CN108774224B - Pyrazolo [3,4-b ] pyridine compound and preparation method and application thereof - Google Patents

Pyrazolo [3,4-b ] pyridine compound and preparation method and application thereof Download PDF

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CN108774224B
CN108774224B CN201810366047.8A CN201810366047A CN108774224B CN 108774224 B CN108774224 B CN 108774224B CN 201810366047 A CN201810366047 A CN 201810366047A CN 108774224 B CN108774224 B CN 108774224B
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崔孙良
侯廷军
陈昌威
邓子阳
汪大海
吴启凡
潘培辰
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Zhejiang University ZJU
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Abstract

The invention provides a preparation method and application of pyrazolo [3,4-b ] pyridine compounds. The pyrazolo [3,4-b ] pyridine is used as a structural parent nucleus, a series of brand-new micromolecular ALK inhibitors are designed and synthesized, ALK kinase inhibition activity tests are carried out on the compounds, and part of the compounds show stronger inhibition activity, so that a new way is provided for research of anticancer drugs.

Description

Pyrazolo [3,4-b ] pyridine compound and preparation method and application thereof
Technical Field
The invention belongs to the field of pharmaceutical chemistry, and particularly relates to a pyrazolo [3,4-b ] pyridine compound, a preparation method thereof, and application thereof as an ALK inhibitor in antitumor drugs.
Background
Malignant tumors have become a serious threat to human health. Among them, lung cancer is the most serious malignant tumor with the highest global morbidity and mortality, including non-small cell lung cancer (NSCLC) and small cell lung cancer, and the proportion of non-small cell lung cancer is 85% of all lung cancers. Pathologically, non-small cell lung cancer in turn includes adenocarcinoma, squamous cell carcinoma and large cell carcinoma. For patients with early confirmed diagnosis, the early diagnosis kit can have good prognosis effect after surgical treatment. However, most patients are diagnosed at an advanced stage, the traditional surgical treatment and the chemotherapy effect cannot effectively control the disease, and the survival rate in five years is very low. Therefore, the development of targeted drugs for non-small cell lung cancer is urgent.
Targeted therapy refers to a method of systemic treatment by blocking a site of a signaling pathway associated with tumor growth or inhibition. The Epidermal Growth Factor Receptor (EGFR) small-molecule inhibitor is the medicament which is applied to the targeted therapy of clinical non-small cell lung cancer at the earliest time. In 2007, fusion oncogenes of echinoderm microtubule-associated protein 4(EML4) and ALK are discovered, and a new target point is provided for molecular therapy of non-small cell lung cancer.
Anaplastic Lymphoma Kinase (ALK) is a receptor tyrosine kinase, structurally belonging to the insulin receptor superfamily. ALK acts as a fusion partner for t (2; 5) chromosomal translocation in anaplastic large cell lymphomas, and the N-terminal part of the normally expressed nucleolar phosphorylated protein is fused to the ALK kinase domain to form an NPM-ALK fusion protein, wherein the NPM part is capable of dimerizing and activating the ALK kinase domain. In addition, partial inversion of the interior of chromosome 2p was found in some cancer tissue samples, resulting in the formation of a fusion gene of part of echinoderm microtubule-associated protein 4(EML4) and ALK. The EML4-ALK fusion gene has the characteristic of 'oncogene dependence' and is closely related to most non-small cell lung cancers.
The human ALK gene is located at chromosome 2p23 at positions from base pairs 29, 192 and 773 to 29, 921 and 610. The ALK gene has 26 exons, a cDNA consisting of 6226 base pairs encodes a polypeptide with 1620 amino acids and a length of 177422Da, and the mature ALK protein with a length of about 220KDa is obtained through posttranslational modification.
It has now been found that 22 different genes can rearrange with ALK to form fusion proteins, suggesting that the ALK locus may be susceptible to translocation for some presently unknown reason. Although a certain number of fusion proteins have been experimentally confirmed, only a few have been extensively studied, including the NPM-ALK fusion protein in anaplastic large cell lymphoma (ACLC) and the EML4-ALK fusion protein in non-small cell lung cancer (NSCLC).
Due to the intensive research on ALK kinase and ALK fusion protein by scientists over the last decade, ALK has become a drug target for the treatment of non-small cell lung cancer. A number of ALK kinase inhibitors have been developed and are currently in clinical trials. Such as Crizotinib (Crizotinib) developed by the pfeizu pharmaceutical, Ceritinib (Ceritinib) developed by the nova pharmaceutical, idetinib (Alectinib) developed by the roche pharmaceutical, and Entrectinib developed by the Ariad pharmaceutical, and the like. The compounds show strong clinical drug activity in ALK positive non-small cell lung cancer patients. As early as 2010, crizotinib was FDA approved for the treatment of ALK-positive non-small cell lung cancer, the first ALK kinase inhibitor to be marketed. Subsequently, ceritinib was FDA approved for ALK-positive, cancer cell metastatic, and non-small cell lung cancer patients who relapsed after using crizotinib in 2014. In 2015, the FDA accelerated approval of erlotinib for non-small cell lung cancer patients with progression of disease after treatment with crizotinib.
Because the ALK plays a key role in the occurrence and development process of tumors, the ALK inhibitor can be developed to effectively improve the treatment level, remarkably prolong the life of patients and benefit a large number of tumor patients by developing the selective ALK targeting small molecule inhibitor, so the development of the ALK inhibitor has a great application prospect.
Disclosure of Invention
The invention aims to provide a pyrazolo [3,4-b ] pyridine compound and a preparation method thereof. Meanwhile, the enzyme activity test (TR-FRET) and the anti-tumor activity (Karpass-299) of the compound achieve better effects.
The pyrazolo [3,4-b ] pyridine compound provided by the invention has the following structural general formula (I):
Figure BDA0001637289860000031
wherein:
ring A is selected from five-membered or six-membered heterocyclic aryl or aryl, and the substituent is selected from R4A group;
R1selected from hydrogen, halogen atoms, hydroxy, amino, cyano, alkoxy, -OC (═ O) Ra，RaOptionally, the compound is selected from alkyl, alkoxy, alkylamino, a five-membered or six-membered aliphatic heterocyclic ring or an aromatic heterocyclic ring, wherein the heterocyclic ring contains 1-2 heteroatoms of O, N;
R2selected from hydrogen, halogen atoms, alkyl groups;
R3selected from hydrogen, halogen atoms, alkyl groups;
R4one or more selected from the group consisting of hydrogen, alkyl, alkylamino, and a substituted or unsubstituted five-or six-membered aliphatic heterocycle (herein, R4Or a plurality of substituents at different positions), the heterocyclic ring contains 1 to 2 heteroatoms selected from O, N, and the substituents are selected from alkyl, hydroxymethyl and amido.
Further, preferred compounds of the present invention have the structure of formula (ii):
Figure BDA0001637289860000032
wherein:
R1selected from hydrogen, halogen atoms, hydroxy, amino, cyano, alkoxy, -OC (═ O) Ra，RaOptionally, the compound is selected from alkyl, alkoxy, alkylamino, a five-membered or six-membered aliphatic heterocyclic ring or an aromatic heterocyclic ring, wherein the heterocyclic ring contains 1-2 heteroatoms of O, N;
R2selected from hydrogen, halogen atoms, alkyl groups;
R3selected from hydrogen, halogen atoms, alkyl groups;
R4the compound is selected from hydrogen, alkyl, alkylamino, substituted or unsubstituted five-membered or six-membered aliphatic heterocyclic ring, the heterocyclic ring contains 1-2 heteroatoms selected from O, N, and the substituent is selected from alkyl, hydroxymethyl and amido.
Further, preferred compounds of the present invention have the structure of formula (iii):
Figure BDA0001637289860000041
R1selected from hydrogen, halogen atoms, hydroxy, amino, cyano, alkoxy, -OC (C＝O)Ra，RaOptionally, the compound is selected from alkyl, alkoxy, alkylamino, a five-membered or six-membered aliphatic heterocyclic ring or an aromatic heterocyclic ring, wherein the heterocyclic ring contains 1-2 heteroatoms of O, N;
R2selected from hydrogen, halogen atoms, alkyl groups;
R3selected from hydrogen, halogen atoms, alkyl groups;
R4the compound is selected from hydrogen, alkyl, alkylamino, substituted or unsubstituted five-membered or six-membered aliphatic heterocyclic ring, the heterocyclic ring contains 1-2 heteroatoms selected from O, N, and the substituent is selected from alkyl, hydroxymethyl and acylamino;
R5selected from hydrogen, alkyl, alkoxy.
More specifically, the preferred compounds of the structure of formula (II) of the present invention are selected from:
4- (3- (5-Methylfuran-2-yl) -1H-pyrazolo [3,4-b ] pyridin-6-yl) phenol (11a)
4- (3- (5- (morpholinomethyl) furan-2-yl) -1H-pyrazolo [3,4-b ] pyridin-6-yl) phenol (11b)
4- (3- (5- (piperidin-1-ylmethyl) furan-2-yl) -1H-pyrazolo [3,4-b ] pyridin-6-yl) phenol (11c)
4- (3- (5- (pyrrolidin-1-ylmethyl) furan-2-yl) -1H-pyrazolo [3,4-b ] pyridin-6-yl) phenol (11d)
4- (3- (5- ((4-methylpiperazin-1-yl) methyl) furan-2-yl) -1H-pyrazolo [3,4-b ] pyridin-6-yl) phenol (11e)
4- (3- (5- ((dimethylamino) methyl) furan-2-yl) -1H-pyrazolo [3,4-b ] pyridin-6-yl) phenol (11f)
4- (3- (5- ((2- (hydroxymethyl) pyrrolidin-1-yl) methyl) furan-2-yl) -1H-pyrazolo [3,4-b ] pyridin-6-yl) phenol (11g)
4- (3- (5- ((4-isopropylpiperazin-1-yl) methyl) furan-2-yl) -1H-pyrazolo [3,4-b ] pyridin-6-yl) phenol (11H)
4- (3- (5- ((2- (hydroxymethyl) piperidin-1-yl) methyl) furan-2-yl) -1H-pyrazolo [3,4-b ] pyridin-6-yl) phenol (11i)
1- ((5- (6- (4-hydroxyphenyl) -1H-pyrazolo [3,4-b ] pyridin-3-yl) furan-2-yl) methyl) piperidine-3-carboxamide (11j)
4- (3- (5- (((2-hydroxyethyl) (methyl) amino) methyl) furan-2-yl) -1H-pyrazolo [3,4-b ] pyridin-6-yl) phenol (11k)
2-fluoro-4- (3- (5-methylfuran-2-yl) -1H-pyrazolo [3,4-b ] pyridin-6-yl) phenol (11l)
2- (4- (3- (5-methylfuran-2-yl) -1H-pyrazolo [3,4-b ] pyridin-6-yl) phenyl) propan-2-ol (11m)
4- (3- (5-Methylfuran-2-yl) -1H-pyrazolo [3,4-b ] pyridin-6-yl) benzonitrile (11n)
More specifically, the preferred compounds of the structure of formula (III) according to the invention are selected from:
6- (4-fluorophenyl) -3- (4- (4-isopropylpiperazin-1-yl) phenyl) -1H-pyrazolo [3,4-b ] pyridine (18a)
6- (3, 4-difluorophenyl) -3- (4- (4-isopropylpiperazin-1-yl) phenyl) -1H-pyrazolo [3,4-b ] pyridine (18b)
6- (3, 5-difluorophenyl) -3- (4- (4-isopropylpiperazin-1-yl) phenyl) -1H-pyrazolo [3,4-b ] pyridine (18c)
6- (4-chlorophenyl) -3- (4- (4-isopropylpiperazin-1-yl) phenyl) -1H-pyrazolo [3,4-b ] pyridine (18d)
6- (4-aminophenyl) -3- (4- (4-isopropylpiperazin-1-yl) phenyl) -1H-pyrazolo [3,4-b ] pyridine (18e)
6- (4-amino-3-fluoro-phenyl) -3- (4- (4-isopropylpiperazin-1-yl) phenyl) -1H-pyrazolo [3,4-b ] pyridine (18f)
2- (4- (3- (4- (4-isopropylpiperazin-1-yl) phenyl) -1H-pyrazolo [3,4-b ] pyridin-6-yl) phenyl) propan-2-ol (18g)
4- (3- (4- (4-isopropylpiperazin-1-yl) phenyl) -1H-pyrazolo [3,4-b ] pyridin-6-yl) benzonitrile (18H)
2-fluoro-4- (3- (4- (4-isopropylpiperazin-1-yl) phenyl) -1H-pyrazolo [3,4-b ] pyridin-6-yl) phenol (18i)
2, 6-difluoro-4- (3- (4- (4-isopropylpiperazin-1-yl) phenyl) -1H-pyrazolo [3,4-b ] pyridin-6-yl) phenol (18j)
4- (3- (4- (4-isopropylpiperazin-1-yl) -2-methoxyphenyl) -1H-pyrazolo [3,4-b ] pyridin-6-yl) phenol (18k)
2- (4- (3- (4- (4-isopropylpiperazin-1-yl) -2-methoxyphenyl) -1H-pyrazolo [3,4-b ] pyridin-6-yl) phenyl) propan-2-ol (18l)
2-fluoro-4- (3- (4- (4-isopropylpiperazin-1-yl) -2-methoxyphenyl) -1H-pyrazolo [3,4-b ] pyridin-6-yl) phenol (18m)
Another object of the present invention is to provide a process for producing the above pyrazolo [3,4-b ] pyridine compound, which comprises the steps of:
synthesis scheme 1 reaction scheme:
Figure BDA0001637289860000061
R1，R2，R3，R4are respectively described in general formula (II);
reacting 2, 6-dichloropyridine (1) with substituted furfural (2) under the action of N-butyllithium, N, N-diisopropylethylamine to generate a compound 3, oxidizing the compound 3 with manganese dioxide to generate a ketone compound 4, and cyclizing the compound 4 and hydrazine hydrate to form pyrazolo [3,4-b ] with a structural general formula]Pyridine compound 5, and Boc2O protection forms compound 6, and finally the tris (dibenzylideneacetone) dipalladium-chloroform adduct (Pd) over a suitable palladium catalyst2(dba)3-CHCl3) Under the action, the compound reacts with a substituted aryl boric acid compound 7 through Suzuki coupling reaction to generate a compound 8, the compound 8 generates a compound 9 after NBS bromination, the compound 9 generates nucleophilic substitution reaction with a nucleophilic reagent to generate a compound 10, and the compound 10 generates a target compound 11 after trifluoroacetic acid deprotection, namely the compound defined by the general formula (II).
Synthesis scheme 2 reaction scheme:
Figure BDA0001637289860000071
R1，R2，R3，R4，R5are respectively described in the general formula (III);
2, 6-dichloropyridine (1) and substituted benzaldehyde (12) react under the action of n-butyl lithium and diisopropylamine to generate a compound 13, the compound 13 is oxidized by manganese dioxide to generate a ketone compound 14, and the compoundCyclizing 14 with hydrazine hydrate to form pyrazolo [3,4-b]Pyridine compound 15, and Boc2O protection forms compound 16, and finally tris (dibenzylideneacetone) dipalladium-chloroform adduct (Pd) over a suitable palladium catalyst2(dba)3-CHCl3) Under the action, the compound reacts with substituted aryl boric acid compound 7 through Suzuki coupling reaction to generate compound 17, and then the compound is deprotected through trifluoroacetic acid to generate target compound 18, namely the compound defined by the general formula (III).
The invention further aims to provide application of the pyrazolo [3,4-b ] pyridine compound in preparation of antitumor drugs. Pharmacological research proves that the compound provided by the invention can be used for inhibiting the activity of ALK kinase and can also be used for treating cancer.
Detailed Description
The present invention will be further described with reference to examples. The following examples are illustrative of the invention and are not intended to limit the invention in any way.
Preparation example 1(2, 6-dichloropyridin-3-yl) (5-methylfuranyl) methanol (3a)
Figure BDA0001637289860000081
In a two-necked round bottom flask, diisopropylamine (3.4mL,24mmol) and anhydrous tetrahydrofuran were added, cooled to-78 deg.C, and n-BuLi (1.6M in hexane) (13.8mL,22mmol) was slowly added dropwise. After stirring for 30 min, 2, 6-dichloropyridine (2.9g,20mmol) was dissolved in anhydrous tetrahydrofuran and slowly added to the flask, which was stirred at-78 ℃ for 2h after the addition. 5-methylfurfural (2.6g,24mmol) was then dissolved in anhydrous tetrahydrofuran and added slowly dropwise to the flask, after which the refrigeration was turned off and stirring was allowed to stand overnight. After the reaction is finished, adding a saturated ammonium chloride solution for quenching, concentrating, adding water, extracting for three times by using ethyl acetate, combining organic phases, washing for two times by using a saturated sodium chloride solution, drying by using anhydrous sodium sulfate, concentrating, and performing column chromatography to obtain a yellow liquid 3a with the yield of 91%.
Preparation example Synthesis of 2(2, 6-dichloropyridin-3-yl) (5-methylfuranyl) methanone (4a)
Figure BDA0001637289860000082
In a single neck round bottom flask, the product 3a from the above step (4.9g,19mmol) was dissolved in anhydrous dichloromethane, and after addition of activated manganese dioxide (8.3g,95mmol), it was stirred at room temperature for 24 h. The reaction progress is monitored by TLC, after the reaction is completed, manganese dioxide is removed from the reaction liquid through diatomite, dichloromethane is used for washing, the solvent is dried in a spinning mode, column chromatography is carried out to obtain yellow solid 4a, and the yield is 95%.
Preparation example 36-chloro-3- (5-methylfuryl) -1H-pyrazolo [3,4-b ] pyrazole (5a)
Figure BDA0001637289860000083
In a single-neck round-bottom flask, the product 4a from the above step (4.9g,19mmol) was dissolved in a mixed solvent of ethanol and tetrahydrofuran, cooled to 0 ℃, diisopropylethylamine (2.5g,19mmol) was added dropwise, followed by hydrazine hydrate (1.04mL,21mmol) dropwise, warmed to room temperature, and then heated to 70 ℃ and stirred overnight. After the reaction is completed, the reaction product is cooled to room temperature, the solvent is concentrated and then water is added, ethyl acetate is used for extraction for three times, organic phases are combined, the organic phases are washed twice by saturated sodium chloride solution, anhydrous magnesium sulfate is used for drying, the solvent is dried in a spinning mode, column chromatography is carried out to obtain yellow solid 5a, and the yield is 97%.
Preparation example 46-chloro-3- (5-methylfuranyl) pyrazolo [3,4-b ] pyridine-1-carboxylic acid tert-butyl ester (6a)
Figure BDA0001637289860000091
In a two-necked round-bottomed flask, the product of the above step 5a (2.1g,9mmol), DMAP (110mg,0.9mmol), triethylamine (3.8mL,27mmol) and Boc were added2O (3g,27mmol) was dissolved in dichloromethane and added to the reaction flask and stirred at 45 ℃ under reflux overnight. After the reaction is completed, dichloromethane is directly added, the mixture is washed for three times by saturated sodium chloride, dried by anhydrous magnesium sulfate, dichloromethane is dried by spinning, and column chromatography is carried out to obtain light yellow solid 6a with the yield of 94%.
Preparation example 53- (5-Methylfuryl) -6- (4- ((tetrahydro-2H-pyran-2-yl) oxy) phenyl) -pyrazolo [3,4-b ] pyridine-1-carboxylic acid tert-butyl ester (8a)
Figure BDA0001637289860000092
The product of the previous step, 6a (333.5mg,1mmol), 4- ((tetrahydro-2H-pyran-2-yl) oxy) phenylboronic acid (444mg,2mmol), Pd, was added to a two-necked round-bottomed flask2(dba)3(40mg,0.05mmol)，P(t-Bu)3HBF4(50mg,0.2mmol), cesium carbonate (978mg,3mmol), 5mL dioxane, 0.5mL water were added and stirred at 100 ℃ under reflux overnight. The reaction progress is monitored by TLC, ethyl acetate is directly added after the reaction is completed, the mixture is washed for three times by saturated sodium chloride solution, dried by anhydrous magnesium sulfate, the solvent is dried by spinning, and the white solid is obtained by column chromatography, wherein the yield is 71%.
Preparation example Synthesis of tert-butyl 5- (1-bromomethyl) furyl) -6- (4- ((tetrahydro-2H-pyran-2-yl) oxy) phenyl) -pyrazolo [3,4-b ] pyridine-1-carboxylate (9a)
Figure BDA0001637289860000101
To a two-necked round-bottomed flask, the product of the above step 8a, bromodiimide (NBS) (38mg, 0.21mmol), Azobisisobutyronitrile (AIBN) (3.6mg, 0.021mmol) were added (100mg, 0.21mmol), respectively, and after argon exchange, carbon tetrachloride was added and heated under reflux for 2 hours. The reaction progress is monitored by TLC, and after the reaction is completed, the solvent is directly dried by spinning to obtain a crude product 9 a.
Preparation example 73- (5- (1-morpholinomethyl) furyl) -6- (4- ((tetrahydro-2H-pyran-2-yl) oxy) phenyl) -pyrazolo [3,4-b ] pyridine-1-carboxylic acid tert-butyl ester (10a)
Figure BDA0001637289860000102
In a single neck round bottom flask, the crude product from the previous step (0.2mmol) was dissolved in anhydrous toluene and heated at 70 ℃ for 7h using addition of morpholine (74uL, 0.8 mmol). The progress of the reaction was monitored by TLC, after completion of the reaction, the solvent was dried by spinning, and the colorless viscous liquid 10a was obtained by column chromatography with a yield of 54%.
Preparation example 83- (5- (1-morpholinomethyl) furyl) -6- (4-hydroxyphenyl) -1H-pyrazolo [3,4-b ] pyridine (11b)
Figure BDA0001637289860000111
In a single neck round bottom flask, the product 10a from the previous step (57.8mg,0.1mmol) was weighed out in dry dichloromethane, trifluoroacetic acid (TFA) (29uL,0.4mmol) was added and heated under reflux overnight. The reaction progress is monitored by TLC, after the reaction is completed, saturated sodium bicarbonate solution is added, extraction is carried out for three times by ethyl acetate, organic phases are combined, drying is carried out by anhydrous magnesium sulfate, column chromatography is carried out to obtain pure yellow solid 11b, and the yield is 87%.
m.p.>300℃；1H NMR(400MHz,DMSO-d6),:13.71(s,1H),9.91(s,1H),8.47(d,J＝8.8Hz,1H),8.04(d,J＝8.8Hz,2H),7.78(d,J＝8.4Hz,1H),7.01(d,J＝3.2Hz,1H),6.91(d,J＝8.8Hz,2H),6.51(d,J＝3.2Hz,1H),3.63(s,2H),3.59(t,J＝4.4Hz,3H),2.46(t,J＝4.4Hz,3H)；13C NMR(100MHz,DMSO-d6),:159.11,156.13,152.67,151.58,147.64,135.43,130.74,129.39,128.73,115.69,114.07,110.92,109.63,107.87,66.19,54.38,52.80；HRMS(ESI)(m/z):calcd for C21H21N4O3(M+H+):377.1614；Found:377.1613.
Preparation example 94- (3- (5-methylfuran-2-yl) -1H-pyrazolo [3,4-b ] pyridin-6-yl) phenol (11a)
Figure BDA0001637289860000112
The preparation method is the same as that of preparation example 8, 8a and trifluoroacetic acid are heated and refluxed in dichloromethane to react, and column chromatography is carried out to obtain yellow solid 11a with yield of 87%.
m.p.247-251℃；1H NMR(400MHz,Acetone-d6),:8.51(d,J＝8.8Hz,1H),8.31-8.09(m,2H),7.69(d,J＝8.8Hz,1H),7.00-6.97(m,2H),6.88(d,J＝3.2Hz,1H),6.24(dd,J1＝3.2Hz,J2＝0.8Hz,1H),2.43(s,3H)；13C NMR(Acetone-d6,100MHz),:159.88,157.50,154.01,153.04,148.46,137.43,131.72,131.41,129.68,116.42,114.74,110.96,108.52,108.40,13.60；HRMS(ESI)(m/z):calcd for C17H14N3O2(M+H+):292.1086；Found:292.1090.
Preparation example 104- (3- (5- (piperidin-1-ylmethyl) furan-2-yl) -1H-pyrazolo [3,4-b ] pyridin-6-yl) phenol (11c)
Figure BDA0001637289860000121
The preparation method is the same as that of preparation examples 7-8, 9a reacts with piperidine, and after trifluoroacetic acid deprotection, column chromatography is carried out to obtain yellow solid 11c, and the yield is 80%.
m.p.226-230℃；1H NMR(400MHz,DMSO-d6),:13.74(s,1H),9.90(s,1H),8.50(d,J＝8.0Hz,1H),8.05(d,J＝8.4Hz,2H),7.79(d,J＝8.8Hz,1H),7.05(s,1H),6.91(d,J＝8.8Hz,2H),6.61(s,1H),3.79(s,2H),2.60(br,4H),1.59(br,4H),1.42(br,2H)；13C NMR(100MHz,DMSO-d6),:159.16,156.18,152.68,148.14,135.30,130.80,129.35,128.73,124.31.115.70,114.10,112.12,109.67,107.91,53.80,53.00,31.18,24.69.
Preparation example 114- (3- (5- (pyrrolidin-1-ylmethyl) furan-2-yl) -1H-pyrazolo [3,4-b ] pyridin-6-yl) phenol (11d)
Figure BDA0001637289860000122
The preparation method is the same as that of preparation examples 7-8, 9a reacts with pyrrolidine, and yellow solid 11d is obtained after trifluoroacetic acid deprotection by column chromatography, and the yield is 83%.
m.p.210-213℃；1H NMR(400MHz,DMSO-d6),:13.78(s,1H),9.92(s,1H),8.56-8.54(m,1H),8.05(d,J＝8.4Hz,2H),7.79(d,J＝8.8Hz,1H),7.06(d,J＝3.2Hz,1H),6.91(d,J＝8.8Hz,2H),6.70(s,1H),4.20(s,2H),2.98(br,4H),1.84(br,4H)；13C NMR(100MHz,DMSO-d6),:159.18,156.24,152.68,148.67,135.14,130.93,129.32,128.74,124.31,115.71,114.15,112.73,109.69,107.86,53.00,49.94,31.19.
Preparation example 124- (3- (5- ((4-methylpiperazin-1-yl) methyl) furan-2-yl) -1H-pyrazolo [3,4-b ] pyridin-6-yl) phenol (11e)
Figure BDA0001637289860000131
The preparation method is the same as that of preparation examples 7-8, 9a reacts with 1-methylpiperazine, and yellow solid 11e is obtained by column chromatography after trifluoroacetic acid deprotection, and the yield is 75%.
m.p.276-279℃；1H NMR(400MHz,DMSO-d6),:13.71(s,1H),9.93(s,1H),8.47(d,J＝8.8Hz,1H),8.04(d,J＝8.8Hz,2H),7.78(d,J＝8.8Hz,1H),7.01(d,J＝3.2Hz,1H),6.91(d,J＝8.8Hz,2H),6.50(d,J＝3.2Hz,1H),3.65(s,2H),2.54(br,4H),2.42(br,3H),2.28(br,4H)；13C NMR(100MHz,DMSO-d6),:159.15,156.14,152.68,151.68,147.64,135.39,130.73,129.35,128.72,124.38,115.70,114.05,109.62,107.86,54.13,53.75,51.32,44.78.
Preparation example 134- (3- (5- ((dimethylamino) methyl) furan-2-yl) -1H-pyrazolo [3,4-b ] pyridin-6-yl) phenol (11f)
Figure BDA0001637289860000132
The preparation method is the same as that of preparation examples 7-8, 9a reacts with dimethylamine, and after trifluoroacetic acid deprotection, column chromatography is carried out to obtain yellow solid 11f, and the yield is 86%.
m.p.181-184℃；1H NMR(500MHz,DMSO-d6),:13.69(s,1H),9.89(s,1H),8.47(d,J＝8.5Hz,1H),8.04(d,J＝8.5Hz,2H),7.78(d,J＝8.5Hz,1H),7.00(d,J＝3.0Hz,1H),6.91(d,J＝8.5Hz,2H),6.49(d,J＝3.0Hz,1H),3.58(s,2H),2.24(s,6H)；13C NMR(100MHz,DMSO-d6),:159.11,156.14,152.67,147.85,140.25,135.39,130.76,129.37,128.72,123.20,115.68,114.06,109.64,107.71,54.71,44.31.
Preparation example 144- (3- (5- ((2- (hydroxymethyl) pyrrolidin-1-yl) methyl) furan-2-yl) -1H-pyrazolo [3,4-b ] pyridin-6-yl) phenol (11g)
Figure BDA0001637289860000141
The preparation method is the same as that of preparation examples 7-8, 9a reacts with 2-hydroxymethyl pyrrolidine, trifluoroacetic acid is used for deprotection, and column chromatography is carried out to obtain 11g of yellow solid with the yield of 79%.
m.p.145-147℃；1H NMR(400MHz,DMSO-d6),:13.75(s,1H),9.92(s,1H),8.51(d,J＝8.4Hz,1H),8.10(d,J＝7.8Hz,1H),8.05(d,J＝8.4Hz,2H),7.79(d,J＝8.8Hz,1H),7.04(s,1H),6.91(d,J＝8.8Hz,2H),5.21-5.13(m,1H),3.51(s,2H),3.12(br,2H),2.33(br,2H),1.72-1.61(m,4H)；13CNMR(100MHz,DMSO-d6),:159.24,158.30,157.99,156.24,153.73,135.29,130.87,129.36,128.76,118.78,115.76,114.15,109.72,107.86,67.31,53.93,49.64,48.66,31.60,22.37.
Preparation example 154- (3- (5- ((4-isopropylpiperazin-1-yl) methyl) furan-2-yl) -1H-pyrazolo [3,4-b ] pyridin-6-yl) phenol (11H)
Figure BDA0001637289860000142
The preparation method is the same as that of preparation examples 7-8, 9a reacts with 1-isopropylpiperazine, and after trifluoroacetic acid deprotection, column chromatography is carried out to obtain yellow solid 11h with yield of 75%.
m.p.184-187℃；1H NMR(400MHz,DMSO-d6),:13.73(s,1H),9.97(s,1H),8.48(d,J＝8.4Hz,1H),8.05(d,J＝8.8Hz,2H),7.78(d,J＝8.4Hz,1H),7.04(d,J＝3.2Hz,1H),6.91(d,J＝8.4Hz,2H),6.53(d,J＝3.2Hz,1H),3.92-3.83(m,1H),3.71(s,2H),3.16-2.32(m,8H),1.12(s,6H)；13C NMR(100MHz,DMSO-d6),:159.11,156.08,152.61,151.13,147.71,135.27,130.66,129.24,128.63,115.63,113.97,111.13,109.53,107.79,55.66,53.23,50.19,47.59,17.05；HRMS(ESI)(m/z):calcd for C24H28N5O2(M+H+):418.2243；Found:418.2243.
Preparation example 164- (3- (5- ((2- (hydroxymethyl) piperidin-1-yl) methyl) furan-2-yl) -1H-pyrazolo [3,4-b ] pyridin-6-yl) phenol (11i)
Figure BDA0001637289860000151
The preparation method is the same as that of preparation examples 7-8, 9a reacts with 2-hydroxymethyl piperidine, and after trifluoroacetic acid deprotection, column chromatography is carried out to obtain yellow solid 11i with yield of 81%.
m.p.135-138℃；1H NMR(400MHz,DMSO-d6),:13.81(s,1H),10.07(s,1H),8.53(d,J＝8.8Hz,1H),8.05(d,J＝8.8Hz,2H),7.78(d,J＝8.4Hz,1H),7.06(d,J＝3.2Hz,1H),6.92(d,J＝8.8Hz,2H),6.74(s,1H),6.18(s,1H),4.31(s,2H),3.74-3.72(m,1H),3.20-3.02(m,2H),2.84-2.67(m,2H),1.76-1.58(m,6H)；13C NMR(100MHz,DMSO-d6),:159.28,158.20,157.89,156.25,152.71,130.91,129.30,128.74,124.03,118.78,115.76,114.21,109.73,107.85,61.52,59.41,59.07,57.42,24.75,21.96,21.43.
Preparation example 171- ((5- (6- (4-hydroxyphenyl) -1H-pyrazolo [3,4-b ] pyridin-3-yl) furan-2-yl) methyl) piperidine-3-carboxamide (11j)
Figure BDA0001637289860000152
The preparation method is the same as that of preparation examples 7-8, 9a reacts with piperidine-2-formamide, and after trifluoroacetic acid deprotection, column chromatography is carried out to obtain yellow solid 11j, and the yield is 75%.
m.p.192-195℃；1H NMR(400MHz,DMSO-d6),:13.74(s,1H),9.92(s,1H),8.51(s,1H),8.05(d,J＝8.8Hz,2H),7.78(d,J＝8.8Hz,1H),7.04(s,1H),6.91(d,J＝8.8Hz,2H),6.56(s,1H),3.67(s,2H),3.07-2.64(m,3H),2.33-1.99(m,2H),1.72-1.50(m,4H)；13C NMR(100MHz,DMSO-d6),:177.83,159.43,158.41,154.38,153.53,147.42,136.96,131.38,130.97,128.73,115.87,114.40,110.53,109.03,108.99,58.42,54.88,53.90,44.05,27.83,23.94.
Preparation example 184- (3- (5- (((2-hydroxyethyl) (methyl) amino) methyl) furan-2-yl) -1H-pyrazolo [3,4-b ] pyridin-6-yl) phenol (11k)
Figure BDA0001637289860000161
The preparation method is the same as that of preparation examples 7-8, 9a reacts with 2-methylamino ethanol, and after trifluoroacetic acid deprotection, column chromatography is carried out to obtain yellow solid 11k, and the yield is 81%.
m.p.148-152℃；1H NMR(400MHz,DMSO-d6),:13.74(s,1H),9.95(s,1H),8.50(d,J＝8.8Hz,1H),8.05(d,J＝8.8Hz,2H),7.78(d,J＝8.8Hz,1H),7.02(d,J＝3.2Hz,1H),6.91(d,J＝8.8Hz,2H),6.58(d,J＝2.4Hz,1H),3.87(s,2H),3.59-3.57(m,2H),3.16(s,1H),2.65(br,2H),2.38(s,3H)；13CNMR(100MHz,DMSO-d6),:159.24,156.22,153.47,152.72,148.19,135.39,130.85,129.37,128.75,115.75,114.13,111.95,109.71,107.77,58.04,53.07,48.67,41.66；HRMS(ESI)(m/z):calcd for C20H21N4O3(M+H+):365.1614；Found:365.1609.
Preparation example 192-fluoro-4- (3- (5-methylfuran-2-yl) -1H-pyrazolo [3,4-b ] pyridin-6-yl) phenol (11l)
Figure BDA0001637289860000162
The preparation method is the same as that of preparation examples 5 and 8, and the white solid 11l is obtained by carrying out Suzuki coupling reaction on 6a and (3-fluoro-4- ((tetrahydro-2H-pyran-2-yl) oxy)) phenylboronic acid, deprotection by trifluoroacetic acid and column chromatography, and the yield is 80%.
m.p.210-213℃；1H NMR(400MHz,Acetone-d6),:12.73(s,1H),9.03(s,1H),8.53(d,J＝8.4Hz,1H),8.01(dd,J1＝12.8Hz,J2＝2.0Hz,1H),7.92(dd,J1＝8.4Hz,J2＝1.2Hz,1H),7.80(d,J＝8.4Hz,1H),7.14(t,J＝8.8Hz,1H),6.89(d,J＝3.2Hz,1H),6.24(dd,J1＝2.8Hz,J2＝0.8Hz,1H),2.43(s,3H)；13C NMR(150MHz,Acetone-d6),:156.24(d,J＝3Hz),153.73(d,J＝79.5Hz),153.22,151.87,148.43,147.28(d,J＝12Hz),137.61,132.46(d,J＝6Hz),132.05,124.64(d,J＝3Hz),118.84,115.68(d,J＝21Hz),114.85,111.45,108.74,108.50,13.67；HRMS(ESI)(m/z):calcd for C17H13FN3O2(M+H+):310.0992；Found:310.0999.
Preparation example 202- (4- (3- (5-methylfuran-2-yl) -1H-pyrazolo [3,4-b ] pyridin-6-yl) phenyl) propan-2-ol (11m)
Figure BDA0001637289860000171
The preparation method is the same as that of preparation examples 5 and 8, and the white solid 11m is obtained by carrying out Suzuki coupling reaction on 6a and 4- (2- ((tetrahydro-2H-pyran-2-yl) oxy) propan-2-yl) phenylboronic acid, deprotection by trifluoroacetic acid and column chromatography, and the yield is 76%.
m.p.240-243℃；1H NMR(400MHz,CD3Cl),:10.53(s,1H),8.53(d,J＝8.4Hz,1H),8.23-8.21(m,2H),7.85-7.81(m,2H),7.71(d,J＝8.4Hz,1H),6.89(d,J＝3.2Hz,1H),6.20-6.19(m,1H),2.47(s,3H)；13C NMR(150MHz,DMSO-d6),:153.72,152.38,152.10,146.23,142.76,135.75,132.82,131.54,127.92,118.72,115.05,111.82,110.91,108.53,107.89,13.40.
Preparation example 214- (3- (5-methylfuran-2-yl) -1H-pyrazolo [3,4-b ] pyridin-6-yl) benzonitrile (11n)
Figure BDA0001637289860000172
The preparation method is the same as that of preparation examples 5 and 8, 6a and 4-cyanophenylboronic acid are subjected to Suzuki coupling reaction, trifluoroacetic acid is used for deprotection, and column chromatography is carried out to obtain yellow solid 11n, wherein the yield is 82%.
m.p.240-243℃；1H NMR(400MHz,CD3Cl),:10.53(s,1H),8.53(d,J＝8.4Hz,1H),8.23-8.21(m,2H),7.85-7.81(m,2H),7.71(d,J＝8.4Hz,1H),6.89(d,J＝3.2Hz,1H),6.20-6.19(m,1H),2.47(s,3H)；13C NMR(150MHz,DMSO-d6),:153.72,152.38,152.10,146.23,142.76,135.75,132.82,131.54,127.92,118.72,115.05,111.82,110.91,108.53,107.89,13.40.
Preparation example 226- (4-fluorophenyl) -3- (4- (4-isopropylpiperazin-1-yl) phenyl) -1H-pyrazolo [3,4-b ] pyridine (18a)
Figure BDA0001637289860000181
The preparation method is the same as that of preparation examples 1-8, and column chromatography is carried out to obtain yellow solid 18a with the yield of 73%.
m.p.259-262℃；1H NMR(400MHz,DMSO-d6),:13.67(s,1H),8.59(d,J＝8.4Hz,1H),8.27-8.23(m,2H),7.95-7.93(m,2H),7.84(d,J＝8.4Hz,1H),7.40-7.35(m,2H),7.14(s,2H),3.52(br,4H),3.18(br,4H),2.67(br,1H),1.23(s,6H).
Preparation example 236- (3, 4-difluorophenyl) -3- (4- (4-isopropylpiperazin-1-yl) phenyl) -1H-pyrazolo [3,4-b ] pyridine (18b)
Figure BDA0001637289860000182
The preparation method is the same as that of preparation examples 1-8, and column chromatography is carried out to obtain yellow solid 18b with the yield of 75%.
m.p.251-254℃；1H NMR(400MHz,DMSO-d6),:13.69(s,1H),8.61(d,J＝8.4Hz,1H),8.26-8.21(m,1H),8.10-8.07(m,1H),7.91-7.87(m,3H),7.64-7.57(m,1H),7.09-7.06(m,2H),3.22(t,J＝4.8Hz,4H),2.72-2.66-8.07(m,1H),2.60(t,J＝4.8Hz,4H),1.02(d,J＝6.8Hz,6H).
Preparation example 246- (3, 5-difluorophenyl) -3- (4- (4-isopropylpiperazin-1-yl) phenyl) -1H-pyrazolo [3,4-b ] pyridine (18c)
Figure BDA0001637289860000183
The preparation method is the same as that of preparation examples 1-8, and column chromatography is carried out to obtain yellow solid 18c with the yield of 77%.
m.p.238-241℃；1H NMR(400MHz,DMSO-d6),:13.75(s,1H),8.63(d,J＝8.8Hz,1H),7.96-7.91(m,5H),7.42-7.36(m,1H),7.10(s,2H),3.98(br,1H),3.20(br,4H),2.61(br,4H),1.04(br,6H).
Preparation example 256- (4-chlorophenyl) -3- (4- (4-isopropylpiperazin-1-yl) phenyl) -1H-pyrazolo [3,4-b ] pyridine (18d)
Figure BDA0001637289860000191
The preparation method is the same as that of preparation examples 1-8, and column chromatography is carried out to obtain yellow solid 18d with the yield of 70%.
m.p.249-252℃；1H NMR(400MHz,DMSO-d6),:13.67(s,1H),8.60(d,J＝8.4Hz,1H),8.22(d,J＝8.0Hz,2H),7.92-7.84(m,3H),7.61(d,J＝8.0Hz,2H),7.10(s,2H),3.21(br,4H),2.61(br,4H),1.23(s,6H).
Preparation example 266- (4-aminophenyl) -3- (4- (4-isopropylpiperazin-1-yl) phenyl) -1H-pyrazolo [3,4-b ] pyridine (18e)
Figure BDA0001637289860000192
The preparation method is the same as that of preparation examples 1-8, and column chromatography is carried out to obtain yellow solid 18e with yield of 72%.
m.p.271-274℃；1H NMR(400MHz,DMSO-d6),:13.37(s,1H),8.41(d,J＝8.8Hz,1H),7.92-7.86(m,4H),7.64(d,J＝8.8Hz,1H),7.06(d,J＝8.0Hz,2H),6.67(d,J＝8.8Hz,2H),5.55(s,2H),3.21(br,4H),2.63(br,5H),1.05(s,6H)；HRMS(ESI)(m/z):calcd forC25H29N6(M+H+):413.2454；Found:413.2448.
Preparation example 276- (4-amino-3-fluoro-phenyl) -3- (4- (4-isopropylpiperazin-1-yl) phenyl) -1H-pyrazolo [3,4-b ] pyridine (18f)
Figure BDA0001637289860000193
The preparation method is the same as that of preparation examples 1-8, and column chromatography is carried out to obtain yellow solid 18f with the yield of 70%.
m.p.269-272℃；1H NMR(400MHz,DMSO-d6),:13.45(s,1H),8.44(d,J＝8.0Hz,1H),7.87-7.69(m,5H),7.05(d,J＝6.8Hz,2H),6.87(t,J＝8.0Hz,1H),5.62(s,2H),3.20(br,4H),2.62(br,5H),1.03(s,6H).
Preparation example 282- (4- (3- (4- (4-isopropylpiperazin-1-yl) phenyl) -1H-pyrazolo [3,4-b ] pyridin-6-yl) phenyl) propan-2-ol (18g)
Figure BDA0001637289860000201
The preparation method is the same as that of preparation examples 1-8, column chromatography is carried out to obtain 18g of yellow solid, and the yield is 81%.
m.p.221-224℃；1H NMR(400MHz,DMSO-d6),:13.59(s,1H),8.55(d,J＝8.4Hz,1H),8.12(d,J＝8.0Hz,2H),7.89(d,J＝8.4Hz,2H),7.80(d,J＝8.4Hz,1H),7.62(d,J＝8.0Hz,2H),7.07(d,J＝8.4Hz,2H),5.11(s,1H),3.21(br,4H),2.74-2.66(m,1H),2.60(br,4H),1.48(s,6H),1.12(d,J＝6.4Hz,6H)；13C NMR(150MHz,DMSO-d6),:174.68,155.20,151.93,140.46,137.94,131.17,127.17,126.60,125.08,123.43,122.35,118.22,116.74,115.22,70.65,53.68,48.22,48.04,31.85,18.26.
Preparation example 294- (3- (4- (4-isopropylpiperazin-1-yl) phenyl) -1H-pyrazolo [3,4-b ] pyridin-6-yl) benzonitrile (18H)
Figure BDA0001637289860000202
The preparation method is the same as that of preparation examples 1-8, and yellow solid is obtained by column chromatography for 18h, and the yield is 85%.
m.p.253-257℃；1H NMR(400MHz,DMSO-d6),:13.75(s,1H),8.65(d,J＝8.4Hz,1H),8.40-8.38(m,2H),8.02-8.00(m,2H),7.95-7.90(m,3H),7.08(d,J＝8.4Hz,2H),3.21(br,4H),2.67(br,1H),2.60(br,4H),1.02(d,J＝5.2Hz,6H)；13C NMR(150MHz,DMSO-d6),:153.06,150.94,149.62,142.89,132.83,131.75,127.87,127.27,118.75,115.24,114.64,117.71,111.63,53.68,48.14,48.01,18.22.
Preparation example 302-fluoro-4- (3- (4- (4-isopropylpiperazin-1-yl) phenyl) -1H-pyrazolo [3,4-b ] pyridin-6-yl) phenol (18i)
Figure BDA0001637289860000211
The preparation method is the same as that of preparation examples 1-8, and column chromatography is carried out to obtain yellow solid 18i with yield of 80%.
m.p.284-287℃；1H NMR(400MHz,DMSO-d6),:13.53(s,1H),10.30(s,1H),8.51(d,J＝8.4Hz,1H),7.98(d,J＝13.2Hz,1H),7.88(d,J＝8.4Hz,3H),7.76(d,J＝8.4Hz,1H),7.11-7.05(m,3H),3.22(br,4H),2.72(br,1H),2.63(br,4H),1.04(d,J＝5.2Hz,6H)；13CNMR(150MHz,DMSO-d6),:154.05,153.10,152.05,150.45,146.35,131.16,130.30,127.18,123.55,117.93,115.25,114.53,113.51,110.55,54.90,47.98,18.18；HRMS(ESI)(m/z):calcd for C25H27FN5O(M+H+):432.2200；Found:432.2204.
Preparation example 312, 6-difluoro-4- (3- (4- (4-isopropylpiperazin-1-yl) phenyl) -1H-pyrazolo [3,4-b ] pyridin-6-yl) phenol (18j)
Figure BDA0001637289860000212
The preparation method is the same as that of preparation examples 1-8, column chromatography is carried out to obtain brown solid 18j, and the yield is 78%.
m.p.>300℃；1H NMR(400MHz,DMSO-d6),:13.68(s,1H),10.79(s,1H),8.56(d,J＝8.4Hz,1H),7.96-7.90(m,4H),7.86(d,J＝8.4Hz,1H),7.15(d,J＝8.4Hz,2H),3.90(br,1H),3.47(br,4H),3.17(br,4H),1.31(d,J＝5.6Hz,6H)；13C NMR(100MHz,DMSO-d6),:154.33,153.71,152.89,152.85,143.25,142.60,131.34,127.31,115.90,113.70,110.90,110.48,99.49,56.78,47.35,16.52.
Preparation example 324- (3- (4- (4-isopropylpiperazin-1-yl) -2-methoxyphenyl) -1H-pyrazolo [3,4-b ] pyridin-6-yl) phenol (18k)
Figure BDA0001637289860000213
The preparation method is the same as that of preparation examples 1-8, column chromatography is carried out to obtain brown solid 18k, and the yield is 85%.
m.p.215-218℃；1H NMR(400MHz,DMSO-d6),:13.42(s,1H),9.82(s,1H),8.12(d,J＝8.4Hz,1H),8.02(d,J＝8.8Hz,2H),7.64(d,J＝8.8Hz,1H),7.48(d,J＝8.4Hz,1H),6.91(d,J＝8.8Hz,2H),6.69(d,J＝1.6Hz,1H),6.64-6.61(m,1H),3.83(s,3H),3.27(br,4H),2.67(br,5H),1.06(d,J＝5.2Hz,6H)；13C NMR(150MHz,DMSO-d6),:158.79,157.43,155.14,152.88,141.73,132.21,130.66,129.73,128.50,115.58,112.82,112.07,107.32,98.94,55.19,47.95,18.04；HRMS(ESI)(m/z):calcd for C26H30N5O2(M+H+):444.2400；Found:444.2403.
Preparation example 332- (4- (3- (4- (4-isopropylpiperazin-1-yl) -2-methoxyphenyl) -1H-pyrazolo [3,4-b ] pyridin-6-yl) phenyl) propan-2-ol (18l)
Figure BDA0001637289860000221
The preparation method is the same as that of preparation examples 1-8, column chromatography is carried out to obtain 18l of white solid, and the yield is 80%.
m.p.267-271℃；1H NMR(400MHz,DMSO-d6),:13.54(s,1H),8.18(d,J＝8.4Hz,1H),8.10(d,J＝8.4Hz,2H),7.73(d,J＝8.4Hz,1H),7.61(d,J＝8.0Hz,2H),7.49(d,J＝8.4Hz,1H),6.69(s,1H),6.62(d,J＝8.4Hz,1H),5.10(s,1H),3.84(s,3H),3.25(br,4H),2.72(br,1H),2.64(br,4H),1.48(s,6H),1.03(d,J＝6.0Hz,6H)；13C NMR(150MHz,DMSO-d6),:157.45,155.01,152.86,152.67,151.77,136.60,132.44,130.69,128.03,126.56,125.05,113.47,112.65,107.29,98.85,70.65,55.19,53.87,48.01,31.85,18.15.
Preparation example 342-fluoro-4- (3- (4- (4-isopropylpiperazin-1-yl) -2-methoxyphenyl) -1H-pyrazolo [3,4-b ] pyridin-6-yl) phenol (18m)
Figure BDA0001637289860000222
The preparation method is the same as that of preparation examples 1-8, column chromatography is carried out to obtain a yellow solid of 18m, and the yield is 78%.
m.p.219-222℃；1H NMR(400MHz,DMSO-d6),:13.48(s,1H),10.30(s,1H),8.14(d,J＝8.4Hz,1H),7.95(dd,J1＝13.2Hz,J2＝2.0Hz,1H),7.87-7.84(m,1H),7.69(d,J＝8.8Hz,1H),7.48(d,J＝8.4Hz,1H),7.08(t,J＝8.8Hz,1H),6.69(s,1H),6.63(d,J＝8.4Hz,1H),3.83(s,3H),3.28(br,4H),2.67(br,5H),1.06(d,J＝4.0Hz,6H)；13C NMR(150MHz,DMSO-d6),:157.45,153.82,150.46,146.30,132.43,130.68,130.53,130.49,123.47,117.92,114.47,112.92,107.35,98.75,55.21,53.73,51.17,47.95,18.00；HRMS(ESI)(m/z):calcd for C26H29FN5O2(M+H+):462.2305；Found:462.2310.
Preparation example 352 phenyl-fluoro-4- (3- (4- (4-isopropylpiperazin-1-yl) phenyl) -1H-pyrazolo [3,4-b ] pyridin-6-yl) acetate (18n)
Figure BDA0001637289860000231
The preparation method comprises the steps of reacting 18i with acetyl chloride and triethylamine in tetrahydrofuran, adding water after the reaction is completed, extracting with ethyl acetate for three times, combining organic phases, washing twice with saturated sodium chloride solution, drying with anhydrous magnesium sulfate, spin-drying the solvent, and carrying out column chromatography to obtain a yellow solid 18n, wherein the yield is 73%.
m.p.195-198℃；1H NMR(400MHz,DMSO-d6),:13.71(s,1H),8.62(d,J＝8.0Hz,1H),8.17(dd,J1＝11.6Hz,J2＝1.6Hz,1H),8.10(d,J＝8.4Hz,1H),7.91(dd,J1＝8.4Hz,J2＝4.4Hz,3H),7.48(t,J＝8.0Hz,1H),7.09(d,J＝8.4Hz,2H),3.22(s,4H),2.67(s,1H),2.61(s,4H),2.38(s,3H),1.03(m,6H).
Preparation example 362-fluoro-4- (3- (4- (4-isopropylpiperazin-1-yl) phenyl) -1H-pyrazolo [3,4-b ] pyridin-6-yl) phenylmethyl carbonate (18o)
Figure BDA0001637289860000241
The preparation method is the same as that of preparation example 35, 18i reacts with methyl chloroformate, and column chromatography is carried out to obtain yellow solid 18o, wherein the yield is 70%.
m.p.213-216℃；1H NMR(400MHz,DMSO-d6),:13.71(s,1H),8.62(d,J＝8.4Hz,1H),8.20(d,J＝11.2Hz,1H),8.11(d,J＝8.0Hz,1H),7.90(d,J＝7.6Hz,3H),7.60(t,J＝8.4Hz,1H),7.07(d,J＝7.6Hz,2H),3.91(s,3H),3.21(s,4H),2.60(s,5H),1.02(s,6H).
Preparation example 372-fluoro-4- (3- (4- (4-isopropylpiperazin-1-yl) phenyl) -1H-pyrazolo [3,4-b ] pyridin-6-yl) nicotinic acid phenyl ester (18p)
Figure BDA0001637289860000242
The preparation method is the same as that of preparation example 35, 18i reacts with nicotinoyl chloride, and yellow solid 18p is obtained through column chromatography, and the yield is 60%.
m.p.223-226℃；1H NMR(400MHz,DMSO-d6),:13.74(s,1H),9.33(s,1H),8.97(d,J＝4.0Hz,1H),8.65(d,J＝8.4Hz,1H),8.55(d,J＝8.0Hz,1H),8.26(d,J＝12.0Hz,1H),8.18(d,J＝8.4Hz,1H),7.94(t,J＝7.6Hz,3H),7.70(q,J1＝8.9Hz,J2＝7.4Hz,2H),7.10(d,J＝8.4Hz,2H),3.22(s,4H),2.62(s,5H),1.05(s,6H).
Preparation example 382-fluoro-4- (3- (4- (4-isopropylpiperazin-1-yl) phenyl) -1H-pyrazolo [3,4-b ] pyridin-6-yl) phenylpiperidinyl-1-carbonate (18q)
Figure BDA0001637289860000243
The preparation method is the same as preparation example 35, 18i reacts with piperidyl formyl chloride, and yellow solid 18q is obtained through column chromatography, and the yield is 70%.
m.p.262-272℃；1H NMR(400MHz,DMSO-d6),:13.66(s,1H),8.60(d,J＝8.4Hz,1H),8.12(d,J＝12.0Hz,1H),8.05(d,J＝7.6Hz,1H),7.89(t,J＝7.6Hz,3H),7.45(t,J＝8.4Hz,1H),7.08(d,J＝8.4Hz,2H),3.61(s,2H),3.44(s,2H),3.22(s,4H),2.60(s,5H),1.61(s,6H),1.02(d,J＝6.4Hz,6H).
Biological experiment example: test of ALK inhibitory Activity of pyrazolo [3,4-b ] pyridine derivatives
We used the LanthaScreen based on TR-FRET techniqueTMKinase assays to determine kinase inhibitory activity of candidate compounds. All experiments were performed on 384-well plates. 4 sets of solutions of the test compounds at different concentrations were prepared before the start of the enzymatic reaction. After 50mM HEPES (pH 7.5), 0.01% BRIJ-35, 10mM MgCl2，4mM MnCl2Group 4 recombinant human ALK proteins and group 2 substrate/ATP mixtures were prepared in 1mM EGTA and 2mM DTT 4. A final 10uL kinase reaction consisted of 5. mu.L of 1 Xsubstrate/ATP mix (0.2. mu.M substrate and 5. mu.M ATP), 2.5. mu.L of ALK protein (5ng/ml) and 2.5. mu.L of 1 test compound at the desired concentration. The assay plate was shaken on a shaker for 30 seconds to thoroughly mix the reactants. After 1 hour incubation of the kinase reaction at room temperature (20-25 ℃), 10 μ L of a pre-prepared antibody solution labeled with 20mM EDTA and 4nM Tb was then added to stop the kinase reaction and initiate antibody binding, and the assay plates were incubated at room temperature for an additional 1 hour. Subsequently, the assay plate was placed in a fluorescence plate reader (BioTek Synergy)TM4) The fluorescein and terbium emission signals were measured with a delay time of 100 μ s and an integration of 200 μ s (excitation: 340 nm; emission: 520 and 495nm, respectively). Finally, the resulting inhibitory activity, calculated from the TR-FRET emission ratio (i.e., fluorescein emission intensity/terbium emission intensity), is plotted against the concentration of inhibitor, and the data is fit to a concentration-response curve with variable slope, from which the IC is calculated50The values, experimental results are shown in table 1.
TABLE 1 ALK inhibitory Activity of pyrazolo [3,4-b ] pyridine derivatives
Figure BDA0001637289860000251
Figure BDA0001637289860000261
As is clear from Table 1, ALK kinase which is currently on the marketThe inhibitors Crizotinib (Crizotinib) and ceritinib (LDK-378) are used as positive controls, and some compounds in the compounds with brand-new structures show better ALK inhibitory activity, wherein the kinase IC of the compound 18i50Reaches 1.58nM which is higher than positive control crizotinib and ceritinib, and provides a new basis for the research of preparing cancer treatment drugs.

Claims (7)

1. A pyrazolo [3,4-b ] pyridine derivative, wherein the compound is:
4- (3- (5-methylfuran-2-yl) -1H-pyrazolo [3,4-b ] pyridin-6-yl) phenol
4- (3- (5- (morpholinomethyl) furan-2-yl) -1H-pyrazolo [3,4-b ] pyridin-6-yl) phenol
4- (3- (5- (piperidin-1-ylmethyl) furan-2-yl) -1H-pyrazolo [3,4-b ] pyridin-6-yl) phenol
4- (3- (5- (pyrrolidin-1-ylmethyl) furan-2-yl) -1H-pyrazolo [3,4-b ] pyridin-6-yl) phenol
4- (3- (5- ((4-methylpiperazin-1-yl) methyl) furan-2-yl) -1H-pyrazolo [3,4-b ] pyridin-6-yl) phenol
4- (3- (5- ((dimethylamino) methyl) furan-2-yl) -1H-pyrazolo [3,4-b ] pyridin-6-yl) phenol
4- (3- (5- ((2- (hydroxymethyl) pyrrolidin-1-yl) methyl) furan-2-yl) -1H-pyrazolo [3,4-b ] pyridin-6-yl) phenol
4- (3- (5- ((4-isopropylpiperazin-1-yl) methyl) furan-2-yl) -1H-pyrazolo [3,4-b ] pyridin-6-yl) phenol
4- (3- (5- ((2- (hydroxymethyl) piperidin-1-yl) methyl) furan-2-yl) -1H-pyrazolo [3,4-b ] pyridin-6-yl) phenol
1- ((5- (6- (4-hydroxyphenyl) -1H-pyrazolo [3,4-b ] pyridin-3-yl) furan-2-yl) methyl) piperidine-3-carboxamide
4- (3- (5- (((2-hydroxyethyl) (methyl) amino) methyl) furan-2-yl) -1H-pyrazolo [3,4-b ] pyridin-6-yl) phenol
2-fluoro-4- (3- (5-methylfuran-2-yl) -1H-pyrazolo [3,4-b ] pyridin-6-yl) phenol
Or 2- (4- (3- (5-methylfuran-2-yl) -1H-pyrazolo [3,4-b ] pyridin-6-yl) phenyl) propan-2-ol.
2. A pyrazolo [3,4-b ] pyridine derivative, wherein the compound is:
6- (4-aminophenyl) -3- (4- (4-isopropylpiperazin-1-yl) phenyl) -1H-pyrazolo [3,4-b ] pyridine
6- (4-amino-3-fluoro-phenyl) -3- (4- (4-isopropylpiperazin-1-yl) phenyl) -1H-pyrazolo [3,4-b ] pyridine
2-fluoro-4- (3- (4- (4-isopropylpiperazin-1-yl) phenyl) -1H-pyrazolo [3,4-b ] pyridin-6-yl) phenol
2, 6-difluoro-4- (3- (4- (4-isopropylpiperazin-1-yl) phenyl) -1H-pyrazolo [3,4-b ] pyridin-6-yl) phenol
4- (3- (4- (4-isopropylpiperazin-1-yl) -2-methoxyphenyl) -1H-pyrazolo [3,4-b ] pyridin-6-yl) phenol
Or 2-fluoro-4- (3- (4- (4-isopropylpiperazin-1-yl) -2-methoxyphenyl) -1H-pyrazolo [3,4-b ] pyridin-6-yl) phenol.
3. The process for the preparation of pyrazolo [3,4-b ] pyridine derivatives according to claim 1, wherein the process is carried out by:
Figure FDA0002517948780000021
reacting 2, 6-dichloropyridine (1) with furfural (2) under the action of n-butyllithium to generate a compound 3, oxidizing the compound 3 with manganese dioxide to generate a compound 4, cyclizing the compound 4 with hydrazine hydrate to form a pyrazole ring, and then cyclizing the pyrazole ring through Boc2Protecting with O to form compound 6, Suzuki coupling reacting with substituted aryl boric acid compound 7 under the action of palladium catalyst to obtain compound 8, bromizing with NBS to obtain compound 9, nucleophilic substitution reacting with compound 9 to obtain compound 10, deprotecting with trifluoroacetic acid to obtain pyrazolo [3,4-b ]]Pyridine derivatives 11;
the pyrazolo [3,4-b ] pyridine derivative 11 is a compound according to claim 1.
4. The process for the preparation of pyrazolo [3,4-b ] pyridine derivatives according to claim 2, wherein the process is carried out by:
Figure FDA0002517948780000031
2, 6-dichloropyridine (1) and substituted benzaldehyde (12) react under the action of n-butyl lithium to generate a compound 13, the compound 13 is oxidized by manganese dioxide to generate a compound 14, the compound 14 is cyclized into a pyrazole ring under the action of hydrazine hydrate, and the pyrazole ring is cyclized by Boc2Protecting with O to form compound 16, Suzuki coupling reacting with substituted aryl boric acid compound 7 under the action of palladium catalyst to obtain compound 17, deprotecting with trifluoroacetic acid to obtain pyrazolo [3,4-b ]]Pyridine derivatives 18;
the pyrazolo [3,4-b ] pyridine derivative 18 is a compound according to claim 2.
5. A process for the preparation of pyrazolo [3,4-b ] pyridine derivatives according to any one of claims 3 to 4 wherein the palladium catalyst is replaced by a tris (dibenzylideneacetone) dipalladium-chloroform adduct.
6. Use of pyrazolo [3,4-b ] pyridine derivatives as defined in any of claims 1-2 for the preparation of an anti-tumor medicament for the prevention or treatment of lung cancer.
7. Use of pyrazolo [3,4-b ] pyridine derivatives according to claim 6 for the preparation of an anti-tumor medicament for inhibiting ALK kinase activity.
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