomach cancer, pancreatic cancer, skin cancer, head and neck cancer, uterine cancer, ovarian cancer, testicular cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, brain cancer, pituitary adenoma, epidermoid carcinoma, T-cell lymphoma, chronic and acute leukemia, colorectal cancer, renal cancer, esophageal cancer, breast cancer, cervical cancer, bladder cancer, fibrosarcoma, breast cancer, esophageal cancer, bladder cancer, hematopoietic cancer, lymphoma, medulloblastoma, rectal adenocarcinoma, colon cancer, liver cancer, adenoid cystic carcinoma, prostate cancer, head and neck squamous cell carcinoma, brain cancer, hepatocellular carcinoma, melanoma, oligodendroglioma, glioblastoma, ovarian clear cell carcinoma, ovarian serous cystic carcinoma, thyroid cancer, multiple myeloma (AML), mantle cell lymphoma, triple negative breast cancer, non-small cell lung cancer.
A medicament comprising a non-nucleotide PRMT5 small molecule inhibitor as described above, or a pharmaceutically acceptable salt or solvate thereof.
Preferably, the medicament further comprises one or more of a pharmaceutically acceptable carrier, diluent or excipient.
Compared with the prior art, the invention has the following advantages and effects:
the non-nucleotide PRMT5 small molecule inhibitor provided by the invention has the advantages of high selectivity, strong drug effect and good patentability, and can be used for treating tumors, cancers and the like.
Drawings
FIG. 1 is a graph of tumor volume change during dosing;
figure 2 is the change in body weight of nude mice during dosing;
FIG. 3 is a weight map of human malignant melanoma;
FIG. 4 is a graph showing the final volume size of representative tumors in each group 21 days after administration;
FIG. 5 is a graph showing the symmetrical dimethyl pattern of PRMT5 arginine in an in vivo mouse experiment.
Detailed Description
The present invention is further explained below with reference to examples and drawings, but the examples are not intended to limit the present invention in any way. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art.
The reagents and materials used in the present invention are commercially available unless otherwise specified.
Example 1 8- (4- ((benzyloxy) phenyl) -7H-purin-6-amine
1.8- (4- (benzyloxy) phenyl) -6-chloro-9H-purine (1 c)
Compound 1a (457mg, 3.9 mmol) and compound 1b (826.8 mg,3.9 mmol) were dissolved in 15mL of methanol, the reaction was heated at 80℃for 3 hours, then the solvent was removed under reduced pressure, and iodobenzene diacetic acid (1.26 g,3.9 mmol) and 15mL of dichloroethane were added thereto, and the heating reaction was continued at 80℃under nitrogen for overnight. After the reaction was completed, the reaction solution was extracted with dichloromethane and water, and the organic phases were combined, dried over anhydrous sodium sulfate, and purified by column chromatography after spin-drying the solvent to give compound 1c 124.7mg as a pale yellow solid, with a yield of 9.5%.1H NMR (500 MHz, DMSO). Delta.14.13 (s, 1H), 8.68 (s, 1H), 8.23 (d, J=8.4 Hz, 2H), 7.49 (d, J=7.3 Hz, 2H), 7.42 (t, J=7.6 Hz, 2H), 7.36 (t, J=7.3 Hz, 1H), 7.25 (d, J=8.8 Hz, 2H), 5.23 (s, 2H). LCMS: M/z (M+H) + ):337.08.
2.8- (4- (benzyloxy) phenyl) -9H-purin-6-amine (1)
Compound 1c (80 mg,0.24 mmol) was placed in a vial and 3mL of methanol was added to dissolveAmmonia methanol solution (2 mol/L in MeOH) 0.36mL was added and reacted overnight at 100 ℃. After the completion of the reaction, it was cooled to room temperature, and after the solvent was removed under reduced pressure, 58mg of a white solid was purified by column chromatography, and the yield was 76.3%. 1 H NMR(500MHz,DMSO)δ13.21(s,1H),8.13–8.05(m,3H),7.47(d,J＝7.5Hz,2H),7.40(t,J＝7.3Hz,2H),7.34(t,J＝7.2Hz,1H),7.17(d,J＝8.2Hz,2H),7.11(s,2H),5.18(s,2H).HRMS(ESI)calcd for C 18 H 15 N 5 O(M+H + ):318.1349；found 318.1349.
Example 2 8- (4- (benzyloxy) phenyl) -6- (1, 2,3, 6-tetrahydropyridin-4-yl) -9H-purine
1.4- (8- (4- (benzyloxy) phenyl) -9H-purin-6-yl) -3, 6-dihydropyridine-1 (2H) -carboxylic acid tert-butyl ester (2 b)
Compound 1c (135 mg,0.40 mmol), N-t-butoxycarbonyl-1, 2,5, 6-tetrahydropyridine-4-boronic acid pinacol ester (compound 2a,247.2mg,0.80 mmol), pdCl 2 (dppf)CH 2 Cl 2 (32.7mg,0.04mmol)、K 2 CO 3 (165.6 mg,1.20 mmol) in 1,4-dioxane/H 2 In a solution of o=3/1 (16 ml), stirring was carried out at 110℃for 6 hours under nitrogen. After the reaction was completed, it was filtered through celite, the cake was washed with methylene chloride, the organic phases were combined, the filtrate was concentrated under reduced pressure, and purified by column chromatography to give 137.8mg of yellow solid in 71.3% yield. 1 H NMR(500MHz,DMSO)δ13.76(s,1H),8.77(s,1H),8.23(d,J＝8.0Hz,2H),7.97(s,1H),7.49(d,J＝7.2Hz,2H),7.42(t,J＝7.1Hz,2H),7.36(t,J＝7.1Hz,1H),7.22(d,J＝8.0Hz,2H),5.22(s,2H),4.22(s,2H),3.60(s,2H),2.80(s,2H),1.45(s,9H).LCMS:m/z(M+H + ):484.57.
2.8- (4- (benzyloxy) phenyl) -6- (1, 2,3, 6-tetrahydropyridin-4-yl) -9H-purine (2)
Compound 2b (130 mg,0.27 mmol) was dissolved in 10mL of methanol, added with HCl-MeOH solution (4.0 mol/L in MeOH) 0.34mL, stirred at room temperature for reaction for 12h, then added with saturated sodium bicarbonate solution to adjust pH to pH=8, and after most of the solvent was distilled off by rotary evaporation, dichloromethane was used The reaction solution was extracted with alkane and water, the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure, and purified by column chromatography to give 69.3mg of a yellow solid with a yield of 67.1%. 1 H NMR(500MHz,DMSO)δ8.80(s,1H),8.23(d,J＝8.3Hz,2H),8.00(s,1H),7.49(d,J＝7.6Hz,2H),7.42(t,J＝7.4Hz,2H),7.35(t,J＝7.2Hz,1H),7.23(d,J＝8.3Hz,2H),5.22(s,2H),3.92(s,2H),3.33(t,J＝5.8Hz,2H),2.94(s,2H).HRMS(ESI)calcd for C 23 H 21 N 5 O(M+H + ):384.1819；found 384.1812.
Example 3 4- (8- (4- (benzyloxy) phenyl) -9H-purin-6-yl) morpholine
8- (4- (benzyloxy) phenyl) -6-chloro-9H-purine (compound 1c,120mg,0.36 mmol), morpholine (37.6 mg,0.43 mmol) and DIPEA (92.9 mg,0.72 mmol) were added to the tube, then 3mL DMF was added as solvent and the reaction was stirred at 100deg.C overnight. After the completion of the reaction, 15mL of ethyl acetate was added, the mixture was washed with water and a saturated sodium chloride solution, and the organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure, followed by purification by column chromatography to give 96mg of a white solid in 69% yield. 1 H NMR(500MHz,DMSO)δ13.39(s,1H),8.22(s,1H),8.08(d,J＝7.6Hz,2H),7.47(d,J＝6.9Hz,2H),7.40(t,J＝6.9Hz,2H),7.34(m,1H),7.16(d,J＝7.6Hz,2H),5.18(s,2H),4.25(s,4H),3.75(s,4H).HRMS(ESI)calcd for C 22 H 21 N 5 O 2 (M+H + ):388.1768；found 388.1767.
Example 4 8- (4- (benzyloxy) phenyl) -6- (piperazin-1-yl) -9H-purine
Substitution of piperazine for morpholine and the remaining starting materials, reagents and preparation were as described in example 3 to give a white solid. 1 H NMR(500MHz,DMSO)δ8.19(s,1H),8.07(d,J＝8.4Hz,2H),7.47(d,J＝7.4Hz,2H),7.40(t,J＝7.3Hz,2H),7.34(t,J＝7.2Hz,1H),7.16(d,J＝8.4Hz,2H),5.18(s,2H),4.21(s,4H),2.85(s,4H).HRMS(ESI)calcd for C 22 H 22 N 6 O(M+H + ):387.1928；found 387.1924.
Example 5 8- (4- (benzyloxy) phenyl) -6- (1H-pyrazol-5-yl) -9H-purine
Replacement of compound 2a with 1H-pyrazole-3-boronic acid pinacol ester gives a yellow solid, as is the case with the remaining desired starting materials, reagents and preparation method in step 1 of example 2. 1 H NMR(500MHz,DMSO)δ13.73(s,1H),13.41(s,1H),8.78(d,J＝39.0Hz,2H),8.58(s,1H),8.29(d,J＝7.9Hz,2H),7.49(d,J＝7.3Hz,2H),7.41(t,J＝7.2Hz,2H),7.35(m,1H),7.22(d,J＝7.9Hz,2H),5.22(s,2H)HRMS(ESI)calcd for C 21 H 16 N 6 O(M+H + ):369.1458；found 369.1454.
Example 6 6- (4- (benzyloxy) phenyl) -7H-pyrrolo [2,3-d ] pyrimidin-4-amine
1.4-chloro-7- (benzenesulfonyl) -7H-pyrrolo [2,3-d ] pyrimidine (6 b)
In a dry flask was added compound 6a (10 g,65 mmol), t-BuOK (8.75 g,78 mmol) and THF (50 mL), after nitrogen sparge, benzenesulfonyl chloride (12.6 g,68.3 mmol) was added dropwise to the reaction at 0deg.C. The reaction was then stirred at room temperature for 3 hours. After the reaction, adding ice water for quenching reaction, extracting the reaction liquid by ethyl acetate, drying the organic phase anhydrous sodium sulfate, filtering and concentrating under reduced pressure to obtain a white solid compound 6b 17.5g, wherein the yield is 92%. 1 H NMR(500MHz,CDCl 3 )δ8.77(s,1H),8.21(d,J＝7.6Hz,2H),7.78(s,1H),7.64(t,J＝7.3Hz,1H),7.54(t,J＝7.6Hz,2H),6.72(s,1H).LCMS:m/z(M+H + ):294.07.
2.4-chloro-6-iodo-7- (benzenesulfonyl) -7H-pyrrolo [2,3-d ] pyrimidine (6 c)
Compound 6b (5 g,17 mmol) and anhydrous tetrahydrofuran (50 ml) were added to a dry flask under nitrogen, and after cooling the solution to-78℃LDA (2.7 g,25.5 mmol) was slowly added dropwise to the flask. After the addition was completed, the mixture was stirred at-78℃for 1.5 hours, and then I was added dropwise 2 (5.2 g,20.4 mmol) (elemental iodine in anhydrous tetrahydrofuran). After stirring the reaction mixture for 5 hours, the reaction was quenched by slowly adding a saturated sodium bisulphite solution, the mixture was extracted three times with DCM, the organic phases were washed with brine, and the combined organic phases were taken up in anhydrous Na2SO 4 Drying, filtration and concentration gave 6c 5g of crude product as a yellow solid in 72% yield. 1 H NMR(500MHz,CDCl 3 )δ8.67(s,1H),8.21(d,J＝7.4Hz,2H),7.67(t,J＝7.5Hz,1H),7.56(t,J＝7.9Hz,2H),6.45(s,1H).LCMS:m/z(M+H + ):419.90.
3.4-chloro-6-iodo-7H-pyrrolo [2,3-d ] pyrimidine (6 d)
Compound 6c (5 g,12 mmol) was dissolved in 40mL THF and a solution of NaOH (1.9 g,48 mmol) in methanol was added dropwise at 0deg.C. The reaction mixture was stirred at room temperature for 2h. After the reaction, the solvent was removed under reduced pressure, and saturated NH was then added 4 The Cl solution, at this time, white solid is separated out, filtered, and the filter cake is washed with water and dried to obtain 6d 2.3g of white solid compound with 67.2% yield. 1 H NMR(400MHz,DMSO)δ8.51(s,1H),6.87(s,1H).LCMS:m/z(M+H + ):279.91.
4.6- (4- (benzyloxy) phenyl) -4-chloro-7H-pyrrolo [2,3-d ] pyrimidine (6 e)
Compound 6d (2 g,7.2 mmol), 4-benzyloxyphenylboronic acid (compound 6f,2.1g,9.4 mmol), pdCl 2 (dppf)CH 2 Cl 2 (588mg,0.72mmol)、K 2 CO 3 (1.7 g,12 mmol) in 1,4-dioxane/H 2 In a solution of o=3/1 (40 ml), stirring was carried out at 110℃for 4 hours under nitrogen. After the reaction was completed, it was filtered through celite, the cake was washed with methylene chloride, the organic phases were combined, the filtrate was concentrated under reduced pressure, and purified by column chromatography to give 6e 1.67g of a white solid with a yield of 69.1%. 1 H NMR(500MHz,DMSO)δ12.00(s,1H),8.09(s,1H),7.81(d,J＝7.3Hz,2H),7.47(d,J＝5.4Hz,2H),7.40(t,J＝5.8Hz,2H),7.34(t,J＝8.0Hz,1H),7.07(d,J＝7.1Hz,2H),7.00(s,1H),5.15(s,2H).LCMS:m/z(M+H + ):336.08.
5.6- (4- (benzyloxy) phenyl) -7H-pyrrolo [2,3-d ] pyrimidin-4-amine (6)
Compound 6e (100 mg,0.30 mmol) was placed in a vial, 3mL of methanol was added to dissolve, and then 0.45mL of methanolic ammonia solution (2 mol/L in MeOH) was added and reacted overnight at 100 ℃. After the completion of the reaction, the mixture was cooled to room temperature, and after the solvent was removed by rotary evaporation, 61.9mg of a white solid was purified by column chromatography, and the yield was 65.3%. 1 H NMR(500MHz,DMSO)δ 1 H NMR(500MHz,DMSO)δ11.91(s,1H),8.03(s,1H),7.70(d,J＝8.8Hz,2H),7.46(d,J＝7.2Hz,2H),7.40(t,J＝7.5Hz,2H),7.33(t,J＝7.3Hz,1H),7.09(d,J＝8.8Hz,2H),6.93(s,2H),6.78(d,J＝1.9Hz,1H),5.15(s,2H).HRMS(ESI)calcd for C 19 H 16 N 4 O(M+H + ):317.1397；found 317.1397.
Example 7 6- (4- (benzyloxy) phenyl) -4- (1, 2,3, 6-tetrahydropyridin-4-yl) -7H-pyrrolo [2,3-d ] pyrimidine
1.4- (6- (4- (benzyloxy) phenyl) -7H-pyrrolo [2,3-d ] pyrimidin-4-yl) -3, 6-dihydropyridine-1 (2H) -carboxylic acid tert-butyl ester (7 a)
Compound 6e (200 mg,0.60 mmol), compound 2a (375 mg,1.20 mmol), pdCl 2 (dppf)CH 2 Cl 2 (49mg,0.06mmol)、K 2 CO 3 (247 mg,1.80 mmol) in 1,4-dioxane/H 2 In a solution of o=3/1 (16 ml), stirring was carried out at 110℃for 4 hours under nitrogen. After the reaction was completed, it was filtered through celite, the cake was washed with methylene chloride, the organic phases were combined, the filtrate was concentrated under reduced pressure, and purified by column chromatography to give 163.2mg of yellow solid in 71.2% yield. 1 H NMR(400MHz,DMSO)δ12.51(s,1H),8.67(s,1H),7.96(d,J＝8.6Hz,2H),7.47(d,J＝7.3Hz,2H),7.40(t,J＝7.4Hz,2H),7.34(t,J＝7.1Hz,1H),7.23(s,1H),7.13(d,J＝8.7Hz,2H),6.98(s,1H),5.17(s,2H),4.02(d,J＝7.1Hz,2H),3.59(s,2H),2.76(s,2H),1.45(s,9H).LCMS:m/z(M+H + ):483.23.
2.6- (4- (benzyloxy) phenyl) -4- (1, 2,3, 6-tetrahydropyridin-4-yl) -7H-pyrrolo [2,3-d ] pyrimidine (7)
Compound 7a (110 mg,0.23 mmol) was dissolved in 10mL of methanol, 0.29mL of HCl-MeOH solution (4.0 mol/L in MeOH) was added, and after stirring at room temperature for reaction for 12 hours, saturated sodium bicarbonate solution was added to adjust ph=8, after most of the solvent was removed by rotary evaporation, the reaction solution was extracted with dichloromethane and water, the organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by column chromatography to give 60.3mg of yellow solid in 68.5% yield. 1 H NMR(500MHz,DMSO)δ12.64(s,1H),9.17(s,1H),8.72(s,1H),7.97(d,J＝8.8Hz,2H),7.48(d,J＝7.3Hz,2H),7.41(t,J＝7.5Hz,2H),7.35(t,J＝7.3Hz,1H),7.26(d,J＝1.5Hz,1H),7.16(d,J＝8.8Hz,2H),7.03(s,1H),5.19(s,2H),3.95(d,J＝2.1Hz,2H),3.40(t,J＝6.1Hz,2H),2.96(s,2H).HRMS(ESI)calcd for C 24 H 22 N 4 O(M+H + ):383.1866；found 383.1866.
Example 8 6- (4- (benzyloxy) phenyl) -4- (piperazin-1-yl) -7H-pyrrolo [2,3-d ] pyrimidine
Compound 6e (150 mg,0.45 mmol), piperazine (58.5 mg,0.68 mmol) and DIPEA (116 mg,0.9 mmol) were added to the tube, then 3mL DMF was added as solvent and the reaction was stirred at 100deg.C overnight. Ethyl acetate (15 mL) was added, the mixture was washed with water and saturated sodium chloride solution, and the organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain 140.7mg of a white solid by column chromatography, with a yield of 81.2%. 1 H NMR(500MHz,DMSO)δ12.13(s,1H),8.15(s,1H),7.85(d,J＝8.8Hz,2H),7.47(d,J＝7.2Hz,2H),7.40(t,J＝7.5Hz,2H),7.33(t,J＝7.2Hz,1H),7.08(d,J＝8.8Hz,2H),7.02(s,1H),5.15(s,2H),4.83(s,1H),3.92–3.87(m,4H),2.96–2.90(m,4H).HRMS(ESI)calcd for C 23 H 23 N 5 O(M+H + ):386.1975；found 386.1973.
Example 9 4- (6- (4- (benzyloxy) phenyl) -7H-pyrrolo [2,3-d ] pyrimidin-4-yl) morpholine
Substitution of morpholine for piperazine, the remaining desired starting materials, reagents and preparation were as described in example 8 to give a white solid. 1 H NMR(500MHz,DMSO)δ12.18(s,1H),8.18(s,1H),7.85(d,J＝8.7Hz,2H),7.47(d,J＝7.3Hz,2H),7.40(t,J＝7.5Hz,2H),7.33(t,J＝7.3Hz,1H),7.09(s,1H),7.07(m,2H),5.15(s,2H),3.87(m,4H),3.74(m,4H).HRMS(ESI)calcd for C 23 H 22 N 4 O 2 (M+H + ):387.1816；found 387.1817.
Example 10 4- (6- (4- (benzyloxy) phenyl) -7H-pyrrolo [2,3-d ] pyrimidin-4-yl) thiomorpholine
Substitution of piperazine with thiomorpholine provided the remaining starting materials, reagents and preparation method as in example 8 provided a yellow solid. 1 H NMR(500MHz,DMSO)δ12.15(s,1H),8.16(s,1H),7.85(d,J＝8.8Hz,2H),7.46(d,J＝7.2Hz,2H),7.40(t,J＝7.5Hz,2H),7.33(t,J＝7.3Hz,1H),7.08(d,J＝8.8Hz,2H),6.96(s,1H),5.15(s,2H),4.20(m,4H),2.70(dd,J＝5.9,3.9Hz,4H).HRMS(ESI)calcd for C 23 H 22 N 4 OS(M+H + ):403.1587；found 403.1587.
Example 11 6- (4- (benzyloxy) phenyl) -N- (piperidin-4-yl) -7H-pyrrolo [2,3-d ] pyrimidin-4-amine
Piperazine was replaced by piperidin-4-amine, and the remaining required starting materials, reagents and preparation were the same as in example 8 to give a white solid. 1 H NMR(400MHz,DMSO)δ11.91(s,1H),8.09(s,1H),7.71(d,J＝8.7Hz,2H),7.47(d,J＝7.1Hz,2H),7.40(t,J＝7.3Hz,2H),7.34(t,J＝7.2Hz,1H),7.25(d,J＝7.7Hz,1H),7.09(d,J＝8.8Hz,2H),6.88(s,1H),5.15(s,2H),4.14(dd,J＝7.4,3.7Hz,1H),3.05(d,J＝12.2Hz,2H),2.66(d,J＝12.0Hz,2H),1.92(d,J＝12.0Hz,2H),1.81(s,1H),1.54–1.43(m,2H).HRMS(ESI)calcd for C 24 H 25 N 5 O(M+H + ):400.2132；found 400.2133.
Example 12 6- (4- (benzyloxy) phenyl) -4- (1H-pyrazol-4-yl) -7H-pyrrolo [2,3-d ] pyrimidine
The procedure of example 7, step 1, was followed using 4-pyrazoloboronic acid pinacol ester instead of compound 2a, and the remaining desired starting materials, reagents and preparation method, to give a yellow solid. 1 H NMR(500MHz,DMSO)δ13.38(s,1H),12.44(s,1H),8.67(d,J＝33.4Hz,2H),8.42(s,1H),8.02(d,J＝8.5Hz,2H),7.48(d,J＝7.4Hz,2H),7.40(m,3H),7.34(t,J＝7.1Hz,1H),7.15(d,J＝8.5Hz,2H),5.19(s,2H).HRMS(ESI)calcd for C 22 H 17 N 5 O(M+H + ):368.1506；found 368.1507.
Example 13 4- (6- (4- (benzyloxy) phenyl) -7H-pyrrolo [2,3-d ] pyrimidin-4-yl) piperazin-2-one
The piperazine was replaced by 2-piperazinone, and the remaining required starting materials, reagents and preparation method were the same as in example 8 to give a white solid. 1 H NMR(500MHz,DMSO)δ12.17(s,1H),8.18(s,2H),7.86(d,J＝8.5Hz,2H),7.47(d,J＝7.5Hz,2H),7.40(t,J＝7.4Hz,2H),7.33(t,J＝7.2Hz,1H),7.08(d,J＝8.5Hz,2H),7.04(s,1H),5.15(s,2H),4.39(s,2H),4.06(t,J＝5.1Hz,2H),3.37(s,2H).HRMS(ESI)calcd for C 23 H 21 N 5 O 2 (M+H + ):400.1768；found 400.1767.
Example 14 1- (6- (4- (benzyloxy) phenyl) -7H-pyrrolo [2,3-d ] pyrimidin-4-yl) -4-methylpiperidin-4-ol
The piperazine was replaced by 4-methyl-4-hydroxypiperidine, and the remaining desired starting materials, reagents and preparation were the same as in example 8 to give a white solid. 1 H NMR(500MHz,DMSO)δ12.05(s,1H),8.12(s,1H),7.84(s,2H),7.40(m,5H),7.02(d,J＝44.3Hz,3H),5.15(s,2H),4.44(s,1H),4.28(s,2H),3.58(s,2H),1.56(d,J＝17.7Hz,4H),1.16(s,3H).HRMS(ESI)calcd for C 25 H 26 N 4 O 2 (M+H + ):415.2129；found 415.2127.
Example 15 6- (4- (benzyloxy) phenyl) -4- (4- (methylsulfonyl) piperazin-1-yl) -7H-pyrrolo [2,3-d ] pyrimidine
Piperazine was replaced by 1-methanesulfonyl piperazine, and the remaining required starting materials, reagents and preparation were the same as in example 8 to give a white solid. 1 H NMR(500MHz,DMSO)δ12.18(s,1H),8.19(s,1H),7.86(d,J＝8.2Hz,2H),7.47(d,J＝7.2Hz,2H),7.40(t,J＝7.2Hz,2H),7.34(t,J＝7.1Hz,1H),7.09(m,3H),5.16(s,2H),4.02(s,4H),3.33(s,4H),2.91(s,3H).HRMS(ESI)calcd for C 24 H 25 N 5 O 3 S(M+H + ):464.1751；found 464.1751.
Example 16 6- (4- (benzyloxy) phenyl) -4- (4-methylpiperazin-1-yl) -7H-pyrrolo [2,3-d ] pyrimidine
The piperazine was replaced by N-methylpiperazine, and the remaining desired starting materials, reagents and preparation were the same as in example 8 to give a white solid. 1 H NMR(500MHz,DMSO)δ12.10(s,1H),8.14(s,1H),7.84(d,J＝8.6Hz,2H),7.47(d,J＝7.5Hz,2H),7.40(t,J＝7.4Hz,2H),7.33(t,J＝7.2Hz,1H),7.07(d,J＝8.6Hz,2H),7.02(s,1H),5.15(s,2H),4.04–3.67(m,4H),2.47–2.38(m,4H),2.22(s,3H).HRMS(ESI)calcd for C 24 H 25 N 5 O(M+H + ):400.2049；found400.2050.
Example 17N 1 - (6- (4- (benzyloxy) phenyl) -7H-pyrrolo [2, 3-d)]Pyrimidin-4-yl) -N 1 ，N 2 ，N 2 -trimethylethane-1, 2-diamine
Piperazine was replaced by N, N' -trimethylethylenediamine, and the remaining desired starting materials, reagents and preparation methods were the same as in example 8 to give a white solid. 1 H NMR(400MHz,DMSO)δ11.99(s,1H),8.08(s,1H),7.79(d,J＝8.8Hz,2H),7.47(d,J＝7.2Hz,2H),7.40(t,J＝7.3Hz,2H),7.33(t,J＝7.2Hz,1H),7.07(d,J＝8.8Hz,2H),6.92(s,1H),5.15(s,2H),3.84(t,J＝6.8Hz,2H),3.36(s,5H),2.21(s,6H).HRMS(ESI)calcd for C 24 H 27 N 5 O(M+H + ):402.2288；found 402.2288.
Example 18 6- (4- (benzyloxy) phenyl) -4- (1-methyl-1H-pyrazol-4-yl) -7H-pyrrole [2,3-d ] pyrimidine
Replacement of compound 2a with 1-methylpyrazole-4-boronic acid pinacol ester gives a yellow solid with the remaining required starting materials, reagents and preparation method as in step 1 of example 7. 1 H NMR(400MHz,DMSO)δ12.45(s,1H),8.67(d,J＝29.4Hz,2H),8.33(s,1H),8.00(d,J＝6.8Hz,2H),7.41(m,6H),7.15(d,J＝6.9Hz,2H),5.19(s,2H),3.97(s,3H).HRMS(ESI)calcd for C 23 H 19 N 5 O(M+H + ):382.1662；found 382.1661.
Example 19 6- (4- ((4-methoxybenzyl) oxy) phenyl) -4- (1, 2,3, 6-tetrahydropyridin-4-yl) -7H-pyrrolo [2,3-d ] pyrimidine
1.4-iodo-7- (benzenesulfonyl) -7H-pyrrole [2,3-d ] pyrimidine (19 a)
4-chloro-7- (benzenesulfonyl) -7H-pyrrole [2,3-d]Pyrimidine (6 b,11g,37.4 mmol) and NaI (7.3 g,4.9 mmol) were added to 50mL of hydroiodic acid (57% W/W aqueous solution, 1.5% hypophosphorous acid stabilizer) and stirred at 60℃for 12 hours. The reaction mixture was diluted with water, the solids were isolated by suction filtration and the filter cake was washed with water. The crude solid was dissolved in methylene chloride, washed twice with saturated sodium bicarbonate solution, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain 19a 10g of reddish brown solid. LCMS: M/z (M+H) + ):385.53.
2.6-bromo-4-iodo-7- (benzenesulfonyl) -7H-pyrrole [2,3-d ] pyrimidine (19 b)
Replacement of compound 6b with compound 19a, replacement of elemental iodine with 1, 2-dibromotetrachloroethane, and the remaining required starting materials, reagents, and preparation were the same as in example 6, step 2, to give yellow solid 19b. 1 H NMR(500MHz,CDCl 3 )δ8.67(s,1H),8.21(d,J＝7.4Hz,2H),7.67(t,J＝7.5Hz,1H),7.56(t,J＝7.9Hz,2H),6.45(s,1H).LCMS:m/z(M+H + ):465.10.
3.6-bromo-4-iodo-7H-pyrrole [2,3-d ] pyrimidine (19 c)
Replacement of compound 6c with compound 19b gives yellow solid 19c as well as the remaining desired starting materials, reagents and preparation method as in example 6, step 3. 1 H NMR(400MHz,DMSO)δ8.51(s,1H),6.87(s,1H).LCMS:m/z(M+H + ):325.96.
4.4- (6-bromo-7H-pyrrolo [2,3-d ] pyrimidin-4-yl) -3, 6-dihydropyridine-1 (2H) -carboxylic acid tert-butyl ester (19 d)
Compound 19c (3 g,9.23 mmol), compound 2a (2.2 g,7.2 mmol), pdCl 2 (dppf)CH 2 Cl 2 (589m,0.72mmol)、K 2 CO 3 (3.0 g,21.6 mmol) in 1,4-dioxane/H 2 In a solution of o=3/1 (40 ml), stirring was carried out at 110℃for 4 hours under nitrogen. After the reaction was completed, it was filtered through celite, the cake was washed with methylene chloride, the organic phases were combined, the filtrate was concentrated under reduced pressure, and purified by column chromatography to give 19d 2.01g of a yellow solid in 57.1% yield. 1 H NMR(500MHz,DMSO)δ12.97(s,1H),8.67(s,1H),6.97(s,1H),6.86(s,1H),4.11(s,2H),3.55(t,J＝5.4Hz,2H),2.69(s,2H),1.43(s,9H).LCMS:m/z(M+H + ):379.36.
5.6-bromo-4- (1, 2,3, 6-tetrahydropyridin-4-yl) -7H-pyrrole [2,3-d ] pyrimidine (19 e)
Compound 19d (2.0 g,0.23 mmol) was dissolved in 5mL dioxane, HCl-dioxane solution (4.0 mol/L in dioxane) was added to 5.3mL, the reaction was stirred at room temperature overnight, a yellow solid was precipitated, and compound 19e 1.2g was obtained by filtration, with a yield of 81.6%. LCMS: M/z (M+H) + ):279.32.
6.6- (4- ((4-methoxybenzyl) oxy) phenyl) -4- (1, 2,3, 6-tetrahydropyridin-4-yl) -7H-pyrrole [2,3-d ] pyrimidine (19)
Compound 19e (100 mg,0.36 mmol), pinacol 4- (4-methoxybenzyloxy) phenylboronic acid ester (compound 19f,193.8mg,0.54 mmol), pdCl 2 (dppf)CH 2 Cl 2 (29.5mg,0.036mmol)、K 2 CO 3 (149.0 g,1.08 mmol) in 1,4-dioxane/H 2 In a solution of o=3/1 (8 ml), stirring was carried out at 110℃for 4 hours under nitrogen. After the reaction was completed, it was filtered through celite, the filter cake was washed with methylene chloride, the organic phases were combined, the filtrate was concentrated under reduced pressure, and purified by column chromatography to give 35mg of yellow solid in 23.7% yield. 1 H NMR(400MHz,DMSO)δ13.32(s,1H),9.85(s,2H),8.84(s,1H),8.03(d,J＝8.7Hz,2H),7.43(s,1H),7.36(d,J＝7.9Hz,2H),7.21(d,J＝7.8Hz,2H),7.16(d,J＝8.8Hz,2H),7.10(s,1H),5.15(s,2H),,4.31(s,3H),3.93(s,2H),3.34(s,2H),3.04(s,2H).HRMS(ESI)calcd for C 25 H 24 N 4 O 2 (M+H + ):413.1899；found 413.1893.
Example 20 6- (4- (benzyloxy) phenyl) -4- (4-methyl-1, 4-diaza-1-yl) -7H-pyrrolo [2,3-d ] pyrimidine
The piperazine was replaced by N-methyl homopiperazine, and the remaining required raw materials, reagents and preparation method were the same as in example 8 to give a white solid. 1 H NMR(400MHz,DMSO)δ12.02(s,1H),8.09(s,1H),7.82(d,J＝8.7Hz,2H),7.47(d,J＝7.2Hz,2H),7.40(t,J＝7.4Hz,2H),7.34(d,J＝7.2Hz,1H),7.07(d,J＝8.8Hz,2H),6.90(s,1H),5.15(s,2H),3.96(m,4H),2.70(s,2H),2.26(s,3H),1.97(s,2H),1.23(d,J＝7.8Hz,2H).HRMS(ESI)calcd for C 25 H 27 N 5 O(M+H + ):414.2288；found 414.2287.
Example 21 6- (4- (benzyloxy) phenyl) -4- (1, 4-diaza-1-yl) -7H-pyrrolo [2,3-d ] pyrimidine
The piperazine was replaced by homopiperazine, and the remaining required starting materials, reagents and preparation method were the same as in example 8 to give a white solid. 1 H NMR(500MHz,DMSO)δ12.02(s,1H),8.10(s,1H),7.82(d,J＝7.9Hz,2H),7.47(d,J＝7.1Hz,2H),7.40(t,J＝7.2Hz,2H),7.34(s,1H),7.07(d,J＝7.9Hz,2H),6.90(s,1H),5.15(s,2H),3.96(s,4H),3.02(s,2H),2.79(s,2H),1.90(s,2H).HRMS(ESI)calcd for C 24 H 25 N 5 O(M+H + ):400.2132；found 400.2130.
Example 22 6- (4- (benzyloxy) phenyl) -4- (4- (oxetan-3-yl) piperazin-1-yl) -7H-pyrrolo [2,3-d ] pyrimidine
The piperazine was replaced by 1- (3-oxetanyl) piperazine and the remaining starting materials, reagents and preparation were the same as in example 8 to give a white solid. 1 H NMR(400MHz,DMSO)δ12.11(s,1H),8.14(s,1H),7.84(d,J＝8.8Hz,2H),7.46(d,J＝7.1Hz,2H),7.40(t,J＝7.3Hz,2H),7.33(t,J＝7.2Hz,1H),7.07(d,J＝8.8Hz,2H),7.01(d,J＝1.8Hz,1H),5.15(s,2H),4.57(t,J＝6.5Hz,2H),4.49(t,J＝6.0Hz,2H),3.91(m,4H),3.44(dd,J＝12.6,6.3Hz,1H),2.39(m,4H).HRMS(ESI)calcd for C 26 H 27 N 5 O 2 (M+H + ):442.2238；found 442.2232.
Example 23 6- (4- (benzyloxy) phenyl) -4- (4-cyclopropylpiperazin-1-yl) -7H-pyrrolo [2,3-d ] pyrimidine
The piperazine was replaced by 1-cyclopropylpiperazine, and the remaining desired starting materials, reagents and preparation were the same as in example 8 to give a white solid. 1 H NMR(400MHz,DMSO)δ13.30(s,1H),8.39(s,1H),7.94(d,J＝8.8Hz,2H),7.47(d,J＝7.0Hz,2H),7.45–7.37(m,3H),7.34(t,J＝7.2Hz,1H),7.13(d,J＝8.9Hz,2H),5.17(s,2H),4.83(s,2H),3.91(s,2H),3.57(d,J＝29.3Hz,4H),2.97–2.85(m,1H),1.25(d,J＝2.3Hz,2H),0.83(d,J＝7.0Hz,2H).HRMS(ESI)calcd for C 26 H 27 N 5 O(M+H + ):426.2288；found 426.2287.
Example 24 7- (6- (4- (benzyloxy) phenyl) -7H-pyrrolo [2,3-d ] pyrimidin-4-yl) -2-oxa-7-azaspiro [3.5] nonane
With 2-oxazol-7-azaspiro [3.5]]Nonane replaces piperazine, and the remaining required raw materials, reagents and preparation method are the same as in example 8, giving a white solid. 1 H NMR(400MHz,DMSO)δ12.09(s,1H),8.13(s,1H),7.85(d,J＝8.7Hz,2H),7.47(d,J＝7.3Hz,2H),7.40(t,J＝7.4Hz,2H),7.34(t,J＝7.1Hz,1H),7.08(d,J＝8.7Hz,2H),6.99(s,1H),5.15(s,2H),4.37(s,4H),3.82(m,4H),1.87(m,4H).HRMS(ESI)calcd for C 26 H 26 N 4 O 2 (M+H + ):427.2129；found427.2121.
Example 25 6- (4- (benzyloxy) phenyl) -4- (4-ethylpiperazin-1-yl) -7H-pyrrolo [2,3-d ] pyrimidine
The piperazine was replaced by N-ethylpiperazine, and the remaining desired starting materials, reagents and preparation method were the same as in example 8 to give a white solid. 1 H NMR(500MHz,DMSO)δ12.10(s,1H),8.15(s,1H),7.85(d,J＝7.7Hz,2H),7.47(d,J＝6.0Hz,2H),7.41(s,2H),7.35(d,J＝6.3Hz,1H),7.08(d,J＝7.7Hz,2H),7.02(s,1H),5.16(s,2H),3.89(s,4H),2.49(d,J＝5.7Hz,4H),2.38(d,J＝6.5Hz,2H),1.05(s,3H).HRMS(ESI)calcd for C 25 H 27 N 5 O(M+H + ):414.2288；found 414.2287.
EXAMPLE 26 6- (4- (benzyloxy) phenyl) -4- (3, 5-dimethylpiperazin-1-yl) -7H-pyrrolo [2,3-d ] pyrimidine
The piperazine was replaced by 2, 6-dimethylpiperazine, and the remaining required starting materials, reagents and preparation method were the same as in example 8 to give a white solid. 1 H NMR(400MHz,DMSO)δ12.08(s,1H),8.12(s,1H),7.86(d,J＝8.6Hz,2H),7.47(d,J＝7.3Hz,2H),7.40(t,J＝7.4Hz,2H),7.33(t,J＝7.1Hz,1H),7.07(d,J＝8.7Hz,2H),6.96(s,1H),5.15(s,2H),4.60(d,J＝11.8Hz,2H),2.81–2.72(m,2H),2.56(d,J＝11.8Hz,2H),1.07(d,J＝6.1Hz,6H).HRMS(ESI)calcd for C 25 H 27 N 5 O(M+H + ):414.2288；found 414.2286.
Example 27 6- (4- ((4-methylbenzyl) oxy) phenyl) -4- (1, 2,3, 6-tetrahydropyridin-4-yl) -7H-pyrrolo [2,3-d ] pyrimidine
The procedure of step 6 of example 19 was repeated using 4- (4-methylbenzyloxy) phenylboronic acid pinacol ester instead of compound 19f to give a yellow solid. 1 H NMR(400MHz,DMSO)δ13.32(s,1H),9.85(s,2H),8.84(s,1H),8.03(d,J＝8.7Hz,2H),7.43(s,1H),7.36(d,J＝7.9Hz,2H),7.21(d,J＝7.8Hz,2H),7.16(d,J＝8.8Hz,2H),7.10(s,1H),5.15(s,2H),3.93(s,2H),3.34(s,2H),3.04(s,2H),2.31(s,3H).HRMS(ESI)calcd for C 25 H 24 N 4 O(M+H + ):397.2023；found 397.2021.
Example 28 6- (4- ((3-nitrobenzyl) oxy) phenyl) -4- (1, 2,3, 6-tetrahydropyridin-4-yl) -7H-pyrrolo [2,3-d ] pyrimidine
The procedure of step 6 of example 19 was followed using 4- (3-nitrobenzyl oxy) phenylboronic acid pinacol ester instead of compound 19f to give a yellow solid. 1 H NMR(400MHz,DMSO)δ12.98(s,1H),8.75(s,1H),8.34(s,1H),8.21(d,J＝7.6Hz,1H),8.04(d,J＝8.3Hz,1H),7.95(d,J＝7.5Hz,1H),7.72(t,J＝7.8Hz,1H),7.36(s,1H),7.20(d,J＝8.3Hz,1H),7.07(s,1H),5.37(s,2H),3.88(s,2H),3.31(s,2H),3.00(s,2H).HRMS(ESI)calcd for C 24 H 21 N 5 O 3 (M+H + ):428.1717；found 428.1718.
Example 29 4-chloro-6- (4- (((4-cyanobenzyl) oxy) phenyl) -7H-pyrrolo [2,3-d ] pyrimidine
The procedure of step 6 of example 19 was repeated using 4- (4-cyanobenzyloxy) phenylboronic acid pinacol ester instead of compound 19f to give a yellow solid. 1 H NMR(500MHz,DMSO)δ8.99(s,1H),7.95(d,J＝6.8Hz,2H),7.82(d,J＝7.1Hz,2H),7.63(d,J＝7.0Hz,2H),7.44(s,1H),7.15(d,J＝7.2Hz,2H),7.00(s,1H),5.26(s,2H),3.95(s,2H),3.39(s,2H),2.97(s,2H).HRMS(ESI)calcd for C 25 H 21 N 5 O(M+H + ):408.1819；found408.1814.
Example 30 6- (4- ((4-fluorobenzyl) oxy) phenyl) -4- (1, 2,3, 6-tetrahydropyridin-4-yl) -7H-pyrrolo [2,3-d ] pyrimidine
With 4- (4-fluorobenzyloxy) phenylboronic acidThe pinacol ester was substituted for compound 19f and the remaining desired starting materials, reagents and preparation were the same as in step 6 of example 19 to give a yellow solid. 1 HNMR(400MHz,DMSO)δ13.39(s,1H),8.97(s,1H),8.04(d,J＝7.9Hz,2H),7.89(d,J＝7.8Hz,2H),7.69(d,J＝7.8Hz,2H),7.48(s,1H),7.20(d,J＝8.0Hz,2H),7.10(s,1H),5.33(s,2H),3.98(s,2H),3.41(s,2H),3.05(s,2H).HRMS(ESI)calcd for C 24 H 21 N 5 OF(M+H + ):401.1770；found 401.1774.
Example 31 4- (1, 2,3, 6-tetrahydropyridin-4-yl) -6- (4- ((4- (trifluoromethyl) benzyl) oxy) phenyl) -7H-pyrrolo [2,3-d ] pyrimidine
The procedure of step 6 of example 19 was followed using 4- (4-trifluoromethylbenzyloxy) phenylboronic acid pinacol ester instead of compound 19f to give a yellow solid. 1 H NMR(400MHz,DMSO)δ13.54(s,1H),8.89(s,1H),8.06(d,J＝8.8Hz,2H),7.78(d,J＝8.2Hz,2H),7.71(d,J＝8.1Hz,2H),7.49(s,1H),7.20(d,J＝8.8Hz,2H),7.12(s,1H),5.33(s,2H),3.97(s,2H),3.36(s,2H),3.06(s,2H).HRMS(ESI)calcd for C 25 H 21 N 4 OF 3 (M+H + ):451.1740；found 451.1735.
Example 32 6- (4- ((3-fluorobenzyl) oxy) phenyl) -4- (1, 2,3, 6-tetrahydropyridin-4-yl) -7H-pyrrolo [2,3-d ] pyrimidine
The procedure of step 6 of example 19 was repeated using 4- (3-fluorobenzyloxy) phenylboronic acid pinacol ester instead of compound 19f to give a yellow solid. 1 HNMR(400MHz,DMSO)δ13.64(s,1H),8.89(s,1H),8.05(d,J＝8.5Hz,2H),7.46(m,2H),7.31(d,J＝8.1Hz,2H),7.16(m,4H),5.22(s,2H),3.97(s,2H),3.36(s,2H),3.07(s,2H).HRMS(ESI)calcd for C 24 H 21 N 4 OF(M+H + ):401.1772；found 401.1774.
Example 33 (R) -6- (4- (1-Phenylethoxy) phenyl) -4- (1, 2,3, 6-tetrahydropyridin-4-yl) -7H-pyrrolo [2,3-d ] pyrimidine
1. (R) -4- (4' -methylbenzyloxy) phenylboronic acid pinacol ester (33 c)
4-Hydroxyphenylboronic acid pinacol ester (33 a,500mg,2.70 mmol), S- (-) -phenethyl alcohol (33 b,659mg,5.40 mmol), triphenylphosphine (1.41 g,5.40 mmol) were placed in a 50mL round bottom flask, 15mL anhydrous tetrahydrofuran was added under nitrogen protection, diisopropyl azodicarboxylate (1.09 g,5.40 mmol) was slowly added dropwise under ice bath conditions, and stirring was resumed at room temperature overnight after the addition. After the reaction was completed, the organic phase was concentrated by spin-drying, and the residue was purified by column chromatography to give 630mg of a white solid with a yield of 72.0%. 1 H NMR(400MHz,CDCl 3 )δ7.71(d,J＝8.6Hz,2H),7.40(d,J＝7.0Hz,2H),7.35(t,J＝7.5Hz,2H),7.27(t,J＝6.2Hz,1H),6.90(d,J＝8.6Hz,2H),5.41(q,J＝6.4Hz,1H),1.68(d,J＝6.4Hz,3H),1.34(s,12H).
2. (R) -6- (4- (1-Phenylethoxy) phenyl) -4- (1, 2,3, 6-tetrahydropyridin-4-yl) -7H-pyrrolo [2,3-d ] pyrimidine (33)
Replacement of compound 19f with compound 33c gives a yellow solid as in step 6 of example 19, as well as the remaining required starting materials, reagents and preparation methods. 1 H NMR(500MHz,DMSO)δ8.63(s,1H),7.84(d,J＝8.8Hz,2H),7.42(d,J＝7.3Hz,2H),7.35(t,J＝7.6Hz,2H),7.25(t,J＝7.3Hz,1H),7.11(s,1H),7.00(d,J＝8.9Hz,3H),5.59(q,1H),3.56(s,2H),3.00(t,J＝5.5Hz,2H),2.65(s,2H),1.57(d,J＝6.3Hz,3H).HRMS(ESI)calcd for C 25 H 24 N 4 O(M+H + ):397.2023；found 397.2020.
Example 34 8- (4- (1-Phenylethoxy) phenyl) -6- (1, 2,3, 6-tetrahydropyridin-4-yl) -9H-purine
1.4- (1-Phenylethoxy) benzaldehyde (34 c)
Compound 34a (500 mg,4.1 mmol), compound 34b (910 mg,4.90 mmol), and potassium carbonate (1.13 g,8.20 mmol) were placed in a bottle, 15mL of acetonitrile as a solvent was added, and the mixture was heated at 60℃for 4 hours. After the completion of the reaction, most of the solvent was removed by rotary evaporation, the reaction solution was extracted with ethyl acetate and water, and the organic phase was collected, dried over anhydrous sodium sulfate, and purified by column chromatography to give 830mg of a colorless oil, with a yield of 89.6%. 1 H NMR(400MHz,CDCl 3 )δ9.82(s,1H),7.74(d,J＝8.8Hz,2H),7.38–7.32(m,4H),7.30–7.26(m,1H),6.96(d,J＝8.7Hz,2H),5.41(q,J＝6.4Hz,1H),1.68(d,J＝6.4Hz,3H).
2.6-chloro-8- (4- (1-phenylethoxy) phenyl) -9H-purine (34 d)
Replacement of compound 1b with compound 34c gives a white solid as in step 1 of example 1, with the remaining required starting materials, reagents and preparation methods. 1 H NMR(400MHz,DMSO)δ8.65(s,1H),8.12(d,J＝8.8Hz,2H),7.44(d,J＝7.3Hz,2H),7.35(t,J＝7.6Hz,2H),7.26(t,J＝7.3Hz,1H),7.11(d,J＝8.8Hz,2H),5.66(q,J＝6.2Hz,1H),1.59(d,J＝6.3Hz,3H).LCMS:m/z(M+H + ):351.47.
3.4- (8- (4- (1- (phenylethoxy) phenyl) -9H-purin-6-yl) -3, 6-dihydropyridine-1 (2H) -carboxylic acid tert-butyl ester (34 e)
Replacement of compound 1c with compound 34d gives a yellow solid as in step 1 of example 2, with the remaining required starting materials, reagents and preparation method. 1 H NMR(400MHz,DMSO)δ8.73(s,1H),8.10(d,J＝8.7Hz,2H),7.96(s,1H),7.42(d,J＝7.3Hz,2H),7.34(t,J＝7.5Hz,2H),7.25(t,J＝7.3Hz,1H),7.07(d,J＝8.8Hz,2H),5.61(q,J＝6.1Hz,1H),4.18(s,2H),3.56(t,J＝5.3Hz,2H),2.76(s,2H),1.57(d,J＝6.3Hz,3H),1.42(s,9H).LCMS:m/z(M+H + ):498.57.
4.8- (4- (1-Phenylethoxy) phenyl) -6- (1, 2,3, 6-tetrahydropyridin-4-yl) -9H-purine (34)
Replacement of compound 2b with compound 34e gives a yellow solid as in step 2 of example 2, with the remaining required starting materials, reagents and preparation method. 1 H NMR(500MHz,DMSO)δ8.76(s,1H),8.12(d,J＝8.8Hz,2H),7.96(s,1H),7.43(d,J＝7.3Hz,2H),7.35(t,J＝7.6Hz,2H),7.26(t,J＝7.3Hz,1H),7.09(d,J＝8.8Hz,2H),5.64(q,J＝6.2Hz,1H),3.84(s,2H),3.26(t,J＝5.7Hz,2H),2.88(s,2H),1.59(d,J＝6.3Hz,3H).HRMS(ESI)calcd for C 24 H 23 N 5 O(M+H + ):398.1975；found 398.1968.
Example 35 (R) -8- (4- (1-Phenylethoxy) phenyl) -6- (1, 2,3, 6-tetrahydropyridin-4-yl) -9H-purine
1. (R) -4- (1-Phenylethoxy) benzaldehyde (35 a)
Compound 34a (330 mg,2.70 mmol), S- (-) -phenethyl alcohol (compound 33b,659mg,5.40 mmol), triphenylphosphine (1.41 g,5.40 mmol) were placed in a 50mL round bottom flask, 15mL anhydrous tetrahydrofuran was added under nitrogen protection, diisopropyl azodicarboxylate (1.09 g,5.40 mmol) was slowly added dropwise under ice bath conditions, and stirring was resumed at room temperature overnight after the addition. After the completion of the reaction, the organic phase was concentrated by spin-drying, and the residue was purified by column chromatography to give 396mg of a colorless oil, yield 65.0%. 1 H NMR(400MHz,CDCl 3 )δ9.84(s,1H),7.80–7.73(m,2H),7.41–7.34(m,4H),7.32–7.28(m,1H),6.99(d,J＝8.7Hz,2H),5.44(q,J＝6.4Hz,1H),1.70(d,J＝6.4Hz,3H).
2. (R) -6-chloro-8- (4- (1-phenylethoxy) phenyl) -9H-purine (35 b)
Replacement of compound 1b with compound 35a gives a white solid as in step 1 of example 1, with the remaining required starting materials, reagents and preparation methods. 1 H NMR(500MHz,CDCl 3 )δ11.78(s,1H),8.74(s,1H),8.38(t,J＝9.0Hz,1H),7.37(d,J＝4.4Hz,4H),7.30(m,J＝8.6,4.4Hz,1H),6.88(dd,J＝8.9,2.2Hz,1H),6.73(dd,J＝14.2,2.2Hz,1H),5.39(q,J＝6.4Hz,1H),1.69(d,J＝2.9Hz,3H).LCMS:m/z(M+H + ):351.65.
3. (R) -4- (8- (4- (1- (phenylethoxy) phenyl) -9H-purin-6-yl) -3, 6-dihydropyridine-1 (2H) -carboxylic acid tert-butyl ester (35 c)
With Compound 35bReplacement of compound 1c, the remaining required starting materials, reagents and preparation were as in step 1 of example 2, giving a yellow solid. 1 H NMR(400MHz,DMSO)δ8.74(s,1H),8.11(d,J＝8.8Hz,2H),7.96(s,1H),7.43(d,J＝7.3Hz,2H),7.35(t,J＝7.6Hz,2H),7.26(t,J＝7.3Hz,1H),7.08(d,J＝8.8Hz,2H),5.64(q,J＝6.2Hz,1H),4.19(s,2H),3.57(t,2H),2.76(s,2H),1.58(d,J＝6.3Hz,3H),1.43(s,9H).LCMS:m/z(M+H + ) 498.32.4 (R) -8- (4- (1-Phenylethoxy) phenyl) -6- (1, 2,3, 6-tetrahydropyridin-4-yl) -9H-purine (35)
Replacement of compound 2b with compound 35c gives a yellow solid as in step 2 of example 2, with the remaining required starting materials, reagents and preparation method. 1 H NMR(400MHz,DMSO)δ8.78(s,1H),8.13(d,J＝8.9Hz,2H),7.96(s,1H),7.45(d,J＝7.2Hz,2H),7.36(t,J＝7.6Hz,2H),7.27(t,J＝7.3Hz,1H),7.11(d,J＝8.9Hz,2H),5.66(q,J＝6.3Hz,1H),3.91(s,2H),3.32(t,J＝5.8Hz,2H),2.93(s,2H),1.60(d,J＝6.3Hz,3H).HRMS(ESI)calcd for C 24 H 23 N 5 O(M+H + ):398.1975；found 398.1971.
Example 36 (S) -8- (4- (1-Phenylethoxy) phenyl) -6- (1, 2,3, 6-tetrahydropyridin-4-yl) -9H-purine
1. (S) -4- (1-Phenylethoxy) benzaldehyde (36 b)
Replacement of compound 33b with compound 36a gives a colorless oil, as is step 1 of example 35, as well as the remaining desired starting materials, reagents and preparation method. 1 H NMR(400MHz,CDCl 3 )δ9.84(s,1H),7.80–7.73(m,2H),7.41–7.34(m,4H),7.32–7.28(m,1H),6.99(d,J＝8.7Hz,2H),5.44(q,J＝6.4Hz,1H),1.70(d,J＝6.4Hz,3H).
2. (S) -6-chloro-8- (4- (1-phenylethoxy) phenyl) -9H-purine (36 c)
Replacement of compound 1b with compound 36b gives a white solid as in step 1 of example 1, with the remaining required starting materials, reagents and preparation methods. 1 H NMR(400MHz,DMSO)δ8.66(s,1H),8.13(d,J＝8.7Hz,2H),7.44(d,J＝7.4Hz,2H),7.36(t,J＝7.5Hz,2H),7.27(t,J＝7.2Hz,1H),7.11(d,J＝8.8Hz,2H),5.66(q,J＝6.2Hz,1H),1.59(d,J＝6.3Hz,3H).LCMS:m/z(M+H + ):351.43.
3. (S) -4- (8- (4- (1- (phenylethoxy) phenyl) -9H-purin-6-yl) -3, 6-dihydropyridine-1 (2H) -carboxylic acid tert-butyl ester (36 d)
Replacement of compound 1c with compound 36c gives a yellow solid as in step 1 of example 2, with the remaining required starting materials, reagents and preparation method. 1 H NMR(400MHz,DMSO)δ8.73(s,1H),8.10(d,J＝8.7Hz,2H),7.96(s,1H),7.42(d,J＝7.3Hz,2H),7.34(t,J＝7.5Hz,2H),7.25(t,J＝7.3Hz,1H),7.07(d,J＝8.8Hz,2H),5.61(q,J＝6.1Hz,1H),4.18(s,2H),3.56(t,J＝5.3Hz,2H),2.76(s,2H),1.57(d,J＝6.3Hz,3H),1.42(s,9H).LCMS:m/z(M+H + ):498.52.
4. (S) -8- (4- (1-Phenylethoxy) phenyl) -6- (1, 2,3, 6-tetrahydropyridin-4-yl) -9H-purine (36)
Replacement of compound 2b with compound 36d gives a yellow solid as in step 2 of example 2, with the remaining required starting materials, reagents and preparation method. 1 H NMR(400MHz,DMSO)δ8.79(s,1H),8.13(d,J＝8.4Hz,2H),7.97(s,1H),7.45(d,J＝7.3Hz,2H),7.36(t,J＝7.3Hz,2H),7.27(t,J＝7.1Hz,1H),7.11(d,J＝8.4Hz,2H),5.67(q,J＝11.6,5.4Hz,1H),3.94(s,2H),3.36(s,2H),2.95(s,2H),1.59(d,J＝6.0Hz,3H).HRMS(ESI)calcd for C 24 H 23 N 5 O(M+H + ):398.1975；found 398.1970.
Example 37 6- (4- (1-Phenylethoxy) phenyl) -4- (1, 2,3, 6-tetrahydropyridin-4-yl) -7H-pyrrolo [2,3-d ] pyrimidine
1. (4- (1-Phenylethoxy) phenyl) boronic acid pinacol ester (37 a)
Compound 33a (500 mg,2.70 mmol), compound 34b (624 mg,2.84 mmol), potassium carbonate (745 mg,5.40 mmol) were placed in a bottle, 15mL of acetonitrile as a solvent was added, and the mixture was heated at 60℃for 4 hours. After the reaction was completed, most of the solvent was removed by rotary evaporation, and ethyl acetate andthe reaction solution was extracted with water, and the organic phase was collected, dried over anhydrous sodium sulfate, and purified by column chromatography to give 814mg of a white solid with a yield of 88.2%. 1 H NMR(500MHz,DMSO)δ7.52(d,J＝8.6Hz,2H),7.39(d,J＝7.3Hz,2H),7.34(t,J＝7.4Hz,2H),7.24(t,1H),6.90(d,J＝8.6Hz,2H),5.56(q,J＝6.3Hz,1H),1.55(d,J＝6.4Hz,3H),1.24(s,12H).
2.6- (4- (1-Phenylethoxy) phenyl) -4- (1, 2,3, 6-tetrahydropyridin-4-yl) -7H-pyrrolo [2,3-d ] pyrimidine (37)
Replacement of compound 19f with compound 37a gives a yellow solid as in step 6 of example 19, as well as the remaining required starting materials, reagents and preparation methods. 1 H NMR(500MHz,DMSO)δ12.42(s,1H),8.65(s,1H),7.85(d,J＝8.6Hz,2H),7.44(d,J＝7.4Hz,2H),7.36(t,J＝7.4Hz,2H),7.26(t,J＝7.0Hz,1H),7.14(s,1H),7.02(d,J＝8.8Hz,3H),5.61(q,J＝12.0,5.8Hz,1H),3.63(s,2H),3.07(t,J＝5.2Hz,2H),2.70(s,2H),1.58(d,J＝6.1Hz,3H).HRMS(ESI)calcd for C 25 H 24 N 4 O(M+H + ):397.2023；found 397.2026.
Example 38 8- (2-fluoro-4- (1-phenylethoxy) phenyl) -6- (1, 2,3, 6-tetrahydropyridin-4-yl) -9H-purine
1.2-fluoro-4- (1-phenylethoxy) benzaldehyde (38 b)
Replacement of compound 34a with compound 38a gives a yellow oil, as is the case with the remaining desired starting materials, reagents and preparation method in step 1 of example 34. 1 H NMR(500MHz,DMSO)δ10.04(s,1H),7.74(t,J＝8.7Hz,1H),7.46(d,J＝7.5Hz,2H),7.38(t,J＝7.6Hz,2H),7.30(t,J＝7.3Hz,1H),6.97–6.95(m,1H),6.94(s,1H),5.73(q,J＝6.3Hz,1H),1.61(d,J＝6.4Hz,3H).
2.6-chloro-8- (2-fluoro-4- (1-phenylethoxy) phenyl) -9H-purine (38 c)
Replacement of compound 1b with compound 38b gives a white solid as in step 1 of example 1, with the remaining required starting materials, reagents and preparation methods. 1 H NMR(500MHz,CDCl 3 )δ11.85(s,1H),8.56(s,1H),7.60(t,J＝8.8Hz,1H),7.37(m,J＝8.1,5.5Hz,4H),7.29(m,J＝5.8,2.7Hz,1H),6.80(dd,J＝9.0,2.1Hz,1H),6.72(dd,J＝13.5,2.0Hz,1H),5.35(q,J＝6.4Hz,1H),1.69(d,J＝6.4Hz,3H).LCMS:m/z(M+H + ):369.84.
3.4- (8- (2-fluoro-4- (1-phenylethoxy) phenyl) -9H-purin-6-yl) -3, 6-dihydropyridine-1 (2H) -carboxylic acid tert-butyl ester (38 d)
Replacement of compound 1c with compound 38c gives a yellow solid as in step 1 of example 2, with the remaining required starting materials, reagents and preparation method. 1 H NMR(500MHz,CDCl 3 )δ12.71(s,1H),8.87(s,1H),8.30(t,J＝8.8Hz,1H),7.99(s,1H),7.37(m,4H),7.29(m,1H),6.85(d,J＝8.8Hz,1H),6.71(d,J＝13.7Hz,1H),5.36(q,J＝6.3Hz,1H),4.30(s,2H),3.71(s,2H),2.91(s,2H),1.70(d,J＝6.4Hz,3H),1.51(s,9H).LCMS:m/z(M+H + ):516.59.
4.8- (2-fluoro-4- (1-phenylethoxy) phenyl) -6- (1, 2,3, 6-tetrahydropyridin-4-yl) -9H-purine (38)
Replacement of compound 2b with compound 38d gives a yellow solid as in step 2 of example 2, with the remaining required starting materials, reagents and preparation method. 1 H NMR(500MHz,DMSO)δ8.83(s,1H),8.04(t,J＝8.7Hz,1H),7.96(s,1H),7.47(d,J＝7.6Hz,2H),7.39(t,J＝7.5Hz,2H),7.29(t,J＝7.2Hz,1H),7.04(d,J＝13.2Hz,1H),6.99(d,J＝8.8Hz,1H),5.71(q,J＝6.1Hz,1H),3.91(s,2H),3.32(t,J＝4.6Hz,2H),2.98(s,2H),1.61(d,J＝6.3Hz,3H).HRMS(ESI)calcd for C 24 H 22 N 5 OF(M+H + ):416.1881；found 416.1873.
Example 39 (R) -8- (2-fluoro-4- (1-phenylethoxy) phenyl) -6- (1, 2,3, 6-tetrahydropyridin-4-yl) -9H-purine
1. (R) -2-fluoro-4- (1-phenylethoxy) benzaldehyde (39 a)
Replacement of compound 34a with compound 38a gives a colourless oil, as is the case with the remaining required starting materials, reagents and preparation method in step 1 of example 35. 1 H NMR(500MHz,CDCl 3 )δ10.12(s,1H),7.70(t,J＝8.5Hz,1H),7.33(m,J＝4.5Hz,4H),7.29–7.23(m,1H),6.73(dd,J＝8.8,2.1Hz,1H),6.57(dd,J＝12.5,2.2Hz,1H),5.35(q,J＝6.4Hz,1H),1.65(d,J＝6.5Hz,3H).
2. (R) -6-chloro-8- (2-fluoro-4- (1-phenylethoxy) phenyl) -9H-purine (39 b)
Replacement of compound 1b with compound 39a gives a white solid as in step 1 of example 1, with the remaining required starting materials, reagents and preparation methods. 1 H NMR(500MHz,CDCl 3 )δ10.14(s,1H),7.73(t,J＝8.5Hz,1H),7.38–7.32(m,4H),7.30(m,J＝6.4,2.2Hz,1H),6.75(dd,J＝8.8,2.2Hz,1H),6.58(dd,J＝12.5,2.3Hz,1H),5.37(q,J＝6.4Hz,1H),1.67(d,J＝6.4Hz,3H).LCMS:m/z(M+H + ):369.75.
3. (R) -4- (8- (2-fluoro-4- (1-phenylethoxy) phenyl) -9H-purin-6-yl) -3, 6-dihydropyridine-1 (2H) -carboxylic acid tert-butyl ester (39 c)
Replacement of compound 1c with compound 39b gives a yellow solid as in step 1 of example 2, with the remaining required starting materials, reagents and preparation method. 1 H NMR(500MHz,CDCl 3 )δ12.71(s,1H),8.89(s,1H),8.30(t,J＝8.8Hz,1H),7.99(s,1H),7.37(m,4H),7.29(m,1H),6.85(d,J＝8.8Hz,1H),6.71(d,J＝13.7Hz,1H),5.37(q,J＝6.3Hz,1H),4.30(s,2H),3.71(s,2H),2.92(s,2H),1.70(d,J＝6.4Hz,3H),1.51(s,9H).LCMS:m/z(M+H + ):516.71.
4. (R) -8- (2-fluoro-4- (1-phenylethoxy) phenyl) -6- (1, 2,3, 6-tetrahydropyridin-4-yl) -9H-purine (39)
Replacement of compound 2b with compound 39c gives a yellow solid as in step 2 of example 2, with the remaining required starting materials, reagents and preparation method. 1 H NMR(400MHz,DMSO)δ8.84(s,1H),8.02(t,J＝8.7Hz,1H),7.94(s,1H),7.46(d,J＝7.4Hz,2H),7.37(t,J＝7.5Hz,2H),7.28(t,J＝7.4Hz,1H),7.04(dd,J＝13.1,2.1Hz,1H),7.00(dd,J＝8.8,2.3Hz,1H),5.71(q,J＝6.3Hz,1H),3.95(s,2H),3.38(t,J＝5.5Hz,2H),2.98(s,2H),1.60(d,J＝6.3Hz,3H).HRMS(ESI)calcd for C 24 H 22 N 5 OF(M+H + ):416.1881；found 416.1876.
Example 40 (S) -8- (2-fluoro-4- (1-phenylethoxy) phenyl) -6- (1, 2,3, 6-tetrahydropyridin-4-yl) -9H-purine
1. (S) -2-fluoro-4- (1-phenylethoxy) benzaldehyde (40 a)
Replacement of compound 34a with compound 38a and compound 33b with compound 36a gave a colorless oil, as well as the remaining desired starting materials, reagents and preparation method as in step 1 of example 35. 1 H NMR(400MHz,CDCl 3 )δ10.14(s,1H),7.72(t,J＝8.5Hz,1H),7.41–7.27(m,5H),6.74(dd,J＝8.8,2.1Hz,1H),6.57(dd,J＝12.5,2.3Hz,1H),5.36(q,J＝6.4Hz,1H),1.67(d,J＝6.4Hz,3H).
2. (S) -6-chloro-8- (2-fluoro-4- (1-phenylethoxy) phenyl) -9H-purine (40 b)
Replacement of compound 1b with compound 40a gives a white solid as in step 1 of example 1, with the remaining required starting materials, reagents and preparation methods. 1 H NMR(500MHz,DMSO)δ8.71(s,1H),8.01(t,J＝8.8Hz,1H),7.46(d,J＝7.2Hz,2H),7.38(t,J＝7.6Hz,2H),7.29(t,J＝7.3Hz,1H),7.05(dd,J＝13.1,2.4Hz,1H),7.00(dd,J＝8.8,2.4Hz,1H),5.72(q,J＝6.3Hz,1H),1.61(d,J＝6.4Hz,3H).LCMS:m/z(M+H + ):369.67.
3. (S) -4- (8- (2-fluoro-4- (1-phenylethoxy) phenyl) -9H-purin-6-yl) -3, 6-dihydropyridine-1 (2H) -carboxylic acid tert-butyl ester (40 c)
Replacement of compound 1c with compound 40b gives a yellow solid as in step 1 of example 2, with the remaining required starting materials, reagents and preparation method. 1 H NMR(500MHz,CDCl 3 )δ12.72(s,1H),8.90(s,1H),8.31(t,J＝8.8Hz,1H),8.00(s,1H),7.44–7.35(m,4H),7.34–7.27(m,1H),6.86(d,J＝8.8Hz,1H),6.71(d,J＝13.7Hz,1H),5.38(q,J＝6.3Hz,1H),4.31(s,2H),3.71(s,2H),2.93(s,2H),1.70(d,J＝6.4Hz,3H),1.52(s,9H).LCMS:m/z(M+H + ):516.64.
4. (S) -8- (2-fluoro-4- (1-phenylethoxy) phenyl) -6- (1, 2,3, 6-tetrahydropyridin-4-yl) -9H-purine (40)
Compound 40c was substituted for compound 2a,the remaining required starting materials, reagents and preparation method were the same as in step 2 of example 2, giving a yellow solid. 1 H NMR(400MHz,DMSO)δ8.84(s,1H),8.02(t,J＝8.7Hz,1H),7.94(s,1H),7.46(d,J＝7.4Hz,2H),7.37(t,J＝7.5Hz,2H),7.28(t,J＝7.4Hz,1H),7.04(dd,J＝13.1,2.1Hz,1H),7.00(dd,J＝8.8,2.3Hz,1H),5.71(q,J＝6.3Hz,1H),3.95(s,2H),3.38(t,J＝5.5Hz,2H),2.98(s,2H),1.60(d,J＝6.3Hz,3H).HRMS(ESI)calcd for C 24 H 22 N 5 OF(M+H + ):416.1881；found 416.1878.
Example 41 6- (2-fluoro-4- (1-phenylethoxy) phenyl) -4- (1, 2,3, 6-tetrahydropyridin-4-yl) -7H-pyrrolo [2,3-d ] pyrimidine
1. (2-fluoro-4- (1-phenylethoxy) phenyl) boronic acid pinacol ester (41 b)
Replacement of compound 33a with compound 41a gives a white solid as in step 1 of example 37, as well as the remaining required starting materials, reagents and preparation methods. 1 H NMR(400MHz,CDCl 3 )δ7.55(t,J＝7.8Hz,1H),7.35–7.30(m,4H),7.26(m,1H),6.64(dd,J＝8.4,2.2Hz,1H),6.51(dd,J＝11.4,2.1Hz,1H),5.32(q,J＝6.4Hz,1H),1.63(d,J＝6.4Hz,3H),1.31(s,12H).
2.6- (2-fluoro-4- (1-phenylethoxy) phenyl) -4- (1, 2,3, 6-tetrahydropyridin-4-yl) -7H-pyrrolo [2,3-d ] pyrimidine (41)
Replacement of compound 19f with compound 41b gives a yellow solid as in step 6 of example 19, as well as the remaining required starting materials, reagents and preparation methods. 1 H NMR(500MHz,DMSO)δ8.72(s,1H),7.81(t,J＝8.9Hz,1H),7.44(d,J＝7.3Hz,2H),7.36(t,J＝7.6Hz,2H),7.27(t,J＝7.3Hz,1H),7.03(d,J＝2.0Hz,1H),6.98(dd,J＝13.5,2.3Hz,1H),6.95–6.89(m,2H),5.66(q,J＝6.2Hz,1H),3.80(s,2H),3.26(s,2H),2.84(s,2H),1.58(d,J＝6.3Hz,3H).HRMS(ESI)calcd for C 25 H 23 N 4 OF(M+H + ):415.1929；found 415.1931.
Example 42 (R) -6- (2-fluoro-4- (1-phenylethoxy) phenyl) -4- (1, 2,3, 6-tetrahydropyridin-4-yl) -7H-pyrrolo [2,3-d ] pyrimidine
1. (R) - (2-fluoro-4- (1-phenylethoxy) phenyl) boronic acid pinacol ester (42 a)
Replacement of compound 33a with compound 41a gives a white solid as in step 1 of example 33, as well as the remaining required starting materials, reagents and preparation methods. 1 H NMR(500MHz,CDCl 3 )δ7.61(d,J＝8.3Hz,1H),7.35(d,J＝7.5Hz,2H),7.30(t,J＝7.6Hz,2H),7.23(m,J＝9.7,3.6Hz,1H),6.70(d,J＝2.2Hz,1H),6.63(dd,J＝8.3,2.3Hz,1H),5.35(q,J＝6.4Hz,1H),2.45(s,3H),1.62(d,J＝6.4Hz,3H),1.29(s,12H).
2. (R) -6- (2-fluoro-4- (1-phenylethoxy) phenyl) -4- (1, 2,3, 6-tetrahydropyridin-4-yl) -7H-pyrrolo [2,3-d ] pyrimidine (42)
Replacement of compound 19f with compound 42a gives a yellow solid as in step 6 of example 19, as well as the remaining required starting materials, reagents and preparation methods. 1 H NMR(500MHz,DMSO)δ8.75(s,1H),7.81(t,J＝8.9Hz,1H),7.42(d,J＝7.5Hz,2H),7.35(t,J＝7.6Hz,2H),7.26(t,J＝7.3Hz,1H),7.05(s,1H),6.96(dd,J＝13.4,2.0Hz,1H),6.91(dd,J＝8.9,2.0Hz,2H),5.63(q,J＝6.3Hz,1H),3.89(s,2H),3.35(t,J＝5.9Hz,2H),2.94(s,2H),1.56(d,J＝6.3Hz,3H).HRMS(ESI)calcd for C 25 H 23 N 4 OF(M+H + ):415.1929；found 415.1930.
Example 43 (S) -6- (2-fluoro-4- (1-phenylethoxy) phenyl) -4- (1, 2,3, 6-tetrahydropyridin-4-yl) -7H-pyrrolo [2,3-d ] pyrimidine
1. (S) - (2-fluoro-4- (1-phenylethoxy) phenyl) boronic acid pinacol ester (43 a)
Replacement of Compound 33a with Compound 43a and replacement of Compound 33b with Compound 36a, the remaining starting materials, reagents and preparation were carried out in the same manner as in example 33Step 1, a white solid was obtained. 1 H NMR(500MHz,CDCl 3 )δ7.58–7.53(m,1H),7.36–7.29(m,4H),7.26–7.21(m,1H),6.64(dd,J＝8.4,2.2Hz,1H),6.51(dd,J＝11.4,2.2Hz,1H),5.32(q,J＝6.4Hz,1H),1.63(d,J＝6.4Hz,3H),1.31(s,12H).
2. (S) -6- (2-fluoro-4- (1-phenylethoxy) phenyl) -4- (1, 2,3, 6-tetrahydropyridin-4-yl) -7H-pyrrolo [2,3-d ] pyrimidine (43)
Replacement of compound 19f with compound 43a gives a yellow solid as in step 6 of example 19, as well as the remaining required starting materials, reagents and preparation methods. 1 H NMR(500MHz,DMSO)δ12.54(s,1H),8.74(s,1H),7.81(t,J＝8.9Hz,1H),7.45(d,J＝7.6Hz,2H),7.37(t,J＝7.6Hz,2H),7.28(t,J＝7.2Hz,1H),7.04(s,1H),7.00(dd,J＝13.6,1.8Hz,1H),6.93(dd,J＝8.8,2.0Hz,1H),6.90(s,1H),5.67(q,J＝6.2Hz,1H),3.90(s,2H),3.35(s,2H),2.92(s,2H),1.58(d,J＝6.3Hz,3H).HRMS(ESI)calcd for C 25 H 23 N 4 OF(M+H + ):415.1929；found 415.1923.
Example 44 (S) -6- (4- (1-Phenylethoxy) phenyl) -4- (1, 2,3, 6-tetrahydropyridin-4-yl) -7H-pyrrolo [2,3-d ] pyrimidine
1. (S) - (4- (1-Phenylethoxy) phenyl) boronic acid pinacol ester (44 a)
Replacement of compound 33b with compound 36a gives a white solid as in step 1 of example 33, as well as the remaining required starting materials, reagents and preparation methods. 1 H NMR(400MHz,CDCl 3 )δ7.66(d,J＝8.5Hz,2H),7.32(m,J＝15.0,7.4Hz,4H),7.23(m,1H),6.85(d,J＝8.6Hz,2H),5.36(q,J＝6.4Hz,1H),1.63(d,J＝6.4Hz,3H),1.29(s,12H).
2. (S) -6- (4- (1-Phenylethoxy) phenyl) -4- (1, 2,3, 6-tetrahydropyridin-4-yl) -7H-pyrrolo [2,3-d ] pyrimidine (44)
Replacement of compound 19f with compound 44a gives a yellow solid as in step 6 of example 19, as well as the remaining required starting materials, reagents and preparation methods. 1 H NMR(400MHz,DMSO)δ12.43(s,1H),8.65(s,1H),7.84(d,J＝8.6Hz,2H),7.43(d,J＝7.4Hz,2H),7.35(t,J＝7.5Hz,2H),7.25(t,J＝7.2Hz,1H),7.15(s,1H),7.01(d,J＝8.4Hz,3H),5.60(q,J＝12.2,5.9Hz,1H),3.65(s,2H),3.10(s,2H),2.74(s,2H),1.57(d,J＝6.3Hz,3H).HRMS(ESI)calcd for C 25 H 24 N 4 O(M+H + ):397.2023；found 397.2020.
Example 45 (S) -6- (2-methyl-4- (1-phenylethoxy) phenyl) -4- (1, 2,3, 6-tetrahydropyridin-4-yl) -7H-pyrrolo [2,3-d ] pyrimidine
1. (S) - (2-methyl-4- (1-phenylethoxy) phenyl) boronic acid pinacol ester tike (45 b)
Replacement of compound 33a with compound 45a and replacement of compound 33b with compound 36a gave a white solid, as was step 1 of example 33, with the remaining starting materials, reagents and preparation methods required. 1 H NMR(400MHz,CDCl 3 )δ7.61(d,J＝8.3Hz,1H),7.39–7.21(m,5H),6.70(s,1H),6.63(d,J＝8.2Hz,1H),5.35(q,J＝10.6,6.2Hz,1H),2.45(s,3H),1.61(d,J＝6.6Hz,3H),1.29(s,12H).
2. (S) -6- (2-methyl-4- (1-phenylethoxy) phenyl) -4- (1, 2,3, 6-tetrahydropyridin-4-yl) -7H-pyrrolo [2,3-d ] pyrimidine (45)
Replacement of compound 19f with compound 45b gives a yellow solid as in step 6 of example 19, as well as the remaining required starting materials, reagents and preparation methods. 1 H NMR(400MHz,DMSO)δ12.30(s,1H),8.71(s,2H),7.43(d,J＝7.3Hz,1H),7.36(t,J＝7.5Hz,2H),7.26(t,J＝7.3Hz,1H),6.95(d,J＝2.1Hz,1H),6.93(s,1H),6.85(dd,J＝8.5,2.2Hz,1H),6.81(s,1H),5.60(q,J＝6.2Hz,1H),3.88(s,2H),3.36(s,2H),2.93(s,2H),2.37(s,3H),1.57(d,J＝6.3Hz,3H).HRMS(ESI)calcd for C 26 H 26 N 4 O(M+H + ):411.2179；found 411.2176.
Example 46 (R) -6- (2-methyl-4- (1-phenylethoxy) phenyl) -4- (1, 2,3, 6-tetrahydropyridin-4-yl) -7H-pyrrolo [2,3-d ] pyrimidine
1. (R) - (2-methyl-4- (1-phenylethoxy) phenyl) boronic acid pinacol ester (46 a)
Replacement of compound 33a with compound 45a gives a white solid as in step 1 of example 33, as well as the remaining required starting materials, reagents and preparation methods. 1 H NMR(400MHz,CDCl 3 )δ7.61(d,J＝8.3Hz,1H),7.33(dd,J＝12.8,7.6Hz,4H),7.26–7.18(m,1H),6.70(s,1H),6.63(d,J＝8.3Hz,1H),5.34(q,J＝6.4Hz,1H),2.45(s,3H),1.65–1.57(d,3H),1.29(s,12H).
2. (R) -6- (2-methyl-4- (1-phenylethoxy) phenyl) -4- (1, 2,3, 6-tetrahydropyridin-4-yl) -7H-pyrrolo [2,3-d ] pyrimidine (46)
Replacement of compound 19f with compound 46a gives a yellow solid as in step 6 of example 19, as well as the remaining required starting materials, reagents and preparation methods. 1 H NMR(500MHz,DMSO)δ12.18(s,1H),8.66(s,1H),7.44(d,J＝7.7Hz,2H),7.37(m,J＝15.3,8.0Hz,3H),7.26(t,J＝7.3Hz,1H),6.94(s,2H),6.84(dd,J＝8.5,2.0Hz,1H),6.73(s,1H),5.59(q,J＝6.2Hz,1H),3.51(s,2H),2.97(t,J＝5.4Hz,2H),2.64(s,2H),2.37(s,3H),1.57(d,J＝6.3Hz,3H).HRMS(ESI)calcd for C 26 H 26 N 4 O(M+H + ):411.2179；found 411.2172.
Example 47 6- (2-methyl-4- (1-phenylethoxy) phenyl) -4- (1, 2,3, 6-tetrahydropyridin-4-yl) -7H-pyrrolo [2,3-d ] pyrimidine
1. (2-methyl-4- (1-phenylethoxy) phenyl) boronic acid pinacol ester (47 a)
Replacement of compound 33a with compound 45a gives a white solid as in step 1 of example 37, as well as the remaining required starting materials, reagents and preparation methods. 1 H NMR(400MHz,CDCl 3 )δ7.61(d,J＝8.3Hz,1H),7.33(dd,J＝12.8,7.6Hz,4H),7.22(m,1H),6.70(s,1H),6.63(d,J＝8.3Hz,1H),5.34(q,J＝6.4Hz,1H),2.45(s,3H),1.61(d,3H),1.29(s,12H).
2.6- (2-methyl-4- (1-phenylethoxy) phenyl) -4- (1, 2,3, 6-tetrahydropyridin-4-yl) -7H-pyrrolo [2,3-d ] pyrimidine
Replacement of compound 19f with compound 47a gives a yellow solid as in step 6 of example 19, as well as the remaining required starting materials, reagents and preparation methods. 1 H NMR(400MHz,DMSO)δ12.30(s,1H),8.71(s,2H),7.43(d,J＝7.3Hz,1H),7.36(t,J＝7.5Hz,2H),7.26(t,J＝7.3Hz,1H),6.95(d,J＝2.1Hz,1H),6.93(s,1H),6.85(dd,J＝8.5,2.2Hz,1H),6.81(s,1H),5.61(q,J＝6.2Hz,1H),3.88(s,2H),3.37(s,2H),2.94(s,2H),2.37(s,3H),1.58(d,J＝6.3Hz,3H).HRMS(ESI)calcd for C 26 H 26 N 4 O(M+H + ):411.2179；found 411.2172.
Example 48 (S) -6- (3-fluoro-4- (1-phenylethoxy) phenyl) -4- (1, 2,3, 6-tetrahydropyridin-4-yl) -7H-pyrrolo [2,3-d ] pyrimidine
1. (S) - (3-fluoro-4- (1-phenylethoxy) phenyl) boronic acid pinacol ester (48 b)
Replacement of compound 33a with compound 48a and replacement of compound 33b with compound 36a gave a white solid, as was step 1 of example 33, with the remaining starting materials, reagents and preparation methods required. 1 H NMR(400MHz,CDCl 3 )δ7.58(s,1H),7.47(d,J＝8.2Hz,1H),7.38–7.30(m,4H),7.25–7.19(m,1H),6.67(d,J＝8.2Hz,1H),5.39–5.35(m,1H),1.63(d,J＝6.4Hz,3H),1.30(s,12H).
2. (S) -6- (3-fluoro-4- (1-phenylethoxy) phenyl) -4- (1, 2,3, 6-tetrahydropyridin-4-yl) -7H-pyrrolo [2,3-d ] pyrimidine (48)
The (S) - (3-fluoro-4- (1-phenylethoxy) phenyl) boronic acid pinacol ester was used instead of compound 19f and the remaining starting materials, reagents and preparation were the same as in step 6 of example 19 to give a yellow solid. 1 H NMR(500MHz,CDCl 3 )δ13.03(s,1H),8.70(s,1H),7.53(d,J＝11.6Hz,1H),7.41(d,J＝7.3Hz,2H),7.36(t,J＝7.3Hz,3H),7.28(d,J＝7.1Hz,1H),6.92(t,J＝7.9Hz,1H),6.83(s,1H),6.75(s,1H),5.41(q,J＝11.9,5.9Hz,1H),4.24(s,2H),3.70(s,2H),2.84(s,2H),1.72(d,J＝5.9Hz,3H).HRMS(ESI)calcd for C 25 H 23 N 4 OF(M+H + ):415.1929；found 415.1923.
Example 49 (S) -6- (3-methyl-4- (1-phenylethoxy) phenyl) -4- (1, 2,3, 6-tetrahydropyridin-4-yl) -7H-pyrrolo [2,3-d ] pyrimidine
1. (S) - (3-methyl-4- (1-phenylethoxy) phenyl) boronic acid pinacol ester (49 b)
Replacement of compound 33a with compound 49a and replacement of compound 33b with compound 36a gave a white solid, as was step 1 of example 33, with the remaining starting materials, reagents and preparation methods required. 1 H NMR(400MHz,CDCl 3 )δ7.58(s,1H),7.47(d,J＝8.2Hz,1H),7.33(m,4H),7.23(m,1H),6.67(d,J＝8.2Hz,1H),5.37(q,1H),2.30(s,3H),1.63(d,J＝6.4Hz,3H),1.30(s,12H).
2. (S) -6- (3-methyl-4- (1-phenylethoxy) phenyl) -4- (1, 2,3, 6-tetrahydropyridin-4-yl) -7H-pyrrolo [2,3-d ] pyrimidine (49)
Replacement of compound 19f with compound 49b gives a yellow solid as in step 6 of example 19, as well as the remaining required starting materials, reagents and preparation methods. 1 H NMR(500MHz,DMSO)δ12.51(s,1H),8.94(s,1H),8.69(s,1H),7.83(s,1H),7.67(d,J＝8.4Hz,1H),7.44(d,J＝7.5Hz,2H),7.36(t,J＝7.5Hz,2H),7.27(t,J＝7.2Hz,1H),7.19(s,1H),7.02(s,1H),6.94(d,J＝8.7Hz,1H),5.63(q,J＝6.1Hz,1H),3.94(s,2H),3.39(s,2H),2.95(s,2H),2.33(s,3H),1.60(d,J＝6.2Hz,3H).HRMS(ESI)calcd for C 26 H 26 N 4 O(M+H + ):411.2179；found 411.2177.
Example 50 (S) -6- (2-methoxy-4- (1-phenylethoxy) phenyl) -4- (1, 2,3, 6-tetrahydropyridin-4-yl) -7H-pyrrolo [2,3-d ] pyrimidine
1. (S) - (2-methoxy-4- (1-phenylethoxy) phenyl) boronic acid pinacol ester (50 b)
Replacement of compound 33a with compound 50a and replacement of compound 33b with compound 36a gave a white solid, as was step 1 of example 33, with the remaining starting materials, reagents and preparation methods required. 1 H NMR(400MHz,CDCl 3 )δ7.51(d,J＝8.1Hz,1H),7.37–7.28(m,4H),7.25–7.18(m,1H),6.43–6.34(m,2H),5.35(q,J＝6.4Hz,1H),3.74(s,3H),1.63(d,J＝6.4Hz,3H),1.30(s,12H).
2. (S) -6- (2-methoxy-4- (1-phenylethoxy) phenyl) -4- (1, 2,3, 6-tetrahydropyridin-4-yl) -7H-pyrrolo [2,3-d ] pyrimidine (50)
Replacement of compound 19f with compound 50a gives a yellow solid as in step 6 of example 19, as well as the remaining required starting materials, reagents and preparation methods. 1 H NMR(500MHz,DMSO)δ12.01(s,1H),8.63(s,J＝4.8Hz,1H),7.66(d,J＝8.6Hz,1H),7.46(d,J＝7.2Hz,2H),7.37(t,J＝7.6Hz,2H),7.27(t,J＝7.4Hz,1H),7.03(s,1H),6.91(s,1H),6.73(d,J＝2.2Hz,1H),6.58(dd,J＝8.7,2.3Hz,1H),5.64(q,J＝6.4Hz,1H),3.86(s,3H),3.54(s,2H),2.99(t,J＝5.8Hz,2H),2.63(s,2H),1.59(d,J＝6.4Hz,3H).HRMS(ESI)calcd for C 26 H 26 N 4 O 2 (M+H + ):427.2129；found 427.2128.
Example 51 (S) -6- (4- (1-phenethyl) phenyl) -4- (piperazin-1-yl) -7H-pyrrolo [2,3-d ] pyrimidine
1. (S) -4-chloro-6- (4- (1-phenylethoxy) phenyl) -7H-pyrrolo [2,3-d ] pyrimidine (51 a)
The remaining required starting materials, reagents and preparation were the same as in step 4 of example 6, substituting compound 44a for 4-benzyloxyphenylboronic acid, to give a white solid. 1 H NMR(400MHz,DMSO)δ12.85(s,1H),8.53(s,1H),7.85(d,J＝8.8Hz,2H),7.42(d,J＝7.3Hz,2H),7.34(t,J＝7.5Hz,2H),7.25(t,J＝7.3Hz,1H),7.01(d,J＝8.8Hz,2H),6.90(d,J＝1.9Hz,1H),5.59(q,J＝6.3Hz,1H),1.56(d,J＝6.3Hz,3H).
2. (S) -6- (4- (1-phenethyl) phenyl) -4- (piperazin-1-yl) -7H-pyrrole [2,3-d ] pyrimidine (51)
Compound 51a (100 mg,0.29 mmol), piperazine (37.8 mg,0.44 mmol) and DIPEA (74.8 mg,0.58 mmol) were added to the tube, then 3mL DMF was added as solvent and the reaction was stirred at 100deg.C overnight. Ethyl acetate (15 mL) was added, the mixture was washed with water and saturated sodium chloride solution, and the organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain 62.7mg of a white solid by column chromatography, with a yield of 54.2%. 1 H NMR(400MHz,DMSO)δ12.12(s,1H),8.16(s,1H),7.75(d,J＝8.8Hz,2H),7.43(d,J＝7.2Hz,2H),7.35(t,J＝7.5Hz,2H),7.26(t,J＝7.3Hz,1H),7.02–6.88(m,3H),5.58(q,J＝6.2Hz,1H),4.14–3.76(m,4H),3.04–2.95(m,4H),1.57(d,J＝6.3Hz,3H).HRMS(ESI)calcd for C 24 H 25 N 5 O(M+H + ):400.2132；found 400.2129.
Example 52 (S) -4- (1, 4-diaza-1-yl) -6- (4- (1-phenylethoxy) phenyl) -7H-pyrrolo [2,3-d ] pyrimidine
The piperazine was replaced with homopiperazine, and the remaining required starting materials, reagents and preparation method were the same as in example 51, step 2, to give a white solid. 1 H NMR(400MHz,DMSO)δ12.08(s,1H),9.08(s,1H),8.14(s,1H),7.75(d,J＝8.7Hz,2H),7.43(d,J＝7.3Hz,2H),7.35(t,J＝7.5Hz,2H),7.26(t,J＝7.3Hz,1H),6.96(d,J＝8.8Hz,2H),6.92(s,1H),5.58(q,J＝6.1Hz,1H),4.19–4.10(m,2H),4.02(t,J＝5.8Hz,2H),3.34–3.22(m,2H),3.21–3.07(m,2H),2.15(s,2H),1.57(d,J＝6.3Hz,3H).HRMS(ESI)calcd for C 25 H 27 N 5 O(M+H + ):414.2288；found 414.2287.
Example 53 (S) -4- (4-methylpiperazin-1-yl) -6- (4- (1-phenylethoxy) phenyl) -7H-pyrrolo [2,3-d ] pyrimidine
The piperazine was replaced with N-methylpiperazine, and the remaining required starting materials, reagents and preparation method were the same as in example 51, step 2, to give a white solid. 1 H NMR(500MHz,CDCl 3 )δ13.32(s,1H),8.30(s,1H),7.64(d,J＝8.5Hz,2H),7.40(d,J＝7.7Hz,2H),7.35(t,J＝7.5Hz,2H),7.26(t,J＝7.1Hz,1H),6.97(d,J＝8.5Hz,2H),6.58(s,1H),5.38(q,J＝6.4Hz,1H),4.29–3.49(m,4H),2.75–2.45(m,4H),2.37(s,3H),1.67(d,J＝6.4Hz,3H).HRMS(ESI)calcd for C 25 H 27 N 5 O(M+H + ):414.2288；found 414.2285.
Example 54 (S) -4- (4-methyl-1, 4-diaza-1-yl) -6- (4- (1-phenethyl) phenyl) -7H-pyrrolo [2,3-d ] pyrimidine
The piperazine was replaced with N-methyl homopiperazine, and the remaining required raw materials, reagents, and preparation method were the same as in example 51, step 2, to give a white solid. 1 H NMR(500MHz,DMSO)δ12.06(s,1H),8.12(s,1H),7.74(d,J＝7.4Hz,2H),7.42(d,J＝6.5Hz,2H),7.34(t,J＝6.3Hz,2H),7.30–7.21(m,1H),6.95(d,J＝7.5Hz,2H),6.89(s,1H),5.56(q,J＝11.9,6.7Hz,1H),4.13(s,2H),3.99(s,2H),3.26(s,2H),3.12(s,2H),2.64(s,3H),2.25(s,2H),1.56(d,J＝4.8Hz,3H).HRMS(ESI)calcd for C 26 H 29 N 5 O(M+H + ):428.2445；found 428.2440.
Example 55 (S) -6- (4- (1-phenethyl) phenyl) -N- (piperidin-4-yl) -7H-pyrrolo [2,3-d ] pyrimidin-4-amine
The piperazine was replaced by 4-aminopiperidine, and the remaining required starting materials, reagents and preparation were the same as in example 51, step 2, to give a white solid. 1 H NMR(500MHz,DMSO)δ11.82(s,1H),8.06(s,1H),7.60(d,J＝8.0Hz,2H),7.42(d,J＝7.5Hz,2H),7.35(t,J＝7.4Hz,2H),7.25(t,J＝7.2Hz,1H),7.17(d,J＝7.6Hz,1H),6.96(d,J＝8.1Hz,2H),6.81(s,1H),5.55(q,J＝6.0Hz,1H),4.10(d,J＝6.8Hz,1H),2.99(d,J＝11.8Hz,2H),2.57(t,J＝11.8Hz,2H),1.87(d,J＝11.6Hz,2H),1.56(d,J＝6.2Hz,3H),1.40(dd,J＝21.1,11.3Hz,2H).HRMS(ESI)calcd for C 25 H 27 N 5 O(M+H + ):414.2288；found414.2289.
Example 56 4- (3, 5-dimethylpiperazin-1-yl) -6- (4- ((S) -1-phenylethoxy) phenyl) -7H-pyrrolo [2,3-d ] pyrimidine
Piperazine was replaced with 2, 6-dimethylpiperazine, and the remaining required raw materials, reagents and preparation method were the same as in example 51, step 2, to obtain a white solid. 1 H NMR(400MHz,DMSO)δ12.03(s,1H),8.11(s,1H),7.76(d,J＝8.8Hz,2H),7.42(d,J＝7.3Hz,2H),7.34(t,J＝7.6Hz,2H),7.25(t,J＝7.3Hz,1H),6.95(d,J＝8.8Hz,2H),6.90(s,1H),5.56(q,J＝6.2Hz,1H),4.59(d,J＝12.2Hz,2H),2.92–2.71(m,2H),2.56(d,J＝11.2Hz,2H),1.56(d,J＝6.3Hz,3H),1.07(d,J＝6.2Hz,6H).HRMS(ESI)calcd for C 26 H 29 N 5 O(M+H + ):428.2445；found 428.2438.
Biological Activity test
Test one: molecular level protein arginine methyltransferase PRMT5 Activity inhibition assay
Test purpose:
determination of the inhibitory Activity of the non-nucleotide PRMT5 Small molecule inhibitors of the invention on PRMT5 protein
The test method comprises the following steps:
1. preparing 1x assay buffer (modified Tris buffer);
2. compounds were transferred to assay plates with Echo in 100% DMSO. The final fraction of DMSO was 1%;
3. adding enzyme into a 1x test buffer to prepare an enzyme solution;
4. adding a substrate to a 1x test buffer to prepare a substrate solution;
5. will [ 3 H]SAM addition to 1x testPreparation in buffer [ 3 H]-SAM solution;
6. remove 15. Mu.L of enzyme solution to assay plate containing compound;
7. incubating for 15 minutes at room temperature;
8. 5 mu L of primer solution is added to each hole;
9. mu.L of each well is added 3 H]The SAM solution starts to react;
10. incubating for 60 minutes at room temperature;
11. adding the cold SAM into a 1x test buffer solution to prepare a stop solution;
12. adding 5 mu L of stop solution into each hole;
13. transferring 25 μl of the reaction mixture solution from each well of the assay plate onto Flashplate;
14. incubating at room temperature for at least 1 hour;
15. by dH 2 Flushing the Flashplate three times by O+0.1% Tween-20;
16. reading data on a MicroBeta liquid scintillation/luminescence counter;
17. And (3) data processing: importing the data into Excel, and obtaining a suppression value by using a formula (1);
formula (1): in% = (maximum signal-compound signal)/(maximum signal-minimum signal) ×100% data was imported into EXCEL-Fit, IC was calculated using formula (2) 50 Value of
Formula (2): y=bottom+ (Top-Bottom)/(1+ (IC) 50 X) ×hillslope) Y is the inhibition rate, X is the compound concentration.
Test results:
the results of the in vitro enzymatic activity of the non-nucleotide PRMT5 small molecule inhibitors of the present invention are shown in Table 1 below.
TABLE 1 inhibition level of protein arginine methyltransferase PRMT5 enzyme activity by non-nucleotide PRMT5 small molecule inhibitors
And (2) testing II: human liver microsome stability experiment
Test purpose:
the stability of human liver microsomes of test compounds was evaluated, and human liver microsome stability experiments were performed using the compounds 33, 36, 37, 40, 43, 44, 45 obtained in examples 33, 36, 37, 40, 43, 44, 45.
The test method comprises the following steps:
0.1M TRIS buffer pH 7.4 (final concentration 0.33 mg/mL), cofactor MgCl 2 (final concentration 5 mM) and test compound (final concentration 0.1. Mu.M, co-solvent (0.01% DMSO) and 0.005% bovine serum albumin) were incubated at 37℃for 10 minutes. The reaction was started by adding NADPH (final concentration 1 mM). Aliquots were sampled at 0, 7, 17, 30 and 60 minutes, respectively, and methanol (cooled at 4 ℃) was added to terminate the reaction. After centrifugation (4000 rpm,5 minutes) the samples were then analyzed by LC-MS/MS. The calculation formula is as follows:
Intrinsic clearance:
in vivo clearance:
liver clearance:
metabolic bioavailability:
p: microsomal protein concentration (mg/mL);
houston: houston factor (45 mg microsomal protein/g liver);
LW: liver weight (g) (per species);
HBF: hepatic blood flow (mL/min) (per species);
fu: unbound fraction (fu=1 is typically used).
Test results:
the test compounds were tested for human liver microsome stability as shown in table 2 below.
TABLE 2 results of human liver microsome stability experiments
And (3) test III: inhibition assay of cellular level histone arginine methyltransferase PRMT5 Activity
Test purpose:
the effect of compound 44 on cell proliferation was evaluated.
Testing cell lines:
AGS, A375, CHL-1, mv (4; 11), MOLM cell line
And (3) observing the indexes:
inhibition of proliferation of Compound 44 on each cell
The detection method comprises the following steps:
CCK-8 cell count kit (Dojindo)
The test principle is as follows:
cell growth may be manifested as an increase in cell number and an increase in cell volume, which is typically within a range, whereas cell number may be increased by continued division. CCK-8 chromogenic agent capable of converting tetrazolium salt by using dehydrogenase possessed by cells themselves
WST-8[2- (2-methoxy-4-nitrophenyl) -3- (4-nitrophenyl) -5- (2, 4-disufo-phenyl) -2H-tetrazolium) reduction produces the water-soluble yellow product formazan, the number of living cells being proportional to the number of formazan dye produced, which can be used to detect the number of living cells in cell proliferation and toxicity analysis experiments.
The action time is as follows:
6Days。
reagent, consumable and instrument:
the experimental water is distilled water produced by national medicine group; all reagents were purchased from national pharmaceutical group chemical reagent limited; the experimental reader is made of Molecular Device company, model number: spectraMax 190.
Compound preparation:
the compound was centrifuged at 12000g for 5min, and 10mM stock solution was prepared by adding DMSO, vortexing well, and then sonicated for 10min for use, and stored at-40 ℃ (the compound with special storage requirements was modified as the case may be). Compounds were diluted from stock solution to the concentrations tested (DMSO concentration in the system did not exceed 0.5%) at the time of testing.
The test method comprises the following steps:
the proliferation inhibition of cell lines such as AGS, A375, CHL-1, mv (4; 11), MOLM and the like by the compounds was detected by the CCK-8 cell counting kit (Dojindo). The method comprises the following specific steps: cells in the logarithmic growth phase were inoculated at the appropriate density into 96-well plates at 190. Mu.L per well, and after overnight incubation, compounds of different concentrations were added to effect 6Days, and a solvent control group (negative control) was set. After the compound acts on the cell 6Days, the effect of the compound on the proliferation of the cell is detected by using a CCK-8 cell counting kit (Dojindo), 20 mu L of CCK-8 reagent is added to each hole, and the mixture is placed in a37 ℃ incubator for 2 to 4 hours, and then is read by a full-wave microplate reader SpectraMax 190, and the measurement wavelength is 450nm.
The inhibition (%) of the compound against tumor cell growth was calculated using the following formula:
inhibition (%) = (OD control well-OD dosing well)/OD control well x 100%
Calculating corresponding IC according to each concentration inhibition rate 50 。
Test results:
the results of inhibition of AGS, A375, CHL-1, mv (4; 11), MOLM and other cell lines by compound 44 are shown in Table 3 below.
TABLE 3 determination of inhibition of Activity of Compound 44 on different cell lines
And (3) testing four: enzyme selectivity assay
Test purpose:
test compound 44 selectivity for PRMT 5.
Testing enzyme species:
PRMT1,PRMT4,PRMT5,EZH2,NSD2,MLL1,MLL4
the test method comprises the following steps:
1. the inhibition of compound 44 on a number of other histone modification enzymes at the molecular level was further examined using established AlphaLISA and HTRF methods to determine the selectivity of the compound for histone modification enzymes. The inhibition detection of the compound on the molecular level activity of histone modification enzymes PRMT1, PRMT4, PRMT5 and NSD2 is carried out by adopting the alpha LISA technology. The compounds, enzymes (PRMT 1, PRMT4, PRMT5, NSD 2) and substrate solutions were prepared in 1 x assay buffer;
2. sequentially adding the following reagents into a 384-microplate whiteboard;
-5 μl of inhibitor;
-2.5 μl enzyme solution;
incubation at 37 c for 10 minutes,
2.5. Mu.L of substrate/S-adenosyl-L-methionine mixture;
3. Sealing the microplate and incubating at room temperature;
4. preparing 1-time concentration detection liquid;
5. preparing anti-histone methylation antibody coupled receptor microbeads (100 mug/mL) with a detection solution of 1-fold concentration, wherein the final concentration is 20 mug/mL;
6. adding 5 mu L of 1-fold concentration anti-histone methylation antibody coupled receptor microbeads, sealing and incubating for 60 minutes;
7. preparing streptavidin donor microbeads (50 mug/mL) with a detection solution of 1-time concentration, wherein the final concentration is 20 mug/mL;
8. adding 10 mu L of diluted streptavidin donor microbeads, and incubating for 30 minutes under sealed light-proof conditions;
9. and reading by an enzyme label instrument.
Two wells were set up for each experiment and a blank control was set up.
Inhibition ratio (%) = { [ (positive control signal value-blank control signal value) - (test compound signal value-blank control signal value) ]/(positive control signal value-blank control signal value) ] }.
The selectivity to EZH2, MLL1, MLL4 was assessed using HTRF assay. In HTFR assays, the peptide substrate in the enzyme, SAM, compound and assay buffer is diluted in 1 x assay buffer prior to use. mu.L of compound and 2. Mu.L of enzyme were added to the wells of white OptiPlate-384 and incubated for 10 min at room temperature. Subsequently, 4. Mu.L of substrate/SAM was mixed with the reaction system and incubated at room temperature for 4 hours. The antibody and SA-XL665 were prepared separately with 1 Xdetection Buffer, which was mixed as a mixture (2X), 10. Mu.L of the mixture was added to each well, and incubated for 1h at room temperature in the absence of light. Two wells were set up for each experiment and a blank control was set up. Inhibition ratio (%) = { [ (positive control signal value-blank control signal value) - (test compound signal value-blank control signal value) ]/(positive control signal value-blank control signal value) }. The inhibition (%) is taken as an ordinate, the concentration of the compound is taken as an abscissa, competition inhibition curves are respectively drawn by using Graphpad prism software, and the concentration (IC) at which the binding rate of the compound and the protein is 50% is calculated by using the obtained regression equation 50 ). Test results:
the results of the enzyme selectivity test are shown in Table 4 below.
Table 4 determination of the selectivity of compound 44 for PRMT5
The results of enzyme selectivity assays (Table 4) indicate that compound 44 has good selectivity for the protein arginine methyltransferase PRMT5 and is a class of inhibitors that specifically target PRMT 5. Test example in vivo protein arginine methyltransferase PRMT5 Activity inhibition assay
Test purpose:
compound 44 was tested for anti-tumor efficacy on an a375 nude mouse engraftment tumor model.
The test method comprises the following steps:
Balb/C nude mice (6 weeks, female, purchased from Shanghai Ling Biotechnology Co., ltd.) were purchased and animals were kept for one week prior to the experiment. In vitro amplification of A375 cells, taking cells in log phase and suspending in serum-freeIn RPMI1640 medium, 100 μl (5.0×10) of the cell suspension was injected in the anterior axilla 6 And (c) a). The animal experiment operation procedure is carried out according to the ethical rule of animal experiment. Average tumor volume of tumor-bearing mice reaches 50-100mm 3 At this time, the mice were divided into two groups by random differentiation: the solvent control group and the compound 44 75mg/kg group, 7 mice per group. The dosing was started 21 days after the grouping, once a day, tumor volumes were tested three times every three days and weighed. Tumor Volume (TV) calculation formula: tv=1/2×a×b 2 Wherein a and b represent length and width, respectively. The relative tumor volume (relative tumor volume, RTV) was calculated from the measurements. The tumor inhibition ratio (tumor growth inhibition, TGI) is calculated according to the measurement result, and the calculation formula is as follows: TGI= [1-RTV (experimental group)/RTV (control group)]*100％。
Western blot method (Western blot)
Mouse tumor tissue frozen in liquid nitrogen (about 20 mg) was removed, thawed on ice, 200 μl of RIPA lysate (containing protease inhibitor and phosphatase inhibitor) was added, and the tissue pieces were cut into small pieces with tissue shears on ice. The tissue mass was thoroughly lysed by on-ice sonication (Apml 45%), the sonication was stopped when no macroscopic tissue mass was present, centrifuged at 15000rpm for 15-20min at 4 ℃, the supernatant was taken into a new centrifuge tube and BCA quantitated. According to the quantitative results, 4 Xprotein loading buffer (200 mM Tris-HCl pH 6.8, 100mM DTT,2%SDS,20% glycerol, 1mM sodium vanadate, 0.1% bromophenol blue) was added to dilute to 1X, the excess was made up with 2% SDS, the protein concentration was set at 20. Mu.g/10. Mu.L, and the mixture was subjected to a water bath at 100℃for 10min. SDS-PAGE was performed on 10. Mu.L samples per well. After electrophoresis, the proteins were transferred to nitrocellulose membranes using a semi-dry electrotransfer system and the cellulose membranes were blocked in 3% BSA for 1h. The approximate position of the protein of interest was determined according to Marker size and the membrane was cut to size below 4℃overnight in primary antibody buffer. The strip was then washed three times with TBST for 10min each time on the converter. Finally, incubating the cellulose membrane printed with the protein with the prepared secondary antibody solution for 1h at room temperature; after washing the strips three times, chromogenic imaging was performed with Millipore (Millipore, USA) reagents in an imagequat imaging system.
Test results:
in vivo levels of protein arginine methyltransferase PRMT5 activity inhibition experiments are shown in fig. 1-5.
As shown in fig. 1, which is a graph of tumor volume change during dosing, compound 44 was shown to significantly inhibit the growth of human malignant melanoma; FIG. 2 is a change in body weight of nude mice during administration showing that compound 44 has no effect on body weight of nude mice, indicating that compound 44 has no toxic side effects; figure 3 shows that compound 44 is capable of significantly reducing the weight of human malignant melanoma; FIG. 4 is a graph showing the final volume size of representative tumors in each group 21 days after administration; figure 5 shows that compound 44 was also able to inhibit PRMT5 arginine symmetrical dimethyl in a mouse in vivo experiment. To sum up: the compound 44 can inhibit the growth of tumors by inhibiting the symmetrical dimethyl of PRMT5 arginine, and is a PRMT5 targeted small molecule inhibitor with high selectivity and good pharmacy.
It should be noted that the above embodiments are merely for illustrating the technical solution of the present invention and not for limiting the scope of the present invention, and that other various changes and modifications can be made by one skilled in the art based on the above description and the idea, and it is not necessary or exhaustive to all embodiments. Any modification, equivalent replacement, improvement, etc. which come within the spirit and principles of the invention are desired to be protected by the following claims.

Claims (5)

1. A non-nucleotide PRMT5 small molecule inhibitor or a pharmaceutically acceptable salt thereof, wherein the non-nucleotide PRMT5 small molecule inhibitor has a structure according to formula (i):
the formula (I) is selected from the following structures:
2. the method for preparing the non-nucleotide PRMT5 small molecule inhibitor according to claim 1, comprising the steps of:
when X is N and Y is CH, S1: the method comprises the steps of (1) mixing a substance shown in a formula (1) and a substance shown in a formula (2) in a solvent, performing ring closure reaction to generate an intermediate shown in a formula (3), and performing substitution reaction on the intermediate shown in the formula (3) and corresponding amine under alkaline conditions to obtain a compound shown in a formula (I); or the intermediate shown in the formula (3) and the corresponding pinacol borate are subjected to SUZUKI coupling reaction to obtain a compound shown in the formula (I);
when X is CH and Y is CH, S2: the method comprises the steps of (1) mixing a substance shown in a formula (4) and a substance shown in a formula (5) in a solvent, carrying out SUZUKI coupling reaction to obtain an intermediate shown in a formula (6), and carrying out substitution reaction on the intermediate shown in the formula (6) and corresponding amine under alkaline conditions to obtain a compound shown in a formula (I);
or when X is CH and Y is CH, S3: dissolving a substance shown in a formula (7) in a solvent, sequentially obtaining a substance shown in a formula (8), a substance shown in a formula (9), a substance shown in a formula (10) and an intermediate shown in a formula (11) through sulfonylation, halogen exchange, bromination and deprotection, then carrying out a first SUZUKI coupling reaction on the intermediate shown in the formula (11) and the corresponding pinacol borate to obtain an intermediate shown in a formula (12), and carrying out a second SUZUKI coupling reaction on the intermediate shown in the formula (12) and the substance shown in the formula (5) to obtain a compound shown in a formula (I);
3. The preparation method according to claim 2, wherein the solvent in S1 to S3 is independently selected from one or more of acetone, 1, 2-dichloroethane, tetrahydrofuran, N-dimethylformamide, dichloromethane, methanol, dioxane or water;
the reaction temperature of the ring closing reaction in S1 is 60-90 ℃; the reaction temperature of the substitution reaction in S1 is 80-110 ℃; the reaction temperature of the SUZUKI coupling reaction in the step S1 is 80-110 ℃;
the reaction temperature of the substitution reaction in S2 is 80-110 ℃; the reaction temperature of the SUZUKI coupling reaction in the S2 is 80-110 ℃;
the reaction temperature of the sulfonylation reaction in the S3 is 0-25 ℃; the reaction temperature of the deprotection reaction in S3 is 0-25 ℃; the reaction temperature of the halogen exchange reaction in the S3 is 50-70 ℃; the reaction temperature of the bromination reaction in the S3 is-78 ℃ to-40 ℃; the reaction temperature of the SUZUKI coupling reaction in the first step in the S3 is 80-110 ℃; the reaction temperature of the SUZUKI coupling reaction in the second step in the step S3 is 80-110 ℃.
4. Use of a non-nucleotide PRMT5 small molecule inhibitor according to claim 1, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the prevention and/or treatment of PRMT5 mediated neoplasms.
5. A medicament comprising the non-nucleotide PRMT5 small molecule inhibitor of claim 1, or a pharmaceutically acceptable salt thereof.
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