CN114014847A - Benzothiophene pyrimidine derivative, preparation method thereof and application thereof in preparation of antitumor drugs - Google Patents
Benzothiophene pyrimidine derivative, preparation method thereof and application thereof in preparation of antitumor drugs Download PDFInfo
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- 239000002246 antineoplastic agent Substances 0.000 title claims abstract description 8
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- DYXNPKNQSCAHFG-UHFFFAOYSA-N 1-benzothiophene pyrimidine Chemical class N1=CN=CC=C1.S1C2=C(C=C1)C=CC=C2 DYXNPKNQSCAHFG-UHFFFAOYSA-N 0.000 title claims abstract description 6
- 150000003230 pyrimidines Chemical class 0.000 claims abstract description 14
- 239000000126 substance Substances 0.000 claims abstract description 3
- FCEHBMOGCRZNNI-UHFFFAOYSA-N 1-benzothiophene Chemical compound C1=CC=C2SC=CC2=C1 FCEHBMOGCRZNNI-UHFFFAOYSA-N 0.000 claims description 24
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D409/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms
- C07D409/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing two hetero rings
- C07D409/04—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing two hetero rings directly linked by a ring-member-to-ring-member bond
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D409/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms
- C07D409/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing three or more hetero rings
Abstract
The invention discloses a derivative containing benzothiophene pyrimidine, a preparation method thereof and application thereof in preparing antitumor drugs. The chemical structural formula is as follows:
Description
Technical Field
The invention belongs to the technical field of medicinal chemistry, and particularly relates to a benzo-thiophene pyrimidine derivative, a preparation method thereof and application thereof in preparation of antitumor drugs.
Background
Malignant tumors are the most common and serious diseases endangering human life and health in the world today. In recent years, with the continuous efforts of scientists, many drugs for treating tumors are on the market, which greatly improve the life quality of tumor patients and make the tumor treatment greatly progress. However, the traditional chemotherapy drugs still have the defects of poor curative effect, strong toxic and side effects, easy generation of drug resistance and the like. Therefore, finding or discovering highly effective, low-toxicity, drug-resistant targeted antitumor drugs is an important issue in current medical research. Protein Tyrosine Kinases (PTK) are important factors in the signal transmission process, can catalyze tyrosine residue phosphorylation of various substrate proteins so as to transmit signals, and play an important role in cell proliferation, differentiation, migration and apoptosis. The abnormal expression of tyrosine kinase can cause the disturbance of cell proliferation regulation, and is also closely related to the invasion and metastasis of tumor, the generation of tumor new vessels and the chemotherapy resistance of tumor.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides a benzo-thiophene-containing pyrimidine derivative, a preparation method thereof and application thereof in preparing antitumor drugs, the prepared benzo-thiophene-containing pyrimidine derivative has excellent in-vivo antitumor activity, and the in-vivo tumor inhibition rate TGI value is 90.24%.
In order to achieve the purpose, the technical scheme adopted by the invention for solving the technical problems is as follows:
a benzo thiophene pyrimidine derivative has the following chemical structural formula:
wherein R is1Is H or F; r2Is a secondary amine, thiol, alkyl piperazine or piperidine.
Further, R2Is composed of
Further, R1Is H, R2Is composed of
Further, R1Is F, R2Is composed of
The preparation method of the benzothiophene-containing pyrimidine derivative is characterized by comprising the following steps of:
(1) reacting benzothiophene-3-boronic acid and (2, 4-dichloropyrimidine) -2b (2, 4-dichloro-6-fluoropyrimidine) through Suzuki;
(2) carrying out aromatic nucleophilic reaction on 4-fluoro-2-methoxy-5-nitroaniline and the product obtained in the step (1) under an acidic condition;
(3) reacting the product obtained in step (2) with R under alkaline conditions2H or hydrochloride thereof;
(4) and (3) mixing and dissolving the product obtained in the step (3), ammonium chloride and a reducing agent, and carrying out nitro reduction and acylation reaction after the reaction is finished to obtain the benzothiophene-containing pyrimidine derivative.
The reaction process is as follows:
further, the aromatic nucleophilic reaction in the step (2) comprises the following specific processes: dissolving 4-fluoro-2-methoxy-5-nitroaniline and the product obtained in the step (1), and heating to 80-100 ℃ overnight.
Furthermore, the dosage of the 4-fluoro-2-methoxy-5-nitroaniline is 1-1.5 eq.
Further, R in the step (3)2In the case of secondary amines: dissolving the product obtained in the step (2), and then adding R2H or hydrochloride thereof and DIPEA react for 10-15H at 80-90 ℃.
Further, R in the step (3)2In the case of thiols or alcohols: r is to be2Dissolving H, adding NaH at 0 ℃, stirring for 10-20min, adding the product obtained in the step (2), stirring to enable the reaction solution to be heated to room temperature, and then reacting overnight.
The benzothiophene-containing pyrimidine derivative is applied to preparation of antitumor drugs.
Further, the tumor is non-small cell lung cancer.
The invention has the beneficial effects that:
the invention designs and synthesizes 13 pyrimidine derivatives with new structures. Research results show that the compound 6a (VI-1) has good inhibition effect on EGFR L858R/T790M high-expression kinase and EGFR L858R/T790M high-expression cell H1975, and the IC50 values are 0.26nM and 2.2nM respectively. Mechanistic studies indicate that 6a can time-dose-dependently inhibit the expression of EGFR and AKT phosphorylated proteins, thereby inducing H1975 apoptosis. In addition, 6a showed less cytotoxicity to all three human normal cell lines and was superior to the positive control drug, oxitinib. Further studies have shown that 6a has good pharmacokinetic properties. Finally, in vivo activity studies show that the compound 6a has good in vivo antitumor activity, and the in vivo tumor inhibition rate TGI value is 90.24%. The above results indicate that 6a is worthy of further investigation in the treatment of non-small cell lung cancer (NSCLC).
Drawings
FIG. 1 shows the comparison of 6a with Afatinib and ositinib for their ability to inhibit EGFR tyrosine kinase.
Detailed Description
The following description of the embodiments of the present invention is provided to facilitate the understanding of the present invention by those skilled in the art, but it should be understood that the present invention is not limited to the scope of the embodiments, and it will be apparent to those skilled in the art that various changes may be made without departing from the spirit and scope of the invention as defined and defined in the appended claims, and all matters produced by the invention using the inventive concept are protected.
EXAMPLE 1 Synthesis of object Compounds VI-1 to VI-13
The synthesis routes of the target compounds VI-1 to VI-13 are as follows:
1. general procedure for the Synthesis of intermediates 3a-3 b:
2, 4-dichloropyrimidine/2, 4-dichloro-6-fluoropyrimidine (2.47mL,20mmol) and bis (triphenylphosphine) palladium dichloride (701.9mg,1mmol) are dissolved in 60mL of toluene, an aqueous solution (20mL) of sodium carbonate (4.24g,40mmol) and a methanol (20mL) solution of 3-hydroxymethylphenylboronic acid (3.34g,22mmol) are added, the mixture is reacted at 90 ℃ for 12 hours under the protection of argon, after the TLC detection reaction is completed, the reaction solution is cooled to room temperature, the mixture is filtered through kieselguhr, the filtrate is extracted through ethyl acetate (100 mL. times.3), the organic phases are combined, dried and filtered, and the mixture is suspended under reduced pressure and purified through column chromatography (petroleum ether: ethyl acetate: 10: 1-3: 1) to obtain the target intermediates 3a-3 b.
Red solid, yield: 62 percent;1H NMR(500MHz,CDCl3)δ8.61(s,2H),8.32(s,1H),8.25(m,1H),7.50–7.40(m,2H),4.74(s,2H).
white solid, yield: 60.5 percent;1H NMR(400MHz,CDCl3)δ8.83(d,J＝8.2Hz,1H),8.53(d,J＝3.0Hz,1H),8.40(d,J＝1.3Hz,1H),7.91(d,J＝8.1Hz,1H),7.53(m,1H),7.45(m,1H).
2. general procedure for the Synthesis of intermediates 4a-4 b:
dissolving the raw material 3a or 3b and 4-fluoro-2-methoxy-5-nitroaniline (1.05eq) in n-butanol, adding 2-3 drops of concentrated hydrochloric acid into the reaction solution, gradually heating the reaction solution to 80 ℃ overnight, detecting by TLC (thin layer chromatography) after the reaction is completed, cooling to room temperature, filtering, washing a filter cake by ethyl acetate, and placing the filter cake in a vacuum drying oven for drying to obtain the target intermediate 4a-4 b.
Yellow solid, yield: 73 percent;1H NMR(500MHz,DMSO-d6)δ9.14(bs,1H),8.82(d,J＝8.3Hz,1H),8.77(s,1H),8.69(d,J＝8.0Hz,1H),8.59(d,J＝5.5Hz,1H),8.09(d,J＝8.0Hz,1H),7.53(d,J＝5.5Hz,1H),7.49–7.32(m,3H).
4 b: grey solid, yield: 50 percent;1H NMR(500MHz,DMSO-d6)δ8.85(bs,1H),8.76(d,J＝8.3Hz,1H),8.72(d,J＝3.0Hz,1H),8.57–8.50(m,2H),8.11(d,J＝8.0Hz,1H),7.47(t,J＝7.5Hz,1H),7.43–7.35(m,2H),4.00(s,3H).
3. general method for synthesizing intermediate 5aa-5al,5ba
(1) When R is2In the case of secondary amine structure, the synthesis method is as follows:
compound 4a or 4b (1eq) was dissolved in DMA and then R was added sequentially2H or hydrochloride (1.2eq) thereof and DIPEA (2eq), placing the reaction solution at 85 ℃ for reaction for 12H, detecting the reaction by TLC, cooling to room temperature after the reaction is completed, quenching the reaction by saturated sodium bicarbonate solution, extracting by dichloromethane, combining organic phases, drying by anhydrous sodium sulfate, filtering, suspending under reduced pressure, and then passing through a column to obtain an intermediate.
(2) When R is2When the structure is mercaptan or alcohol, the synthesis method is as follows:
compound R2Dissolving H (1.5eq) in anhydrous DMF, slowly adding NaH (3eq) at 0 ℃, stirring for 10-20min, slowly dropwise adding 4a or 4b (1eq) DMF solution, slowly heating the reaction solution to room temperature after dropwise adding, reacting overnight, detecting by TLC (thin layer chromatography), adding saturated sodium bicarbonate solution to quench the reaction after the reaction is completed, extracting by dichloromethane, combining organic phases, drying by anhydrous sodium sulfate, filtering, suspending under reduced pressure, and passing through a column to obtain the final productAn intermediate.
4. General method for synthesizing intermediate 6aa-6al,6 ba:
the intermediates 5aa-5al,5ba (1eq), iron powder (6eq) and ammonium chloride (5eq) were dissolved in MeOH H2Reacting in a mixed solvent of O-4: 1 at 80 ℃ for 4h, detecting by TLC after the reaction is finished, filtering the reaction solution while the reaction solution is hot, extracting by dichloromethane, combining organic phases, drying by anhydrous sodium sulfate, filtering, suspending under reduced pressure, and passing through a column to obtain an intermediate.
5. General procedure for synthesis of target compounds:
dissolving the compound 6aa-6al,6ba (1eq) in anhydrous tetrahydrofuran, sequentially adding triethylamine (1.2eq) and acryloyl chloride (1.1eq) under an ice bath condition, continuously reacting for 10min at 0 ℃, adding a saturated sodium bicarbonate solution to quench the reaction after the TLC detection reaction is completed, extracting with dichloromethane, combining organic phases, drying with anhydrous sodium sulfate, filtering, and carrying out reduced pressure suspension drying and column chromatography to obtain a target product.
N-(5-((4-(benzo[b]thiophen-3-yl)pyrimidin-2-yl)amino)-2-((2-(dimethyl-amino)-ethyl)(methyl)amino)-4-methoxyphenyl)acrylamide(Ⅵ-1)
Yellow solid, yield: 44%;1H NMR(500MHz,CDCl3)δ10.09(bs,1H),9.74(s,1H),8.61(bs,1H),8.56(d,J＝4.9Hz,1H),8.42(d,J＝8.0Hz,1H),7.90(d,J＝7.9Hz,1H),7.71(s,1H),7.44(t,J＝7.5Hz,1H),7.38(t,J＝7.4Hz,1H),7.15(d,J＝4.9Hz,1H),3.88(s,3H),2.86(t,J＝5.0Hz 2H),2.70(s,3H),2.32–2.19(m,8H).13C NMR(126MHz,CDCl3)δ163.12,161.62,160.14,158.56,144.79,141.18,136.65,135.72,134.53,132.37,131.47,129.53,126.68,125.88,124.74,124.45,123.64,122.90,111.30,110.08,104.65,57.43,56.56,56.07,45.50,43.56.HRMS(ESI)calculated for C27H30N6O2S[M+H]+:503.2224,found:503.2213.Purity:99.9％(by HPLC).N-(5-((4-(benzo[b]thiophen-3-yl)pyrimidin-2-yl)amino)-2-((2-(diethyl-amino)-ethyl)(methyl)amino)-4-methoxyphenyl)acrylamide(Ⅵ-2)
yellow solid, yield: 35 percent;1H NMR(400MHz,CDCl3)δ9.72(s,1H),9.63(s,1H),8.59(s,1H),8.55(d,J＝5.1Hz,1H),8.42(d,J＝8.0Hz,1H),7.89(d,J＝7.9Hz,1H),7.70(s,1H),7.43(t,J＝7.4Hz,1H),7.37(t,J＝7.4Hz,1H),7.14(d,J＝5.1Hz,1H),6.77(s,1H),6.47(m,2H),5.70(d,J＝11.2Hz,1H),3.87(s,3H),2.90(m,2H),2.66(s,3H),2.61(t,J＝7.1Hz,4H),2.48(m,2H),1.02(t,J＝7.1Hz,6H).13C NMR(101MHz,CDCl3)δ163.12,161.63,160.14,158.60,144.80,141.17,136.64,136.02,134.53,132.30,131.45,128.74,126.41,126.20,124.78,124.49,123.67,122.91,111.55,110.09,104.18,56.09,50.53,47.50,43.13,10.98.HRMS(ESI)calculated for C29H34N6O2S[M+H]+:531.2537,found:531.2561.Purity:99.7％(by HPLC).
N-(5-((4-(benzo[b]thiophen-3-yl)pyrimidin-2-yl)amino)-2-((3-(dimethyl-amino)-propyl)(methyl)amino)-4-methoxyphenyl)acrylamide(Ⅵ-3)
yellow-green solid, yield: 47%;1H NMR(500MHz,CDCl3)δ9.70(s,1H),8.72(s,1H),8.55(d,J＝5.1Hz,2H),8.42(d,J＝8.1Hz,1H),7.90(d,J＝8.0Hz,1H),7.70(s,1H),7.44(t,J＝7.6Hz,1H),7.38(t,J＝7.5Hz,1H),7.15(d,J＝5.1Hz,1H),6.76(s,1H),6.43(d,J＝16.8Hz,1H),6.33(dd,J＝16.8,10.0Hz,1H),5.74(d,J＝9.9Hz,1H),3.88(s,3H),2.89(t,J＝7.2Hz,2H),2.62(s,3H),2.34(t,J＝7.2Hz,4H),2.23(s,6H),1.69–1.59(m,2H).13C NMR(126MHz,CDCl3)δ162.80,161.67,160.09,158.57,144.87,141.17,136.63,135.38,134.53,132.11,131.32,128.03,126.60,126.52,124.79,124.53,123.67,122.92,110.88,110.20,103.95,57.43,56.11,55.15,45.27,43.64,25.64.HRMS(ESI)calculated for C28H32N6O2S[M+H]+:517.2380,found:517.2396.Purity:98.3％(by HPLC).
N-(5-((4-(benzo[b]thiophen-3-yl)pyrimidin-2-yl)amino)-4-methoxy-2-(4-methyl-piperazin-1-yl)phenyl)acrylamide(Ⅵ-4)
yellow solid, yield: 40.8 percent;1H NMR(500MHz,CDCl3)δ9.68(s,1H),8.66–8.49(m,3H),8.42(d,J＝8.0Hz,1H),7.91(d,J＝8.0Hz,1H),7.69(s,1H),7.44(t,J＝7.6Hz,1H),7.38(t,J＝7.5Hz,1H),7.15(d,J＝5.1Hz,1H),6.79(s,1H),6.43(d,J＝16.9Hz,1H),6.29(dd,J＝16.8,10.2Hz,1H),5.76(d,J＝10.1Hz,1H),3.87(s,3H),2.91(t,J＝5.0Hz,4H),2.61(m,4H),2.39(s,3H).13C NMR(126MHz,CDCl3)δ162.61,161.65,160.10,158.57,144.83,141.16,136.63,135.22,134.54,132.16,131.27,126.92,126.49,126.45,124.79,124.52,123.68,122.92,111.04,110.20,103.50,56.04,52.45,46.19.HRMS(ESI)calculated for C27H28N6O2S[M+H]+:501.2067,found:501.2072.Purity:97.4％(by HPLC).
N-(5-((4-(benzo[b]thiophen-3-yl)pyrimidin-2-yl)amino)-2-(4-ethyl-piperazin-1-yl)-4-methoxyphenyl)acrylamide(Ⅵ-5)
yellow solid, yield: 58 percent;1H NMR(500MHz,CDCl3)δ9.68(s,1H),8.61(s,1H),8.55(d,J＝5.0Hz,2H),8.42(d,J＝8.0Hz,1H),7.90(d,J＝8.0Hz,1H),7.69(s,1H),7.44(t,J＝7.6Hz,1H),7.38(t,J＝7.5Hz,1H),7.15(d,J＝5.1Hz,1H),6.81(s,1H),6.42(d,J＝16.9Hz,1H),6.29(dd,J＝16.8,10.2Hz,1H),5.75(d,J＝10.1Hz,1H),3.86(s,3H),2.92(t,J＝4.3Hz,4H),2.63(m,4H),2.51(q,J＝7.2Hz,2H),1.15(t,J＝7.2Hz,3H).13C NMR(126MHz,CDCl3)δ162.62,161.65,160.10,158.57,144.82,141.16,136.63,135.31,134.54,132.18,131.29,126.95,126.45,126.43,124.79,124.52,123.69,122.92,111.00,110.19,103.55,56.00,53.75,52.55,52.43,12.14.HRMS(ESI)calculated for C28H30N6O2S[M+H]+:515.2224,found:515.2239.Purity:99.9％(by HPLC).
N-(5-((4-(benzo[b]thiophen-3-yl)pyrimidin-2-yl)amino)-2-(4-isopropyl-piperazin-1-yl)-4-methoxyphenyl)acrylamide(Ⅵ-6)
white solid, yield: 49 percent;1H NMR(500MHz,CDCl3)δ9.69(s,1H),8.65(s,1H),8.57(d,J＝5.0Hz,2H),8.45(d,J＝8.0Hz,1H),7.92(d,J＝8.0Hz,1H),7.71(s,1H),7.46(t,J＝7.6Hz,1H),7.40(t,J＝7.5Hz,1H),7.17(d,J＝5.1Hz,1H),6.84(s,1H),6.45(d,J＝16.9Hz,1H),6.32(dd,J＝16.8,10.2Hz,1H),5.78(d,J＝10.1Hz,1H),3.88(s,3H),2.94(t,J＝4.4Hz,4H),2.83–2.60(m,5H),1.14(d,J＝6.5Hz,6H).13C NMR(126MHz,CDCl3)δ162.60,161.63,160.10,158.55,144.82,141.15,136.62,135.41,134.53,132.18,131.26,126.92,126.41,126.37,124.77,124.50,123.68,122.90,111.00,110.16,103.59,55.98,54.59,52.90,49.63,18.70.HRMS(ESI)calculated for C29H32N6O2S[M+H]+:529.2380,found:529.2364.Purity:97.2％(by HPLC).N-(2-(4-acetylpiperazin-1-yl)-5-((4-(benzo[b]thiophen-3-yl)pyrimidin-2-yl)-amino)-4-methoxyphenyl)acrylamide(Ⅵ-7)
yellow solid, yield: 48.8 percent;1H NMR(400MHz,CDCl3)δ9.69(s,1H),8.56(d,J＝5.1Hz,2H),8.50–8.37(m,2H),7.91(d,J＝7.8Hz,1H),7.72(s,1H),7.52–7.34(m,2H),7.17(d,J＝5.2Hz,1H),6.71(s,1H),6.43(d,J＝16.7Hz,1H),6.28(dd,J＝16.9,10.1Hz,1H),5.77(d,J＝10.1Hz,1H),3.88(s,3H),3.79(s,2H),3.69–3.56(m,2H),2.86(t,J＝4.8Hz,4H),2.15(s,3H).13C NMR(101MHz,CDCl3)δ169.27,162.76,161.78,160.12,158.67,144.94,141.27,136.70,134.59,132.05,131.47,127.08,126.93,124.92,124.67,123.75,123.06,111.38,110.45,103.36,56.20,52.97,52.27,47.22,42.32,21.53.HRMS(ESI)calculated for C28H28N6O3S[M+H]+:529.2016,found:529.2007.Purity:98.6％(by HPLC).
N-(5-((4-(benzo[b]thiophen-3-yl)pyrimidin-2-yl)amino)-4-methoxy-2-(4-methyl-1,4-diazepan-1-yl)phenyl)acrylamide(Ⅵ-8)
yellow solid, yield: 42%;1H NMR(400MHz,CDCl3)δ9.59(s,1H),8.85(s,1H),8.54(d,J＝5.1Hz,2H),8.43(d,J＝8.0Hz,1H),7.90(d,J＝7.7Hz,1H),7.67(s,1H),7.49–7.41(m,1H),7.41–7.32(m,1H),7.14(d,J＝5.2Hz,1H),6.76(s,1H),6.50–6.29(m,2H),5.75(d,J＝9.8Hz,1H),3.87(s,3H),3.19–3.07(m,4H),2.84–2.72(m,4H),2.47(s,3H),1.97(m,2H).13C NMR(101MHz,CDCl3)δ162.86,161.66,160.12,158.57,145.00,141.15,137.94,136.63,134.56,132.16,131.25,126.96,126.48,126.09,124.80,124.53,123.71,122.91,111.44,110.15,105.17,59.48,57.08,56.07,55.61,54.90,47.41,28.77.HRMS(ESI)calculated for C28H30N6O2S[M+H]+:515.2224,found:515.2199.Purity:99.4％(by HPLC).
N-(5-((4-(benzo[b]thiophen-3-yl)pyrimidin-2-yl)amino)-2-(4-(dimethyl-amino)-piperidin-1-yl)-4-methoxyphenyl)acrylamide(Ⅵ-9)
yellow solid, yield: 54 percent;1H NMR(500MHz,CDCl3)δ9.63(s,1H),8.54(d,J＝4.9Hz,2H),8.48(s,1H),8.42(d,J＝8.0Hz,1H),7.91(d,J＝8.0Hz,1H),7.68(s,1H),7.44(t,J＝7.5Hz,1H),7.38(t,J＝7.4Hz,1H),7.15(d,J＝5.1Hz,1H),6.72(s,1H),6.42(d,J＝16.8Hz,1H),6.30(dd,J＝16.8,10.1Hz,1H),5.75(d,J＝10.0Hz,1H),3.88(s,3H),3.06(d,J＝11.5Hz,2H),2.71(t,J＝11.5Hz,2H),2.46(m,7H),2.08(d,J＝11.9Hz,2H),1.76(m,2H).13C NMR(126MHz,CDCl3)δ162.75,161.64,160.07,158.57,144.82,141.15,136.60,135.67,134.50,132.08,131.31,126.66,126.62,126.32,124.81,124.55,123.68,122.93,111.35,110.20,103.15,62.25,56.10,52.11,41.54,29.37.HRMS(ESI)calculated for C29H32N6O2S[M+H]+:529.2380,found:529.2375.Purity:97.3％(by HPLC).
N-(5-((4-(benzo[b]thiophen-3-yl)pyrimidin-2-yl)amino)-4-methoxy-2-(4-(4-methylpiperazin-1-yl)piperidin-1-yl)phenyl)acrylamide(Ⅵ-10)
yellow-green solid, yield: 41.5 percent;1H NMR(500MHz,CDCl3)δ9.65(s,1H),8.63–8.46(m,3H),8.42(d,J＝8.0Hz,1H),7.90(d,J＝8.0Hz,1H),7.68(s,1H),7.44(t,J＝7.6Hz,1H),7.38(t,J＝7.5Hz,1H),7.15(d,J＝5.1Hz,1H),6.74(s,1H),6.42(d,J＝16.9Hz,1H),6.27(dd,J＝16.8,10.2Hz,1H),5.75(d,J＝10.1Hz,1H),3.88(s,3H),3.04(d,J＝11.4Hz,2H),2.72(t,J＝11.7Hz,4H),2.65–2.35(m,5H),2.34(m,4H),2.23(m,1H),2.06(d,J＝11.8Hz,2H),1.66(q,J＝11.6Hz,2H).13C NMR(126MHz,CDCl3)δ162.68,161.64,160.10,158.56,144.80,141.16,136.62,135.95,134.53,132.10,131.29,126.73,126.53,126.20,124.78,124.51,123.69,122.91,111.20,110.15,103.16,61.44,56.08,55.35,52.59,49.43,45.97,29.92.HRMS(ESI)calculated for C32H37N7O2S[M+H]+:584.2802,found:584.2802.Purity:99.8％(by HPLC).
N-(5-((4-(benzo[b]thiophen-3-yl)pyrimidin-2-yl)amino)-2-((2-(dimethyl-amino)-ethyl)-thio)-4-methoxyphenyl)acrylamide(Ⅵ-11)
yellow solid, yield 68.1%;1H NMR(500MHz,CDCl3)δ10.19(s,1H),9.73(s,1H),8.62(bs,1H),8.58(d,J＝5.1Hz,1H),8.40(d,J＝8.1Hz,1H),7.96–7.90(m,2H),7.46(t,J＝7.6Hz,1H),7.39(t,J＝7.5Hz,1H),7.20(d,J＝5.1Hz,1H),7.10(s,1H),6.50(d,J＝16.9Hz,1H),6.31(dd,J＝16.9,10.2Hz,1H),5.75(d,J＝10.2Hz,1H),3.92(s,3H),2.84(t,J＝6.0Hz,2H),2.31(t,J＝6.0Hz,2H),2.24(s,6H).13C NMR(126MHz,CDCl3)δ163.25,161.63,159.82,158.58,144.32,141.19,136.56,136.06,134.36,132.09,131.76,131.11,126.57,124.85,124.55,123.50,122.99,117.58,113.42,111.54,110.62,56.31,56.10,44.75,35.43.HRMS(ESI)calculated for C26H27N5O2S2[M+H]+:506.1679,found:506.1659.Purity:97.5％(by HPLC).
N-(5-((4-(benzo[b]thiophen-3-yl)pyrimidin-2-yl)amino)-4-methoxy-2-(2-mor-pholinoethoxy)phenyl)acrylamide(Ⅵ-12)
yellow solid, yield: 38.7 percent;1H NMR(500MHz,CDCl3)δ9.57(s,1H),8.56–8.47(m,2H),8.44(m,2H),7.90(d,J＝8.0Hz,1H),7.58(s,1H),7.43(t,J＝7.5Hz,1H),7.38(t,J＝7.4Hz,1H),7.12(d,J＝5.0Hz,1H),6.60(s,1H),6.47(d,J＝16.8Hz,1H),6.37(dd,J＝16.8,10.0Hz,1H),5.76(d,J＝9.9Hz,1H),4.17(t,J＝5.1Hz,2H),3.87(s,3H),3.75(m,4H),2.69(t,J＝4.9Hz,2H),2.54(m,4H).13C NMR(126MHz,CDCl3)δ162.91,161.70,160.20,158.55,145.39,142.75,141.12,136.64,134.60,131.88,131.07,126.93,124.78,124.54,123.79,123.73,122.90,122.52,112.98,110.04,99.30,67.82,66.76,57.70,56.17,53.78.HRMS(ESI)calculated for C28H29N5O4S[M+H]+:532.2013,found:532.2006.Purity:99.8％(by HPLC).
N-(5-((4-(benzo[b]thiophen-3-yl)-5-fluoropyrimidin-2-yl)amino)-2-((2-(dimethyl-amino)ethyl)(methyl)amino)-4-methoxyphenyl)acrylamide(Ⅵ-13)
yellow solid, yield: 32 percent;1H NMR(500MHz,CDCl3)δ10.11(s,1H),9.55(s,1H),8.50(m,2H),8.40(s,1H),7.90(d,J＝7.9Hz,1H),7.64(s,1H),7.41(m,2H),6.79(s,1H),6.43(d,J＝16.9Hz,1H),6.31(dd,J＝16.9,10.1Hz,1H),5.69(d,J＝10.1Hz,1H),3.88(s,3H),2.88(t,J＝5.3Hz,2H),2.70(s,3H),2.29(t,J＝5.3Hz,3H),2.26(s,6H).13C NMR(126MHz,CDCl3)δ163.30,156.41,150.08(1J＝252.5Hz),149.63(3J＝11.25Hz),146.34(2J＝25.0Hz),144.95,140.08,136.98,136.04,132.97(3J＝10.0Hz),132.28,129.41,128.92,128.88,126.52,126.02,124.85,124.82,124.80,124.76,122.46,111.22,104.63,57.38,56.48,56.04,45.48,43.52.HRMS(ESI)calculated for C27H29FN6O2S[M+H]+:521.2129,found:521.2122.Purity:98.3％(by HPLC).
example 2 kinase assay experiment
To determine targeting of 6a and to evaluate its anti-tumor potential, we performed in vitro enzymatic assays. Afatinib and Osimetinib were used as positive controls. As shown in fig. 1, fig. 1(a) is that 6a induces dose-dependent inhibition of EGFR phosphorylation; (B) dose-dependent inhibition of EGFR phosphorylation was induced for Afatinib and Osimertinib. Data are presented as mean ± standard deviation of triplicate independent experiments.
Of these, 6a showed strong inhibitory activity at nanomolar concentration levels and was dose-dependent (IC50 ═ 1.33 ± 0.02nM for EGFR-L858R), similar to IC50 ═ 1.03 ± 0.03nM for the positive control Afatinib. The inhibitory effect of 6a (IC50 ═ 0.26 ± 0.01nM) was slightly stronger than that of Osimertinib (IC50 ═ 0.37 ± 0.04nM) for EGFR T790M/L858R mutation, suggesting that 6a has the potential to overcome resistance to EGFR mutations.
Example 3 antitumor Activity study of benzothiophenopyrimidine derivative
The in vitro antiproliferative activity of the synthesized 13 compounds on H1975, PC9 and H229 cells is determined by taking the Osimetinib as a positive control, and the specific process is as follows:
collecting cells in logarithmic phase, adjusting the concentration of cell suspension,at 5X 103Inoculating one cell/well into 96-well plate, and incubating at 37 deg.C in 5% carbon dioxide incubator for 12-24 hr. After the cells adhere to the wall, the old culture medium is sucked away, and a culture medium without FBS is added; six concentration gradients (10, 1, 0.1, 0.05, 0.01, 0.001 μ M) are set for each compound, three sets of multiple wells are set for each concentration, after adding, the mixture is cultured for 48h, then CCK8 solution is added into each well in a dark place, and the mixture is put into a carbon dioxide incubator to be continuously incubated for 2-4 h. The absorbance (OD value) of light of each well was measured at an emission wavelength of 450nM in a microplate reader. And finally, carrying out data statistics, and calculating the inhibition rate under each concentration according to the following formula:
the half maximal inhibitory amount (IC) was then calculated using the software GraphPad Prism 5.0250) Each set of experiments was independently repeated three times and averaged to obtain the final data. The results of the specific experiments are shown in table 1.
TABLE 1 VI-1 to VI-13 antitumor Activity
EGFR kinase L858R/T790M double mutant cells; egfr kinase L858R single mutant cells; EGFR kinase wild-type cells.
The results of in vitro antiproliferation experiments show that: the antiproliferative activity of the compound VI-1 on H1975 cells is 2.2nM, which is 1.58 times of that of the positive drug Osimetinib; meanwhile, compared with a positive drug, the compound VI-1 has higher selectivity on double-mutant H1975 cells. The in vitro antiproliferative activity of the compounds VI-2 to VI-12 and VI-13 is weaker than that of VI-1.
The results of in vitro antiproliferative activity research on 13 Osimetinib derivatives show that N-methylindole ring is substituted by heterocyclic ring with larger hydrophobicity, which is beneficial to resistingThe increase of the proliferation activity indicates that the combination of the partial structure and the target site needs hydrophobic effect, and in addition, the high selectivity of the compound VI-1 to H1975 cells further verifies that the toxicity of the compound is partially derived from N-methylindole demethylation reaction; to R2Partial structure modification, whether the volume, the electrical property or the lipid solubility of the chain is changed, reduces the activity, which probably leads small molecules not to be effectively combined with target protein due to the change of the space conformation of the molecules, and also shows that the side chain structure has higher conservation; r is to be1Following substitution with fluorine atoms, the antiproliferative activity is reduced, and it is likely that the increased volume will not be able to effectively bind to the target protein due to the smaller binding site to the target.
Claims (10)
1. A benzo thiophene pyrimidine-containing derivative is characterized by having the following chemical structural formula:
wherein R is1Is H or F; r2Is a secondary amine, thiol, alkyl piperazine or piperidine containing substituent.
2. The benzothiophene-containing pyrimidine derivative of claim 1, wherein R is2Is composed of
3. The benzothiophene-containing pyrimidine derivative of claim 2, wherein R is1Is H, R2Is composed of
4. The benzothiophene-containing pyrimidine derivative of claim 2, wherein R is1Is F, R2Is composed of
5. The method for producing a benzothiophene-containing pyrimidine derivative as claimed in any one of claims 1 to 4, which comprises the steps of:
(1) reacting benzothiophene-3-boronic acid and (2, 4-dichloropyrimidine) -2b (2, 4-dichloro-6-fluoropyrimidine) through Suzuki;
(2) carrying out aromatic nucleophilic reaction on 4-fluoro-2-methoxy-5-nitroaniline and the product obtained in the step (1) under an acidic condition;
(3) reacting the product obtained in step (2) with R under alkaline conditions2H or hydrochloride thereof;
(4) and (3) mixing and dissolving the product obtained in the step (3), ammonium chloride and a reducing agent, and carrying out nitro reduction and acylation reaction after the reaction is finished to obtain the benzothiophene-containing pyrimidine derivative.
6. The preparation method according to claim 5, wherein the aromatic nucleophilic reaction in the step (2) is carried out by the following specific steps: dissolving 4-fluoro-2-methoxy-5-nitroaniline and the product obtained in the step (1), and heating to 80-100 ℃ overnight.
7. The method according to claim 5 or 6, wherein the 4-fluoro-2-methoxy-5-nitroaniline is used in an amount of 1 to 1.5 eq.
8. The method according to claim 5, wherein R in the step (3)2In the case of secondary amines: dissolving the product obtained in the step (2), and then adding R2H or hydrochloride thereof and DIPEA react for 10-15H at 80-90 ℃.
9. Root of herbaceous plantThe method according to claim 5, wherein R in the step (3)2In the case of thiols or alcohols: r is to be2Dissolving H, adding NaH at 0 ℃, stirring for 10-20min, adding the product obtained in the step (2), stirring to enable the reaction solution to be heated to room temperature, and then reacting overnight.
10. The benzo-thiophene-containing pyrimidine derivative of any one of claims 1 to 4 or the benzo-thiophene-containing pyrimidine derivative prepared by the method of any one of claims 5 to 9 is applied to preparation of an anti-tumor drug.
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| CN101039919A (en) * | 2004-10-13 | 2007-09-19 | 惠氏公司 | N-benzenesulfonyl substituted anilino-pyrimidine analogs |
| WO2009046416A1 (en) * | 2007-10-05 | 2009-04-09 | Targegen Inc. | Anilinopyrimidines as jak kinase inhibitors |
| CN101460491A (en) * | 2006-04-12 | 2009-06-17 | 惠氏公司 | Anilino-pyrimidine phenyl and benzothiophene analogs |
| CN103702990A (en) * | 2011-07-27 | 2014-04-02 | 阿斯利康(瑞典)有限公司 | 2-(2,4,5-substituted -anilino) pyrimidine derivatives as egfr modulators useful for treating cancer |
| CN106559991A (en) * | 2014-06-19 | 2017-04-05 | 阿里亚德医药股份有限公司 | For the heteroaryl compound of kinase inhibition |
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| CN101039919A (en) * | 2004-10-13 | 2007-09-19 | 惠氏公司 | N-benzenesulfonyl substituted anilino-pyrimidine analogs |
| CN101460491A (en) * | 2006-04-12 | 2009-06-17 | 惠氏公司 | Anilino-pyrimidine phenyl and benzothiophene analogs |
| WO2009046416A1 (en) * | 2007-10-05 | 2009-04-09 | Targegen Inc. | Anilinopyrimidines as jak kinase inhibitors |
| CN103702990A (en) * | 2011-07-27 | 2014-04-02 | 阿斯利康(瑞典)有限公司 | 2-(2,4,5-substituted -anilino) pyrimidine derivatives as egfr modulators useful for treating cancer |
| CN106559991A (en) * | 2014-06-19 | 2017-04-05 | 阿里亚德医药股份有限公司 | For the heteroaryl compound of kinase inhibition |
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