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CN110452243B - Pyrrolopyrimidine derivative epidermal growth factor inhibitor and preparation method and application thereof - Google Patents

Pyrrolopyrimidine derivative epidermal growth factor inhibitor and preparation method and application thereof Download PDF

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CN110452243B
CN110452243B CN201910796161.9A CN201910796161A CN110452243B CN 110452243 B CN110452243 B CN 110452243B CN 201910796161 A CN201910796161 A CN 201910796161A CN 110452243 B CN110452243 B CN 110452243B
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梁永宏
曾兆森
凌苑
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Yaoya Technology Shanghai Co ltd
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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 pyrrolopyrimidine 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 on the EGFR of a T790M variant epidermal growth factor receptor, and the compound can be used for treating diseases related to the overexpression of the EGFR of the epidermal growth factor receptor, such as kidney cancer, lung cancer, prostate cancer, pancreatic cancer, breast cancer and glioma.

Description

Pyrrolopyrimidine derivative epidermal growth factor inhibitor as well as preparation method and application thereof
Technical Field
The invention relates to the field of medicines, and in particular relates to a pyrrolopyrimidine 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 epidermal growth factor receptor, function by phosphorylation, and that epidermal growth factor receptor EGFR tyrosine kinase phosphorylates 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 response (50-80%) was shown in humans with these specific activating mutations. 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, further development of EGFR tyrosine kinase inhibitors having strong inhibitory effects on mutant forms, and development of novel highly effective, low-toxic antitumor drugs having drug resistance are very important in the current drug development field.
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 pyrrolopyrimidine 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 pyrrolopyrimidine derivative compound shown in a formula (I), or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, which can be used as an irreversible EGFR inhibitor,
Figure GDA0002221273830000021
wherein:
Figure GDA0002221273830000022
may be any of the following
Figure GDA0002221273830000031
Figure GDA0002221273830000032
Can be of the following structure:
Figure GDA0002221273830000033
R 1 the structure can be as follows:
Figure GDA0002221273830000034
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 801-815
Figure GDA0002221273830000041
Figure GDA0002221273830000051
The invention also provides a preparation method of the pyrrolopyrimidine derivative EGFR protein tyrosine kinase selective inhibitor, as shown in Scheme 1, and 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)
Figure GDA0002221273830000061
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 use of the pyrrolopyrimidine derivative epidermal growth factor inhibitor and pharmaceutically acceptable salts thereof as protein tyrosine kinase inhibitors;
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 T790M variant epidermal growth factor receptor.
Use of pyrrolopyrimidine derivative epidermal growth factor inhibitors and pharmaceutically acceptable salts or pharmaceutical compositions thereof in the preparation of medicaments for the treatment of diseases associated with epidermal growth factor receptor overexpression.
Further, the tumor is liver cancer, lung cancer, prostate cancer, pancreatic cancer, breast cancer, and astrocytoma.
It will be apparent that various other modifications, substitutions and alterations can be made in the present invention without departing from the basic technical concept of the invention as described above, according to the common technical knowledge and practice in the field.
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) -pyrrole [3,4-d ] pyrimidin-1-yl ] -2-azaspiro [3.5] nonan-2-yl } -propenone (compound 801):
Figure GDA0002221273830000071
preparation of intermediate 4-amino-3- (1-methyl-1H-indol-3-yl) -1H-pyrrole [3,4-d ] pyrimidine 2 a:
Figure GDA0002221273830000072
reacting 3-iodo-4-amino-1H-pyrrole [3,4-d]Pyrimidine (293mg, 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 245mg (0.93 mmol) of the objective compound as a pale green solid in yield: 82 percent. LC/MS (ESI): m/z 264 (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-pyrrole [3,4-d ] pyrimidine 3 a:
Figure GDA0002221273830000081
2a (205mg, 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. The organic phase was collected, dried and concentrated in vacuo to give 182mg of the pale green target compound in yield: and 48 percent. LC/MS (ESI): m/z 487 (M + H) + 。
Preparation of intermediate 1- { 2-azaspiro [3.5] nonan-7-yl } -4-amino-3- (1-methyl-1H-indol-3-yl) -1H-pyrrole [3,4-d ] pyrimidine 4 a:
Figure GDA0002221273830000082
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) -pyrrole [3,4-d ] pyrimidin-1-yl ] -2-azaspiro [3.5] nonan-2-yl } -propenone (compound 801):
Figure GDA0002221273830000083
the crude product 4a 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, 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 )δ8.27(s,1H),8.17(d,1H),7.59(d,1H),7.44(m,1H),7.33(m,1H),7.19(s,1H),7.10(s,1H),7.01(s,2H),6.62(m,1H),6.04(m,1H),5.58(m,1H),3.72-3.64(m,8H),1.79-1.54(m,4H),1.32(m,4H).LC/MS(ESI)：m/z 441(M+H) + 。
Example 2: preparation of 1- {7- [ 4-amino-3- (1-methyl-1H-indazol-3-yl) -pyrrole [3,4-d ] pyrimidin-1-yl ] -2-azaspiro [3.5] nonan-2-yl } -propenone (compound 802):
Figure GDA0002221273830000091
preparation of intermediate 4-amino-3- (1-methyl-2-aza-1H-indazol-3-yl) -1H-pyrrole [3,4-d ] pyrimidine 2 b:
Figure GDA0002221273830000092
3-iodo-4-amino-1H-pyrrole [3,4-d]Pyrimidine (294mg, 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 265 (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-pyrrole [3,4-d ] pyrimidine 3 b:
Figure GDA0002221273830000101
2b (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. 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 488 (M + H) + 。
Preparation of intermediate 1- { 2-azaspiro [3.5] nonan-7-yl } -4-amino-3- (1-methyl-1H-indazol-3-yl) -1H-pyrrole [3,4-d ] pyrimidine 4 b:
Figure GDA0002221273830000102
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 taken to the next step without purification.
Preparation of 1- {7- [ 4-amino-3- (1-methyl-1H-indazol-3-yl) -pyrrole [3,4-d ] pyrimidin-1-yl ] -2-azaspiro [3.5] nonan-2-yl } -propenone (compound 802): :
Figure GDA0002221273830000103
the crude product 4b from the previous step was dissolved in 5mL tetrahydrofuran solution, 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 )δ8.32-8.27(m,2H),7.92(d,1H),7.65(m,1H),7.45(m,1H),7.10(s,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),1.79-1.54(m,4H),1.32(m,4H).LC/MS(ESI)：m/z 442(M+H) + 。
Example 3: preparation of 1- {7- [ 4-amino-3- (1-methyl-1H-pyrrole [2,3-b ] pyridin-3-yl) -pyrrole [3,4-d ] pyrimidin-1-yl ] -2-azaspiro [3.5] nonan-2-yl } -propenone (compound 803):
Figure GDA0002221273830000111
preparation of intermediate 4-amino-3- (1-methyl-1H-pyrrole [2,3-b ] pyridin-3-yl) -1H-pyrrole [3,4-d ] pyrimidine 2 c:
Figure GDA0002221273830000112
3-iodo-4-amino-1H-pyrrole [3,4-d]Pyrimidine (294)mg,1.13 mmol) was dissolved in 2mL of a mixed solvent of ethanol and DME (1:3) and 0.25mL of saturated sodium carbonate solution and N-methyl 2-azaindole-3-boronic acid pinacol ester (413mg, 1.6 mmol) were added. 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 232mg (0.88 mmol) of the objective compound as a pale green solid in yield: 78 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-pyrrole [2,3-b ] pyridin-3-yl) -1H-pyrrole [3,4-d ] pyrimidine 3 c:
Figure GDA0002221273830000121
2c (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. 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 488 (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-pyrrole [3,4-d ] pyrimidine 4 c:
Figure GDA0002221273830000122
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) -pyrrole [3,4-d ] pyrimidin-1-yl ] -2-azaspiro [3.5] nonan-2-yl } -propenone (compound 803): :
Figure GDA0002221273830000123
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 )δ8.51(d,1H),8.39(d,1H),8.27(s,1H),7.06-7.17(m,5H),6.62(m,1H),6.04(m,1H),5.58(m,1H),3.72-3.59(m,8H),1.79-1.54(m,4H),1.32(m,4H).LC/MS(ESI)：m/z442(M+H) + 。
Example 4: preparation of 1- {6- [ 4-amino-3- (1-methyl-1H-indol-3-yl) -pyrrole [3,4-d ] pyrimidin-1-yl ] -2-azaspiro [3.3] heptan-2-yl } -propenone (compound 804):
Figure GDA0002221273830000131
preparation of intermediate 1- { N-Boc-2-azaspiro [3.3] heptan-6-yl } -4-amino-3- (1-methyl-1H-indol-3-yl) -1H-pyrrole [3,4-d ] pyrimidine 3 d:
Figure GDA0002221273830000132
2a (205mg, 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 suspended for 16h. After the reaction is finished, the reaction mixture is treated by waterThe mixture was 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 459 (M + H) + 。
Preparation of intermediate 1- { 2-azaspiro [3.3] heptan-6-yl } -4-amino-3- (1-methyl-1H-indol-3-yl) -1H-pyrrole [3,4-d ] pyrimidine 4 d:
Figure GDA0002221273830000141
3d (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-indol-3-yl) -pyrrole [3,4-d ] pyrimidin-1-yl ] -2-azaspiro [3.3] heptan-2-yl } -propenone (compound 804):
Figure GDA0002221273830000142
the crude product 4d 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 45mg of a yellow solid in two yields: 58 percent. 1 H NMR(400MHz,DMSO-d 6 )δ8.27(s,1H),8.17(d,1H),7.59(d,1H),7.44(m,1H),7.33(m,1H),7.19(s,1H),7.10(s,1H),7.01(s,2H),6.62(m,1H),6.04(m,1H),5.58(m,1H),4.20(m,1H),3.74-3.72(m,7H),1.89-1.64(m,4H).LC/MS(ESI)：m/z 413(M+H) + 。
Example 5: preparation of 1- {6- [ 4-amino-3- (1-methyl-1H-indazol-3-yl) -pyrrole [3,4-d ] pyrimidin-1-yl ] -2-azaspiro [3.3] heptan-2-yl } -propenone (compound 805):
Figure GDA0002221273830000151
preparation of intermediate 1- { N-Boc-2-azaspiro [3.3] heptan-6-yl } -4-amino-3- (1-methyl-1H-indazol-3-yl) -1H-pyrrole [3,4-d ] pyrimidine 3 e:
Figure GDA0002221273830000152
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 afford 161mg of the greenish target compound in yield: 45 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-indazol-3-yl) -1H-pyrrole [3,4-d ] pyrimidine 4 e:
Figure GDA0002221273830000153
3e (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 taken to the next step without purification.
Preparation of 1- {6- [ 4-amino-3- (1-methyl-1H-indazol-3-yl) -pyrrole [3,4-d ] pyrimidin-1-yl ] -2-azaspiro [3.3] heptan-2-yl } -propenone (compound 805):
Figure GDA0002221273830000161
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 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 49mg of a yellow solid in two yields: 62 percent. 1 H NMR(400MHz,DMSO-d 6 )δ8.32-8.27(m,2H),7.92(d,1H),7.65(m,1H),7.45(m,1H),7.10(s,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),1.89-1.64(m,4H).LC/MS(ESI)：m/z 414(M+H) + 。
Example 6: preparation of 1- {6- [ 4-amino-3- (1-methyl-1H-pyrrole [2,3-b ] pyridin-3-yl) -pyrrole [3,4-d ] pyrimidin-1-yl ] -2-azaspiro [3.3] heptan-2-yl } -propenone (compound 806):
Figure GDA0002221273830000162
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-pyrrole [3,4-d ] pyrimidine 3 f:
Figure GDA0002221273830000171
2c (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 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 183mg of the pale green title compound in yield: 51 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-pyrrole [2,3-b ] pyridin-3-yl) -1H-pyrrole [3,4-d ] pyrimidine 4 f:
Figure GDA0002221273830000172
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 used in the next step without further purification.
Preparation of 1- {6- [ 4-amino-3- (1-methyl-1H-pyrrole [2,3-b ] pyridin-3-yl) -pyrrole [3,4-d ] pyrimidin-1-yl ] -2-azaspiro [3.3] heptan-2-yl } -propenone (compound 806):
Figure GDA0002221273830000173
the crude product 4f 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 47mg of a yellow solid in two yields: 60 percent. 1 H NMR(400MHz,DMSO-d 6 )δ8.51(d,1H),8.39(d,1H),8.27(s,1H),7.06-7.17(m,5H),6.62(m,1H),6.04(m,1H),5.58(m,1H),4.20(m,1H),3.72(s,4H),3.59(s,3H),1.89-1.64(m,4H).LC/MS(ESI)：m/z 414(M+H) + 。
Example 7: preparation of 1- {2- [ 4-amino-3- (1-methyl-1H-indol-3-yl) -pyrrole [3,4-d ] pyrimidin-1-yl ] -7-azaspiro [3.5] nonan-7-yl } -propenone (compound 807):
Figure GDA0002221273830000181
preparation of intermediate 1- { N-Boc-7-azaspiro [3.5] nonan-2-yl } -4-amino-3- (1-methyl-1H-indol-3-yl) -1H-pyrrole [3,4-d ] pyrimidine 3 g:
Figure GDA0002221273830000182
2a (205mg, 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 487 (M + H) + 。
Preparation of intermediate 1- { 7-azaspiro [3.5] nonan-2-yl } -4-amino-3- (1-methyl-1H-indol-3-yl) -1H-pyrrole [3,4-d ] pyrimidine 4 g:
Figure GDA0002221273830000191
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 used in the next step without further purification.
Preparation of 1- {2- [ 4-amino-3- (1-methyl-1H-indol-3-yl) -pyrrole [3,4-d ] pyrimidin-1-yl ] -7-azaspiro [3.5] nonan-7-yl } -propenone (compound 807):
Figure GDA0002221273830000192
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: 63％。 1 H NMR(400MHz,DMSO-d 6 )δ8.27(s,1H),8.17(d,1H),7.59(d,1H),7.44(m,1H),7.33(m,1H),7.19(s,1H),7.10(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,7H),1.89-1.64(m,4H),1.54-1.52(m,4H).LC/MS(ESI)：m/z 441(M+H) + 。
Example 8: preparation of 1- {2- [ 4-amino-3- (1-methyl-1H-indazol-3-yl) -pyrrole [3,4-d ] pyrimidin-1-yl ] -7-azaspiro [3.5] nonan-7-yl } -propenone (compound 808):
Figure GDA0002221273830000201
preparation of intermediate 1- { N-Boc-7-azaspiro [3.5] nonan-2-yl } -4-amino-3- (1-methyl-1H-indazol-3-yl) -1H-pyrrole [3,4-d ] pyrimidine 3H:
Figure GDA0002221273830000202
2b (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 yield 190mg of the pale green target compound in yield: 50 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-indazol-3-yl) -1H-pyrrole [3,4-d ] pyrimidine 4H:
Figure GDA0002221273830000203
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 taken to the next step without purification.
Preparation of 1- {2- [ 4-amino-3- (1-methyl-1H-indazol-3-yl) -pyrrole [3,4-d ] pyrimidin-1-yl ] -7-azaspiro [3.5] nonan-7-yl } -propenone (compound 808):
Figure GDA0002221273830000211
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 )δ8.32-8.27(m,2H),7.92(d,1H),7.65(m,1H),7.45(m,1H),7.10(s,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),1.89-1.64(m,4H),1.54-1.52(m,4H).LC/MS(ESI)：m/z 442(M+H) + 。
Example 9: preparation of 1- {2- [ 4-amino-3- (1-methyl-1H-pyrrole [2,3-b ] pyridin-3-yl) -pyrrole [3,4-d ] pyrimidin-1-yl ] -7-azaspiro [3.5] nonan-7-yl } -propenone (compound 809):
Figure GDA0002221273830000212
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-pyrrole [3,4-d ] pyrimidine 3 i:
Figure GDA0002221273830000221
2c (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 is finished, treating and mixing with waterThe mixture was 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 488 (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-pyrrole [3,4-d ] pyrimidine 4 i:
Figure GDA0002221273830000222
3i (93mg, 0.19mmol) was dissolved in 2mL 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) -pyrrole [3,4-d ] pyrimidin-1-yl ] -7-azaspiro [3.5] nonan-7-yl } -propenone (compound 809):
Figure GDA0002221273830000223
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, 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 )δ8.51(d,1H),8.39(d,1H),8.27(s,1H),7.06-7.17(m,5H),6.62(m,1H),6.04(m,1H),5.58(m,1H),4.20(m,1H),3.82(s,4H),3.59(s,3H),1.89-1.64(m,4H),1.54-1.52(m,4H).LC/MS(ESI)：m/z 442(M+H) + 。
Example 10: preparation of 1- {2- [ 4-amino-3- (1-methyl-1H-indol-3-yl) -pyrrole [3,4-d ] pyrimidin-1-yl ] -6-azaspiro [3.5] nonan-6-yl } -propenone (compound 810):
Figure GDA0002221273830000231
preparation of intermediate 1- { N-Boc-6-azaspiro [3.5] nonan-2-yl } -4-amino-3- (1-methyl-1H-indol-3-yl) -1H-pyrrole [3,4-d ] pyrimidine 3 j:
Figure GDA0002221273830000232
2a (205mg, 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 give 182mg of the pale green target compound in yield: 48 percent. LC/MS (ESI): m/z 487 (M + H) + 。
Preparation of intermediate 1- { 6-azaspiro [3.5] nonan-2-yl } -4-amino-3- (1-methyl-1H-indol-3-yl) -1H-pyrrole [3,4-d ] pyrimidine 4 j:
Figure GDA0002221273830000241
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) -pyrrole [3,4-d ] pyrimidin-1-yl ] -6-azaspiro [3.5] nonan-6-yl } -propenone (compound 810):
Figure GDA0002221273830000242
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 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 )δ8.27(s,1H),8.17(d,1H),7.59(d,1H),7.44(m,1H),7.33(m,1H),7.19(s,1H),7.10(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),1.89-1.64(m,6H),1.44-1.42(m,2H).LC/MS(ESI)：m/z 441(M+H) + 。
Example 11: preparation of 1- {2- [ 4-amino-3- (1-methyl-1H-indazol-3-yl) -pyrrole [3,4-d ] pyrimidin-1-yl ] -6-azaspiro [3.5] nonan-6-yl } -propenone (compound 811):
Figure GDA0002221273830000251
preparation of intermediate 1- { N-Boc-6-azaspiro [3.5] nonan-2-yl } -4-amino-3- (1-methyl-1H-indazol-3-yl) -1H-pyrrole [3,4-d ] pyrimidine 3 k:
Figure GDA0002221273830000252
2b (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) 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 488 (M + H) + 。
Preparation of intermediate 1- { 6-azaspiro [3.5] nonan-2-yl } -4-amino-3- (1-methyl-1H-indazol-3-yl) -1H-pyrrole [3,4-d ] pyrimidine 4 k:
Figure GDA0002221273830000253
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 taken to the next step without purification.
Preparation of 1- {2- [ 4-amino-3- (1-methyl-1H-indazol-3-yl) -pyrrole [3,4-d ] pyrimidin-1-yl ] -6-azaspiro [3.5] nonan-6-yl } -propenone (compound 811):
Figure GDA0002221273830000261
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 )δ8.32-8.27(m,2H),7.92(d,1H),7.65(m,1H),7.45(m,1H),7.10(s,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),1.89-1.64(m,6H),1.44-1.42(m,2H).LC/MS(ESI)：m/z 442(M+H) + 。
Example 12: preparation of 1- {2- [ 4-amino-3- (1-methyl-1H-pyrrole [2,3-b ] pyridin-3-yl) -pyrrole [3,4-d ] pyrimidin-1-yl ] -6-azaspiro [3.5] nonan-6-yl } -propenone (compound 812):
Figure GDA0002221273830000262
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-pyrrole [3,4-d ] pyrimidine 3 l:
Figure GDA0002221273830000263
2c (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) 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 488 (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-pyrrole [3,4-d ] pyrimidine 4 l:
Figure GDA0002221273830000271
3l (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 taken to the next step without purification.
Preparation of 1- {2- [ 4-amino-3- (1-methyl-1H-pyrrole [2,3-b ] pyridin-3-yl) -pyrrole [3,4-d ] pyrimidin-1-yl ] -6-azaspiro [3.5] nonan-2-yl } -propenone (compound 812):
Figure GDA0002221273830000272
4L of the crude product from the previous step was dissolved in 5mL of tetrahydrofuran, DIPEA (196. Mu.L, 1.2 mmol) was added, and then 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 48mg of a yellow solid in two yields: 57 percent. 1 H NMR(400MHz,DMSO-d 6 )δ8.51(d,1H),8.39(d,1H),8.27(s,1H),7.06-7.17(m,5H),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),1.89-1.64(m,6H),1.44-1.42(m,2H).LC/MS(ESI)：m/z 442(M+H) + 。
Example 13: preparation of 1- {2- [ 4-amino-3- (1-methyl-1H-indol-3-yl) -pyrrole [3,4-d ] pyrimidin-1-yl ] -6-azaspiro [3.4] octan-6-yl } -propenone (compound 813):
Figure GDA0002221273830000281
preparation of intermediate 1- { N-Boc-6-azaspiro [3.4] octan-2-yl } -4-amino-3- (1-methyl-1H-indol-3-yl) -1H-pyrrole [3,4-d ] pyrimidine 3 m:
Figure GDA0002221273830000282
2a (205mg, 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 yield 177mg of the pale green title compound in yield: 48 percent. LC/MS (ESI): m/z 473 (M + H) + 。
Preparation of intermediate 1- { 6-azaspiro [3.4] octan-2-yl } -4-amino-3- (1-methyl-1H-indol-3-yl) -1H-pyrrole [3,4-d ] pyrimidine 4 m:
Figure GDA0002221273830000283
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 taken to the next step without purification.
Preparation of 1- {2- [ 4-amino-3- (1-methyl-1H-indol-3-yl) -pyrrole [3,4-d ] pyrimidin-1-yl ] -6-azaspiro [3.4] octan-6-yl } -propenone (compound 813):
Figure GDA0002221273830000291
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 )δ8.27(s,1H),8.17(d,1H),7.59(d,1H),7.44(m,1H),7.33(m,1H),7.19(s,1H),7.10(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),1.89-1.64(m,6H).LC/MS(ESI)：m/z 427(M+H) + 。
Example 14: preparation of 1- {2- [ 4-amino-3- (1-methyl-1H-indazol-3-yl) -pyrrole [3,4-d ] pyrimidin-1-yl ] -6-azaspiro [3.4] oct-6-yl } -propenone (compound 814):
Figure GDA0002221273830000292
preparation of intermediate 1- { N-Boc-6-azaspiro [3.4] octan-2-yl } -4-amino-3- (1-methyl-1H-indazol-3-yl) -1H-pyrrole [3,4-d ] pyrimidine 3N:
Figure GDA0002221273830000301
2b (206mg, 0.78mmol) and N-Boc-6-azaspiro [3.4] were stirred at 80 deg.C]Octane-2- ((methylsulfonyl) oxy) (1 eq) and potassium carbonate (2 eq) in DMF (3 mL) suspensionThe supernatant was 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 184mg of the target compound in greenish color, yield: 50 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-indazol-3-yl) -1H-pyrrole [3,4-d ] pyrimidine 4 n:
Figure GDA0002221273830000302
3n (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 2 hours, 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-indazol-3-yl) -pyrrole [3,4-d ] pyrimidin-1-yl ] -6-azaspiro [3.4] octan-6-yl } -propenone (compound 814):
Figure GDA0002221273830000303
the crude product 4n 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 46mg of a yellow solid in two yields: 57 percent. 1 H NMR(400MHz,DMSO-d 6 )δ8.32-8.27(m,2H),7.92(d,1H),7.65(m,1H),7.45(m,1H),7.10(s,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),1.89-1.64(m,6H).LC/MS(ESI)：m/z 428(M+H) + 。
Example 15: preparation of 1- {2- [ 4-amino-3- (1-methyl-1H-pyrrole [2,3-b ] pyridin-3-yl) -pyrrole [3,4-d ] pyrimidin-1-yl ] -6-azaspiro [3.4] octane-6-yl } -propenone (compound 815):
Figure GDA0002221273830000311
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-pyrrole [3,4-d ] pyrimidine 3 o:
Figure GDA0002221273830000312
2c (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. The organic phase was collected, dried and concentrated in vacuo to give 184mg of the pale green target compound in yield: 50 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-pyrrole [2,3-b ] pyridin-3-yl) -1H-pyrrole [3,4-d ] pyrimidine 4 o:
Figure GDA0002221273830000321
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) -pyrrole [3,4-d ] pyrimidin-1-yl ] -6-azaspiro [3.4] octan-2-yl } -propenone (compound 815):
Figure GDA0002221273830000322
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 )δ8.51(d,1H),8.39(d,1H),8.27(s,1H),7.06-7.17(m,5H),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),1.89-1.64(m,6H).LC/MS(ESI)：m/z 428(M+H) + 。
Example 16: determination of EGFR kinase inhibitory Activity and binding Rate of related mutation sites
For compounds 811-815 prepared in examples 1-15 above, the FRET technique was used to determine the inhibitory activity of the compounds on EGFR kinase by IC 50 This indicator represents, IC 50 I.e. the concentration of the compound at which the activity of EGFR kinase is inhibited by 50%.
At the same time, the EGFR-T790M kinase activity inhibition of the compounds 811-815 prepared in the compounds of examples 1-15 of the invention was determined by the TR-FRET technique, and IC was also used 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)
801 18.2 0.9
802 27.5 18.6
803 37.2 26.5
804 16.7 1.4
805 26.5 13.3
806 48.9 32.8
807 18.7 1.6
808 23.5 17.3
809 38.6 32.4
810 19.7 2.1
811 26.4 13.5
812 38.9 31.7
813 12.7 1.1
814 26.9 16.5
815 36.6 30.4
Example 17: measurement of inhibitory Activity of the Compound on cancer cell line (MTT method assay)
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: the cell lines NCI-H1975 and A549 were placed in 20% FBS (fetal bovine serum) (Gibco) +1640+1% double antibodyAnd (5) culturing. Then, NCI-H1975 cells in a good growth state were seeded at 5000 cells/well in a 96-well cell plate, and incubated at 37 ℃ for 24 hours in an incubator containing 5% CO2 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 in a cell culture box, continuously incubating for 4h, removing the supernatant, adding 100 mu LDMSO into each hole, oscillating, and measuring the absorbance value of each hole by using a microplate 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)。
Effect of Compounds on Lung cancer cell lines
NCI-H1975 A549
Sample numbering IC 50 (nM) IC 50 (nM)
801 18 1.2
804 26 1.8

Claims (6)

1. A pyrrolopyrimidine derivative having the general structural formula (I) or a pharmaceutically acceptable salt thereof,
Figure FDA0003855785490000011
wherein:
Figure FDA0003855785490000012
is selected from any one of the following:
Figure FDA0003855785490000013
Figure FDA0003855785490000014
selected from the following structures:
Figure FDA0003855785490000021
R 1 selected from the following structures:
Figure FDA0003855785490000022
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. A pyrrolopyrimidine derivative or a pharmaceutically acceptable salt thereof, according to claim 1, wherein the compound of general structural formula (I) is selected from the following structural formulae:
Figure FDA0003855785490000023
Figure FDA0003855785490000031
3. a pharmaceutical composition comprising the pyrrolopyrimidine derivative 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 according to 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 pyrrolopyrimidine derivative or a pharmaceutically acceptable salt thereof as claimed in claim 1 or 2 or a pharmaceutical composition as claimed in any one of claims 3 to 5 in the manufacture of a medicament for the treatment of a disease associated with overexpression of epidermal growth factor receptor;
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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CN106132957A (en) * 2015-08-31 2016-11-16 无锡双良生物科技有限公司 2 aryl amine pyridines, pyrimidine or pyrrolotriazine derivatives and its production and use
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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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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