CN110483523B - Pyrazolopyrimidine derivative epidermal growth factor inhibitor and preparation method and application thereof - Google Patents
Pyrazolopyrimidine derivative epidermal growth factor inhibitor and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a selective inhibitor of a clinical mutant of EGFR (epidermal growth factor receptor) protein tyrosine kinase, which has a structure shown in a formula (I), is a compound containing a pyrazolopyrimidine structure, and also discloses a preparation method of the compound and application of the compound as the selective inhibitor of the clinical mutant of EGFR protein tyrosine kinase, in particular to the inhibition effect of the EGFR of a T790M variant epidermal growth factor, and the selective inhibitor can be used for treating diseases related to the overexpression of the EGFR of the epidermal growth factor, such as kidney cancer, lung cancer, prostate cancer, pancreatic cancer, breast cancer and glioma.
Description
Technical Field
The invention relates to the field of medicines, and in particular relates to a pyrazolopyrimidine derivative epidermal growth factor inhibitor, and a preparation method and application thereof.
Background
The epidermal growth factor EGFR (epidermal growth factor receptor) is a 170kDa transmembrane glycoprotein receptor tyrosine kinase, which is activated by epidermal growth factors and influences the growth and differentiation of cells. Binding of EGF or TGF α to EGFR activates receptor tyrosine kinase activity. Tyrosine residues Tyr1068, tyr1148, and Tyr1173 at the carboxyl terminal of EGFR are the main sites for autophosphorylation to occur after EGF binding. Once activated, phosphorylated tyrosine residues at positions 1068 and 1173 of EGFR mediate the binding of Grb2 to EGFR. In addition, the tyrosine residue phosphorylated at position 1173 is the primary binding site for SHC on EGFR. EGFR is widely distributed in many normal and malignant epithelial cells, and its overexpression and self-activation may be associated with the development of many tumors. At present, the method is mainly used for researching various epitheliogenic malignant tumors including head and neck squamous cell carcinoma, lung cancer, breast cancer, bladder cancer and the like.
Protein Tyrosine Kinases (PTKs) are a class of kinases that catalyze the transfer of gamma-phosphate on ATP to protein tyrosine residues. It is believed that the growth factor receptor proteins, including the epidermal growth factor receptor, function by phosphorylation, and that epidermal growth factor receptor EGFR tyrosine kinase phosphorylates the epidermal growth factor receptor. Clinically, EGFR tyrosine kinase inhibitors have been used in the treatment of cancer, the first generation reversible EGFR tyrosine kinase inhibitors gefitinib and erlotinib. Significant clinical responses (50-80%) were shown in humans with viruses with these specific activating mutations in the body. However, patients who develop secondary mutations to these drugs suffer a recurrence of the cancer within months. Second generation EGFR tyrosine kinase inhibitors include neratinib, dacatinib, afatinib, which all contain electrophilic groups Michael receptors in their structures. In the case of afatinib as a representative drug, the allylamide structure plays a crucial role in the antitumor activity of afatinib, and the allylamide structure is used as a Michael acceptor to perform Michael addition reaction with a catalytic site (a new nuclear sulfhydryl group) of a cysteine residue (Cys 797) on EGFR, so that kinase is inactivated, the activity of tyrosine kinase is irreversibly inhibited, and the alfa-substituted allylamide derivative has good tolerance. Researchers have completely demonstrated the presence of these covalent bonds at the molecular level by growing crystals of inhibitors and receptors, and have found that afatinib strongly inhibits these enzymes by interacting with Cys 805 of HER2 and Cys 803 of HER 4. In vitro tests on wild type EGFR show that afatinib has better effects on the inhibition effect of wild type EGFR and L858R/T790M double mutant compared with gefitinib, erlotinib and lapatinib. In addition, the inhibition effect of afatinib on DER4 is 30 times higher than that of lapatinib. The inhibition effect of the third generation EGFR tyrosine kinase inhibitor oxitinib on the L858R/T790M/C797S triple mutant type also reaches nanomole. In conclusion, it is important in the current drug development field to further develop EGFR tyrosine kinase inhibitors having strong inhibitory effects on mutants and to develop novel anti-tumor drugs having drug resistance with high efficiency and low toxicity.
Disclosure of Invention
The invention designs a series of inhibitor micromolecules for inhibiting T790M variant EGFR tyrosine kinase, finds out a compound with high inhibitory activity to T790M variant EGFR, has obvious inhibitory action to cancer cells, and is a compound containing a pyrazolopyrimidine structure and an alpha, beta-unsaturated carboxylic acid amide structure. The compound can realize high-activity inhibition on T790M/L858R variant EGFR, can inhibit or kill EGFRT790M variant tumor cells, and has high inhibitory activity on wild type EGFR.
The invention provides a pyrazolopyrimidine derivative compound shown in formula (I), or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, which can be used as an irreversible EGFR inhibitor,
wherein:
may be any of the following
R 1 the following structure is possible:
wherein R is 2 ，R 3 And R 4 Each may be H, C 1-12 Alkyl radical, C 1-12 Heteroalkyl group, C 1-12 Heterocycloalkyl radical, C 1-12 Alkenyl radical, C 1-12 Alkynyl, C 1-12 Any one of cycloalkyl groups.
Preferably, the compounds include compounds having the structure as shown in table 1:
TABLE 1 Compounds of the general structural formula (I) include one of the compounds numbered 701-715
The invention also provides a preparation method of the pyrazolopyrimidine derivative EGFR protein tyrosine kinase selective inhibitor, as shown in Scheme 1, the method comprises the following steps: the key intermediate 2 is obtained by Suzuki coupling reaction of the initial raw materials, the intermediate 2 respectively and the bicyclic compound are electrophilic substituted and acylated to finally obtain the corresponding compound (shown as Scheme 1) shown as the general formula (I)
Synthesis and preparation of Scheme 1 compound with general formula (I)
The invention also provides application of the compound or the stereoisomer or the pharmaceutically acceptable salt thereof in preparing antitumor drugs and EFGR kinase inhibitors.
The pharmaceutical composition is in the form of tablets, capsules, granules, sprays or injections.
The pharmaceutically acceptable carrier is selected from one or more of a filler, a disintegrant, a binder and a lubricant. Including, but not limited to, any and all solvents, dispersion media, coatings, absorption delaying agents, and the like, such media and agents for pharmaceutically active substances are well known in the art.
The invention also provides the usage of the pyrazolopyrimidine derivative epidermal growth factor inhibitor and pharmaceutically acceptable salts thereof as a protein tyrosine kinase inhibitor;
further, the protein tyrosine kinase inhibitor is an epidermal growth factor receptor inhibitor;
more preferably, the epidermal growth factor receptor inhibitor is an inhibitor of the epidermal growth factor receptor of the T790M variant.
Use of pyrazolopyrimidine derivative epidermal growth factor inhibitor and pharmaceutically acceptable salt or pharmaceutical composition thereof in preparation of medicines for treating diseases related to epidermal growth factor receptor overexpression.
Further, the tumor is liver cancer, lung cancer, prostate cancer, pancreatic cancer, breast cancer, and astrocytoma.
Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.
The above-described aspects of the present invention will be described in further detail below with reference to specific embodiments of embodiments. It should not be understood that the scope of the above-described subject matter of the present invention is limited to the following examples. All the technologies realized based on the above-mentioned contents of the invention belong to the scope of the invention.
Detailed Description
The invention is further illustrated by the following specific examples.
A process for preparing a compound having the general formula (I) comprising: the key intermediate 2 is obtained by Suzuki coupling reaction of the initial raw material, the intermediate 2 and the bicyclic compound respectively generate electrophilic substitution and acylation, and finally the corresponding compound shown in the general formula (I) is obtained. It is to be noted that the compounds of the general formula (I) include, but are not limited to, the compounds listed below.
Example 1: preparation of 1- {7- [ 4-amino-3- (1-methyl-1H-indol-3-yl) -pyrazolo [3,4-d ] pyrimidin-1-yl ] -2-azaspiro [3.5] nonan-2-yl } -propenone (compound 701):
preparation of intermediate 4-amino-3- (1-methyl-1H-indol-3-yl) -1H-pyrazole [3,4-d ] pyrimidine 2 a:
3-iodo-4-amino-1H-pyrazole [3,4-d]Pyrimidine (295mg, 1.13mmol) was dissolved in 2mL (1:3) of a mixed solvent of ethanol and DME, and 0.25mL of a saturated sodium carbonate solution and N-methylindole-3-boronic acid pinacol ester (411mg, 1.6 mmol) were added thereto. After the whole system was purged three times, tetrakis (triphenylphosphine) palladium (127mg, 0.11mmol) was added to the mixture, and then the whole system was heated to 90 ℃ under nitrogen and stirred for 12 hours. After the reaction was completed, filtration was performed with celite, and the obtained filtrate was washed three times with water and dichloromethane. The organic phase was rotary-distilled under reduced pressure to give a solid, which was separated by means of silica gel column chromatography (eluent was dichloromethane containing 1-2% methanol), to finally obtain 246mg (0.93 mmol) of the objective compound as a pale green solid in yield: 82 percent. LC/MS (ESI): m/z 265 (M + H) + 。
Preparation of intermediate 1- { N-Boc-2-azaspiro [3.5] nonan-7-yl } -4-amino-3- (1-methyl-1H-indol-3-yl) -1H-pyrazole [3,4-d ] pyrimidine 3 a:
2a (206mg, 0.78mmol) and N-Boc-2-azaspiro [3.5] were stirred at 80 deg.C]Nonane-7- ((methylsulfonyl) oxy) (1 eq) and potassium carbonate (2 eq) were suspended in DMF (3 mL) for 16h. After the reaction was complete, the mixture was treated with water and extracted with dichloromethane. Collecting organic phase, drying, and vacuum concentrating to obtain light green targetCompound 182mg, yield: and 48 percent. LC/MS (ESI): m/z 488 (M + H) + 。
Preparation of intermediate 1- { 2-azaspiro [3.5] nonan-7-yl } -4-amino-3- (1-methyl-1H-indol-3-yl) -1H-pyrazole [3,4-d ] pyrimidine 4 a:
3a (92mg, 0.19mmol) was dissolved in 2mL of a dichloromethane solution, an excess of trifluoroacetic acid was added, the mixture was stirred at room temperature for 2h, diluted with dichloromethane, and then washed with saturated aqueous sodium bicarbonate solution. The organic phase was collected, dried over anhydrous sodium sulfate and concentrated in vacuo, and the crude product was taken to the next step without purification.
Preparation of 1- {7- [ 4-amino-3- (1-methyl-1H-indol-3-yl) -pyrazolo [3,4-d ] pyrimidin-1-yl ] -2-azaspiro [3.5] nonan-2-yl } -propenone (compound 701):
the crude product 4a from the previous step was dissolved in 5mL tetrahydrofuran solution, DIPEA (196. Mu.L, 1.2 mmol) was added, acryloyl chloride (17. Mu.L, 0.21 mmol) was added dropwise, and the reaction was stirred for 2 hours. The reaction was complete by TLC. The reaction was stopped, DCM (25 mL) was added, followed by 50mL of saturated NaHCO 3 Washed with water, the aqueous layer was extracted with DCM (2 × 25ml), anhydrous MgSO 4 Drying, concentration and flash column separation of the crude product gave 53mg of a yellow solid in two yields: and 63 percent. 1 H NMR(400MHz,DMSO-d 6 )δ 8.27(s,1H),8.17(d,1H),7.59(d,1H),7.44(m,1H),7.33(m,1H),7.19(s,1H),7.01 (s,2H),6.62(m,1H),6.04(m,1H),5.58(m,1H),3.72-3.64(m,8H),2.00-1.75(m, 4H),1.32(m,4H).LC/MS(ESI)：m/z 442(M+H) + 。
Example 2: preparation of 1- {7- [ 4-amino-3- (1-methyl-1H-indazol-3-yl) -pyrazolo [3,4-d ] pyrimidin-1-yl ] -2-azaspiro [3.5] nonan-2-yl } -propenone (compound 702):
preparation of intermediate 4-amino-3- (1-methyl-2-aza-1H-indazol-3-yl) -1H-pyrazole [3,4-d ] pyrimidine 2 b:
3-iodo-4-amino-1H-pyrazole [3,4-d]Pyrimidine (295mg, 1.13mmol) was dissolved in 2mL (1:3) of a mixed solvent of ethanol and DME, and 0.25mL of a saturated sodium carbonate solution and N-methyl 2-azaindole-3-boronic acid pinacol ester (413mg, 1.6 mmol) were added thereto. After the whole system was purged three times, tetrakis (triphenylphosphine) palladium (127mg, 0.11mmol) was added to the mixture, and then the whole system was heated to 90 ℃ under nitrogen and stirred for 12 hours. After the reaction was completed, filtration was performed with celite, and the obtained filtrate was washed three times with water and dichloromethane. The organic phase was rotary-distilled under reduced pressure to give a solid, which was separated by means of silica gel column chromatography (eluent was dichloromethane containing 1-2% methanol), to finally obtain 234mg (0.88 mmol) of the objective compound as a pale green solid in yield: 78 percent. LC/MS (ESI): m/z 266 (M + H) + 。
Preparation of intermediate 1- { N-Boc-2-azaspiro [3.5] nonan-7-yl } -4-amino-3- (1-methyl-1H-indazol-3-yl) -1H-pyrazole [3,4-d ] pyrimidine 3 b:
2b (207mg, 0.78mmol) and N-Boc-2-azaspiro [3.5] were stirred at 80 deg.C]Nonane-7- ((methylsulfonyl) oxy) (1 eq) and potassium carbonate (2 eq) in DMF (3 mL) were suspended for 16h. After the reaction was complete, the mixture was treated with water and extracted with dichloromethane. The organic phase was collected, dried and concentrated in vacuo to yield 190mg of the pale green title compound in yield: 50 percent. LC/MS (ESI): m/z 489 (M + H) + 。
Preparation of intermediate 1- { 2-azaspiro [3.5] nonan-7-yl } -4-amino-3- (1-methyl-1H-indazol-3-yl) -1H-pyrazole [3,4-d ] pyrimidine 4 b:
3b (93mg, 0.19mmol) was dissolved in 2mL of a dichloromethane solution, an excess of trifluoroacetic acid was added, the mixture was stirred at room temperature for 2h, diluted with dichloromethane, and then washed with saturated aqueous sodium bicarbonate solution. The organic phase was collected, dried over anhydrous sodium sulfate and concentrated in vacuo, and the crude product was used in the next step without further purification.
Preparation of 1- {7- [ 4-amino-3- (1-methyl-1H-indazol-3-yl) -pyrazolo [3,4-d ] pyrimidin-1-yl ] -2-azaspiro [3.5] nonan-2-yl } -propenone (compound 702): :
the crude product 4b from the previous step was dissolved in 5mL tetrahydrofuran, DIPEA (196. Mu.L, 1.2 mmol) was added, followed by dropwise addition of acryloyl chloride (17. Mu.L, 0.21 mmol), and the reaction was stirred for 2 hours. The reaction was complete by TLC. The reaction was stopped, DCM (25 mL) was added, followed by 50mL of saturated NaHCO 3 Washed with water, the aqueous layer was extracted with DCM (2 × 25ml), anhydrous MgSO 4 Drying, concentration and flash column separation of the crude product gave 48mg of a yellow solid in two yields: 57 percent. 1 H NMR(400MHz,DMSO-d 6 )δ 8.32-8.27(m,2H),7.92(d,1H),7.65(m,1H),7.45(m,1H),7.02(s,2H),6.62(m,1H), 6.04(m,1H),5.58(m,1H),3.95(s,3H),3.72-3.64(m,5H),2.00-1.75(m,4H),1.32 (m,4H).LC/MS(ESI)：m/z 443(M+H) + 。
Example 3: preparation of 1- {7- [ 4-amino-3- (1-methyl-1H-pyrrole [2,3-b ] pyridin-3-yl) -pyrazole [3,4-d ] pyrimidin-1-yl ] -2-azaspiro [3.5] nonan-2-yl } -propenone (compound 703):
preparation of intermediate 4-amino-3- ((1-methyl-1H-pyrrole [2,3-b ] pyridin-3-yl) -1H-pyrazole [3,4-d ] pyrimidine 2 c:
3-iodo-4-amino-1H-pyrazole [3,4-d]Pyrimidine (295mg, 1.13mmol) was dissolved in 2mL (1:3) of a mixed solvent of ethanol and DME, and 0.25mL of a saturated sodium carbonate solution and N-methyl 2-azaindole-3-boronic acid pinacol ester (413mg, 1.6 mmol) were added thereto. After the whole system was purged three times, tetrakis (triphenylphosphine) palladium (127mg, 0.11mmol) was added to the mixture, and then the whole system was heated to 90 ℃ under nitrogen and stirred for 12 hours. After the reaction was completed, filtration was performed with celite, and the obtained filtrate was washed three times with water and dichloromethane. The organic phase was rotary-distilled under reduced pressure to give a solid, which was separated by means of silica gel column chromatography (eluent was dichloromethane containing 1-2% methanol), to finally obtain 234mg (0.88 mmol) of the objective compound as a pale green solid in yield: and 78 percent. LC/MS (ESI): m/z 266 (M + H) + 。
Preparation of intermediate 1- { N-Boc-2-azaspiro [3.5] nonan-7-yl } -4-amino-3- (1-methyl-1H-pyrrole [2,3-b ] pyridin-3-yl) -1H-pyrazole [3,4-d ] pyrimidine 3 c:
2c (207mg, 0.78mmol) and N-Boc-2-azaspiro [3.5] were stirred at 80 deg.C]Nonane-7- ((methylsulfonyl) oxy) (1 eq) and potassium carbonate (2 eq) in DMF (3 mL) were suspended for 16h. After the reaction was complete, the mixture was treated with water and extracted with dichloromethane. The organic phase was collected, dried and concentrated in vacuo to yield 190mg of the pale green title compound in yield: 50 percent. LC/MS (ESI): m/z 489 (M + H) + 。
Preparation of intermediate 1- { 2-azaspiro [3.5] nonan-7-yl } -4-amino-3- (1-methyl-1H-pyrrole [2,3-b ] pyridin-3-yl) -1H-pyrazole [3,4-d ] pyrimidine 4 c:
3c (93mg, 0.19mmol) was dissolved in 2mL of a dichloromethane solution, an excess of trifluoroacetic acid was added, the mixture was stirred at room temperature for 2h, diluted with dichloromethane, and then washed with saturated aqueous sodium bicarbonate solution. The organic phase was collected, dried over anhydrous sodium sulfate and concentrated in vacuo, and the crude product was taken to the next step without purification.
Preparation of 1- {7- [ 4-amino-3- (1-methyl-1H-pyrrole [2,3-b ] pyridin-3-yl) -pyrazole [3,4-d ] pyrimidin-1-yl ] -2-azaspiro [3.5] nonan-2-yl } -propenone (compound 703): :
the crude product 4b from the previous step was dissolved in 5mL tetrahydrofuran, DIPEA (196. Mu.L, 1.2 mmol) was added, followed by dropwise addition of acryloyl chloride (17. Mu.L, 0.21 mmol), and the reaction was stirred for 2 hours. The reaction was complete by TLC. The reaction was stopped, DCM (25 mL) was added, followed by 50mL saturated NaHCO 3 Washed with water, the aqueous layer was extracted with DCM (2 × 25ml), anhydrous MgSO 4 Drying, concentration and flash column separation of the crude product gave 48mg of a yellow solid in two yields: 57 percent. 1 H NMR(400MHz,DMSO-d 6 )δ 8.51(d,1H),8.39(d,1H),8.27(s,1H),7.17(m,1H),7.06-7.12(m,3H),6.62(m,1H), 6.04(m,1H),5.58(m,1H),3.72-3.59(m,8H),2.00-1.75(m,4H),1.32(m,4H). LC/MS(ESI)：m/z 443(M+H) + 。
Example 4: preparation of 1- {6- [ 4-amino-3- (1-methyl-1H-indol-3-yl) -pyrazolo [3,4-d ] pyrimidin-1-yl ] -2-azaspiro [3.3] heptan-2-yl } -propenone (compound 704):
preparation of intermediate 1- { N-Boc-2-azaspiro [3.3] heptan-6-yl } -4-amino-3- (1-methyl-1H-indol-3-yl) -1H-pyrazole [3,4-d ] pyrimidine 3 d:
2a (206mg, 0.78mmol) and N-Boc-2-azaspiro [3.3] were stirred at 80 deg.C]A suspension of heptane-6- ((methylsulfonyl) oxy) (1 eq) and potassium carbonate (2 eq) in DMF (3 mL) was left for 16h. After the reaction was complete, the mixture was treated with water and extracted with dichloromethane. The organic phase was collected, dried and concentrated in vacuo to give 168mg of the pale green title compound in yield: and 47 percent. LC/MS (ESI): m/z 460 (M + H) + 。
Preparation of intermediate 1- { 2-azaspiro [3.3] heptan-6-yl } -4-amino-3- (1-methyl-1H-indol-3-yl) -1H-pyrazole [3,4-d ] pyrimidine 4 d:
3d (87mg, 0.19mmol) was dissolved in 2mL of dichloromethane solution, excess trifluoroacetic acid was added, the mixture was stirred at room temperature for 2h, the mixture was diluted with dichloromethane and washed with saturated aqueous sodium bicarbonate solution. The organic phase was collected, dried over anhydrous sodium sulfate and concentrated in vacuo, and the crude product was used in the next step without further purification.
Preparation of 1- {6- [ 4-amino-3- (1-methyl-1H-indol-3-yl) -pyrazolo [3,4-d ] pyrimidin-1-yl ] -2-azaspiro [3.3] heptan-2-yl } -propenone (compound 704):
the crude product 4d from the previous step was dissolved in 5mL tetrahydrofuran solution, DIPEA (196. Mu.L, 1.2 mmol) was added, acryloyl chloride (17. Mu.L, 0.21 mmol) was added dropwise, and the reaction was stirred for 2 hours. The reaction was complete by TLC. The reaction was stopped, DCM (25 mL) was added, followed by 50mL of saturated NaHCO 3 Washed with water, the aqueous layer was extracted with DCM (2 × 25ml), anhydrous MgSO 4 Drying, concentration and flash column separation of the crude product gave 46mg of a yellow solid in two yields: 58 percent. 1 H NMR(400MHz,DMSO-d 6 )δ 8.27(s,1H),8.17(d,1H),7.59(d,1H),7.44(m,1H),7.33(m,1H),7.19(s,1H),7.01 (s,2H),6.62(m,1H),6.04(m,1H),5.58(m,1H),4.20(m,1H),3.72(m,7H),2.09- 1.85(m,4H).LC/MS(ESI)：m/z 414(M+H) + 。
Example 5: preparation of 1- {6- [ 4-amino-3- (1-methyl-1H-indazol-3-yl) -pyrazolo [3,4-d ] pyrimidin-1-yl ] -2-azaspiro [3.3] heptan-2-yl } -propenone (compound 705):
preparation of intermediate 1- { N-Boc-2-azaspiro [3.3] heptan-6-yl } -4-amino-3- (1-methyl-1H-indazol-3-yl) -1H-pyrazole [3,4-d ] pyrimidine 3 e:
2a (207mg, 0.78mmol) and N-Boc-2-azaspiro [3.3] were stirred at 80 deg.C]A suspension of heptane-6- ((methylsulfonyl) oxy) (1 eq) and potassium carbonate (2 eq) in DMF (3 mL) was left for 16h. After the reaction was complete, the mixture was treated with water and extracted with dichloromethane. The organic phase was collected, dried and concentrated in vacuo to give 161mg of the pale green title compound in yield: 45 percent. LC/MS (ESI): m/z 461 (M + H) + 。
Preparation of intermediate 1- { 2-azaspiro [3.3] heptan-6-yl } -4-amino-3- (1-methyl-1H-indazol-3-yl) -1H-pyrazole [3,4-d ] pyrimidine 4 e:
3e (87mg, 0.19mmol) was dissolved in 2mL of a dichloromethane solution, an excess of trifluoroacetic acid was added, the mixture was stirred at room temperature for 2h, diluted with dichloromethane, and then washed with saturated aqueous sodium bicarbonate solution. The organic phase was collected, dried over anhydrous sodium sulfate and concentrated in vacuo, and the crude product was used in the next step without further purification.
Preparation of 1- {6- [ 4-amino-3- (1-methyl-1H-indazol-3-yl) -pyrazolo [3,4-d ] pyrimidin-1-yl ] -2-azaspiro [3.3] heptan-2-yl } -propenone (compound 705):
the crude product 4e from the previous step was dissolved in 5mL tetrahydrofuran, DIPEA (196. Mu.L, 1.2 mmol) was added, followed by dropwise addition of acryloyl chloride (17. Mu.L, 0.21 mmol), and the reaction was stirred for 2 hours. The reaction was complete by TLC. The reaction was stopped, DCM (25 mL) was added, followed by 50mL saturated NaHCO 3 Washed with water, the aqueous layer was extracted with DCM (2 × 25ml), anhydrous MgSO 4 Drying, concentration and flash column separation of the crude product gave 49mg of a yellow solid in two yields: 62 percent. 1 H NMR(400MHz,DMSO-d 6 )δ 8.32-8.27(m,2H),7.92(d,1H),7.65(m,1H),7.45(m,1H),7.02(s,2H),6.62(m,1H), 6.04(m,1H),5.58(m,1H),4.20(m,1H),3.95(s,3H),3.72(s,4H),2.09-1.85(m,4H). LC/MS(ESI)：m/z 415(M+H) + 。
Example 6: preparation of 1- {6- [ 4-amino-3- (1-methyl-1H-pyrrole [2,3-b ] pyridin-3-yl) -pyrazole [3,4-d ] pyrimidin-1-yl ] -2-azaspiro [3.3] heptan-2-yl } -propenone (compound 706):
preparation of intermediate 1- { N-Boc-2-azaspiro [3.3] heptan-6-yl } -4-amino-3- (1-methyl-1H-pyrrole [2,3-b ] pyridin-3-yl) -1H-pyrazole [3,4-d ] pyrimidine 3 f:
2c (207mg, 0.78mmol) and N-Boc-2-azaspiro [3.3] were stirred at 80 deg.C]Heptane-6- ((methylsulfonyl) oxy) (1 eq) and carbonSuspension of potassium (2 eq) in DMF (3 mL) for 16h. After the reaction was complete, the mixture was treated with water and extracted with dichloromethane. The organic phase was collected, dried and concentrated in vacuo to yield 183mg of the pale green title compound in yield: 51 percent. LC/MS (ESI): m/z 461 (M + H) + 。
Preparation of intermediate 1- { 2-azaspiro [3.3] heptan-6-yl } -4-amino-3- (1-methyl-1H-pyrrole [2,3-b ] pyridin-3-yl) -1H-pyrazole [3,4-d ] pyrimidine 4 f:
3f (87mg, 0.19mmol) was dissolved in 2mL of a dichloromethane solution, an excess of trifluoroacetic acid was added, the mixture was stirred at room temperature for 2h, diluted with dichloromethane, and then washed with saturated aqueous sodium bicarbonate solution. The organic phase was collected, dried over anhydrous sodium sulfate and concentrated in vacuo, and the crude product was taken to the next step without purification.
Preparation of 1- {6- [ 4-amino-3- (1-methyl-1H-pyrrole [2,3-b ] pyridin-3-yl) -pyrazole [3,4-d ] pyrimidin-1-yl ] -2-azaspiro [3.3] heptan-2-yl } -propenone (compound 706):
the crude product 4f from the previous step was dissolved in 5mL tetrahydrofuran solution, DIPEA (196. Mu.L, 1.2 mmol) was added, acryloyl chloride (17. Mu.L, 0.21 mmol) was added dropwise, and the reaction was stirred for 2 hours. The reaction was complete by TLC. The reaction was stopped, DCM (25 mL) was added, followed by 50mL of saturated NaHCO 3 Washed with water, the aqueous layer was extracted with DCM (2 × 25ml), anhydrous MgSO 4 Drying, concentration and flash column separation of the crude product gave 47mg of a yellow solid in two yields: 60 percent. 1 H NMR(400MHz,DMSO-d 6 )δ 8.51(d,1H),8.39(d,1H),8.27(s,1H),7.17(m,1H),7.06-7.12(m,3H),6.62(m,1H), 6.04(m,1H),5.58(m,1H),4.20(m,1H),3.72(s,4H),3.59(s,3H),2.00-1.75(m,4H). LC/MS(ESI)：m/z 415(M+H) + 。
Example 7: preparation of 1- {2- [ 4-amino-3- (1-methyl-1H-indol-3-yl) -pyrazolo [3,4-d ] pyrimidin-1-yl ] -7-azaspiro [3.5] nonan-7-yl } -propenone (compound 707):
preparation of intermediate 1- { N-Boc-7-azaspiro [3.5] nonan-2-yl } -4-amino-3- (1-methyl-1H-indol-3-yl) -1H-pyrazole [3,4-d ] pyrimidine 3 g:
2a (206mg, 0.78mmol) and N-Boc-7-azaspiro [3.5] were stirred at 80 deg.C]Nonane-2- ((methylsulfonyl) oxy) (1 eq) and potassium carbonate (2 eq) were suspended in DMF (3 mL) for 16h. After the reaction was complete, the mixture was treated with water and extracted with dichloromethane. The organic phase was collected, dried and concentrated in vacuo to give 182mg of the pale green target compound in yield: 48 percent. LC/MS (ESI): m/z 488 (M + H) + 。
Preparation of intermediate 1- { 7-azaspiro [3.5] nonan-2-yl } -4-amino-3- (1-methyl-1H-indol-3-yl) -1H-pyrazole [3,4-d ] pyrimidine 4 g:
3g (92mg, 0.19mmol) was dissolved in 2mL of a dichloromethane solution, an excess of trifluoroacetic acid was added, the mixture was stirred at room temperature for 2h, diluted with dichloromethane, and then washed with saturated aqueous sodium bicarbonate solution. The organic phase was collected, dried over anhydrous sodium sulfate and concentrated in vacuo, and the crude product was taken to the next step without purification.
Preparation of 1- {2- [ 4-amino-3- (1-methyl-1H-indol-3-yl) -pyrazolo [3,4-d ] pyrimidin-1-yl ] -7-azaspiro [3.5] nonan-7-yl } -propenone (compound 707):
4g of the crude product from the previous step was dissolved in 5mL of tetrahydrofuran solution, DIPEA (196. Mu.L, 1.2 mmol) was added, acryloyl chloride (17. Mu.L, 0.21 mmol) was added dropwise, and the reaction was stirred for 2 hours. The reaction was complete by TLC. The reaction was stopped, DCM (25 mL) was added, followed by 50mL saturated NaHCO 3 Washed with water, the aqueous layer was extracted with DCM (2 × 25ml), anhydrous MgSO 4 Drying, concentration and flash column separation of the crude product gave 53mg of a yellow solid in two yields: and 63 percent. 1 H NMR(400MHz,DMSO-d 6 )δ 8.27(s,1H),8.17(d,1H),7.59(d,1H),7.44(m,1H),7.33(m,1H),7.19(s,1H),7.01 (s,2H),6.62(m,1H),6.04(m,1H),5.58(m,1H),4.20(m,1H),3.82(m,7H),2.09- 1.85(m,4H),1.54-1.52(m,4H).LC/MS(ESI)：m/z 442(M+H) + 。
Example 8: preparation of 1- {2- [ 4-amino-3- (1-methyl-1H-indazol-3-yl) -pyrazolo [3,4-d ] pyrimidin-1-yl ] -7-azaspiro [3.5] nonan-7-yl } -propenone (compound 708):
preparation of intermediate 1- { N-Boc-7-azaspiro [3.5] nonan-2-yl } -4-amino-3- (1-methyl-1H-indazol-3-yl) -1H-pyrazole [3,4-d ] pyrimidine 3H:
2b (207mg, 0.78mmol) and N-Boc-7-azaspiro [3.5] were stirred at 80 deg.C]Nonane-2- ((methylsulfonyl) oxy) (1 eq) and potassium carbonate (2 eq) were suspended in DMF (3 mL) for 16h. After the reaction was complete, the mixture was treated with water and extracted with dichloromethane. The organic phase was collected, dried and concentrated in vacuo to yield 190mg of the pale green title compound in yield: 50 percent. LC/MS (ESI): m/z 489 (M + H) + 。
Preparation of intermediate 1- { 2-azaspiro [3.5] nonan-7-yl } -4-amino-3- (1-methyl-1H-indazol-3-yl) -1H-pyrazole [3,4-d ] pyrimidine 4H:
3h (93mg, 0.19mmol) was dissolved in 2mL of dichloromethane solution, excess trifluoroacetic acid was added, the mixture was stirred at room temperature for 2h, the mixture was diluted with dichloromethane and washed with saturated aqueous sodium bicarbonate solution. The organic phase was collected, dried over anhydrous sodium sulfate and concentrated in vacuo, and the crude product was used in the next step without further purification.
Preparation of 1- {2- [ 4-amino-3- (1-methyl-1H-indazol-3-yl) -pyrazolo [3,4-d ] pyrimidin-1-yl ] -7-azaspiro [3.5] nonan-7-yl } -propenone (compound 708):
the crude product from the previous step was dissolved in 5mL tetrahydrofuran solution for 4h, DIPEA (196. Mu.L, 1.2 mmol) was added, followed by dropwise addition of acryloyl chloride (17. Mu.L, 0.21 mmol), and the reaction was stirred for 2 h. The reaction was complete by TLC. The reaction was stopped, DCM (25 mL) was added, followed by 50mL of saturated NaHCO 3 Washed with water, the aqueous layer was extracted with DCM (2 × 25ml), anhydrous MgSO 4 Drying, concentration and flash column separation of the crude product gave 48mg of a yellow solid in two yields: 57 percent. 1 H NMR(400MHz,DMSO-d 6 )δ 8.32-8.27(m,2H),7.92(d,1H),7.65(m,1H),7.45(m,1H),7.02(s,2H),6.62(m,1H), 6.04(m,1H),5.58(m,1H),4.20(m,1H),3.95(s,3H),3.82(s,4H),2.09-1.85(m,4H), 1.54-1.52(m,4H).LC/MS(ESI)：m/z 443(M+H) + 。
Example 9: preparation of 1- {2- [ 4-amino-3- (1-methyl-1H-pyrrole [2,3-b ] pyridin-3-yl) -pyrazole [3,4-d ] pyrimidin-1-yl ] -7-azaspiro [3.5] nonan-7-yl } -propenone (compound 709):
preparation of intermediate 1- { N-Boc-7-azaspiro [3.5] nonan-2-yl } -4-amino-3- (1-methyl-1H-pyrrole [2,3-b ] pyridin-3-yl) -1H-pyrazole [3,4-d ] pyrimidine 3 i:
2c (207mg, 0.78mmol) and N-Boc-7-azaspiro [3.5] were stirred at 80 deg.C]Nonane-2- ((methylsulfonyl) oxy) (1 eq) and potassium carbonate (2 eq) were suspended in DMF (3 mL) for 16h. After the reaction was complete, the mixture was treated with water and extracted with dichloromethane. The organic phase was collected, dried and concentrated in vacuo to yield 190mg of the pale green title compound in yield: 50 percent. LC/MS (ESI): m/z 489 (M + H) + 。
Preparation of intermediate 1- { 7-azaspiro [3.5] nonan-2-yl } -4-amino-3- (1-methyl-1H-pyrrole [2,3-b ] pyridin-3-yl) -1H-pyrazole [3,4-d ] pyrimidine 4 i:
3i (92mg, 0.19mmol) was dissolved in 2mL of a dichloromethane solution, an excess of trifluoroacetic acid was added, the mixture was stirred at room temperature for 2h, diluted with dichloromethane, and then washed with saturated aqueous sodium bicarbonate solution. The organic phase was collected, dried over anhydrous sodium sulfate and concentrated in vacuo, and the crude product was taken to the next step without purification.
Preparation of 1- {2- [ 4-amino-3- (1-methyl-1H-pyrrole [2,3-b ] pyridin-3-yl) -pyrazole [3,4-d ] pyrimidin-1-yl ] -7-azaspiro [3.5] nonan-7-yl } -propenone (compound 709):
the crude product 4i from the previous step was dissolved in 5mL tetrahydrofuran solution, DIPEA (196. Mu.L, 1.2 mmol) was added, followed by dropwise addition of acryloyl chloride (17. Mu.L, 0.21 mmol), and the reaction was stirred for 2 hours. The reaction was complete by TLC. The reaction was stopped and DCM (25 mL) was added, thenThen 50mL of saturated NaHCO 3 Washed with water, the aqueous layer was extracted with DCM (2 × 25ml), anhydrous MgSO 4 Drying, concentration and flash column separation of the crude product gave 48mg of a yellow solid in two yields: 57 percent. 1 H NMR(400MHz,DMSO-d 6 )δ 8.51(d,1H),8.39(d,1H),8.27(s,1H),7.17(m,1H),7.06-7.12(m,3H),6.62(m,1H), 6.04(m,1H),5.58(m,1H),4.20(m,1H),3.82(s,4H),3.59(s,3H),2.09-1.85(m,4H), 1.54-1.52(m,4H).LC/MS(ESI)：m/z 443(M+H) + 。
Example 10: preparation of 1- {2- [ 4-amino-3- (1-methyl-1H-indol-3-yl) -pyrazolo [3,4-d ] pyrimidin-1-yl ] -6-azaspiro [3.5] nonan-6-yl } -propenone (compound 710):
preparation of intermediate 1- { N-Boc-6-azaspiro [3.5] nonan-2-yl } -4-amino-3- (1-methyl-1H-indol-3-yl) -1H-pyrazole [3,4-d ] pyrimidine 3 j:
2a (206mg, 0.78mmol) and N-Boc-6-azaspiro [3.5] were stirred at 80 deg.C]Nonane-2- ((methylsulfonyl) oxy) (1 eq) and potassium carbonate (2 eq) in DMF (3 mL) were suspended for 16h. After the reaction was complete, the mixture was treated with water and extracted with dichloromethane. The organic phase was collected, dried and concentrated in vacuo to give 182mg of the pale green target compound in yield: 48 percent. LC/MS (ESI): m/z 488 (M + H) + 。
Preparation of intermediate 1- { 6-azaspiro [3.5] nonan-2-yl } -4-amino-3- (1-methyl-1H-indol-3-yl) -1H-pyrazole [3,4-d ] pyrimidine 4 j:
3j (92mg, 0.19mmol) was dissolved in 2mL of a dichloromethane solution, an excess of trifluoroacetic acid was added, the mixture was stirred at room temperature for 2h, diluted with dichloromethane, and then washed with saturated aqueous sodium bicarbonate solution. The organic phase was collected, dried over anhydrous sodium sulfate and concentrated in vacuo, and the crude product was taken to the next step without purification.
Preparation of 1- {2- [ 4-amino-3- (1-methyl-1H-indol-3-yl) -pyrazolo [3,4-d ] pyrimidin-1-yl ] -6-azaspiro [3.5] nonan-6-yl } -propenone (compound 710):
the crude product 4j from the previous step was dissolved in 5mL tetrahydrofuran, DIPEA (196. Mu.L, 1.2 mmol) was added, followed by dropwise addition of acryloyl chloride (17. Mu.L, 0.21 mmol), and the reaction was stirred for 2 hours. The reaction was complete by TLC. The reaction was stopped, DCM (25 mL) was added, followed by 50mL saturated NaHCO 3 Washed with water, the aqueous layer was extracted with DCM (2 × 25ml), anhydrous MgSO 4 Drying, concentration and flash column separation of the crude product gave 53mg of a yellow solid in two yields: and 63 percent. 1 H NMR(400MHz,DMSO-d 6 )δ 8.27(s,1H),8.17(d,1H),7.59(d,1H),7.44(m,1H),7.33(m,1H),7.19(s,1H),7.01 (s,2H),6.62(m,1H),6.04(m,1H),5.58(m,1H),4.20(m,1H),3.82-3.74(m,5H), 3.27(s,2H),2.09-1.85(m,4H),1.68(m,2H),1.44-1.42(m,2H).LC/MS(ESI)：m/z 442(M+H) + 。
Example 11: preparation of 1- {2- [ 4-amino-3- (1-methyl-1H-indazol-3-yl) -pyrazolo [3,4-d ] pyrimidin-1-yl ] -6-azaspiro [3.5] nonan-6-yl } -propenone (compound 711):
preparation of intermediate 1- { N-Boc-6-azaspiro [3.5] nonan-2-yl } -4-amino-3- (1-methyl-1H-indazol-3-yl) -1H-pyrazole [3,4-d ] pyrimidine 3 k:
2b (207mg, 0.78mmol) and N-Boc-6-azaspiro [3.5] were stirred at 80 deg.C]Nonane-2- ((methylsulfonyl) oxy) (1 eq) and potassium carbonate (2 eq) were suspended in DMF (3 mL) for 16h. After the reaction was complete, the mixture was treated with water and extracted with dichloromethane. The organic phase was collected, dried and concentrated in vacuo to yield 190mg of the pale green target compound in yield: 50 percent. LC/MS (ESI): m/z 489 (M + H) + 。
Preparation of intermediate 1- { 6-azaspiro [3.5] nonan-2-yl } -4-amino-3- (1-methyl-1H-indazol-3-yl) -1H-pyrazole [3,4-d ] pyrimidine 4 k:
3k (93mg, 0.19mmol) was dissolved in 2mL of a dichloromethane solution, an excess of trifluoroacetic acid was added, the mixture was stirred at room temperature for 2h, diluted with dichloromethane, and then washed with saturated aqueous sodium bicarbonate solution. The organic phase was collected, dried over anhydrous sodium sulfate and concentrated in vacuo, and the crude product was used in the next step without further purification.
Preparation of 1- {2- [ 4-amino-3- (1-methyl-1H-indazol-3-yl) -pyrazolo [3,4-d ] pyrimidin-1-yl ] -6-azaspiro [3.5] nonan-6-yl } -propenone (compound 711):
the crude product 4k from the previous step was dissolved in 5mL tetrahydrofuran, DIPEA (196. Mu.L, 1.2 mmol) was added, followed by the dropwise addition of acryloyl chloride (17. Mu.L, 0.21 mmol), and the reaction was stirred for 2 hours. The reaction was complete by TLC. The reaction was stopped, DCM (25 mL) was added, followed by 50mL of saturated NaHCO 3 Washed with water, the aqueous layer was extracted with DCM (2 × 25ml), anhydrous MgSO 4 Drying, concentration and flash column separation of the crude product gave 48mg of a yellow solid in two yields: 57 percent. 1 H NMR(400MHz,DMSO-d 6 )δ 8.32-8.27(m,2H),7.92(d,1H),7.65(m,1H),7.45(m,1H),7.02(s,2H),6.62(m,1H), 6.04(m,1H),5.58(m,1H),4.20(m,1H),3.95(s,3H),3.82(m,2H),3.27(s,2H),2.09- 1.85(m,4H),1.68(m,2H),1.44-1.42(m,2H).LC/MS(ESI)：m/z 443(M+H) + 。
Example 12: preparation of 1- {2- [ 4-amino-3- (1-methyl-1H-pyrrole [2,3-b ] pyridin-3-yl) -pyrazole [3,4-d ] pyrimidin-1-yl ] -6-azaspiro [3.5] nonan-6-yl } -propenone (compound 712):
preparation of intermediate 1- { N-Boc-6-azaspiro [3.5] nonan-2-yl } -4-amino-3- (1-methyl-1H-pyrrole [2,3-b ] pyridin-3-yl) -1H-pyrazole [3,4-d ] pyrimidine 3 l:
2c (207mg, 0.78mmol) and N-Boc-6-azaspiro [3.5] were stirred at 80 deg.C]Nonane-2- ((methylsulfonyl) oxy) (1 eq) and potassium carbonate (2 eq) were suspended in DMF (3 mL) for 16h. After the reaction was complete, the mixture was treated with water and extracted with dichloromethane. The organic phase was collected, dried and concentrated in vacuo to yield 190mg of the pale green title compound in yield: 50 percent. LC/MS (ESI): m/z 489 (M + H) + 。
Preparation of intermediate 1- { 6-azaspiro [3.5] nonan-2-yl } -4-amino-3- (1-methyl-1H-pyrrole [2,3-b ] pyridin-3-yl) -1H-pyrazole [3,4-d ] pyrimidine 4 l:
3l (92mg, 0.19mmol) was dissolved in 2mL of a dichloromethane solution, an excess of trifluoroacetic acid was added, the mixture was stirred at room temperature for 2h, diluted with dichloromethane, and then washed with saturated aqueous sodium bicarbonate solution. The organic phase was collected, dried over anhydrous sodium sulfate and concentrated in vacuo, and the crude product was taken to the next step without purification.
Preparation of 1- {2- [ 4-amino-3- (1-methyl-1H-pyrrole [2,3-b ] pyridin-3-yl) -pyrazole [3,4-d ] pyrimidin-1-yl ] -6-azaspiro [3.5] nonan-2-yl } -propenone (compound 712):
4L of the crude product from the previous step was dissolved in 5mL of tetrahydrofuran solution, DIPEA (196. Mu.L, 1.2 mmol) was added, acryloyl chloride (17. Mu.L, 0.21 mmol) was added dropwise, and the reaction was stirred for 2 hours. The reaction was complete by TLC. The reaction was stopped, DCM (25 mL) was added, followed by 50mL saturated NaHCO 3 Washed with water, the aqueous layer was extracted with DCM (2 × 25ml), anhydrous MgSO 4 Drying, concentration and flash column separation of the crude product gave 48mg of a yellow solid in two yields: 57 percent. 1 H NMR(400MHz,DMSO-d 6 )δ 8.51(d,1H),8.39(d,1H),8.27(s,1H),7.17(m,1H),7.06-7.12(m,3H),6.62(m,1H), 6.04(m,1H),5.58(m,1H),4.20(m,1H),3.82(m,2H),3.59(s,3H),3.27(s,2H),2.09- 1.85(m,4H),1.68(m,2H),1.44-1.42(m,2H).LC/MS(ESI)：m/z 443(M+H) + 。
Example 13: preparation of 1- {2- [ 4-amino-3- (1-methyl-1H-indol-3-yl) -pyrazolo [3,4-d ] pyrimidin-1-yl ] -6-azaspiro [3.4] octan-6-yl } -propenone (compound 713):
preparation of intermediate 1- { N-Boc-6-azaspiro [3.4] octan-2-yl } -4-amino-3- (1-methyl-1H-indol-3-yl) -1H-pyrazole [3,4-d ] pyrimidine 3 m:
2a (206mg, 0.78mmol) and N-Boc-6-azaspiro [3.4] were stirred at 80 deg.C]Suspension of octane-2- ((methylsulfonyl) oxy) (1 eq) and potassium carbonate (2 eq) in DMF (3 mL) for 16h. After the reaction was complete, the mixture was treated with water and extracted with dichloromethane. Collecting organic phase, drying, vacuum concentrating to obtain light green target compound 177mg, and collectingRate: and 48 percent. LC/MS (ESI): m/z 474 (M + H) + 。
Preparation of intermediate 1- { 6-azaspiro [3.4] octan-2-yl } -4-amino-3- (1-methyl-1H-indol-3-yl) -1H-pyrazole [3,4-d ] pyrimidine 4 m:
3m (90mg, 0.19mmol) was dissolved in 2mL of a dichloromethane solution, an excess of trifluoroacetic acid was added, and the mixture was stirred at room temperature for 2h, diluted with dichloromethane, and then washed with saturated aqueous sodium bicarbonate solution. The organic phase was collected, dried over anhydrous sodium sulfate and concentrated in vacuo, and the crude product was used in the next step without further purification.
Preparation of 1- {2- [ 4-amino-3- (1-methyl-1H-indol-3-yl) -pyrazolo [3,4-d ] pyrimidin-1-yl ] -6-azaspiro [3.4] octan-6-yl } -propenone (compound 713):
the crude product of the previous step (4 m) was dissolved in 5mL tetrahydrofuran, DIPEA (196. Mu.L, 1.2 mmol) was added, acryloyl chloride (17. Mu.L, 0.21 mmol) was added dropwise, and the reaction was stirred for 2 hours. The reaction was complete by TLC. The reaction was stopped, DCM (25 mL) was added, followed by 50mL of saturated NaHCO 3 Washed with water, the aqueous layer was extracted with DCM (2 × 25ml), anhydrous MgSO 4 Drying, concentration and flash column separation of the crude product gave 51mg of a yellow solid in two yields: and 63 percent. 1 H NMR(400MHz,DMSO-d 6 )δ 8.27(s,1H),8.17(d,1H),7.59(d,1H),7.44(m,1H),7.33(m,1H),7.19(s,1H),7.01 (s,2H),6.62(m,1H),6.04(m,1H),5.58(m,1H),4.20(m,1H),3.74(s,3H),3.33-3.28 (s,4H),2.09-1.85(m,4H),1.67(m,2H).LC/MS(ESI)：m/z 428(M+H) + 。
Example 14: preparation of 1- {2- [ 4-amino-3- (1-methyl-1H-indazol-3-yl) -pyrazolo [3,4-d ] pyrimidin-1-yl ] -6-azaspiro [3.4] octan-6-yl } -propenone (compound 714):
preparation of intermediate 1- { N-Boc-6-azaspiro [3.4] octan-2-yl } -4-amino-3- (1-methyl-1H-indazol-3-yl) -1H-pyrazole [3,4-d ] pyrimidine 3N:
2b (207mg, 0.78mmol) and N-Boc-6-azaspiro [3.4] were stirred at 80 deg.C]Suspension of octane-2- ((methylsulfonyl) oxy) (1 eq) and potassium carbonate (2 eq) in DMF (3 mL) for 16h. After the reaction was complete, the mixture was treated with water and extracted with dichloromethane. The organic phase was collected, dried and concentrated in vacuo to give 185mg of the pale green title compound in yield: 50 percent. LC/MS (ESI): m/z 475 (M + H) + 。
Preparation of intermediate 1- { 6-azaspiro [3.4] octan-2-yl } -4-amino-3- (1-methyl-1H-indazol-3-yl) -1H-pyrazole [3,4-d ] pyrimidine 4 n:
3n (90mg, 0.19mmol) was dissolved in 2mL of dichloromethane solution, excess trifluoroacetic acid was added, the mixture was stirred at room temperature for 2h, the mixture was diluted with dichloromethane and washed with saturated aqueous sodium bicarbonate solution. The organic phase was collected, dried over anhydrous sodium sulfate and concentrated in vacuo, and the crude product was taken to the next step without purification.
Preparation of 1- {2- [ 4-amino-3- (1-methyl-1H-indazol-3-yl) -pyrazolo [3,4-d ] pyrimidin-1-yl ] -6-azaspiro [3.4] octan-6-yl } -propenone (compound 714):
the crude product 4n from the previous step was dissolved in 5mL of tetrahydrofuran solutionDIPEA (196. Mu.L, 1.2 mmol) was added, followed by dropwise addition of acryloyl chloride (17. Mu.L, 0.21 mmol), and the reaction was stirred for 2 hours. The reaction was complete by TLC. The reaction was stopped, DCM (25 mL) was added, followed by 50mL saturated NaHCO 3 Washed with water, the aqueous layer was extracted with DCM (2 × 25ml), anhydrous MgSO 4 Drying, concentration and flash column separation of the crude product gave 46mg of a yellow solid in two yields: 57 percent. 1 H NMR(400MHz,DMSO-d 6 )δ 8.32-8.27(m,2H),7.92(d,1H),7.65(m,1H),7.45(m,1H),7.02(s,2H),6.62(m,1H), 6.04(m,1H),5.58(m,1H),4.20(m,1H),3.95(s,3H),3.33-3.28(s,4H),2.09-1.85(m, 4H),1.67(m,2H).LC/MS(ESI)：m/z 429(M+H) + 。
Example 15: preparation of 1- {2- [ 4-amino-3- (1-methyl-1H-pyrrole [2,3-b ] pyridin-3-yl) -pyrazole [3,4-d ] pyrimidin-1-yl ] -6-azaspiro [3.4] octane-6-yl } -propenone (compound 715):
preparation of intermediate 1- { N-Boc-6-azaspiro [3.4] octan-2-yl } -4-amino-3- (1-methyl-1H-pyrrole [2,3-b ] pyridin-3-yl) -1H-pyrazole [3,4-d ] pyrimidine 3 o:
2c (207mg, 0.78mmol) and N-Boc-6-azaspiro [3.4] were stirred at 80 deg.C]Suspension of octane-2- ((methylsulfonyl) oxy) (1 eq) and potassium carbonate (2 eq) in DMF (3 mL) for 16h. After the reaction was complete, the mixture was treated with water and extracted with dichloromethane. The organic phase was collected, dried and concentrated in vacuo to give 185mg of the pale green target compound in yield: 50 percent. LC/MS (ESI): m/z 475 (M + H) + 。
Preparation of intermediate 1- { 6-azaspiro [3.4] octan-2-yl } -4-amino-3- (1-methyl-1H-pyrrole [2,3-b ] pyridin-3-yl) -1H-pyrazole [3,4-d ] pyrimidine 4 o:
3o (90mg, 0.19mmol) was dissolved in 2mL of dichloromethane solution, excess trifluoroacetic acid was added, the mixture was stirred at room temperature for 2h, the mixture was diluted with dichloromethane and washed with saturated aqueous sodium bicarbonate solution. The organic phase was collected, dried over anhydrous sodium sulfate and concentrated in vacuo, and the crude product was taken to the next step without purification.
Preparation of 1- {2- [ 4-amino-3- (1-methyl-1H-pyrrole [2,3-b ] pyridin-3-yl) -pyrazole [3,4-d ] pyrimidin-1-yl ] -6-azaspiro [3.4] octan-2-yl } -propenone (compound 715):
the crude product 4o from the previous step was dissolved in 5mL tetrahydrofuran, DIPEA (196. Mu.L, 1.2 mmol) was added, followed by the dropwise addition of acryloyl chloride (17. Mu.L, 0.21 mmol), and the reaction was stirred for 2 hours. The reaction was complete by TLC. The reaction was stopped, DCM (25 mL) was added, followed by 50mL of saturated NaHCO 3 Washed with water, the aqueous layer was extracted with DCM (2 × 25ml), anhydrous MgSO 4 Drying, concentration and flash column separation of the crude product gave 46mg of a yellow solid in two yields: 57 percent. 1 H NMR(400MHz,DMSO-d 6 )δ 8.51(d,1H),8.39(d,1H),8.27(s,1H),7.17(m,1H),7.06-7.12(m,3H),6.62(m,1H), 6.04(m,1H),5.58(m,1H),4.20(m,1H),3.59(s,3H),3.33-3.28(m,4H),2.09-1.85 (m,4H),1.67(m,2H).LC/MS(ESI)：m/z 429(M+H) + 。
Example 16: determination of EGFR kinase inhibitory Activity and binding Rate of related mutation sites
For compounds 711-715 prepared in examples 1-15 above, FRET technique was used to determine the inhibitory activity of the compounds on EGFR kinase, which is IC 50 This index pair represents, IC 50 I.e. the concentration of the compound at which the activity of the EGFR kinase is inhibited by 50%.
At the same time, TR-FRET technology is adopted to inhibit the EGFR-T790M kinase activity of the compounds 711-715 prepared in the compounds of examples 1-15 of the inventionMeasurement, also using IC 50 This index pair is expressed. The method employs Life technologies Z' -LYTE TM The Kinase Assay kit (PV 3193) was used and the EGFR-T790M Kinase solution from Tyrosine 4Peptide was selected for activity determination. The results are shown in Table 2
Table 2 EGFR inhibitory activity and EGFR of the compounds of the examples T790M Determination of binding Rate
| EGFR | EGFR T790M | |
| Sample numbering | IC 50 (nM) | IC 50 (nM) |
| 701 | 13.2 | 1.3 |
| 702 | 67.5 | 28.6 |
| 703 | 87.2 | 46.5 |
| 704 | 14.7 | 1.4 |
| 705 | 36.5 | 23.3 |
| 706 | 68.9 | 32.8 |
| 707 | 19.8 | 2.1 |
| 708 | 35.4 | 17.3 |
| 709 | 87.4 | 45.7 |
| 710 | 21.1 | 1.7 |
| 711 | 32.4 | 15.4 |
| 712 | 62.3 | 38.8 |
| 713 | 21.8 | 1.9 |
| 714 | 45.7 | 23.1 |
| 715 | 86.4 | 53.8 |
Example 17: the inhibitory activity of the compound on cancer cell lines is measured (MTT method detection)
Cell lines: human non-small cell lung cancer adenocarcinoma cell line NCI-H1975 (EGFR-T790M high expression), and human non-small cell lung cell line A549 (EFGR high expression).
The method comprises the following steps: cell lines NCI-H1975 and A549 were cultured in 20% FBS (fetal bovine serum) (Gibco) +1640+1% double antibody. Then, NCI-H1975 cells in good growth state were taken, inoculated into a 96-well cell plate at 5000 cells/well, and incubated at 37 ℃ in an incubator containing 5% CO2 for 24 hours to complete cell adhesion. Discarding old culture solution, sequentially adding 100 μ L culture solution containing 0.3, 1, 3, 10, 30, 100, 300, 1000, 3000 and 10000nmol/L compounds to be tested into each well, adding 100 μ L culture solution containing 0.1% DMSO into each well of solvent control group, repeating the wells for 3 times, discarding old culture solution after 72 hr, adding 100 μ L culture solution containing 0.5 mg/mL into each well under dark condition -1 And (3) placing the MTT culture solution into a cell culture box to be incubated for 4h, removing the supernatant, adding 100 mu of LDMSO into each hole, oscillating, and measuring the absorbance value of each hole by using an enzyme-linked immunosorbent assay (ELIAS) reader at the wavelength of 492 nm. The inhibition rate of each compound on the growth of the prepared cells is converted to obtain the IC in the following table 50 (nM)。
TABLE 3 Effect of Compounds on Lung cancer cell lines
| NCI-H1975 | A549 | |
| Sample numbering | IC 50 (nM) | IC 50 (nM) |
| 710 | 28 | 1.8 |
| 704 | 36 | 2.1 |
Claims (6)
1. A pyrazolopyrimidine derivative epidermal growth factor inhibitor having a general structural formula (I) or a pharmaceutically acceptable salt thereof,
wherein:
is selected from any one of the following:
R 1 selected from the following structures:
wherein R is 2 ，R 3 And R 4 Each is selected from H, C 1-12 Alkyl radical, C 1-12 Alkenyl radical, C 1-12 Alkynyl.
2. The pyrazolopyrimidine derivative epidermal growth factor inhibitor or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that the compound of the general structural formula (I) is selected from the following structural formulae:
3. a pharmaceutical composition comprising the pyrazolopyrimidine derivative epidermal growth factor inhibitor or a pharmaceutically acceptable salt thereof according to claim 1 or 2, and a pharmaceutically acceptable carrier.
4. The pharmaceutical composition according to claim 3, wherein the pharmaceutical composition is in the form of a tablet, capsule, granule, spray or injection.
5. The pharmaceutical composition of claim 3, wherein the pharmaceutically acceptable carrier is selected from one or more of a filler, a disintegrant, a binder, and a lubricant.
6. Use of a pyrazolopyrimidine epidermal growth factor inhibitor or a pharmaceutically acceptable salt thereof according to claim 1 or 2 or a pharmaceutical composition according to any one of claims 3 to 5 for the preparation of a medicament for the treatment of a disease associated with epidermal growth factor receptor overexpression;
wherein, the diseases related to the epidermal growth factor receptor overexpression are selected from one or more of renal cancer, lung cancer, prostatic cancer, pancreatic cancer, breast cancer and glioma.
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| CN1259950A (en) * | 1997-03-19 | 2000-07-12 | Basf公司 | Pyrrolo [2,3D] pyrimidines and their use as tyrosine kinase inhibitors |
| WO2000017202A1 (en) * | 1998-09-18 | 2000-03-30 | Basf Aktiengesellschaft | 4-aminopyrrolopyrimidines as kinase inhibitors |
| CN108191861A (en) * | 2018-03-01 | 2018-06-22 | 天津大学 | N- [5- (pyrimidine -2- amino) -2,4- di-substituted-phenyls]-trans- -2,4- Pentadienamides |
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