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CN106946896B - Furans simultaneously [2,3-d] pyrimidine -4- amine derivative - Google Patents

Furans simultaneously [2,3-d] pyrimidine -4- amine derivative Download PDF

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CN106946896B
CN106946896B CN201710204740.0A CN201710204740A CN106946896B CN 106946896 B CN106946896 B CN 106946896B CN 201710204740 A CN201710204740 A CN 201710204740A CN 106946896 B CN106946896 B CN 106946896B
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CN106946896A (en
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王康敏
赵刚
刘继峰
蒲林
陈伟
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Chengdu Zhipulai Biomedicine Technology Co Ltd
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Abstract

The invention discloses a kind of formula (I) compound represented or its stereoisomers or its pharmaceutically acceptable salt or its solvate.The present invention also provides aforesaid compounds to prepare the purposes in anti-tumor drug, angiogenesis inhibitors, EFGR kinase inhibitor or AUR A kinase inhibitor.

Description

Furo [2,3-d ] pyrimidin-4-amine derivatives
Technical Field
The present invention relates to furo [2,3-d ] pyrimidin-4-amine derivatives.
Background
Along with the increasingly worsening of the living environment of people and the continuous improvement of mental stress, the number of tumor patients increases year by year, but the traditional commonly used chemical drugs for treating tumors have poor selectivity, strong toxic and side effects and serious drug resistance problem, and can not meet the treatment requirements far away. Therefore, the research and development of new antitumor drugs with high efficiency and low toxicity are very important in the current drug research and development field.
In recent years, with the development of molecular biology technology and the further understanding of pathogenesis from cell and molecular level, the discovery of high-efficiency and low-toxicity anticancer drugs by taking key enzymes in the signal path for tumor development as targets has become an important research direction. The molecular target treatment of tumor is different from traditional tumor treatment method, and is based on the research of tumor molecular biology, and the key pathway for tumor growth adopts small molecular compound to block its key enzyme, so as to achieve the effect of blocking tumor cell growth. The molecular targeting therapy has better selectivity and reduces the damage to normal tissues, which is difficult to realize by the traditional chemical drug therapy.
Among the numerous anti-tumor drug targets, Epidermal Growth Factor Receptor (EGFR), a protein tyrosine kinase (RTK) receptor, is an important factor in the regulation of signaling pathways for cell growth, proliferation, survival and migration. In tumor tissues, nrPTKs are often activated, and then downstream signaling pathways are activated, promoting cell proliferation, resisting apoptosis, and promoting tumor development and development [ summmy, j.m.; gallick, g.e.clin.cancer rev.2006,12,1398.]. the most common activating mutations are represented by L858R, single point substitution of exon 21 and deletion of exon 19 (del e746-a 750). The first generation of reversible epidermal growth factor receptor inhibitors gefitinib and erlotinib. Significant clinical responses (50-80%) were shown in patients with these specific activating mutations in the body. However, patients who develop secondary resistance mutations to these drugs suffer recurrence of the cancer within months. Second generation epidermal growth factor receptor inhibitors include neratinib, dacatinib, afatinib, all of which contain electrophilic groups in the Michael-receptor structure. 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 receptor to perform michael addition reaction with the catalytic site (nucleophilic sulfhydryl) of cysteine residue (Cys797) on EGFR, so that kinase is inactivated, and the activity of tyrosine kinase is irreversibly inhibited, thereby having good tolerance. The presence of these covalent bonds was further demonstrated by extensive spectroscopic analysis and afatinib was found to strongly inhibit these enzymes by acting with Cys805 of HER2 and Cys803 of HER 4. In vitro tests on wild-type EGFR show that afatinib has better effects on the inhibition of the wild-type EGFR and the L858R/T790M double mutant compared with gefitinib, erlotinib and lapatinib. In addition, the inhibition effect of afatinib on HER4 is 30 times higher than that of lapatinib, 300 times higher than that of gefitinib, and 500 times higher than that of erlotinib.
Although the curative effect of afatinib is better than that of gefitinib, erlotinib and the like, the adverse reaction is improved. Afatinib has many adverse reactions, wherein diarrhea, rash, oral inflammation, paronychia, appetite decrease, epistaxis, pruritus and dry skin are common, dehydration, taste change, cystitis, cheilitis, fever, nasal obstruction, hypokalemia, conjunctivitis, transaminase increase, hand-foot syndrome, muscle spasm and kidney injury are common, and keratitis and pneumonia are occasionally seen.
Disclosure of Invention
In order to solve the above problems, the present invention provides a compound represented by formula (i), or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, or a solvate thereof:
wherein,
R1selected from the group A, R2Selected from group B groups; or, R1Selected from groups B, R2Selected from group A groups;
the group A is selected from phenyl or heteroaryl, wherein the phenyl or heteroaryl are each independently optionally further substituted by halogen, C1-C6Alkyl of (C)1-C6Alkoxy group of (C)1-C6Substituted with haloalkyl groups of (a);
the group B is selected from
Wherein m is 0 or 1, n is 0 or 1, and m and n are not 0 at the same time;
x representsThe phenyl end of X is linked to the furan ring of the pyrimidofuran ring;
y representsWhen m is 0, the phenyl end of Y is attached to the furan ring of the pyrimidofuran ring; when m is not 0, the phenyl end of Y is linked to X;
z is selected from hydrogen, halogen, aryl or-N (R)a)(Rb)；
RaAnd RbAre each independently selected from C1-C6Alkyl groups of (a); or, RaAnd RbTogether with the nitrogen atom to which they are attached form a saturated heterocyclic group optionally further substituted by hydroxy or C1-C6Alkyl of (a);
R3、R4each independently selected from hydrogen, phenyl or-C (O) Rc, wherein said phenyl is optionally further substituted with one or more halogens, RCIs selected from C1-C6Alkyl groups of (a);
R5selected from hydrogen or-SO2-RdWherein R isdIs selected from C1-C6Alkyl group of (1).
Further, R3And R4And is also hydrogen.
Further, R5Is hydrogen.
Further, the compound is represented by the following formula (Ia):
further, when the group a is selected from heteroaryl, the heteroaryl is a 5-or 6-membered ring.
Further, the heteroaryl group is selected from pyridyl or pyrrolyl.
Further, when the group A is selected from substituted phenyl, the substituents are selected from fluoro, chloro, bromo, C1-C3Any one or more of alkyl, methoxy, trifluoromethyl.
Further, in the structure represented by X or Y, the substituent on the phenyl group is located at the 3-position or 4-position of the phenyl group.
Further, when the group A is selected from substituted phenyl or substituted heteroaryl, the number of the substituent groups is 1-3.
Further, when Z is selected from aryl, the aryl is phenyl, amino-substituted phenyl or nitro-substituted phenyl.
Further, when R isaAnd RbTaken together with the nitrogen atom to which they are attached to form a saturated heterocyclic group, the saturated heterocyclic group is a 6-membered ring.
Further, the saturated heterocyclic group is a piperidyl group, a morpholinyl group or a piperazinyl group.
Further, when R isaAnd RbWhen taken together with the atoms to which they are attached to form a saturated heterocyclic group, the saturated heterocyclic group is further substituted with a hydroxyl group, C1-C6or-NH-Pg, wherein Pg represents an amino protecting group.
Further, the compound is one of the following compounds:
the invention also provides application of the compound, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, or a solvate thereof in preparation of antitumor drugs, angiogenesis inhibitors, EFGR kinase inhibitors or AUR A kinase inhibitors.
Further, the tumor is liver cancer, lung cancer, glioma, astrocytoma, cervical cancer, colon cancer or breast cancer.
In the invention:
the "stereoisomers" include the presence of stereocenters (e.g., carbons with 4 different substituents), axial asymmetries such as critical, planar asymmetries, and mixtures thereof. Stereoisomers include enantiomers, diastereomers, epimers, racemates and meso compounds with an internal symmetry plane.
By "pharmaceutically acceptable" is meant that the carrier, cargo, diluent, excipient, and/or salt formed is generally chemically or physically compatible with the other ingredients comprising a pharmaceutical dosage form and physiologically compatible with the recipient.
Said C is1-C6Alkyl of (A) means C1、C2、C3、C4、C5、C6The alkyl group of (1) is a straight or branched alkyl group having 1 to 6 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, sec-butyl, pentyl, hexyl, and the like.
An "amino protecting group" will refer to a group that can be attached to a nitrogen atom on an amino group to protect the amino group from reaction and which can be easily removed in a subsequent reaction. Suitable amino protecting groups include, but are not limited to, the following:
a carbamate group of the formula-C (O) O-R, wherein R is, for example, methyl, ethyl, tert-butyl, benzyl, phenethyl, CH2＝CH-CH2-, etc.; amide groups of the formula-C (O) -R ', wherein R' is for example methyl,Ethyl, phenyl, trifluoromethyl, and the like; formula-SO2The N-sulfonyl derivative-group of-R ', wherein R' is, for example, tolyl, phenyl, trifluoromethyl, 2, 5, 7, 8-pentamethylchroman-6-yl-, 2,3, 6-trimethyl-4-methoxybenzene, and the like.
Test results show that the compound can be used for preparing antitumor drugs, angiogenesis inhibitors or EFGR kinase inhibitors.
Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.
The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.
Drawings
FIG. 1 shows the results of the test of inhibitory activity of the compounds of the present invention on liver cancer HepG2 cells, as shown in FIG. 1.
FIG. 2 shows the results of the test of the lung cancer A549 cell inhibitory activity of the compounds of the present invention in FIG. 2
Detailed Description
The compound and the intermediate are mainly prepared according to the following routes:
the first scheme is as follows:
for the synthesis of intermediates I and II, reference is made to Bioorganic & Medicinal Chemistry Letters15(2005) 2203-2207.
Synthesis of intermediate (E) -4-bromobut-2-enoyl chloride III:
methyl (E) -4-bromocrotonate (6g, 33.5mmol) was dissolved in THF (60mL) and cooled to 0 deg.C, and an aqueous solution (20mL) of lithium hydroxide monohydrate (1.83g, 43.6mmol) was added dropwise with protection of N2. After 15min, stirring at 0 ℃ for 3 h. Cold water (150mL) and petroleum ether (200mL) were then added to the system and stirring was continued at 0 ℃ for 10 min. The aqueous phase was separated, adjusted to pH 1 with concentrated hydrochloric acid at 0 ℃ and extracted with dichloromethane (80mLx 3). The combined organic phases were dried over anhydrous sodium sulfate and concentrated to give E-4-bromocrotonic acid III as a yellow solid (4.5g, yield 82%).
Oxalyl chloride (250mg, 1.96mmol) was added dropwise to a solution of E-4-bromocrotonic acid III in 1 drop of DMF in DCM (3 mL). After the dropwise addition, the temperature was raised to room temperature and stirred for 1 hour. After the reaction is finished, the solvent is concentrated to obtain an intermediate (E) -4-bromobut-2-enoyl chloride IV which is directly used for the next reaction without further treatment.
Scheme II:
the third scheme is as follows:
the intermediate XII can be synthesized according to WO2005121149A 1.
And the scheme is as follows:
scheme five
EXAMPLE 1 preparation of Compound C-14
Intermediate I (1g,3.01mmol) was dissolved in THF, triethylamine (0.6g,6.02 mmol) was added, and chloroacetyl chloride (0.37g,3.31mmol) was added dropwise. After the reaction was completed by liquid phase monitoring, 3 drops of water were added dropwise to quench the reaction, and the solvent was concentrated. Water was added to the residue, extracted with dichloromethane, and the organic phases were combined. The organic phase was washed with saturated ammonium chloride, dried over anhydrous sodium sulfate and the solvent was concentrated. The residue was subjected to column chromatography to give 0.88g of the compound as a white solid (yield 72%).
1H NMR(400MHz，CDCl3)δppm:3.80(s,3H),4.25(s,2H),6.81-6.83(d，J＝9.2Hz,2H),7.46-7.51(m，4H)，7.73-7.76(d，J＝7.6Hz,1H)，8.37(s，1H)，8.3,9(s，1H)。
EXAMPLE 2 preparation of Compound C-13
Potassium carbonate (0.89g,6.47mmol) was added to compound c-14(0.88g, 2.15mmol) in acetonitrile followed by dimethylamine hydrochloride (0.17g,2.15mmol) and the reaction monitored by TLC and stirred for 5 h. After the reaction, the solid was washed with a small amount of acetonitrile and the mother liquor was concentrated. The residue was subjected to column chromatography to obtain 0.55g of a compound (yield 61%).
1HNMR(DMSO)δ:9.33(s,1H),8.36(s,1H),7.76-7.78(m，2H),7.45-7.49(m，4H),6.80-6.84(m,2H),4.93(s,2H),3.80(s,3H),3.14(s,2H),2.43(s,6H)；
EXAMPLE 3 preparation of Compound C-11
The preparation was carried out as described in example 2 starting from c-14 and N-methylpiperazine and giving a white solid in 60% yield.1HNMR(DMSO)δ:9.33(s,1H),8.36(s,1H),7.74-7.76(m，2H),7.46-7.52(m，4H),6.81-6.83(m,2H),4.92(s,2H),3.80(s,3H),3.20(s,2H),2.51-2.71(m,8H),2.35(s,3H)；
EXAMPLE 4 preparation of Compound C-5
0.5g (1.08mmol) of intermediate I is dissolved in THF, 0.22g (2.18mmol) of triethylamine is added, and then 0.195g (1.08mmol) of the (E) -4-bromobut-2-enoyl chloride IV prepared above is added dropwise. After the reaction was completed by liquid phase monitoring, the solvent was concentrated. Water was added to the residue, extracted with dichloromethane, and the organic phases were combined. The organic phase was washed with saturated ammonium chloride, dried over anhydrous sodium sulfate and the solvent was concentrated to give intermediate IV as a white solid which was used directly in the next step without further treatment.
Potassium carbonate 0.60g (4.32mmol) was added to a solution of intermediate V prepared as described above in acetonitrile followed by dimethylamine hydrochloride 0.089g (1.08mmol) and the reaction monitored by tlc and stirred for 5 h. After the reaction, the solid was washed with a small amount of acetonitrile and the mother liquor was concentrated. The residue was subjected to column chromatography (dichloromethane/methanol as eluent) to give 0.40g of a white solid (c-5) in 64% yield.
1H NMR(400MHz，DMSO-d6)δppm:2.45(s,6H),3.44(s,2H),3.75(s,3H),6.38-6.42(d，J＝15.2Hz,1H)，6.75-6.79(m，1H),6.93-6.95(d，J＝8.8Hz,1H),7.37-7.39(d，J＝8.8Hz,2H),7.43-7.45(d，J＝8.4Hz,2H)，7.84-7.86(d,J＝8.8Hz，2H)，8.23(s，1H),10.40(s，1H)。
EXAMPLE 5 preparation of Compound C-1
The preparation method refers to the synthesis of intermediate V in example 4, the raw materials are intermediate I and acryloyl chloride, the product is white solid, and the yield is 72%.
1H NMR(400MHz，DMSO-d6)δppm:3.80(s,3H),4.92(s,2H),5.84-5.87(dd，J＝10.0Hz,1.2Hz,1H)，6.31-6.32(dd，J＝16.8Hz,10.0Hz,1H),6.47(d，J＝2.0Hz,1H),6.49-6.51(dd，J＝16.8Hz,1.2Hz,1H),6.80-6.83(d，J＝12.0Hz，2H),7.42(s，1H),7.46-7.50(m，4H)，7.76-7.78(d,J＝8.8Hz，2H)，8.36(s，1H)。
EXAMPLE 6 preparation of Compound C-6
The preparation was as described in example 4, starting from intermediate II and piperidine, and the product was a white solid in 59% yield.
1H NMR(400MHz，CDCl3)δppm:1.47(m,2H),1.61-1.64(m,4H),2.45(s,4H),3.17-3.19(dd,J＝5.6Hz,2H),3.80(s,3H),4.90(s,2H),6.13-6.17(d，J＝15.2Hz,1H)，6.80-6.82(d，J＝8.0Hz,2H),6.99-7.07(m，1H),7.36(s，1H),7.45-7.49(m，4H)，7.73-7.75(d,J＝8.4Hz，2H)，8.36(s，1H)。
EXAMPLE 7 preparation of Compound C-4
The preparation method refers to the synthesis of intermediate V in example 4, the raw materials are intermediate II and acryloyl chloride, the product is white solid, and the yield is 66%.
1H NMR(400MHz，CDCl3)δppm:3.80(s,3H),5.17(s,2H),5.81-5.84(d，J＝11.2Hz,1H)，6.23-6.29(dd，J＝16.8Hz,10.0Hz,1H),6.44-6.48(d，J＝16.8Hz,1H),6.81-6.83(d，J＝8.8Hz,2H),7.24(s，1H),7.42(s，1H),7.44-7.51(m，4H)，7.65(s,1H)，7.74-7.76(d，J＝7.6Hz,1H)，8.35(s，1H)。
EXAMPLE 8 preparation of Compound C-7
The preparation was as described in example 4, starting from intermediate II and N-methylpiperazine, and the product was a white solid in 63% yield.
1H NMR(400MHz，DMSO-d6)δppm:2.15bs,3H),2.34(s,6H),3.10-3.11(d，J＝4.8Hz,2H)，3.75(s,3H),6.24-6.28(d，J＝15.6Hz,1H)，6.68-6.74(m，1H),6.93-6.95(d，J＝9.2Hz,2H),7.15-7.17(d，J＝7.6Hz,1H),7.38-7.41(d，J＝9.2Hz,2H)，7.48-7.52(d，J＝8.4Hz,7.6Hz,1H)，7.78-7.80(m,2H)，8.24(s，1H),10.25(s，1H)。
EXAMPLE 9 preparation of Compound C-6
The preparation was as described in example 4, starting from intermediate II and dimethylamine, and the product was a white solid in 58% yield.
1H NMR(400MHz，DMSO-d6)δppm:2.72(s,6H),3.75(s,3H),3.87-3.88(d,J＝5.2Hz,2H),6.47-6.51(d，J＝15.2Hz,1H)，6.75-6.80(m，1H),6.93-6.95(d，J＝9.2Hz,2H),7.19-7.21(d，J＝7.6Hz,1H),7.38-7.41(d，J＝9.2Hz,2H)，7.51-7.55(t，J＝8.0Hz,1H)，7.82-7.85(m，2H)，8.25(s，1H),10.67(s，1H)。
EXAMPLE 10 preparation of Compound C-16
The preparation was as described in example 4, starting from intermediate II and piperidine, and the product was a white solid in 58% yield.
1H NMR(400MHz，DMSO-d6)δppm:1.38-1.52(s,6H),2.34(s,4H),3.10-3.11(d，J＝4.8Hz,2H)，3.75(s,3H),6.23-6.27(d，J＝15.2Hz,1H)，6.68-6.72(m，1H),6.93-6.95(d，J＝8.8Hz,2H),7.15-7.17(d，J＝7.6Hz,1H),7.39-7.41(d，J＝8.8Hz,2H)，7.48-7.52(d，J＝8.4Hz,8.0Hz,1H)，7.79(s,2H)，8.25(s，1H),10.25(s，1H)。
EXAMPLE 11 preparation of Compound C-18
The preparation was as described in example 4, starting from intermediate II and morpholine, the product being a white solid in 60% yield.
1H NMR(400MHz，DMSO-d6)δppm:2.40(s,6H),2.34(s,2H),3.10-3.11(t，J＝4.4Hz,4H)，3.74(s,3H),6.31-6.35(d，J＝15.6Hz,1H)，6.74-6.79(m，1H),6.92-6.94(d，J＝8.8Hz,2H),7.37-7.38(d，J＝9.2Hz,2H),7.42-7.44(d，J＝8.4Hz,2H)，7.83-7.85(d，J＝8.8Hz,2H)，8.23(s,2H)，10.30(s，1H)。
EXAMPLE 12 preparation of Compound C-21
The preparation was as described in example 4, starting from intermediate I and morpholine, the product being a white solid in 61% yield.
1H NMR(400MHz，DMSO-d6)δppm:10.31(s,1H),8.24(s,1H),7.86-7.83(d,J＝8.4Hz,2H),7.44-7.37(m,4H),6.95-6.92(d,J＝8.8Hz,2H),6.79-6.74(m,1H),6.35-6.31(d,J＝15.2Hz,1H),3.75(s,3H),3.63-3.60(J＝4.4Hz,J＝4.4Hz,4H),3.15-3.14(d,J＝5.6Hz,2H),2.41(m,4H)。
EXAMPLE 13 preparation of Compound C-20
2.53g (25mmol) of ethylamine and 1.64g (10mmol) of m-nitrophenylisocyanate were added to a solution of the intermediate (I) (3.32g,10mmol) in 50ml of tetrahydrofuran in this order, and after completion of the addition, the reaction was carried out at room temperature for 30min, and the solvent was concentrated to obtain a crude product. The crude product, ethyl acetate, was slurried, filtered and dried to give the compound as a pale yellow solid 4.46g with a yield of 90%.
1H NMR(400MHz，DMSO-d6)δppm:3.75(s,3H),6.96-6.94(d,J＝9.2Hz,2H),7.44-7.40(m,4H),7.61-7.57(q,J＝8.4Hz,J＝8.0Hz,1H),7.69-7.66(d,J＝8.4Hz,2H),7.76-7.74(d,J＝8.0Hz,1H),7.86-7.84(d,J＝8.4Hz,1H),8.24(s,1H),8.35(s,1H),8.59-8.58(q,J＝2.4Hz,J＝2Hz,1H),9.11(s,1H),9.33(s,1H).
EXAMPLE 14 preparation of Compound C-9
The preparation method refers to the synthesis of example 13, the raw materials are intermediate II and m-nitrobenzene isocyanate, the product is light yellow solid, and the yield is 83%.
1H NMR(400MHz，DMSO-d6)δppm:3.76(s,3H),6.94-6.96(d,J＝8.8Hz,2H),7.06-7.15(m,3H),7.41-7.63(m,4H),7.70-7.73(d,J＝8.8Hz,2H),8.27(s,1H),8.59-8.58(t,J＝2.4Hz,J＝2Hz,1H),9.07(s,1H),9.29(s,1H)。
EXAMPLE 15 preparation of Compound C-22
The compound from example 13 (1.32g, 2.66mmol) was added to a mixture of 10ml ethanol, 10ml water and 1.2ml acetic acid, the temperature was raised to 70 ℃ and 0.62g iron powder was added and stirring continued at this temperature for 2 h. After the reaction, the reaction solution was filtered, and the mother liquor was concentrated. The residue was adjusted to pH 9 with aqueous sodium hydroxide and extracted with ethyl acetate 50ml x 3. And (4) combining organic phases, washing with saturated sodium chloride, drying the organic phases, and concentrating to dryness to obtain a crude product. The crude product was purified by column chromatography (dichloromethane/methanol as eluent) to give 0.99g of a white solid in 80% yield.
1H NMR(400MHz，DMSO-d6)δppm:3.75(s,3H),5.05(s,2H),6.21-6.20(d,J＝8.0Hz,1H),6.59-6.57(d,J＝8.0Hz),6.79(s,1H),6.96-6.89(m,3H),7.42-7.37(m,4H),7.64-7.62(d,J＝8.4Hz,2H),8.23(s,1H),8.49(s,1H),8.84(s,1H).
EXAMPLE 16 preparation of Compound C-10
The preparation was carried out according to the synthesis of example 15, starting from example 14 and giving the product a white solid with a yield of 71%.
1H NMR(400MHz，DMSO-d6)δppm:3.76(s,3H),5.02(s,2H),6.17-6.19(d，J＝8.0Hz,,1H),6.52-6.55(d，J＝9.2Hz,1H),6.75(s,1H),6.86-6.90(t，J＝8.0Hz,1H),6.94-6.96(d，J＝9.2Hz,2H),7.05-7.07(d,J＝7.6Hz,1H),7.42-7.47(m，3H),7.54-7.56(d，J＝8.4Hz,1H)，7.62(s，1H)，8.25(s，1H),8.42(s，1H),8.76(s，1H)。
EXAMPLE 17 preparation of Compound C-24
Preparation method referring to example 4, starting from example 15 and dimethylamine, the product is a white solid with a yield of 53%.
1H NMR(400MHz，DMSO-d6)δppm:10.15(s,1H),9.30(m,1H),9.15(m,1H),8.24(s,1H),7.85(s,1H),7.66-7.64(d,J＝8.8Hz,2H),7.42-7.39(m,4H),7.35(m,1H),7.22-7.21(d,J＝5.2Hz,2H),6.96-6.94(d,J＝7.6Hz,2H),6.77-6.70(m,1H),6.35-6.31(d,J＝14.4Hz,1H),3.75(s,3H),3,17(s,2H),2.26(s,6H)。
EXAMPLE 18 preparation of Compound C-23
Preparation method reference example 17, starting from example 15 and piperidine, gave a white solid in 55% yield.
1H NMR(400MHz，DMSO-d6)δppm:1.54-1.40(m,6H),2.37-2.33(m,4H),3.10(s,2H),3.75(s,3H),6.31-6.27(d,J＝16.0Hz,1H),6.76-6.70(m,1H),6.96-6.94(d,J＝8.8Hz,2H),7.22-7.20(d,J＝6.4Hz,2H),7.30(m,1H),7.42-7.39(m,4H),7.66-7.64(d,J＝8.4Hz,2H),7.89(s,1H),8.24(s,1H),8.96-8.91(m,2H),10.08(s,1H)。
EXAMPLE 19 preparation of Compound C-19
Preparation method referring to example 17, starting from example 16 and piperidine, the product is a white solid with a yield of 53%.
1H NMR(400MHz，DMSO-d6)δppm:1H NMR(400MHz，DMSO-d6)δppm:1.39-1.52(m,6H),14.2.39(s,4H),3.07(s,2H),3.75(s,3H),6.24-6.28(d,J＝14.8Hz,1H),6.68-6.75(m,1H),6.94-6.96(d,J＝8.8Hz,2H),7.07-7.20(m,3H),7.28-7.30(d,J＝14.8Hz,1H),7.41-7.48(m,3H),7.55-7.57(d,J＝8.4Hz,2H),7.63(s,1H),7.84(s,1H),8.24(s,1H),8.78-8.82(d,J＝14Hz,2H),10.08(s,1H)。
EXAMPLE 20 preparation of Compound C-12
Preparation method referring to example 17, starting from example 16 and acryloyl chloride, the product was a white solid with a yield of 53%.
1H NMR(400MHz，DMSO-d6)δppm:3.76(s,3H),5.73-5.76(m,1H),6.22-6.27(dd，J＝16.8Hz,2.0Hz,1H),6.41-6.47(dd，J＝16.8Hz,10.0Hz,1H),6.94-6.96(d，J＝7.2Hz,2H),6.94-6.96(d，J＝7.6Hz,1H),7.14-7.22(m,2H),7.31-7.33(d，J＝7.6Hz,1H),7.41-7.49(m，3H)，7.56-7.58(d，J＝9.2Hz,1H)，7.64(s，1H)，7.87(s，1H),7.87(s，1H),8.25(s，1H),8.84-8.87(d，J＝14.8Hz,2H),10.12(s，1H)。
EXAMPLE 21 preparation of Compound C-45
Method 1
According to the synthesis of example 4, the raw materials are 4-fluoro bromo acetophenone and dimethylamine hydrochloride, and the product is a white solid.
1H NMR(400MHz，DMSO-d6)δppm:2.22(s,6H),3.09-3.10(d，J＝4.8Hz,2H),6.30-6.34(d，J＝15.2Hz,1H)，6.74-6.80(m，1H),7.21-7.25(t，J＝9.2,6.8Hz,2H),7.43-7.48(m，4H)，7.85-7.87(d,J＝8.8Hz，2H)，8.26(s，1H),10.32(s，1H)。
Method two
Method two reference W03022852A2 can be synthesized to obtain intermediate XI, and reference can be made to the synthesis method of example 4 to obtain example 22 from intermediate XI.
EXAMPLE 22 preparation of Compound C-32
The raw materials, 4-bromoacetylpyridine and dimethylamine hydrochloride, were white solids with a yield of 41%.
1H NMR(400MHz，DMSO-d6)δppm:2.31(s,6H),3.26(s,2H),6.36-6.40(d,J＝15.2Hz,1H),6.76-6.83(m,1H),7.41-7.44(m,1H),7.46-7.49(d,J＝12.4Hz,2H),7.81-7.84(m,1H),7.88-7.90(d,J＝8.4Hz,2H),8.30(s,1H),8.50-8.51(m,1H),8.57-8.58(d,J＝1.6Hz,1H),10.45(s,1H)；
EXAMPLE 23 preparation of Compound C-25
Preparation method referring to example 21, starting materials 4-fluorobromoacetophenone and 3-tert-butoxycarbonylaminopiperidine, the product was a white solid with a yield of 50%.
1H NMR(400MHz，DMSO-d6)δppm:1.05-1.178(m，2H),1.38(s，9H),1.49-1.88(m,5H),2.69-2.81(m,2H),3.14-3.15(d,J＝5.2Hz，2H),6.28-6.32(d，J＝15.2Hz,1H)，6.74-6.80(m，2H),7.21-7.25(t，J＝9.2Hz，8.8Hz,1H),7.49-7.50(m，4H)，7.85-7.87(d,J＝8.4Hz，2H)，8.26(s，1H),10.31(s，1H)。
EXAMPLE 24 preparation of Compound C-27
Preparation method referring to example 21, bromoacetophenone and dimethylamine hydrochloride were used as raw materials, and the product was white solid with a yield of 50%.
1H NMR(400MHz，DMSO-d6)δppm:2.72(s,6H),3.87(d，J＝4.8Hz,2H),6.55-6.59(d，J＝15.2Hz,1H)，6.83-6.87(m，1H),7.32-7.46(m，7H),7.91-7.93(d,J＝8.4Hz，2H)，8.27(s，1H),10.82(s，1H)。
EXAMPLE 25 preparation of Compound C-28
Preparation method referring to example 8, starting materials bromoacetophenone and piperidine, the product was a white solid in 51% yield.
1H NMR(400MHz，DMSO-d6)δppm:1.40-1.532(m,6H),2.33-2.36(m,4H),3.10-3.11(d，J＝4.4Hz,2H)，6.24-6.28(d,J＝15.6Hz,1H),6.72-6.76(m，1H)，7.17-7.19(d，J＝7.6Hz，1H),7.33-7.37(m,3H),7.46-7.53(m,3H),7.80(d，J＝6.4Hz，2H),8.28(s，1H)，10.28(s，1H)。
EXAMPLE 26 preparation of Compound C-29
Preparation method referring to example 8, starting materials 2-bromo-1- (1-methyl-1H-pyrazol-4-yl) ethanone and piperidine, the product was a white solid in 43% yield.
1H NMR(400MHz，DMSO-d6)δppm:1.50-1.70(m,6H),2.87(m,4H),3.65(m,2H),3.83(s,3H),6.46-6.50(d,J＝15.2Hz,1H),6.82-6.87(m,1H),7.34(s,1H),7.46-7.48(d,J＝8.4Hz,2H),7.88-7.91(q,J＝4.8Hz,J＝4.8Hz,3H),8.21(s,1H),10.59(s,1H)；
EXAMPLE 27 preparation of Compound C-37
Preparation method referring to example 21, starting materials 4-fluoro bromoacetophenone and piperidine, the product was a white solid with a yield of 50%.
1H NMR(400MHz，DMSO-d6)δppm:1.42-1.56(m,6H),2.49(s,4H),3.22(s,2H),6.32-6.35(d，J＝15.2Hz,1H)，6.76-6.81(m，1H),7.21-7.25(t，J＝8.8Hz,1H),7.44-7.48(m，4H)，7.85-7.87(d,J＝8.4Hz，2H)，8.26(s，1H),10.34(s，1H)。
EXAMPLE 28 preparation of Compound C-31
Preparation method referring to example 8, starting materials bromoacetophenone and 4-hydroxypiperidine, the product was a white solid with a yield of 50%.
1H NMR(400MHz，DMSO-d6)δppm:1.35-1.45(m,2H),1.65-1.78(m,2H),1.95-2.15(m,2H),2.63-2.75(m,2H),3.16-3.17(d，J＝5.0Hz,2H),4.60(m,2H),6.24-6.28(d，J＝15.6Hz,1H)，6.71-6.77(m，1H),7.17-7.19(d，7.2Hz,1H),7.33-7.53(m，5H)，7.80-7.81(m，2H),10.28(s，1H)。
EXAMPLE 29 preparation of Compound C-30
Preparation method referring to example 22, starting material 4-bromoacetylpyridine and piperidine, the product was a white solid in 43% yield.
1H NMR(400MHz，DMSO-d6)δppm:1.40(m,2H),1.52-1.55(m,4H),2.37(m,4H),3.10-3.12(d,J＝5.2Hz,2H),6.29-6.33(d,J＝15.6Hz,1H),6.75-6.82(m,1H),7.41-7.44(m,1H),7.45-7.49(d,J＝12.8Hz,2H),7.82-7.84(m,1H),7.86-7.89(d,J＝8.8Hz,2H),8.30(s,1H),8.50-8.51(m,1H),8.57-8.58(d,J＝1.6Hz,1H),10.33(s,1H)。
EXAMPLE 30 preparation of Compound C-33
Preparation method referring to example 8, starting materials 3-bromoacetophenone and dimethylamine hydrochloride, the product was a white solid with a yield of 41%.
1H NMR(400MHz，DMSO-d6)δppm:2.18(s,6H),3.05-3.06(d,J＝4.8Hz,2H),3.86(s,3H),6.26-6.30(d,J＝15.6Hz,1H),6.54(s,1H),6.70-6.76(m,1H),7.06-7.08(m,1H),7.17-7.19(d,J＝8.8Hz,2H),7.25-7.30(m,2H),7.40(s,2H),7.65-7.67(d,J＝8.8Hz,2H),8.09(s,1H),10.13(s,1H)。
EXAMPLE 31 preparation of Compound C-35
Preparation method referring to example 22, starting materials 3, 5-bistrifluoromethylbromoacetophenone and piperidine, the product was a white solid with a yield of 41%.
1H NMR(400MHz，DMSO-d6)δppm:1.40(m,2H),1.50-1.51(m,4H),2.35(m,4H),3.08-3.09(d,J＝5.2Hz,2H),6.23-6.27(d,J＝15.6Hz,1H),6.71-6.76(m,1H),7.22-7.24(d,J＝7.6Hz,1H),7.53-7.56(q,J＝7.6Hz,J＝8.0Hz,1H),7.79-7.81(d,J＝8.4Hz,1H),7.91-7.94(m,3H),8.08(s,1H),8.34(s,1H),10.29(s,1H)。
EXAMPLE 32 preparation of Compound C-43
The preparation method refers to example 21, the raw materials are 3,4, 5-trimethoxy bromoacetophenone and dimethylamine, the product is white solid, and the yield is 41%.
1H NMR(400MHz，DMSO-d6)δppm:2.19(s，6H)，3.07-3.08(d，J＝5.2Hz，2H),3.58(s,6H),3.65(s,3H),6.29-6.33(d,J＝15.6Hz,1H),6.72(s,2H),6.76-6.80(m,1H),7.47-7.49(d,J＝8.8Hz,2H),7.86-7.88(d,J＝8.4Hz,2H),8.25(s,1H),10.30(s,1H)；
EXAMPLE 33 preparation of Compound C-44
The preparation method refers to example 21, the raw materials are 3,4, 5-trimethoxy bromoacetophenone and piperidine, the product is white solid, and the yield is 41%.
1H NMR(400MHz，DMSO-d6)δppm:1.30(m,2H),1.52-1.55(m,4H),2.37(m,4H),3.10-3.12(d,J＝5.2Hz,2H),3.58(s,6H),3.65(s,3H),6.28-6.32(d,J＝15.2Hz,1H),6.72(s,2H),6.74-6.81(m,1H),7.47-7.49(d,J＝8.4Hz,2H),7.86-7.88(d,8.4Hz,2H),8.25(s,1H),10.29(s,1H)；
EXAMPLE 34 preparation of Compound C-46
Preparation method referring to example 21, starting materials bromoacetophenone and piperidine, the product was a white solid with a yield of 50%.
1H NMR(400MHz，DMSO-d6)δppm:1.35-1.55(m,6H),2.29-2.39(m,4H),3.11(d，J＝4.8Hz,2H),6.28-6.32(d，J＝15.2Hz,1H)，6.75-6.80(m，1H),7.32-7.44(m，7H),7.85-7.87(d,J＝8.8Hz，2H)，8.26(s，1H),10.30(s，1H)。
EXAMPLE 35 preparation of Compound C-8
The preparation method refers to example 21, the raw materials are 3,4, 5-trimethoxy bromoacetophenone and piperidine, the product is white solid, and the yield is 41%.
1H NMR(400MHz，DMSO-d6)δppm:1.40-1.70(m,6H),2.87(m,4H),3.58(s，6H)，3.66(s，3H)，3.88(s,2H),6.43-6.47(d，J＝14.8Hz,1H)，6.74(s，2H),6.77-6.82(m，1H),7.26-7.28(d，J＝7.6Hz,1H),7.55-7.59(t，J＝8.07.6Hz,7.6Hz,1H)，7.80-7.82(d,J＝8.0Hz，1H)，7.85(s，1H),8.28(s，1H),10.58(s，1H)。
EXAMPLE 36 preparation of Compound C-15
The preparation method refers to example 22, the raw materials are 3, 5-bis (trifluoromethyl) bromoacetophenone and dimethylamine hydrochloride, the product is white solid, and the yield is 41%.
1H NMR(400MHz，DMSO-d6)δppm:2.17(s,6H),3.05-3.06(d,J＝4.8Hz,2H),6.24-6.28(d,J＝15.2Hz,1H)6.71-6.75(m,1H),7.22-7.24(d,J＝7.6Hz,1H),7.52-7.56(q,J1＝J2＝8.0Hz,1H),7.78-7.80(d,J＝8.0Hz,1H),7.91-7.94(d,J＝12.4Hz,3H),8.08(s,1H),8.34(s,1H),10.30(s,1H)。
EXAMPLE 37 preparation of Compound C-17
Preparation method referring to example 22, starting materials 3, 5-bistrifluoromethylbromoacetophenone and piperidine, the product was a white solid with a yield of 41%.
1H NMR(400MHz，DMSO-d6)δppm:1.39-1.51(m,6H),2.25-2.39(m,4H),3.07-3.08(d,J＝5.2Hz,2H),6.23-6.27(d,J＝15.2Hz,1H)，6.72-6.75(m,1H),7.22-7.24(d,J＝7.6Hz,1H),7.52-7.56(q,J＝8.0Hz,7.6Hz,1H),7.78-7.80(d,J＝8.4Hz,1H),7.90-7.93(d,J＝12.4Hz,3H),8.34(s,1H),8.34(s,1H),10.29(s,1H)。
EXAMPLE 38 preparation of Compound C-40
Preparation method referring to example 22, starting materials 3-bromothienylethanone and piperidine, the product was a white solid with a yield of 41%.
1H NMR(400MHz，DMSO-d6)δppm:1.43-1.54(m,6H),2.30-2.40(m,4H),3.14(s,2H),6.30-6.34(d,J＝15.2Hz,1H)，6.75-6.82(m,1H),6.88-6.89(d,J＝5.2Hz,1H)，7.45-7.47(d,J＝8.8Hz,2H)，7.55-7.57(m,1H),7.69-7.70(m,1H),7.86-7.88(d,J＝8.8Hz,2H),8.34(s,1H),8.24(s,1H),10.29(s,1H)。
EXAMPLE 39 preparation of Compound C-2
Preparation method referring to example 22, starting materials 3-bromothienylethanone and piperidine, the product was a white solid with a yield of 41%.1H NMR(400MHz，DMSO-d6)δppm:1.40-1.65(m,9H),1.91(s,3H),2.20-2.25(m,3H),2.37-2.46(m,4H),2.99-3.02(t,J＝6.0Hz,5.2Hz,3H),2.56-3.59(m,4H),6.32-6.35(d,J＝15.2Hz,1H)，6.76-6.85(m,1H),7.42-7.53(d,J＝8.4Hz,2H),7.50-7.51(d,J＝9.2Hz,2H),7.84-7.86(d,J＝8.4Hz,1H),8.14-8.15(d,J＝2.4Hz,1H),8.22(s,1H),10.29(s,1H)。
EXAMPLE 40 preparation of Compound C-47
Preparation method referring to example 21, starting materials 3, 4-dimethoxy bromoacetophenone and piperidine, the product was a white solid with a yield of 41%.
1H NMR(400MHz，DMSO-d6)δppm:2.19(s,6H),3.08-3.09(d,J＝5.6Hz,2H),3.55(s,3H),3.74(s,3H),6.29-6.33(d,J＝15.2Hz,1H),6.74-6.79(m,1H),6.94-7.03(m,3H),7.44-7.46(q,J＝8.4Hz,2H),7.84-7.86(d,J＝8.4Hz,2H),8.24(s,1H),10.31(s,1H)；
EXAMPLE 41 preparation of Compound C-36
Preparation method referring to example 21, starting materials 3, 5-dimethoxy bromoacetophenone and piperidine, the product was a white solid with a yield of 41%.
EXAMPLE 42 preparation of Compound C-39
After intermediate XII (100mg, 0.26mmol) was dissolved in 14mL of isopropanol, (69mg, 0.31mmol) of (R) - (-) -1-benzyl-3-aminopyrrolidine and (0.66mg, 0.65mmol) of triethylamine were added, and the mixture was stirred at 80 ℃ for 2.5 h. Liquid phase detection reaction is complete, and the solvent is concentrated. Water was added to the residue, extracted with ethyl acetate, and the organic phases were combined. The organic phase was washed with saturated ammonium chloride, dried over anhydrous sodium sulfate and the solvent was concentrated. The residue was subjected to column chromatography to give compound 77mg as a white solid (yield 62%).
1H NMR(400MHz，DMSO-d6)δppm:2.95(s,3H),3.08(s,3H),3.86(s,3H),7.14-7.16(d,J＝8.8Hz，2H),7.79(s,1H),8.04-8.06(d,J＝8.8Hz,2H)。
The preparation was carried out in 69% yield from example 42 starting with aqueous ammonia and intermediate XII.
1H NMR(400MHz，DMSO-d6)δppm:2.82(s,3H),3.83(s,3H),7.09-7.11(dd,J＝6.8Hz，2.0Hz，2H),7.25(s,1H),7.73-7.77(dd,J＝6.8Hz,2.0Hz,2H),7.98(s,4H)。
EXAMPLE 43 preparation of Compound C-38
Referring to the synthesis of example 4, starting from intermediate XIII and dimethylamine hydrochloride.
1H NMR(400MHz，DMSO-d6)δppm:2.21(s,6H),3.09-3.10(d,J＝3.6Hz,2H),3.76(s,3H),6.31-6.34(d,J＝15.2Hz,1H),6.76-6.80(m,1H),6.95-6.97(d,＝8.8Hz,2H),7.40-7.45(m,3H),7.85-7.87(q,J＝8.8Hz,2H),10.32(s,1H)。
EXAMPLE 44 preparation of Compound C-26
Intermediate XIV was synthesized according to W03022852A2, and the product was obtained according to the synthesis method of example 4.
1H NMR(400MHz，DMSO-d6)δppm:10.21(s,1H),8.25(s,1H),7.64-7.62(d,J＝8.8Hz,2H),7.44-7.39(m,4H),7.15-7.13(d,J＝8.8Hz,2H),6.76-6.72(m,1H),6.28-6.25(d,J＝15.6Hz,1H),3.85(s,3H),3.06-3.05(d,J＝4.8Hz,2H),3.17(s,6H)；
EXAMPLE 45 preparation of Compound C-34
Preparation method referring to example 44, 4-methoxy bromoacetophenone and piperidine were used as raw materials, and the product was a white-like solid.
1H NMR(400MHz，DMSO-d6)δppm:1.40-1.53(m，6H),2.37(m,4H),3.11(m,2H),3.86(s,3H),6.24-6.28(d,J＝15.6Hz,1H),6.71-6.77(m,1H),7.13-7.15(d,J＝8.8Hz,2H),7.39-7.44(m,4H),7.62-7.64(d,J＝8.8Hz,2H),8.25(s,1H),10.22(s,1H)；
EXAMPLE 46 preparation of Compound C-41
Intermediate XV can be synthesized according to W03022852A2, and the product can be obtained according to the synthesis method of example 4.
1H NMR(400MHz，DMSO-d6)δppm:1.41(m,2H),1.51-1.55(m,4H),2.33-2.36(m,4H),3.10-3.11(d,J＝4.4Hz,2H),3.77(s,3H),6.30-6.33(d,J＝15.6Hz,1H),6.75-6.83(m,1H),6.97-6.99(d,J＝8.8Hz,2H),7.15-7.18(m,1H),7.25(s,1H),7.34-7.38(q,J＝9.2Hz,J＝8.8Hz,1H),7.43-7.46(d,J＝9.2Hz,2H),7.53-7.56(d,J＝8.8Hz,2H),7.87-7.90(m,3H),8.52(s,1H),10.32(s,1H)；
EXAMPLE 47 preparation of Compound C-42
Preparation method referring to example 46, starting materials 4-methoxy bromoacetophenone, m-aminobenzoic acid and piperidine, the product was a white solid.
1H NMR(400MHz，DMSO-d6)δppm:1.38(m,2H),1.50-1.82(m,4H),2.34(m,4H),3.07-3.08(d,J＝5.2Hz,2H),6.24-6.28(d,J＝15.6Hz,1H),6.72-6.79(m,1H),6.97-6.99(d,J＝8.8Hz,2H),7.22-7.34(m,4H),7.46-7.48(d,J＝8.8Hz,2H),7.53-7.57(q,J＝7.6Hz,J＝8.0Hz,1H),7.79-7.84(m,2H),7.95(s,1H),8.52(s,1H),10.30(s,1H)；
EXAMPLE 48 biological Activity of Compounds of the invention
The first part
First, experimental material
1. Cell lines: human glioma cell U251, human brain astrocytoma cell U87, cervical carcinoma cell Hela, human colon carcinoma cell HCT116, human breast cancer cell line MCF-7, and human umbilical vein endothelial cell line HUVEC.
2. Experimental reagent: MTT (3- (4, 5-dimethylthiazole-2) -2, 5-diphenyl tetrazolium bromide), PBS buffer solution and DMEM culture medium: gibco Corp., fetal bovine serum: ilex ltd, DMSO: sigma, trypsin: gibco Corp
3. An experimental instrument: a cell culture box: japan ocean electric company, microplate reader: MOLECULAR, optical microscope: LEICA, superclean bench: beijing Weida institute of purification technology.
4. Preparation of a reagent:
(1) PBS solution: NaCl 8g, KCl 0.2g, Na2HPO4 & 2H2O 3.62.62 g and KH2PO40.24g, adding the mixture into 900ml of distilled water for dissolving, adjusting the pH value to 7.0, and adding the distilled water to 1000 ml.
(2) MTT solution: MTT powder was dissolved in PBS (5mg/ml), sterilized by filtration and stored at 4 ℃.
5. The tested drugs are example 4, example 5, example 6, example 7, example 12, example 15, example 16, example 18, example 20, example 21 and example 22.
Second, the experimental procedure
1. Cells were collected in log phase, adjusted to 25000 cells/ml and 100ul of cell suspension (2500 cells per well) was added per well. Typically, 6 duplicate wells are provided and control wells are provided.
2. Cells are placed in an incubator for culture and administered the next day after adherence (typically plating in the afternoon or evening of the previous day and administration in the morning of day 2).
3. The administration method comprises the following steps: the drugs are prepared first, then the 96-well plate is taken out, the original culture solution is discarded, and the drugs are added. The medicines are prepared by a culture medium, and each medicine is provided with eight concentration gradients of 0ug/ml, 2ug/ml, 4ug/ml, 6ug/ml, 8ug/ml, 10ug/ml, 20ug/ml and 25 ug/ml.
4. The cells are put into an incubator to be cultured for 72 h.
5. After the drug action is finished, 20ul of MTT (5mg/ml) is added into each hole, and the culture is carried out for 3-4 h.
6. The culture was terminated and the culture medium in the wells was carefully aspirated. 150ul of DMSO was added to each well and incubated at 37 ℃ for 10 minutes in an incubator or shaking at low speed on a shaker for 10 minutes. The absorbance of each well at OD-490 nm (also 570 nm) was then measured using a microplate reader.
7. Zero-setting wells (serum-free medium, MTT, DMSO), control wells (cells, maximum concentration drug dissolution medium, serum-free medium, MTT, DMSO) were also set.
8. Cell viability (cell viability):
cell viability of control (drug group a value-zero well a value)/(control well a value-zero well a value) 100%.
Third, experimental results
IC50 values (μ M) for the compounds of Table 1 against cancer cell lines MCF-7, cervical carcinoma cells Hela and 231C
Compound (I) hela MCF7 231C
c-4 100.544 45.527 194.23
c-6 6.585 5.368 26.963
c-16 3.07 4.48 3.65
c-18 Is free of 75.678 Is free of
c-19 9.216 9.3 10.316
c-20 8.567 10.025 11.434
c-21 17.943 50.617 51.998
c-22 9.472 15.984 21.206
c-23 4.052 6.841 7.285
c-24 14.695 10.65 10.931
c-39 9.165 12.063 13.539
TABLE 2 IC50 values (μ M) of the compounds on human glioma cells U251 and U87, etc
Compound (I) U87 U251
Afatinib 7.429 6.849
c-3 13.931 3.905
c-5 8.119 4.503
c-7 13.52 9.498
TABLE 3 IC50 values (μ M) for HCT-116 for the compounds
Compound (I) HCT-116
Afatinib 2.668
c-1 7.715
c-3 0.7882
c-4 61.038
c-5 1.1548
c-7 3.008
c-9 8.959
c-10 15.083
c-11 5.288
c-12 13.677
c-13 6.827
c-14 10.401
c-6 13.984
c-16 4.642
c-18 138.268
c-19 11.729
c-20 10.468
c-21 19.216
c-22 10.165
c-23 3.761
c-24 8.507
c-39 6.508
TABLE 4 IC50 values (μ M) for compound A549
Compound (I) A549
Afatinib 3.13
c-3 6.39
c-5 14.59
c-16 10.06
c-25 7.32
c-26 6.6
c-34 7.89
c-35 2.49
c-36 15.46
c-37 2.47
c-41 2.07
c-42 6.25
c-45 3.79
c-46 2.46
Ibrutinib 61.4
IC50 values (μ M) for HepG2 for the compounds of Table 5
Compound (I) HepG2
Afatinib 1.69
c-3 4.2
c-5 13.43
c-16 8.63
c-25 5.33
c-26 6.6
c-34 7.89
c-35 2.49
c-36 15.83
c-37 2.47
c-41 2.07
c-42 6.25
c-45 2.2
c-46 2.46
Ibrutinib 61.4
TABLE 6 IC50 values (μ M) of the compounds on human umbilical vein endothelial cell line HUVEC
Name of Compound HUVEC
Afatinib 1.436
c-3 4.283
c-5 4.605
c-7 12.054
c-4 49.671
c-6 11.392
c-16 3.957
c-19 15.902
c-20 12.609
c-21 34.191
c-22 28.036
c-23 11.782
The results of the test of the inhibitory activity of the compound on liver cancer HepG2 cells are shown in figure 1. The results of the test for the inhibitory activity of lung cancer a549 cells are shown in fig. 2.
Second partial kinase Activity assay
In this experiment we screened 2 compounds for the in vitro kinases AXL and TRKA using the method of Mobility Shift Assay, starting at 1 μ M, 3-fold dilution, 10 concentrations, single well Assay. The compound afatinib was used as a standard control.
Experimental methods
I. Preparing 1 time of kinase buffer solution and stop solution
1.1-fold kinase buffer
25Mm HEPES,pH 7.5
0.001％Brij-35
0.01％Triton
0.5Mm EGTA
10Mm MgCl2
2Mm DTT
2. Stopping liquid
100mM HEPES,pH 7.5
0.015％Brij-35
0.2％Coating Reagent#3
50mM EDTA
Preparation of the Compounds
1) Dilution of Compounds
3. Compound preparation 50-fold: the final concentration of compound tested was 1 μ M, configured to 50 fold concentration, i.e., 50 μ M, and 100 μ l of 50 μ M compound was added to the second well of a 96-well plate, and 60 μ l of 100% DMSO was added to the other wells. Mu.l of compound from well 2 was added to well 3, and 3-fold dilutions were made sequentially down to a total of 10 concentrations.
2) Transfer 5 times the compound to the reaction plate
4. Mu.l of each of the above 96-well plates was transferred to another 96-well plate, and 90. mu.l of kinase buffer was added. Thus, the second to eleventh wells were compounds dissolved in 10% DMSO, and the first and twelfth wells were 10% DMSO.
5. From the above 96-well plate, 5. mu.l was taken out to a 384-well reaction plate. Thus, 5. mu.l of 5-fold compound dissolved in 10% DMSO and 5. mu.l of 10% DMSO were present in 384-well plates.
Kinase reaction
1) Preparing 2.5 times of enzyme solution
6. The kinase was added to 1 fold kinase buffer to form a 2.5 fold enzyme solution.
2) Preparing 2.5 times of substrate solution
7. FAM-labeled polypeptide and ATP were added to 1-fold kinase buffer to form a 2.5-fold substrate solution.
3) Adding enzyme solution to 384-well plate
5 μ l of 5-fold compound dissolved in 10% DMSO was present in an 8.384 well plate.
9. Mu.l of a 2.5-fold enzyme solution was added to the 384-well reaction plate.
10. Incubate for 10 minutes at room temperature.
4) Adding substrate solution to 384-well plate
11. Mu.l of a 2.5-fold substrate solution was added to the 384-well reaction plate.
5) Kinase reaction and termination
Incubate at 12.28 ℃ for 3 hr.
13. The reaction was stopped by adding 25. mu.l of stop solution.
Caliper read data
Conversion data were read on the Caliper.
Inhibition ratio calculation
15. Conversion data was copied from the Caliper.
16. The conversion was converted to inhibition data. Wherein max refers to the conversion rate of a DMSO control, and min refers to the conversion rate of an enzyme-free control.
Percent inhibition＝(max-conversion)/(max-min)*100.
17. IC50 values were fitted with XLFit excel add-in version 4.3.1.
Fitting formula of Y ═ Bottom + (Top-Bottom)/(1+ (IC50/X) ^ HillSlope)
The results are shown in the following table.
TABLE 7 IC50 results (nM)
From the obtained cell activity test results, most of the compounds show good activity on anti-tumor cells such as liver cancer, lung cancer, human colon cancer cell HCT116, cervical cancer cells and the like, and even have better effect than the reference of afatinib and ibrutinib; table 6 shows that most compounds are less toxic than afatinib to normal cells of human umbilical vein endothelial cell line HUVEC. According to results of EGFR and AUR A kinase activity tests, the tested compound has a good inhibitory effect on EGRF kinase, and the inhibitory effect on AUR A kinase is stronger than that of afatinib.

Claims (3)

1. The following compounds or pharmaceutically acceptable salts thereof:
2. use of a compound of claim 1, or a pharmaceutically acceptable salt thereof, in the manufacture of an anti-neoplastic drug, an angiogenesis inhibitor, an EFGR kinase inhibitor or an AUR a kinase inhibitor.
3. Use according to claim 2, characterized in that: the tumor is liver cancer, lung cancer, glioma, astrocytoma, cervical cancer, colon cancer or breast cancer.
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