CN110483523A - A kind of Pyrazolopyrimidine derivative egf inhibitor and preparation method thereof and purposes - Google Patents
A kind of Pyrazolopyrimidine derivative egf inhibitor and preparation method thereof and purposes Download PDFInfo
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- C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
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Abstract
The present invention discloses a kind of selective depressant of the clinical mutant of EGFR protein tyrosine kinase, and with the structure such as formula (I), it is a kind of compound containing pyrazolopyrimidine structure, also disclose the preparation method and its application as the selective depressant of the clinical mutant of EGFR protein tyrosine kinase of such compound, especially for the inhibiting effect of the EGF-R ELISA EGFR of T790M variation, related disease such as kidney is over-expressed in treatment and EGF-R ELISA EGFR, lung cancer, prostate cancer, cancer of pancreas, breast cancer and spongiocytoma.
Description
Technical field
The present invention relates to field of medicaments, and in particular to a kind of Pyrazolopyrimidine derivative egf inhibitor and its
Preparation method and purposes.
Background technique
Skin growth factor EGFR (epithelial growth factor receptor) is the cross-film sugar of a 170kDa
Protein receptor tyrosine kinase, is activated by epidermal growth factor, influences the growth and differentiation of cell.EGF or TGFα are to EGFR's
In conjunction with the tyrosine-kinase enzyme activity of activated receptor.Tyrosine residue Tyr1068, Tyr1148 of EGFR carboxyl terminal and
Tyr1173 is the major site of the autophosphorylation occurred after EGF is combined.Once it is activated, EGFR1068 and 1173 phosphorus
The tyrosine residue of acidification can mediate combination of the Grb2 to EGFR.In addition, the tyrosine residue of 1173 phosphorylations is SHC
Principal binding sites on EGFR.EGFR is widely distributed in many normal and malignant epithelial cells, overexpression and oneself
I activates may be related with the occurrence and development of many tumours.Being currently used primarily in various human cancers includes incidence
The research of squamous carcinoma, lung cancer, breast cancer and bladder cancer etc..
Protein tyrosine kinase (protein tyrosine kinase, PTK) is γ-phosphoric acid transfer on a kind of catalysis ATP
Kinases onto protein-tyrosine residue.Perhaps my growth factor receptor protein includes that EGF-R ELISA passes through phosphorylation
It works, EGF-R ELISA EGFR tyrosine kinase makes EGF-R ELISA phosphorylation.Clinically, EGFR junket ammonia
Acid kinase inhibitor has been used for the treatment of cancer, the first generation reversible EGFR tyrosine kinase inhibitor Gefitinib and Ai Luo
For Buddhist nun.Significant clinical response (50-80%) is shown to the viral people for there are these specific activated mutants in body.So
And the patient for generating the mutation of secondary and medicine for these drugs understands the recurrence in some months by cancer.Second generation EGFR junket
Histidine kinase inhibitor includes linatinib, replaces Buddhist nun up to gram, Afatinib all contains electrophilic group in the structure of these drugs
Michael receptor.Wherein by taking Afatinib is pharmaceutical representative as an example, allyl amide structure plays the anti-tumor activity of Afatinib
Vital effect, it is (new as the catalytic site of Michael receptor and EGFR cysteine residue (Cys797)
The sulfydryl of core) Michael addition reaction occurs, to make kinase-dead, irreversibly inhibit the activity of tyrosine kinase, thus
With good tolerance.Researchers demonstrate this from molecular level by the crystal of culture inhibitor and receptor completely
The presence of a little covalent bonds, and it was found that Afatinib is by the way that the Cys 803 of Cys 805 and HER4 with HER2 are acted on and then
Strength inhibits these enzymes.Afatinib is to Wild type EGFR and L858R/ to be shown to Wild type EGFR testing in vitro
The inhibiting effect of T790M double-mutant has better effect compared with Gefitinib, Erlotinib and Lapatinib.This
Outside, Afatinib is higher than 30 times of Lapatinib to the inhibiting effect of DER4.Third generation EGFR tyrosine kinase inhibitor Austria is uncommon to be replaced
Buddhist nun also reaches nanomole to the inhibiting effect of tri- saltant type of L858R/T790M/C797S.In short, further developing to saltant type
EGFR tyrosine kinase inhibitor with high inhibition effect, meanwhile, the antineoplastic new with drug resistance of research and development efficiently, less toxic
Medicine is particularly significant in current medical research and development field.
Summary of the invention
The present invention devises a series of inhibitor small molecules for the EGFR tyrosine kinase for inhibiting T790M to make a variation, and finds
There is the compound of high inhibitory activity to T790M variation EGFR, and the compound has cancer cell and apparent inhibits to make
With the compound is compound and α containing pyrazolopyrimidine structure, β-unsaturated carboxylic acid amide structure.The compound can be with
High activity, which inhibits, to be realized to T790M/L858R variant EGFR, and can inhibit or kill the tumour cell of EGFRT790M variation,
There is very high inhibitory activity effect to Wild type EGFR simultaneously.
The present invention provides the compound of Pyrazolopyrimidine derivative shown in formula (I) or its stereoisomer or its pharmacy
Upper acceptable salt, can be used as a kind of irreversible EGFR inhibitor,
Wherein:It can be following any
It can be for such as flowering structure:
R1Can be following structure:
Wherein R2, R3And R4It respectively can be H, C1-12Alkyl, C1-12Miscellaneous alkyl, C1-12Heterocyclylalkyl, C1-12Alkenyl, C1-12
Alkynyl, C1-12Any one in naphthenic base.
Preferably, the compound includes with the compound such as 1 structure of table:
The compound of 1 general structure of table (I) includes one kind of number 701-715
The present invention also provides the one of above-mentioned Pyrazolopyrimidine derivative EGFR protein tyrosine kinase selective depressant
Kind preparation method, as shown in Scheme 1, method includes the following steps: starting material is closed through Suzuki coupling reaction
Key intermediate 2, parental materials respectively occur with dicyclic compound for intermediate 2, acylated, finally obtain accordingly shown in logical formula (I)
Compound (shown in Scheme 1)
Scheme 1 leads to the synthesis and preparation of formula (I) compound
The present invention also provides compounds above-mentioned or its stereoisomer or its pharmaceutically acceptable salt to prepare
Purposes in anti-tumor drug, EFGR kinase inhibitor.
Described pharmaceutical composition is the form of tablet, capsule, granule, spray or injection.
The pharmaceutically acceptable carrier is selected from one of filler, disintegrating agent, adhesive and lubricant or a variety of.
Including but not limited to any and whole solvent, decentralized medium, coating, absorption delaying agent etc., such medium and medicament are used for
Pharmaceutically active substances are in the well-known of this field.
The present invention also provides the Pyrazolopyrimidine derivative egf inhibitors and pharmaceutically acceptable
Purposes of the salt as protein tyrosine kinase inhibitor；
Further, the protein tyrosine kinase inhibitor is epidermal growth factor receptor inhibitor；
It is highly preferred that the epidermal growth factor receptor inhibitor is to the T790M EGF-R ELISA to make a variation
Inhibitor.
Pyrazolopyrimidine derivative egf inhibitor and pharmaceutically acceptable salt or its pharmaceutical composition
Purposes in drug of the preparation for treating EGF-R ELISA overexpression related disease.
Further, the tumour is liver cancer, lung cancer, prostate cancer, cancer of pancreas, breast cancer and astrocytoblast
Tumor.
Obviously, above content according to the present invention is not being departed from according to the ordinary technical knowledge and customary means of this field
Under the premise of the above-mentioned basic fundamental thought of the present invention, the modification of other diversified forms, replacement or change can also be made.
Below by way of the specific embodiment of form of implementation, above content of the invention is remake further specifically
It is bright.But this should not be understood the range of the above-mentioned theme of the hair present invention only in example below.It is all real based on invention above content institute
Existing technology all belongs to the scope of the present invention.
Specific embodiment
The present invention is further illustrated below by way of specific embodiment.
It includes: that starting material obtains among key through Suzuki coupling reaction that preparing, which has the method for logical formula (I) compound,
Body 2, parental materials respectively occur with dicyclic compound for intermediate 2, acylated, finally obtain chemical combination shown in logical formula (I) accordingly
Object.It is worth noting that logical formula (I) compound includes but is not limited to following compounds enumerated.
Embodiment 1:1- { 7- [4- amino -3- (1- Methyl-1H-indole -3- base)-pyrazoles [3,4-d] pyrimidine -1- base] -2-
Azaspiro [3.5] nonane -2- base }-propenone (compound 701) preparation:
The preparation of intermediate 4- amino -3- (1- Methyl-1H-indole -3- base) -1H- pyrazoles [3,4-d] pyrimidine 2a:
Iodo- 4- amino -1H- pyrazoles [3, the 4-d] pyrimidine (295mg, 1.13mmol) of 3- is dissolved in ethyl alcohol and the mixing of DME
In solvent 2mL in (1:3), 0.25mL saturated sodium carbonate solution and N- methyl indol -3- pinacol borate are added
(411mg, 1.6mmol).After whole system substitutes gas three times, into mixture be added tetrakis triphenylphosphine palladium (127mg,
0.11mmol), then whole system is heated under a nitrogen atmosphere to 90 DEG C and stirred 12 hours.To after reaction, use silicon
Diatomaceous earth is filtered, and gained filtrate water and methylene chloride washing are three times.Organic phase is obtained into solid by vacuum rotary steam, and is led to
It crosses silica gel column chromatography and is separated (methylene chloride that eluent is the methanol containing 1-2%), finally obtain target compound 246mg
(0.93mmol) is light green solid, yield: 82%.LC/MS (ESI): m/z 265 (M+H)+。
Intermediate 1- { N-Boc-2- azaspiro [3.5] nonane -7- base } -4- amino -3- (1- Methyl-1H-indole -3-
Base) -1H- pyrazoles [3,4-d] pyrimidine 3a preparation:
2a (206mg, 0.78mmol) and N-Boc-2- azaspiro [3.5] nonane -7- ((sulfonyloxy methyl is stirred at 80 DEG C
Base) oxygroup) (1eq) and potassium carbonate (2eq) DMF (3mL) suspension 16h.After reaction, with water treatment and with two
Chloromethanes extraction.Organic phase is collected, is concentrated in vacuo after dry and obtains light green target compound 182mg, yield: 48%.LC/MS
(ESI): m/z 488 (M+H)+。
Intermediate 1- { 2- azaspiro [3.5] nonane -7- base } -4- amino -3- (1- Methyl-1H-indole -3- base) -1H-
The preparation of pyrazoles [3,4-d] pyrimidine 4a:
3a (92mg, 0.19mmol) is dissolved in 2mL dichloromethane solution, excessive trifluoroacetic acid is added, at room temperature
Mixture 2h is stirred, with methylene chloride diluted mixture, then uses in saturated sodium bicarbonate aqueous solution and washs.It collects organic
Phase is concentrated in vacuo after being dried with anhydrous sodium sulfate, and gained crude product does not have to purification and directly throws in next step.
1- { 7- [4- amino -3- (1- Methyl-1H-indole -3- base)-pyrazoles [3,4-d] pyrimidine -1- base] -2- azaspiro
[3.5] nonane -2- base }-propenone (compound 701) preparation:
Previous step crude product 4a is dissolved in 5mL tetrahydrofuran solution, is added DIPEA (196 μ L, 1.2mmol), then
It is added dropwise acryloyl chloride (17 μ L, 0.21mmol), is stirred to react 2 hours.Fully reacting is detected through TLC.Stop reaction, DCM is added
(25mL) is then saturated NaHCO with 50mL3Washing, water layer are extracted with DCM (2*25mL), anhydrous MgSO4Dry, concentration is thick to produce
Product obtain yellow solid 53mg, two step yields: 63% by quick post separation.1H NMR(400MHz,DMSO-d6)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)+。
Embodiment 2:1- { 7- [4- amino -3- (1- methyl-1 H- indazole -3- base)-pyrazoles [3,4-d] pyrimidine -1- base] -2-
Azaspiro [3.5] nonane -2- base }-propenone (compound 702) preparation:
The system of intermediate 4- amino -3- (1- methyl -2- azepine -1H- indazole -3- base) -1H- pyrazoles [3,4-d] pyrimidine 2b
It is standby:
Iodo- 4- amino -1H- pyrazoles [3, the 4-d] pyrimidine (295mg, 1.13mmol) of 3- is dissolved in ethyl alcohol and the mixing of DME
In solvent 2mL in (1:3), 0.25mL saturated sodium carbonate solution and N- methyl 2- azaindole -3- pinacol borate are added
(413mg, 1.6mmol).After whole system substitutes gas three times, into mixture be added tetrakis triphenylphosphine palladium (127mg,
0.11mmol), then whole system is heated under a nitrogen atmosphere to 90 DEG C and stirred 12 hours.To after reaction, use silicon
Diatomaceous earth is filtered, and gained filtrate water and methylene chloride washing are three times.Organic phase is obtained into solid by vacuum rotary steam, and is led to
It crosses silica gel column chromatography and is separated (methylene chloride that eluent is the methanol containing 1-2%), finally obtain target compound 234mg
(0.88 mmol) is light green solid, yield: 78%.LC/MS (ESI): m/z 266 (M+H)+。
Intermediate 1- { N-Boc-2- azaspiro [3.5] nonane -7- base } -4- amino -3- (1- methyl-1 H- indazole -3-
Base) -1H- pyrazoles [3,4-d] pyrimidine 3b preparation:
2b (207mg, 0.78mmol) and N-Boc-2- azaspiro [3.5] nonane -7- ((sulfonyloxy methyl is stirred at 80 DEG C
Base) oxygroup) (1eq) and potassium carbonate (2eq) DMF (3mL) suspension 16h.After reaction, with water treatment and with two
Chloromethanes extraction.Organic phase is collected, is concentrated in vacuo after dry and obtains light green target compound 190mg, yield: 50%.LC/MS
(ESI): m/z 489 (M+H)+。
Intermediate 1- { 2- azaspiro [3.5] nonane -7- base } -4- amino -3- (1- methyl-1 H- indazole -3- base) -1H-
The preparation of pyrazoles [3,4-d] pyrimidine 4b:
3b (93mg, 0.19mmol) is dissolved in 2mL dichloromethane solution, excessive trifluoroacetic acid is added, at room temperature
Mixture 2h is stirred, with methylene chloride diluted mixture, then uses in saturated sodium bicarbonate aqueous solution and washs.It collects organic
Phase is concentrated in vacuo after being dried with anhydrous sodium sulfate, and gained crude product does not have to purification and directly throws in next step.
1- { 7- [4- amino -3- (1- methyl-1 H- indazole -3- base)-pyrazoles [3,4-d] pyrimidine -1- base] -2- azaspiro
[3.5] nonane -2- base }-propenone (compound 702) preparation::
Previous step crude product 4b is dissolved in 5mL tetrahydrofuran solution, is added DIPEA (196 μ L, 1.2mmol), then
It is added dropwise acryloyl chloride (17 μ L, 0.21mmol), is stirred to react 2 hours.Fully reacting is detected through TLC.Stop reaction, DCM is added
(25mL) is then saturated NaHCO with 50mL3Washing, water layer are extracted with DCM (2*25mL), anhydrous MgSO4Dry, concentration is thick to produce
Product obtain yellow solid 48mg, two step yields: 57% by quick post separation.1H NMR(400MHz,DMSO-d6)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)+。
{ [4- amino -3- (1- methyl-1 H- pyrroles [2,3-b] pyridin-3-yl)-pyrazoles [3,4-d] is phonetic by 7- by embodiment 3:1-
Pyridine -1- base] -2- azaspiro [3.5] nonane -2- base-propenone (compound 703) preparation:
Intermediate 4- amino -3- ((1- methyl-1 H- pyrroles [2,3-b] pyridin-3-yl) -1H- pyrazoles [3,4-d] pyrimidine
The preparation of 2c:
Iodo- 4- amino -1H- pyrazoles [3, the 4-d] pyrimidine (295mg, 1.13mmol) of 3- is dissolved in ethyl alcohol and the mixing of DME
In solvent 2mL in (1:3), 0.25mL saturated sodium carbonate solution and N- methyl 2- azaindole -3- pinacol borate are added
(413mg, 1.6mmol).After whole system substitutes gas three times, into mixture be added tetrakis triphenylphosphine palladium (127mg,
0.11mmol), then whole system is heated under a nitrogen atmosphere to 90 DEG C and stirred 12 hours.To after reaction, use silicon
Diatomaceous earth is filtered, and gained filtrate water and methylene chloride washing are three times.Organic phase is obtained into solid by vacuum rotary steam, and is led to
It crosses silica gel column chromatography and is separated (methylene chloride that eluent is the methanol containing 1-2%), finally obtain target compound 234mg
(0.88 mmol) is light green solid, yield: 78%.LC/MS (ESI): m/z 266 (M+H)+。
Intermediate 1- { N-Boc-2- azaspiro [3.5] nonane -7- base } -4- amino -3- (1- methyl-1 H- pyrroles [2,3-
B] pyridin-3-yl) -1H- pyrazoles [3,4-d] pyrimidine 3c preparation:
2c (207mg, 0.78mmol) and N-Boc-2- azaspiro [3.5] nonane -7- ((sulfonyloxy methyl is stirred at 80 DEG C
Base) oxygroup) (1eq) and potassium carbonate (2eq) DMF (3mL) suspension 16h.After reaction, with water treatment and with two
Chloromethanes extraction.Organic phase is collected, is concentrated in vacuo after dry and obtains light green target compound 190mg, yield: 50%.LC/MS
(ESI): m/z 489 (M+H)+。
Intermediate 1- { 2- azaspiro [3.5] nonane -7- base } -4- amino -3- (1- methyl-1 H- pyrroles [2,3-b] pyridine
- 3- base) -1H- pyrazoles [3,4-d] pyrimidine 4c preparation:
3c (93mg, 0.19mmol) is dissolved in 2mL dichloromethane solution, excessive trifluoroacetic acid is added, at room temperature
Mixture 2h is stirred, with methylene chloride diluted mixture, then uses in saturated sodium bicarbonate aqueous solution and washs.It collects organic
Phase is concentrated in vacuo after being dried with anhydrous sodium sulfate, and gained crude product does not have to purification and directly throws in next step.
1- { 7- [4- amino -3- (1- methyl-1 H- pyrroles [2,3-b] pyridin-3-yl)-pyrazoles [3,4-d] pyrimidine -1-
Base] -2- azaspiro [3.5] nonane -2- base-propenone (compound 703) preparation::
Previous step crude product 4b is dissolved in 5mL tetrahydrofuran solution, is added DIPEA (196 μ L, 1.2mmol), then
It is added dropwise acryloyl chloride (17 μ L, 0.21mmol), is stirred to react 2 hours.Fully reacting is detected through TLC.Stop reaction, DCM is added
(25mL) is then saturated NaHCO with 50mL3Washing, water layer are extracted with DCM (2*25mL), anhydrous MgSO4Dry, concentration is thick to produce
Product obtain yellow solid 48mg, two step yields: 57% by quick post separation.1H NMR(400MHz,DMSO-d6)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)+。
Embodiment 4:1- { 6- [4- amino -3- (1- Methyl-1H-indole -3- base)-pyrazoles [3,4-d] pyrimidine -1- base] -2-
Azepine spiroheptane -2- base }-propenone (compound 704) preparation:
Intermediate 1- { N-Boc-2- azepine spiroheptane -6- base } -4- amino -3- (1- Methyl-1H-indole -3-
Base) -1H- pyrazoles [3,4-d] pyrimidine 3d preparation:
2a (206mg, 0.78mmol) and N-Boc-2- azepine spiroheptane -6- ((sulfonyloxy methyl are stirred at 80 DEG C
Base) oxygroup) (1eq) and potassium carbonate (2eq) DMF (3mL) suspension 16h.After reaction, with water treatment and with two
Chloromethanes extraction.Organic phase is collected, is concentrated in vacuo after dry and obtains light green target compound 168mg, yield: 47%.LC/MS
(ESI): m/z 460 (M+H)+。
Intermediate 1- { 2- azepine spiroheptane -6- base } -4- amino -3- (1- Methyl-1H-indole -3- base) -1H-
The preparation of pyrazoles [3,4-d] pyrimidine 4d:
3d (87mg, 0.19mmol) is dissolved in 2mL dichloromethane solution, excessive trifluoroacetic acid is added, at room temperature
Mixture 2h is stirred, with methylene chloride diluted mixture, then uses in saturated sodium bicarbonate aqueous solution and washs.It collects organic
Phase is concentrated in vacuo after being dried with anhydrous sodium sulfate, and gained crude product does not have to purification and directly throws in next step.
1- { 6- [4- amino -3- (1- Methyl-1H-indole -3- base)-pyrazoles [3,4-d] pyrimidine -1- base] -2- azaspiro
[3.3] heptane -2- base }-propenone (compound 704) preparation:
Previous step crude product 4d is dissolved in 5mL tetrahydrofuran solution, is added DIPEA (196 μ L, 1.2mmol), so
Acryloyl chloride (17 μ L, 0.21mmol) is added dropwise afterwards, is stirred to react 2 hours.Fully reacting is detected through TLC.Stop reaction, is added
DCM (25mL) is then saturated NaHCO with 50mL3Washing, water layer are extracted with DCM (2*25mL), anhydrous MgSO4It is dry, concentration, slightly
Product obtains yellow solid 46mg, two step yields: 58% by quick post separation.1H NMR(400MHz,DMSO-d6)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)+。
Embodiment 5:1- { 6- [4- amino -3- (1- methyl-1 H- indazole -3- base)-pyrazoles [3,4-d] pyrimidine -1- base] -2-
Azepine spiroheptane -2- base }-propenone (compound 705) preparation:
Intermediate 1- { N-Boc-2- azepine spiroheptane -6- base } -4- amino -3- (1- methyl-1 H- indazole -3-
Base) -1H- pyrazoles [3,4-d] pyrimidine 3e preparation:
2a (207mg, 0.78mmol) and N-Boc-2- azepine spiroheptane -6- ((sulfonyloxy methyl are stirred at 80 DEG C
Base) oxygroup) (1eq) and potassium carbonate (2eq) DMF (3mL) suspension 16h.After reaction, with water treatment and with two
Chloromethanes extraction.Organic phase is collected, is concentrated in vacuo after dry and obtains light green target compound 161mg, yield: 45%.LC/MS
(ESI): m/z 461 (M+H)+。
Intermediate 1- { 2- azepine spiroheptane -6- base } -4- amino -3- (1- methyl-1 H- indazole -3- base) -1H-
The preparation of pyrazoles [3,4-d] pyrimidine 4e:
3e (87mg, 0.19mmol) is dissolved in 2mL dichloromethane solution, excessive trifluoroacetic acid is added, at room temperature
Mixture 2h is stirred, with methylene chloride diluted mixture, then uses in saturated sodium bicarbonate aqueous solution and washs.It collects organic
Phase is concentrated in vacuo after being dried with anhydrous sodium sulfate, and gained crude product does not have to purification and directly throws in next step.
1- { 6- [4- amino -3- (1- methyl-1 H- indazole -3- base)-pyrazoles [3,4-d] pyrimidine -1- base] -2- azaspiro
[3.3] heptane -2- base }-propenone (compound 705) preparation:
Previous step crude product 4e is dissolved in 5mL tetrahydrofuran solution, is added DIPEA (196 μ L, 1.2mmol), so
Acryloyl chloride (17 μ L, 0.21mmol) is added dropwise afterwards, is stirred to react 2 hours.Fully reacting is detected through TLC.Stop reaction, is added
DCM (25mL) is then saturated NaHCO with 50mL3Washing, water layer are extracted with DCM (2*25mL), anhydrous MgSO4It is dry, concentration, slightly
Product obtains yellow solid 49mg, two step yields: 62% by quick post separation.1H NMR(400MHz,DMSO-d6)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)+。
{ [4- amino -3- (1- methyl-1 H- pyrroles [2,3-b] pyridin-3-yl)-pyrazoles [3,4-d] is phonetic by 6- by embodiment 6:1-
Pyridine -1- base] -2- azepine spiroheptane -2- base-propenone (compound 706) preparation:
Intermediate 1- { N-Boc-2- azepine spiroheptane -6- base } -4- amino -3- (1- methyl-1 H- pyrroles [2,3-
B] pyridin-3-yl) -1H- pyrazoles [3,4-d] pyrimidine 3f preparation:
2c (207mg, 0.78mmol) and N-Boc-2- azepine spiroheptane -6- ((sulfonyloxy methyl are stirred at 80 DEG C
Base) oxygroup) (1eq) and potassium carbonate (2eq) DMF (3mL) suspension 16h.After reaction, with water treatment and with two
Chloromethanes extraction.Organic phase is collected, is concentrated in vacuo after dry and obtains light green target compound 183mg, yield: 51%.LC/MS
(ESI): m/z 461 (M+H)+。
Intermediate 1- { 2- azepine spiroheptane -6- base } -4- amino -3- (1- methyl-1 H- pyrroles [2,3-b] pyridine -
3- yl) -1H- pyrazoles [3,4-d] pyrimidine 4f preparation:
3f (87mg, 0.19mmol) is dissolved in 2mL dichloromethane solution, excessive trifluoroacetic acid is added, at room temperature
Mixture 2h is stirred, with methylene chloride diluted mixture, then uses in saturated sodium bicarbonate aqueous solution and washs.It collects organic
Phase is concentrated in vacuo after being dried with anhydrous sodium sulfate, and gained crude product does not have to purification and directly throws in next step.
1- { 6- [4- amino -3- (1- methyl-1 H- pyrroles [2,3-b] pyridin-3-yl)-pyrazoles [3,4-d] pyrimidine -1-
Base] -2- azepine spiroheptane -2- base-propenone (compound 706) preparation:
Previous step crude product 4f is dissolved in 5mL tetrahydrofuran solution, is added DIPEA (196 μ L, 1.2mmol), then
It is added dropwise acryloyl chloride (17 μ L, 0.21mmol), is stirred to react 2 hours.Fully reacting is detected through TLC.Stop reaction, DCM is added
(25mL) is then saturated NaHCO with 50mL3Washing, water layer are extracted with DCM (2*25mL), anhydrous MgSO4Dry, concentration is thick to produce
Product obtain yellow solid 47mg, two step yields: 60% by quick post separation.1H NMR(400MHz,DMSO-d6)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)+。
Embodiment 7:1- { 2- [4- amino -3- (1- Methyl-1H-indole -3- base)-pyrazoles [3,4-d] pyrimidine -1- base] -7-
Azaspiro [3.5] nonane -7- base }-propenone (compound 707) preparation:
Intermediate 1- { N-Boc-7- azaspiro [3.5] nonane -2- base } -4- amino -3- (1- Methyl-1H-indole -3-
Base) -1H- pyrazoles [3,4-d] pyrimidine 3g preparation:
2a (206mg, 0.78mmol) and N-Boc-7- azaspiro [3.5] nonane -2- ((sulfonyloxy methyl is stirred at 80 DEG C
Base) oxygroup) (1eq) and potassium carbonate (2eq) DMF (3mL) suspension 16h.After reaction, with water treatment and with two
Chloromethanes extraction.Organic phase is collected, is concentrated in vacuo after dry and obtains light green target compound 182mg, yield: 48%.LC/MS
(ESI): m/z 488 (M+H)+。
Intermediate 1- { 7- azaspiro [3.5] nonane -2- base } -4- amino -3- (1- Methyl-1H-indole -3- base) -1H-
The preparation of pyrazoles [3,4-d] pyrimidine 4g:
3g (92mg, 0.19mmol) is dissolved in 2mL dichloromethane solution, excessive trifluoroacetic acid is added, at room temperature
Mixture 2h is stirred, with methylene chloride diluted mixture, then uses in saturated sodium bicarbonate aqueous solution and washs.It collects organic
Phase is concentrated in vacuo after being dried with anhydrous sodium sulfate, and gained crude product does not have to purification and directly throws in next step.
1- { 2- [4- amino -3- (1- Methyl-1H-indole -3- base)-pyrazoles [3,4-d] pyrimidine -1- base] -7- azaspiro
[3.5] nonane -7- base }-propenone (compound 707) preparation:
Previous step crude product 4g is dissolved in 5mL tetrahydrofuran solution, is added DIPEA (196 μ L, 1.2mmol), so
Acryloyl chloride (17 μ L, 0.21mmol) is added dropwise afterwards, is stirred to react 2 hours.Fully reacting is detected through TLC.Stop reaction, is added
DCM (25mL) is then saturated NaHCO with 50mL3Washing, water layer are extracted with DCM (2*25mL), anhydrous MgSO4It is dry, concentration, slightly
Product obtains yellow solid 53mg, two step yields: 63% by quick post separation.1H NMR(400MHz,DMSO-d6)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)+。
Embodiment 8:1- { 2- [4- amino -3- (1- methyl-1 H- indazole -3- base)-pyrazoles [3,4-d] pyrimidine -1- base] -7-
Azaspiro [3.5] nonane -7- base }-propenone (compound 708) preparation:
Intermediate 1- { N-Boc-7- azaspiro [3.5] nonane -2- base } -4- amino -3- (1- methyl-1 H- indazole -3-
Base) -1H- pyrazoles [3,4-d] pyrimidine 3h preparation:
2b (207mg, 0.78mmol) and N-Boc-7- azaspiro [3.5] nonane -2- ((sulfonyloxy methyl is stirred at 80 DEG C
Base) oxygroup) (1eq) and potassium carbonate (2eq) DMF (3mL) suspension 16h.After reaction, with water treatment and with two
Chloromethanes extraction.Organic phase is collected, is concentrated in vacuo after dry and obtains light green target compound 190mg, yield: 50%.LC/MS
(ESI): m/z 489 (M+H)+。
Intermediate 1- { 2- azaspiro [3.5] nonane -7- base } -4- amino -3- (1- methyl-1 H- indazole -3- base) -1H-
The preparation of pyrazoles [3,4-d] pyrimidine 4h:
3h (93mg, 0.19mmol) is dissolved in 2mL dichloromethane solution, excessive trifluoroacetic acid is added, at room temperature
Mixture 2h is stirred, with methylene chloride diluted mixture, then uses in saturated sodium bicarbonate aqueous solution and washs.It collects organic
Phase is concentrated in vacuo after being dried with anhydrous sodium sulfate, and gained crude product does not have to purification and directly throws in next step.
1- { 2- [4- amino -3- (1- methyl-1 H- indazole -3- base)-pyrazoles [3,4-d] pyrimidine -1- base] -7- azaspiro
[3.5] nonane -7- base }-propenone (compound 708) preparation:
Previous step crude product 4h is dissolved in 5mL tetrahydrofuran solution, is added DIPEA (196 μ L, 1.2mmol), so
Acryloyl chloride (17 μ L, 0.21mmol) is added dropwise afterwards, is stirred to react 2 hours.Fully reacting is detected through TLC.Stop reaction, is added
DCM (25mL) is then saturated NaHCO with 50mL3Washing, water layer are extracted with DCM (2*25mL), anhydrous MgSO4It is dry, concentration, slightly
Product obtains yellow solid 48mg, two step yields: 57% by quick post separation.1H NMR(400MHz,DMSO-d6)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)+。
{ [4- amino -3- (1- methyl-1 H- pyrroles [2,3-b] pyridin-3-yl)-pyrazoles [3,4-d] is phonetic by 2- by embodiment 9:1-
Pyridine -1- base] -7- azaspiro [3.5] nonane -7- base-propenone (compound 709) preparation:
Intermediate 1- { N-Boc-7- azaspiro [3.5] nonane -2- base } -4- amino -3- (1- methyl-1 H- pyrroles [2,3-
B] pyridin-3-yl) -1H- pyrazoles [3,4-d] pyrimidine 3i preparation:
2c (207mg, 0.78mmol) and N-Boc-7- azaspiro [3.5] nonane -2- ((sulfonyloxy methyl is stirred at 80 DEG C
Base) oxygroup) (1eq) and potassium carbonate (2eq) DMF (3mL) suspension 16h.After reaction, with water treatment and with two
Chloromethanes extraction.Organic phase is collected, is concentrated in vacuo after dry and obtains light green target compound 190mg, yield: 50%.LC/MS
(ESI): m/z 489 (M+H)+。
Intermediate 1- { 7- azaspiro [3.5] nonane -2- base } -4- amino -3- (1- methyl-1 H- pyrroles [2,3-b] pyridine -
3- yl) -1H- pyrazoles [3,4-d] pyrimidine 4i preparation:
3i (92mg, 0.19mmol) is dissolved in 2mL dichloromethane solution, excessive trifluoroacetic acid is added, at room temperature
Mixture 2h is stirred, with methylene chloride diluted mixture, then uses in saturated sodium bicarbonate aqueous solution and washs.It collects organic
Phase is concentrated in vacuo after being dried with anhydrous sodium sulfate, and gained crude product does not have to purification and directly throws in next step.
1- { 2- [4- amino -3- (1- methyl-1 H- pyrroles [2,3-b] pyridin-3-yl)-pyrazoles [3,4-d] pyrimidine -1-
Base] -7- azaspiro [3.5] nonane -7- base-propenone (compound 709) preparation:
Previous step crude product 4i is dissolved in 5mL tetrahydrofuran solution, is added DIPEA (196 μ L, 1.2mmol), then
It is added dropwise acryloyl chloride (17 μ L, 0.21mmol), is stirred to react 2 hours.Fully reacting is detected through TLC.Stop reaction, DCM is added
(25mL) is then saturated NaHCO with 50mL3Washing, water layer are extracted with DCM (2*25mL), anhydrous MgSO4Dry, concentration is thick to produce
Product obtain yellow solid 48mg, two step yields: 57% by quick post separation.1H NMR(400MHz,DMSO-d6)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)+。
Embodiment 10:1- { 2- [4- amino -3- (1- Methyl-1H-indole -3- base)-pyrazoles [3,4-d] pyrimidine -1- base] -
6- azaspiro [3.5] nonane -6- base }-propenone (compound 710) preparation:
Intermediate 1- { N-Boc-6- azaspiro [3.5] nonane -2- base } -4- amino -3- (1- Methyl-1H-indole -3-
Base) -1H- pyrazoles [3,4-d] pyrimidine 3j preparation:
2a (206mg, 0.78mmol) and N-Boc-6- azaspiro [3.5] nonane -2- ((sulfonyloxy methyl is stirred at 80 DEG C
Base) oxygroup) (1eq) and potassium carbonate (2eq) DMF (3mL) suspension 16h.After reaction, with water treatment and with two
Chloromethanes extraction.Organic phase is collected, is concentrated in vacuo after dry and obtains light green target compound 182mg, yield: 48%.LC/MS
(ESI): m/z 488 (M+H)+。
Intermediate 1- { 6- azaspiro [3.5] nonane -2- base } -4- amino -3- (1- Methyl-1H-indole -3- base) -1H-
The preparation of pyrazoles [3,4-d] pyrimidine 4j:
3j (92mg, 0.19mmol) is dissolved in 2mL dichloromethane solution, excessive trifluoroacetic acid is added, at room temperature
Mixture 2h is stirred, with methylene chloride diluted mixture, then uses in saturated sodium bicarbonate aqueous solution and washs.It collects organic
Phase is concentrated in vacuo after being dried with anhydrous sodium sulfate, and gained crude product does not have to purification and directly throws in next step.
1- { 2- [4- amino -3- (1- Methyl-1H-indole -3- base)-pyrazoles [3,4-d] pyrimidine -1- base] -6- azaspiro
[3.5] nonane -6- base }-propenone (compound 710) preparation:
Previous step crude product 4j is dissolved in 5mL tetrahydrofuran solution, is added DIPEA (196 μ L, 1.2mmol), so
Acryloyl chloride (17 μ L, 0.21mmol) is added dropwise afterwards, is stirred to react 2 hours.Fully reacting is detected through TLC.Stop reaction, is added
DCM (25mL) is then saturated NaHCO with 50mL3Washing, water layer are extracted with DCM (2*25mL), anhydrous MgSO4It is dry, concentration, slightly
Product obtains yellow solid 53mg, two step yields: 63% by quick post separation.1H NMR(400MHz,DMSO-d6)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)+。
Embodiment 11:1- { 2- [4- amino -3- (1- methyl-1 H- indazole -3- base)-pyrazoles [3,4-d] pyrimidine -1- base] -
6- azaspiro [3.5] nonane -6- base }-propenone (compound 711) preparation:
Intermediate 1- { N-Boc-6- azaspiro [3.5] nonane -2- base } -4- amino -3- (1- methyl-1 H- indazole -3-
Base) -1H- pyrazoles [3,4-d] pyrimidine 3k preparation:
2b (207mg, 0.78mmol) and N-Boc-6- azaspiro [3.5] nonane -2- ((sulfonyloxy methyl is stirred at 80 DEG C
Base) oxygroup) (1eq) and potassium carbonate (2eq) DMF (3mL) suspension 16h.After reaction, with water treatment and with two
Chloromethanes extraction.Organic phase is collected, is concentrated in vacuo after dry and obtains light green target compound 190mg, yield: 50%.LC/MS
(ESI): m/z 489 (M+H)+。
Intermediate 1- { 6- azaspiro [3.5] nonane -2- base } -4- amino -3- (1- methyl-1 H- indazole -3- base) -1H-
The preparation of pyrazoles [3,4-d] pyrimidine 4k:
3k (93mg, 0.19mmol) is dissolved in 2mL dichloromethane solution, excessive trifluoroacetic acid is added, at room temperature
Mixture 2h is stirred, with methylene chloride diluted mixture, then uses in saturated sodium bicarbonate aqueous solution and washs.It collects organic
Phase is concentrated in vacuo after being dried with anhydrous sodium sulfate, and gained crude product does not have to purification and directly throws in next step.
1- { 2- [4- amino -3- (1- methyl-1 H- indazole -3- base)-pyrazoles [3,4-d] pyrimidine -1- base] -6- azaspiro
[3.5] nonane -6- base }-propenone (compound 711) preparation:
Previous step crude product 4k is dissolved in 5mL tetrahydrofuran solution, is added DIPEA (196 μ L, 1.2mmol), so
Acryloyl chloride (17 μ L, 0.21mmol) is added dropwise afterwards, is stirred to react 2 hours.Fully reacting is detected through TLC.Stop reaction, is added
DCM (25mL) is then saturated NaHCO with 50mL3Washing, water layer are extracted with DCM (2*25mL), anhydrous MgSO4It is dry, concentration, slightly
Product obtains yellow solid 48mg, two step yields: 57% by quick post separation.1H NMR(400MHz,DMSO-d6)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)+。
Embodiment 12:1- { 2- [4- amino -3- (1- methyl-1 H- pyrroles [2,3-b] pyridin-3-yl)-pyrazoles [3,4-d]
Pyrimidine -1- base] -6- azaspiro [3.5] nonane -6- base-propenone (compound 712) preparation:
Intermediate 1- { N-Boc-6- azaspiro [3.5] nonane -2- base } -4- amino -3- (1- methyl-1 H- pyrroles [2,3-
B] pyridin-3-yl) -1H- pyrazoles [3,4-d] pyrimidine 3l preparation:
2c (207mg, 0.78mmol) and N-Boc-6- azaspiro [3.5] nonane -2- ((sulfonyloxy methyl is stirred at 80 DEG C
Base) oxygroup) (1eq) and potassium carbonate (2eq) DMF (3mL) suspension 16h.After reaction, with water treatment and with two
Chloromethanes extraction.Organic phase is collected, is concentrated in vacuo after dry and obtains light green target compound 190mg, yield: 50%.LC/MS
(ESI): m/z 489 (M+H)+。
Intermediate 1- { 6- azaspiro [3.5] nonane -2- base } -4- amino -3- (1- methyl-1 H- pyrroles [2,3-b] pyridine -
3- yl) -1H- pyrazoles [3,4-d] pyrimidine 4l preparation:
3l (92mg, 0.19mmol) is dissolved in 2mL dichloromethane solution, excessive trifluoroacetic acid is added, at room temperature
Mixture 2h is stirred, with methylene chloride diluted mixture, then uses in saturated sodium bicarbonate aqueous solution and washs.It collects organic
Phase is concentrated in vacuo after being dried with anhydrous sodium sulfate, and gained crude product does not have to purification and directly throws in next step.
1- { 2- [4- amino -3- (1- methyl-1 H- pyrroles [2,3-b] pyridin-3-yl)-pyrazoles [3,4-d] pyrimidine -1-
Base] -6- azaspiro [3.5] nonane -2- base-propenone (compound 712) preparation:
Previous step crude product 4l is dissolved in 5mL tetrahydrofuran solution, is added DIPEA (196 μ L, 1.2mmol), so
Acryloyl chloride (17 μ L, 0.21mmol) is added dropwise afterwards, is stirred to react 2 hours.Fully reacting is detected through TLC.Stop reaction, is added
DCM (25mL) is then saturated NaHCO with 50mL3Washing, water layer are extracted with DCM (2*25mL), anhydrous MgSO4It is dry, concentration, slightly
Product obtains yellow solid 48mg, two step yields: 57% by quick post separation.1H NMR(400MHz,DMSO-d6)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)+。
Embodiment 13:1- { 2- [4- amino -3- (1- Methyl-1H-indole -3- base)-pyrazoles [3,4-d] pyrimidine -1- base] -
6- azaspiro [3.4] octane -6- base }-propenone (compound 713) preparation:
Intermediate 1- { N-Boc-6- azaspiro [3.4] octane -2- base } -4- amino -3- (1- Methyl-1H-indole -3-
Base) -1H- pyrazoles [3,4-d] pyrimidine 3m preparation:
2a (206mg, 0.78mmol) and N-Boc-6- azaspiro [3.4] octane -2- ((sulfonyloxy methyl is stirred at 80 DEG C
Base) oxygroup) (1eq) and potassium carbonate (2eq) DMF (3mL) suspension 16h.After reaction, with water treatment and with two
Chloromethanes extraction.Organic phase is collected, is concentrated in vacuo after dry and obtains light green target compound 177mg, yield: 48%.LC/MS
(ESI): m/z 474 (M+H)+。
Intermediate 1- { 6- azaspiro [3.4] octane -2- base } -4- amino -3- (1- Methyl-1H-indole -3- base) -1H-
The preparation of pyrazoles [3,4-d] pyrimidine 4m:
3m (90mg, 0.19mmol) is dissolved in 2mL dichloromethane solution, excessive trifluoroacetic acid is added, at room temperature
Mixture 2h is stirred, with methylene chloride diluted mixture, then uses in saturated sodium bicarbonate aqueous solution and washs.It collects organic
Phase is concentrated in vacuo after being dried with anhydrous sodium sulfate, and gained crude product does not have to purification and directly throws in next step.
1- { 2- [4- amino -3- (1- Methyl-1H-indole -3- base)-pyrazoles [3,4-d] pyrimidine -1- base] -6- azaspiro
[3.4] octane -6- base }-propenone (compound 713) preparation:
Previous step crude product 4m is dissolved in 5mL tetrahydrofuran solution, is added DIPEA (196 μ L, 1.2mmol), so
Acryloyl chloride (17 μ L, 0.21mmol) is added dropwise afterwards, is stirred to react 2 hours.Fully reacting is detected through TLC.Stop reaction, is added
DCM (25mL) is then saturated NaHCO with 50mL3Washing, water layer are extracted with DCM (2*25mL), anhydrous MgSO4It is dry, concentration, slightly
Product obtains yellow solid 51mg, two step yields: 63% by quick post separation.1H NMR(400MHz,DMSO-d6)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)+。
Embodiment 14:1- { 2- [4- amino -3- (1- methyl-1 H- indazole -3- base)-pyrazoles [3,4-d] pyrimidine -1- base] -
6- azaspiro [3.4] octane -6- base }-propenone (compound 714) preparation:
Intermediate 1- { N-Boc-6- azaspiro [3.4] octane -2- base } -4- amino -3- (1- methyl-1 H- indazole -3-
Base) -1H- pyrazoles [3,4-d] pyrimidine 3n preparation:
2b (207mg, 0.78mmol) and N-Boc-6- azaspiro [3.4] octane -2- ((sulfonyloxy methyl is stirred at 80 DEG C
Base) oxygroup) (1eq) and potassium carbonate (2eq) DMF (3mL) suspension 16h.After reaction, with water treatment and with two
Chloromethanes extraction.Organic phase is collected, is concentrated in vacuo after dry and obtains light green target compound 185mg, yield: 50%.LC/MS
(ESI): m/z 475 (M+H)+。
Intermediate 1- { 6- azaspiro [3.4] octane -2- base } -4- amino -3- (1- methyl-1 H- indazole -3- base) -1H-
The preparation of pyrazoles [3,4-d] pyrimidine 4n:
3n (90mg, 0.19mmol) is dissolved in 2mL dichloromethane solution, excessive trifluoroacetic acid is added, at room temperature
Mixture 2h is stirred, with methylene chloride diluted mixture, then uses in saturated sodium bicarbonate aqueous solution and washs.It collects organic
Phase is concentrated in vacuo after being dried with anhydrous sodium sulfate, and gained crude product does not have to purification and directly throws in next step.
1- { 2- [4- amino -3- (1- methyl-1 H- indazole -3- base)-pyrazoles [3,4-d] pyrimidine -1- base] -6- azaspiro
[3.4] octane -6- base }-propenone (compound 714) preparation:
Previous step crude product 4n is dissolved in 5mL tetrahydrofuran solution, is added DIPEA (196 μ L, 1.2mmol), so
Acryloyl chloride (17 μ L, 0.21mmol) is added dropwise afterwards, is stirred to react 2 hours.Fully reacting is detected through TLC.Stop reaction, is added
DCM (25mL) is then saturated NaHCO with 50mL3Washing, water layer are extracted with DCM (2*25mL), anhydrous MgSO4It is dry, concentration, slightly
Product obtains yellow solid 46mg, two step yields: 57% by quick post separation.1H NMR(400MHz,DMSO-d6)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)+。
Embodiment 15:1- { 2- [4- amino -3- (1- methyl-1 H- pyrroles [2,3-b] pyridin-3-yl)-pyrazoles [3,4-d]
Pyrimidine -1- base] -6- azaspiro [3.4] octane -6- base-propenone (compound 715) preparation:
Intermediate 1- { N-Boc-6- azaspiro [3.4] octane -2- base } -4- amino -3- (1- methyl-1 H- pyrroles [2,3-
B] pyridin-3-yl) -1H- pyrazoles [3,4-d] pyrimidine 3o preparation:
2c (207mg, 0.78mmol) and N-Boc-6- azaspiro [3.4] octane -2- ((sulfonyloxy methyl is stirred at 80 DEG C
Base) oxygroup) (1eq) and potassium carbonate (2eq) DMF (3mL) suspension 16h.After reaction, with water treatment and with two
Chloromethanes extraction.Organic phase is collected, is concentrated in vacuo after dry and obtains light green target compound 185mg, yield: 50%.LC/MS
(ESI): m/z 475 (M+H)+。
Intermediate 1- { 6- azaspiro [3.4] octane -2- base } -4- amino -3- (1- methyl-1 H- pyrroles [2,3-b] pyridine -
3- yl) -1H- pyrazoles [3,4-d] pyrimidine 4o preparation:
3o (90mg, 0.19mmol) is dissolved in 2mL dichloromethane solution, excessive trifluoroacetic acid is added, at room temperature
Mixture 2h is stirred, with methylene chloride diluted mixture, then uses in saturated sodium bicarbonate aqueous solution and washs.It collects organic
Phase is concentrated in vacuo after being dried with anhydrous sodium sulfate, and gained crude product does not have to purification and directly throws in next step.
1- { 2- [4- amino -3- (1- methyl-1 H- pyrroles [2,3-b] pyridin-3-yl)-pyrazoles [3,4-d] pyrimidine -1-
Base] -6- azaspiro [3.4] octane -2- base-propenone (compound 715) preparation:
Previous step crude product 4o is dissolved in 5mL tetrahydrofuran solution, is added DIPEA (196 μ L, 1.2mmol), so
Acryloyl chloride (17 μ L, 0.21mmol) is added dropwise afterwards, is stirred to react 2 hours.Fully reacting is detected through TLC.Stop reaction, is added
DCM (25mL) is then saturated NaHCO with 50mL3Washing, water layer are extracted with DCM (2*25mL), anhydrous MgSO4It is dry, concentration, slightly
Product obtains yellow solid 46mg, two step yields: 57% by quick post separation.1H NMR(400MHz,DMSO-d6)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)+。
The measurement of embodiment 16:EGFR kinase inhibiting activity and relevant mutational site Percentage bound
To compound 711-715 made from above-described embodiment 1-15, aforesaid compound pair is measured using FRET technology
EGFR kinase inhibiting activity, the inhibitory activity use IC50This index is to expression, IC50That is the activity inhibited of EGFR kinases
The concentration of compound when 50%.
Simultaneously using TR-FRET technology to compound 711-'s 715 made from the compounds of this invention embodiment 1-15
EGFR-T790M kinase activity inhibiting effect is measured, and also uses IC50This index is to expression.Using Life
Technologies company Z '-LYTETMKinase Assay Kits (PV3193), and select in Tyrosine 4Peptide
EGFR-T790M kinases solution carry out active determination test.Measurement result is shown in Table 2
The EGFR inhibitory activity and EGFR of 2 embodiment compound of tableT790MPercentage bound measurement
| EGFR | EGFRT790M | |
| Sample number into spectrum | IC50(nM) | IC50(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 |
Embodiment 17: chemical combination of the present invention measures (mtt assay detection) to the inhibitory activity of cancer cell line
Cell strain: Non-small cell lung carcinoma adenocarcinoma cell strain NCI-H1975 (EGFR-T790M high expression), people's non-small cell
Pneumonocyte strain A549 (EFGR high expression).
Method: it is double that cell strain NCI-H1975 and A549 are placed in 20%FBS (fetal calf serum) (Gibco)+1640+1%
It is anti-to be cultivated.Then the good NCI-H1975 cell of growth conditions is taken, 5000/hole is inoculated in 96 porocyte plates respectively, sets
Hatch to educate in 37 DEG C, the incubator containing 5%CO2 makes cell adherent completely for 24 hours.Old culture solution is discarded, every hole sequentially adds 100
The culture solution of μ L untested compound Han 0.3,1,3,10,30,100,300,1000,3000 and 10000nmol/L, solvent control
The every hole of group is added 100 culture solutions of the μ L containing 0.1%DMSO, every group of 3 multiple holes, old culture solution is discarded after 72h, under the conditions of being protected from light
Every hole is added 100 μ L and contains 0.5mgmL-1The culture solution of MTT is placed in cell incubator and continues to be incubated for 4h, discards supernatant, every hole
100 μ LDMSO are added, vibrates, measures each hole absorbance value under 492nm wavelength with microplate reader.According to each compound various concentration
To the inhibiting rate of detailed intracellular growth, the IC in following table is obtained through conversion50(nM)。
3 compound of table acts on lung cancer cell line
| NCI-H1975 | A549 | |
| Sample number into spectrum | IC50(nM) | IC50(nM) |
| 710 | 28 | 1.8 |
| 704 | 36 | 2.1 |
Claims (10)
1. one kind is with the Pyrazolopyrimidine derivative egf inhibitor of general structure (I) and pharmaceutically acceptable
Salt
Wherein:It can be following any
It can be for such as flowering structure:
R1Can be following structure:
Wherein R2, R3And R4It respectively can be H, C1-12Alkyl, C1-12Miscellaneous alkyl, C1-12Heterocyclylalkyl, C1-12Alkenyl, C1-12Alkynyl,
C1-12Naphthenic base.
2. Pyrazolopyrimidine derivative egf inhibitor according to claim 1 and pharmaceutically acceptable
Salt, which is characterized in that the compound of the general structure (I) includes one kind of number 701-715
3. a kind of Pyrazolopyrimidine derivative egf inhibitor described in claims 1 or 2 and pharmaceutically of preparing
The method of acceptable salt.It is characterized by comprising following steps: starting material obtains among key through Suzuki coupling reaction
Body 2, parental materials respectively occur with dicyclic compound for intermediate 2, acylated, finally obtain chemical combination shown in logical formula (I) accordingly
Object
4. a kind of pharmaceutical composition, which is characterized in that include Pyrazolopyrimidine derivative epidermis described in claims 1 or 2
Growth factor receptor inhibitors and pharmaceutically acceptable salt and pharmaceutically acceptable carrier.
5. pharmaceutical composition according to claim 4, which is characterized in that described pharmaceutical composition is tablet, capsule, particle
The form of agent, spray or injection.
6. pharmaceutical composition according to claim 4, which is characterized in that the pharmaceutically acceptable carrier is selected from filling
One of agent, disintegrating agent, adhesive and lubricant are a variety of.
7. claim 1 and Pyrazolopyrimidine derivative egf inhibitor described in 2 and pharmaceutically acceptable
Purposes of the salt as protein tyrosine kinase inhibitor.
8. purposes according to claim 7, it is characterised in that: the protein tyrosine kinase inhibitor is epidermal growth factor
Sub- acceptor inhibitor.
9. claim 1 and pyrazolopyrimidine egf inhibitor and pharmaceutically acceptable salt described in 2 or
Pharmaceutical composition described in any one of person's claim 4 to 6 is in preparation for treating EGF-R ELISA overexpression
Purposes in the drug of related disease.
10. purposes according to claim 9, it is characterised in that: the EGF-R ELISA over-expresses related disease
Disease is selected from one of kidney, lung cancer, prostate cancer, cancer of pancreas, breast cancer and spongiocytoma or a variety of.
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