Novel pyrimidine derivative of sulfinyl anilino and application of novel pyrimidine derivative in preparation of antitumor drugs
阅读说明：本技术 一种新型亚磺酰基苯胺基的嘧啶衍生物及其在制备抗肿瘤药物中的应用 (Novel pyrimidine derivative of sulfinyl anilino and application of novel pyrimidine derivative in preparation of antitumor drugs ) 是由 黎兴术 姚晗 安佰娇 陈新滋 任媛媛 吴峰 李卫 于 2021-06-04 设计创作，主要内容包括：本发明涉及药物领域,公开了一种新型亚磺酰基苯胺基的嘧啶衍生物,所述新型亚磺酰基苯胺基的嘧啶衍生物结构式如下所示。本发明中含有亚砜基团的苯胺基嘧啶衍生物,通过细胞和动物水平上的抗肿瘤活性实验(L858R/T790M突变基因的非小细胞肺癌H1975肿瘤细胞,ALK高表达的BaF3细胞等)证明,与现有ALK抑制剂及EGFR/ALK双靶点参照物相比,有更好的抗肿瘤活性。通过人肝微粒体实验证明,本发明中含有亚砜基团的苯胺基嘧啶衍生物与现有的含有异丙硫酰基的苯胺基嘧啶衍生物相比,稳定性有较大幅度的提高。(The invention relates to the field of medicines, and discloses a novel pyrimidine derivative of sulfinyl anilino, wherein the structural formula of the novel pyrimidine derivative of sulfinyl anilino is shown as follows. The anilinopyrimidine derivative containing a sulfoxide group has better antitumor activity compared with the existing ALK inhibitor and EGFR/ALK double-target reference substances as proved by antitumor activity experiments on a cellular and animal level (non-small cell lung cancer H1975 tumor cells of L858R/T790M mutant genes, BaF3 cells with high ALK expression and the like). Proved by human liver microsome experimentsCompared with the prior anilinopyrimidine derivative containing isopropylthioacyl, the anilinopyrimidine derivative containing sulfoxide groups has greatly improved stability.)
1. A novel pyrimidine derivative of sulfinyl anilino is characterized in that the structural formula is shown as a formula I:
in formula I, R1 is an alkyl group containing 1 to 4 carbon atoms; r2, R3 and R4 are respectively halogen atoms, chain alkyl containing 1-4 carbon atoms, naphthenic base and alkoxy containing 1-4 carbon atoms; r5 is an alkyl group containing 1 to 4 carbon atoms; r6 is hydrogen, methyl, or acrylamido; r7 is piperidyl, piperazinyl or N, N, N-trimethylethylenediamine, wherein hydrogen bonded to a nitrogen atom in piperidyl or piperazinyl may be substituted with a straight-chain alkyl group having 1 to 3 carbon atoms.
2. A novel pyrimidine derivative according to claim 1, wherein R1 is a straight chain alkyl group having 1 to 4 carbon atoms, and the hydrogen atom in the straight chain alkyl group may be substituted by an alkyl group having 1 to 4 carbon atoms.
3.A novel pyrimidine derivative according to claim 2 wherein when the hydrogen atom of R1 is substituted by an alkyl group having from 1 to 4 carbon atoms, the carbon atom is deuterium with hydrogen or the hydrogen isotope attached.
4. A novel pyrimidine derivative of the sulfinyl anilino group according to claim 1, characterised in that the halogen atom is fluorine or chlorine.
5. A novel pyrimidine derivative of the sulfinyl anilino group as claimed in claim 1, wherein R1 is-CD (CD)3)2Cyclopropyl, cyclopropylmethyl, isopropyl, ethyl, -CD2CD3(ii) a R2, R3 and R4 are respectively halogen atoms and chain alkyl containing 1-2 carbon atoms; r5 is methyl, ethyl or isopropyl; r7 is piperidinyl, N-methylpiperidinyl or N, N, N-trimethylethylenediamino.
6. An ALK inhibitor comprising a pyrimidine derivative of the sulfinyl anilino group of claim 1.
7. A dual target inhibitor of ALK and EGFR comprising a pyrimidine derivative of the sulfinyl anilino group of claim 1.
8. The ALK and EGFR double-target inhibitor according to claim 7, wherein when R6 is an acrylamide group, a pyrimidine derivative containing the sulfinyl anilino group according to claim 1 is prepared as the ALK and EGFR double-target inhibitor.
9. Use of a pyrimidine derivative as defined in claim 1 as an anticancer agent.
10. The use according to claim 9, in the preparation of a medicament against lung adenocarcinoma and lung cancer.
Technical Field
The invention relates to the field of medicines, in particular to a novel pyrimidine derivative of sulfinyl anilino and application thereof in preparing antitumor medicines.
Background
Anaplastic Lymphoma Kinase (ALK) is a highly conserved receptor-type tyrosine kinase. The research shows that the abnormal expression of ALK, such as gene mutation, rearrangement and amplification, is closely related to various human malignant tumors (such as neuroblastoma, non-small cell lung cancer and the like). In non-small cell lung cancer, about 5% of patients are accompanied by gene fusion mutation of ALK and Echinoderm microtubule-associated protein-like 4 (EML 4), which activates ALK and various downstream signal transduction pathways thereof, promotes rapid proliferation and differentiation of cells, and finally causes tumor. Therefore, the ALK inhibitor is also one of the hot spots in the research and development of antitumor drugs in recent years. In 2011, crizotinib (crizotinib), the first global drug for treating ALK-dependent non-small cell lung cancer, was marketed. Next, second-generation ALK inhibitors having stronger specificity and affinity for ALK kinase, ceritinib (ceritinib), brigatinib (brigatinib), and the like are also successively marketed. Although these drugs play an important role in saving the life of a patient, resistance after long-term use is inevitable for a variety of reasons.
Epidermal Growth Factor Receptor (EGFR) tyrosine kinases are one class of receptor tyrosine kinases. EGFR is widely distributed in epithelial cell membranes other than vascular tissue. In normal cells, EGFR tyrosine kinase is regulated by its ligand, and plays a role in regulating the growth and proliferation of normal cells. When the regulatory gene of the pathway is mutated or amplified, the downstream pathway mediated by the regulatory gene is abnormally activated, thereby inducing various cancers. Targeting EGFR tyrosine kinase is also one of important means for developing antitumor drugs. To date, first to third generation EGFR inhibitors such as gefitinib, afatinib and ocitinib (Osimertinib, AZD9291) are also marketed in succession, and have good therapeutic effects on patients with EGFR gene sensitive mutation, non-small cell lung cancer, or patients with first generation EGFR inhibitors resistant to drugs. Although these new targeted drugs (e.g., ocitinib) are encouraging in the treatment of non-small cell lung cancer patients, tumor cells have a very strong screening capacity and it is still possible that new drug resistance is developed after adaptation to third generation EGFR inhibitors. In addition, EGFR activation has been shown to be a compensatory resistance mechanism following the use of small molecule ALK inhibitors such as crizotinib, elotinib and ceritinib. Meanwhile, in clinical practice, cancer patients accompanied by both EGFR mutation and ALK rearrangement (simply referred to as "double mutation") are successively discovered as detection techniques advance. Lee et al reported that the incidence of double mutations of EGFR and ALK in Korean lung adenocarcinoma was 0.9%. 977 Chinese NSCLC patients are continuously detected by Yang and the like, 13 EGFR and ALK double-mutation patients are found together, and the double-mutation incidence rate is 1.3 percent.
In 2017, the literature (European Journal of Medicinal Chemistry2017,136, 497-one 510) reports the design synthesis and activity research of double-target (EGFR and ALK) antitumor drugs with structural characteristics containing sulfonyl anilinopyrimidine derivatives. The preferred compounds have excellent tumor cell inhibiting activity, but the bioavailability is not high (24%), and are to be further optimized. Patent WO2017/101803a also reports on studies containing sulfonylanilinopyrimidine derivatives, and similar situations exist. Therefore, it is an urgent problem to find an antitumor compound having a good activity and few side effects and to study the pharmaceutical properties.
Disclosure of Invention
To overcome the above-mentioned disadvantages and drawbacks of the prior art, the present invention provides a novel pyrimidine derivative as a sulfinyl anilino group.
The invention also provides application of the novel pyrimidine derivative of sulfinyl anilino in preparation of antitumor drugs.
In order to achieve the purpose, the invention adopts the technical scheme that:
a novel pyrimidine derivative of sulfinyl anilino has a structural formula I:
in formula I, R1 is an alkyl group containing 1 to 4 carbon atoms; r2, R3 and R4 are respectively halogen atoms, chain alkyl containing 1-4 carbon atoms, naphthenic base and alkoxy containing 1-4 carbon atoms; r5 is an alkyl group containing 1 to 4 carbon atoms; r6 is hydrogen, methyl, or acrylamido; r7 is piperidyl, piperazinyl or N, N, N-trimethylethylenediamine, wherein hydrogen bonded to a nitrogen atom in piperidyl or piperazinyl may be substituted with a straight-chain alkyl group having 1 to 3 carbon atoms.
As a preferred embodiment, R1 is a straight chain alkyl group having 1 to 4 carbon atoms, and the hydrogen atom in the straight chain alkyl group may be substituted with an alkyl group having 1 to 4 carbon atoms.
As a preferred embodiment, when the hydrogen atom on R1 is substituted with an alkyl group of 1 to 4 carbon atoms, the carbon atom is attached to hydrogen or deuterium, which is an isotope of hydrogen.
As a preferred embodiment, the halogen atom is fluorine or chlorine.
As a preferred technical scheme, R1 is-CD (CD)3)2Cyclopropyl, cyclopropylmethyl, isopropyl, ethyl, -CD2CD3(ii) a R2, R3 and R4 are respectively halogen atoms and chain alkyl containing 1-2 carbon atoms; r5 is methyl, ethyl or isopropyl; r7 is piperidinyl, N-methylpiperidinyl or N, N, N-trimethylethylenediamino.
The invention also discloses an ALK inhibitor which comprises the pyrimidine derivative of the sulfinyl anilino.
In addition, the invention also protects an ALK and EGFR double-target inhibitor, which comprises the pyrimidine derivative of the sulfinyl anilino.
Further, when R6 is hydrogen or an alkyl group such as methyl, the formula I is a sulfinyl anilinopyrimidine derivative ALK inhibitor. When R6 is an acrylamide group, the formula I is a double-target inhibitor containing sulfinyl anilinopyrimidine derivative ALK and EGFR, and can also be used as an ALK inhibitor.
The synthesis route and method of the novel ALK inhibitor containing sulfinyl anilinopyrimidine derivative, R6 is hydrogen or alkyl such as methyl, and R7 is piperidinyl is described as follows, taking as an example the synthesis of a representative target A-1:
in the synthetic route, 2-aminothiophenol as a starting material reacts with bromoethane to obtain a thioether intermediate M-1, and then the thioether intermediate M-1 reacts with hydrogen peroxide to produce an intermediate M-2 with a sulfoxide structure, and the intermediate M-2 reacts with 2,4, 5-trichloropyrimidine to obtain a key intermediate M-3. On the other hand, 2-isopropoxy-4-chloro-5-methylnitrobenzene is coupled with 4-pyridineboronic acid in the presence of a catalyst to give intermediate M-4, which is reacted with iodomethane to give M-5. And reducing the M-5 by sodium borohydride, and then carrying out catalytic hydrogenation reduction by palladium carbon or platinum dioxide to obtain a key intermediate M-7. M-7 and the key intermediate M-3 react under the catalysis of acid to obtain the target compound A-1. Compound A-1 was separated by hand (AD-H column; mobile phase: isopropanol/n-hexane, 40/60; flow rate: 0.5mL/min.) to give optically active two enantiomers, A-1a (retention time: 8.38 min.) and A-1b (retention time: 10.85 min.).
The synthesis method of the novel ALK/EGFR double-target inhibitor containing sulfinyl anilinopyrimidine derivative, wherein R6 is acrylamide, R7 is piperazinyl, homopiperazinyl or N, N, N-trimethylethylenediamine and the like, takes the synthesis example of a representative target object B-1 as follows:
in the synthetic route, 2-amino-5-fluoro-phenethyl sulfide reacts with hydrogen peroxide to obtain an intermediate M-9 with a sulfoxide structure. The latter reacts with 2,4, 5-trichloropyrimidine under the action of sodium hydride to obtain an intermediate M-10. Commercial 2-methoxy-4-fluoro-5-nitroaniline was reacted with intermediate M-10 in the presence of acid to give M-11. And (3) substituting fluorine atoms at the ortho-position of the nitro group of the M-11 by trimethylethylenediamine to obtain an intermediate M-12, reducing the nitro group by stannous chloride, and reacting with acryloyl chloride to obtain a target compound B-1. The synthesis steps and methods of the other target compounds of the series are similar to those of the target compound B-1. The compound B-1 was subjected to manual separation (AD-H chiral column; mobile phase: isopropanol/n-hexane, 40/60; flow rate: 0.5mL/min.) to give optically active two enantiomers (-) B-1a (retention time: 24.6 min.) and (+) B-1B (retention time: 27.0 min.). Chiral separation of the compound B-1 can also be carried out using other chiral columns, e.g.Separation was performed with Y352546.
The novel anilinopyrimidine derivative containing the chiral sulfoxide group has excellent proliferation inhibition effect on tumor cells, particularly on ALK abnormal expression such as gene mutation, rearrangement and amplification human malignant tumors and EGFR gene T7900M mutant non-small cell lung cancer cells, and is remarkably superior to pyrimidine derivatives containing sulfonyl groups reported in the literature.
The invention also provides pharmaceutically acceptable salts of the anilinopyrimidine derivative (the novel sulfinyl anilino pyrimidine derivative) of the novel chiral sulfoxide group, namely corresponding salts formed by the pyrimidine derivative of the novel chiral sulfoxide group and pharmaceutically acceptable anions such as chloride ions, methanesulfonate ions and the like, and the anilinopyrimidine derivative is prepared by adopting a known salt forming method.
The anilino pyrimidine derivatives of the novel chiral sulfoxide group are used as active ingredients in the preparation of antitumor drugs, and tumors are effectively treated.
The anilinopyrimidine derivatives of the above novel chiral sulfoxide groups are used in the form of pharmaceutically acceptable solvates, preferably hydrates.
The invention also provides a pharmaceutical composition for treating tumors, which contains therapeutically effective amount of anilinopyrimidine derivatives of the novel chiral sulfoxide groups and pharmaceutically acceptable auxiliary agents. The pharmaceutical composition can be prepared into injection, tablet, capsule, pill, suspension or emulsion for use; the administration route can be oral, percutaneous, intravenous or intramuscular injection.
Compared with the prior art, the invention has the beneficial effects that:
(1) the anilinopyrimidine derivative containing a sulfoxide group has better antitumor activity compared with the existing ALK inhibitor and an EGFR/ALK double-target reference substance as proved by an antitumor activity experiment on a cell level (non-small cell lung cancer H1975 tumor cells of L858R/T790M mutant genes, and BaF3 cells with high ALK expression).
(2) Human liver microsome experiments prove that the anilinopyrimidine derivative containing a sulfoxide group has greatly improved stability compared with anilinopyrimidine derivative containing isopropylthioacyl in the literature.
Drawings
FIG. 1 shows the results of the anti-tumor experiments of transplanted nude mice (non-small cell lung cancer H1975 tumor cell modeling with L858R/T790M mutation) with compounds B3a (P1) and B3B (P2). FIG. 1 is a graph A showing the change in tumor volume in groups of nude mice during the experiment; b is a bar chart showing the change of tumor volume of each group of nude mice after administration; the C picture is the weight and the tumor inhibition rate of the tumors of each group of nude mice after the experiment is finished; d represents the weight change of each group of nude mice during the experiment.
FIG. 2 is a graph of in vivo antitumor assay.
Detailed Description
The following further describes the embodiments of the present invention. It should be noted that the description of the embodiments is provided to help understanding of the present invention, but the present invention is not limited thereto. In addition, the technical features involved in the embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.
Example 1: synthesis of Compound A-1.
(1) Synthesis of intermediate M-1
2-Aminothiophenol (10mmol, 1.25g) was dissolved in 20ml of methanol, and potassium hydroxide solid (20mmol, 5.6g) and bromoethane (12mmol,1.3 g) were added in this order and stirred at room temperature overnight. And after TLC plate counting is carried out to confirm that the reaction is finished, removing methanol by spinning, adding 30ml of dichloromethane and 15ml of water, extracting twice, combining organic phases, washing with saturated saline solution, drying by anhydrous sodium sulfate, and carrying out reduced pressure concentration to obtain a crude product intermediate-1 which is directly used in the next step.1H NMR(400MHz,Chloroform-d)δ7.41(dd,J＝7.6,1.3Hz,1H),7.14(td,J＝8.0,1.5Hz,1H),6.80–6.62(m,2H),4.37(s,2H),2.79(q,J＝7.3Hz,2H),1.26(t,J＝7.3Hz,3H).
(2) Synthesis of intermediate M-2
Dissolving the intermediate M-1(10mmol, 1.53g) in 5ml of glacial acetic acid, slowly dropwise adding a 30% hydrogen peroxide solution (1.25g, 11mmol) under ice bath, stirring for 30 minutes under ice bath, reacting for 4-5 hours at room temperature, and confirming the completion of the reaction by TLC point plates. After 3.5g of sodium hydroxide was dissolved in crushed ice, the solution was added to the reaction system and stirred for 5 minutes. The mixture was extracted twice with 30ml of methylene chloride, and the organic phases were combined, washed with saturated brine and dried over anhydrous sodium sulfate. The solvent was removed under reduced pressure, purified by silica gel column chromatography, petroleum ether: eluting with ethyl acetate 8:1 to 2:1, and concentrating to obtain light yellow oil 156 g. Yield: 92 percent.1H NMR(400MHz,DMSO-d6)δ7.27(d,J＝7.9Hz,1H),7.20(t,J＝7.8Hz,1H),6.71(dd,J＝18.1,8.1Hz,2H),5.63(s,2H),2.94(tq,J＝13.2,6.6Hz,2H),1.06(t,J＝7.6Hz,3H).
(3) Synthesis of intermediate M-3
The intermediate 2- (ethyl) sulfinylaniline (10mmol, 1.7g) from the previous reaction was dissolved in dry DMF (30mL) and 60 wt% sodium hydride (25mmol, 1g, 2.5eq) was added under ice-bath and stirred for 15 min. A solution of 2,4, 5-trichloropyrimidine (20mmol, 3.62g) in dry DMF (10mL) was added slowly dropwise over 1 hour, the ice bath was removed and the reaction was allowed to proceed overnight at ambient temperature. After the TLC detection reaction is finished, 30mL of water is slowly added to quench the reaction, ethyl acetate is extracted for 3 times, 30mL of the reaction is carried out each time, organic phases are combined, the organic phases are washed by water and saturated saline solution, the mixture is dried by anhydrous sodium sulfate and concentrated to obtain a crude product, and the crude product is purified by silica gel column chromatography (petroleum ether: ethyl acetate: 10:1 to 4:1) to obtain 1.92g of a light yellow solid product. Yield: 61 percent.1H NMR(400MHz,Chloroform-d)δ11.03(s,1H),8.62(d,J＝8.5Hz,1H),8.25(d,J＝1.3Hz,1H),7.58(t,J＝7.9Hz,1H),7.31(dd,J＝7.7,1.7Hz,1H),7.19(t,J＝7.5Hz,1H),3.29–2.81(m,2H),1.27(td,J＝7.6,1.2Hz,3H).
(4) Synthesis of intermediate M-4
To an isopropanol solution (25ml) containing 1-chloro-5-fluoro-2-methyl-4-nitrobenzene (20mmol, 3.78g) was added cesium carbonate (30mmol, 9.77g), and the mixture was refluxed for 10 hours. After completion of the TLC detection reaction, the solvent was removed by concentration under reduced pressure, the residue was extracted with methylene chloride (30ml) and water (20ml), and the organic phases obtained by 2-fold extraction were combined, washed with saturated brine and dried over anhydrous sodium sulfate. Removal of the solvent gave 3.8g of product as an orange solid. Yield: 83 percent.1H NMR(400MHz,Chloroform-d)δ7.72(d,J＝7.5Hz,1H),7.09(d,J＝7.9Hz,1H),4.63(dt,J＝13.8,6.1Hz,1H),2.36(d,J＝7.9Hz,3H),1.41(t,J＝7.0Hz,6H).
(5) Synthesis of intermediate M-5
The product of the previous step, 1-chloro-5-isopropoxy-2-methyl-4-nitrobenzene (10mmol, 2.29g), was dissolved in 30ml of 1, 4-dioxane, and potassium carbonate solid (25mmol, 3.46g), bis triphenylphosphine palladium dichloride (210.6mg, 0.3mmol), 4-pyridineboronic acid (12mmol, 1.48g), and 10ml of water were added in this order. The reaction is refluxed for 8 hours under the protection of nitrogen, and after the TLC detection reaction is finished, the solvent is removed by decompression and concentration. The residue was extracted with 15ml of water and 40ml of methylene chloride, and the combined 3-fold extracts were washed with saturated brine and dried over anhydrous sodium sulfate. The solvent was removed under reduced pressure and the crude product was purified by silica gel column chromatography (petroleum ether: ethyl acetate: dichloromethane/8: 2: 1) to give 2.55g of a yellow solid. Yield: 93 percent.1H NMR(400MHz,Chloroform-d)δ8.61(d,J＝6.1Hz,2H),7.27(d,J＝6.1Hz,2H),6.68(d,J＝18.5Hz,2H),4.53(p,J＝6.0Hz,1H),2.21(s,3H),1.38(d,J＝6.1Hz,6H).
(6) Synthesis of intermediate M-6
Dissolving the product 2-isopropoxy-4-pyridyl-5-methylnitrobenzene (10mmol, 2.72g) in 15ml acetonitrile, adding iodomethane (15mmol, 2.15g), reacting at 50 ℃ for 3 hours, after TLC determination of reaction, removing the solvent under reduced pressure, and directly carrying out the next reaction on the crude product. The crude product was dissolved in 20ml of methanol, and sodium borohydride (40mmol, 1.51g) was added in small portions each time in ice bath, and the reaction was stirred for half an hour and continued at room temperature for 2 hours. The reaction system was quenched by adding saturated ammonium chloride, the solvent was removed under reduced pressure, and the residue was extracted with 30ml of dichloromethane and 30ml of water. The combined 3 extracts were washed with saturated brine and dried over anhydrous sodium sulfate. Removing the solvent under reduced pressure to obtain a crude product column layerPurification by chromatography (dichloromethane: methanol 50:1 to 20:1 gave 2.1g of product as a pale yellow oil: 72% yield.1H NMR(400MHz,Chloroform-d)δ7.61(s,1H),6.81(s,1H),5.60(s,1H),4.59(dq,J＝12.1,6.2Hz,1H),3.10(q,J＝2.8Hz,2H),2.66(t,J＝5.6Hz,2H),2.41(d,J＝16.5Hz,4H),2.24(s,3H),1.64(s,1H),1.36(d,J＝6.0Hz,6H).
(7) Synthesis of intermediate M-7
The product of the previous step (5mmol, 1.45g) was dissolved in 5ml of methanol, 10% palladium on charcoal (150mg) was added, and the reaction was hydrogenated at 50 ℃ under a reactor pressure of 45psi for 24 hours. Cooling to room temperature, filtering with diatomite to remove palladium carbon, rinsing with 10ml of absolute ethanol, filtering with the diatomite twice, spin-drying the filtrate, and purifying with silica gel column chromatography to obtain colorless oily substance.1H NMR(400MHz,Chloroform-d)δ6.73(s,1H),6.53(s,1H),4.44(p,J＝6.1Hz,1H),3.62(s,2H),2.98(d,J＝11.5Hz,2H),2.60(p,J＝8.1,7.6Hz,1H),2.34(s,3H),2.21(s,3H),2.07(dd,J＝15.0,11.2Hz,2H),1.83–1.64(m,4H),1.33(d,J＝6.1Hz,6H).
(8) Synthesis of target A-1
Intermediate-7 (1mmol, 262mg) was dissolved in 10ml of isopropanol, and intermediate M-3(2mmol, 630mg) and trifluoroacetic acid (2mmol) were added thereto, followed by heating and refluxing for 8 hours. The TLC detection shows that the reaction is finished. The isopropanol was removed by concentration, the pH was adjusted to 10 with 10% sodium hydroxide solution and extracted three times with dichloromethane (30 ml). The organic phases were combined, washed with brine, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by column chromatography on silica gel (ethyl acetate: petroleum ether: 1:2 to 3:1) to give a pale yellow solid.1H NMR(400MHz,Chloroform-d)δ9.98(s,1H),8.53–8.35(m,1H),8.00(d,J＝37.2Hz,2H),7.57–7.36(m,2H),7.32(d,J＝7.6Hz,1H),7.09(t,J＝7.5Hz,1H),6.72(s,1H),4.50(dq,J＝12.1,6.0Hz,1H),3.26(d,J＝9.9Hz,2H),3.17–2.93(m,2H),2.74–2.58(m,1H),2.45(s,3H),2.34(t,J＝12.2Hz,2H),2.08(s,3H),1.91(d,J＝12.0Hz,2H),1.73(t,J＝12.9Hz,2H),1.29(d,J＝6.0Hz,6H),1.17(t,J＝7.5Hz,3H).
Example 2: synthesis of target product A-2
(1) Synthesis of intermediate M-8
Intermediate-4 (1-chloro-5-isopropoxy-2-methyl-4-nitrobenzene, 10mmol, 2.29g) was dissolved in 30ml of 1, 4-dioxane, and potassium carbonate solid (25mmol, 3.46g), bis triphenylphosphine palladium dichloride (210.6mg, 0.3mmol), 1-Boc- (11mmol, 3.4g), and 10ml of water were added in that order. The reaction was refluxed for 8 hours under nitrogen protection. After completion of the TLC detection reaction, the solvent was removed under reduced pressure, and the residue was extracted 3 times with 40ml of methylene chloride and 15ml of water, and the combined extracts were washed with saturated brine and dried over anhydrous sodium sulfate. The solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether: ethyl acetate: dichloromethane ═ 8: 2: 1) to give 3.1g of a white solid. Yield; 82 percent.1H NMR(400MHz,Chloroform-d)δ7.61(s,1H),6.79(s,1H),5.62(s,1H),4.62(p,J＝6.1Hz,1H),4.07(s,2H),3.64(t,J＝5.6Hz,2H),2.33(s,2H),2.24(s,3H),1.51(s,9H),1.38(d,J＝6.1Hz,6H).
(2) Synthesis of target product A-2
Intermediate M-8(5mmol, 1.88g) was dissolved in methanol (5ml), and 10% palladium on charcoal (190mg) was added to conduct hydrogenation reaction at 50 ℃ under a pressure of 45psi in the reaction vessel for 24 hours. Cooling to room temperature, filtering with diatomite to remove palladium carbon, rinsing with 10ml of absolute ethanol, filtering with the diatomite twice, spin-drying the filtrate, and purifying with silica gel column chromatography to obtain colorless oily substance.
Dissolving the colorless oily product (1mmol, 350mg) in 10ml isopropanol, adding intermediate M-3(2mmol, 630mg) and trifluoroacetic acid (2mmol), heating under reflux for 8 hr, and detecting by TLCAfter the reaction, the reaction mixture was concentrated to remove isopropanol, and extracted with 30ml of dichloromethane. The extract was dried over anhydrous sodium sulfate. Concentrated under reduced pressure and purified by silica gel column chromatography (ethyl acetate: petroleum ether: 1:2 to 3:1) to give a pale yellow solid.1H NMR(400MHz,Chloroform-d)δ10.06(s,1H),8.54(d,J＝8.3Hz,1H),8.08(d,J＝36.3Hz,2H),7.57–7.33(m,3H),7.16(t,J＝7.5Hz,1H),6.80(s,1H),4.54(p,J＝6.0Hz,1H),3.13(ddd,J＝43.9,12.9,7.4Hz,4H),2.77(ddt,J＝11.2,6.0,2.9Hz,3H),2.18(s,3H),1.75(d,J＝9.4Hz,5H),1.36(d,J＝6.1Hz,6H),1.26(t,J＝7.5Hz,3H).
The synthesis of the other target compounds of this series is analogous to that of examples 1 and 2. Wherein, the deutero-compound of the intermediate M-1, o-aminophenyl deuteroethyl thioether is obtained by the reaction of p-methyl benzenesulfonic acid deuteroethyl ester and amino thiophenol. The structural formula and the spectrum data of the A series of compounds are shown in Table 1.
Table 1 synthesis of ALK inhibitors containing chiral sulfoxide structure
The synthetic procedure for the ALK/EGFR dual-target inhibitor is illustrated by the synthetic example of compound B-1. Example 3: synthesis of ALK/EGFR double-target inhibitor B-1
(1) Synthesis of intermediate M-9
In a 50mL round bottom flask, 2-amino-5-fluorophenethyl sulfide (10mmol, 1.76g), glacial acetic acid (6mL), 30% H2O2(12mmol,1.36 g). The reaction mixture was stirred at 0 ℃ for 3 hours, and after TLC detection of the starting material, the reaction was quenched with 1M sodium hydroxide solution and extracted 3 times with ethyl acetate. The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed, and the residue was subjected to silica gel column chromatography to give intermediate M-9, an oily liquid, 87% yield.1H NMR(400MHz,CDCl3)δ7.05–6.89(m,2H),6.72–6.53(m,1H),4.74(s,2H)，3.40–2.94(m,2H),1.22(t,J＝7.5Hz,3H).。Ms(M+1),192。
(2) Synthesis of intermediate M-10
To a solution of intermediate M-9(5mmol,0.96g,5mmol) in DMF (12mL) was added sodium hydride (10mmol, 0.4g, 60%). After stirring at 0 ℃ for 30 minutes, 10mL of a DMF solution of 2,4, 5-trichloropyrimidine (7.5mmol, 1.37g) was slowly added dropwise. After the reaction system was warmed to room temperature and stirred for 12 hours, an appropriate amount of ice water was poured in. Extracting with ethyl acetate for 3 times. The organic phases are combined, washed by saturated saline solution, dried by anhydrous sodium sulfate, decompressed and concentrated, and the crude product is chromatographed by a silica gel column to obtain an intermediate M-10, colorless solid. Yield: 60 percent.1H NMR(400MHz,CDCl3)δ10.68(s,1H),8.57(dd,J＝9.3,4.7Hz,1H),8.24(s,1H),7.33–7.27(m,1H),7.07(dd,J＝7.2,3.0Hz,1H),3.33–2.89(m,2H),1.29(t,J＝7.5Hz,3H)。
(2) Synthesis of intermediate M-11
Concentrated hydrochloric acid (3mmol, 0.248mL) was added dropwise to a solution of intermediate M-10(1mmol, 332mg) and 4-fluoro-2-methoxy-5-nitroaniline (1.1mmol, 205mg) in n-butanol (12 mL). Stirring and reacting for 6 hours at 80 ℃, and cooling to room temperatureConcentrating, and purifying by silica gel column chromatography to obtain intermediate M-11 as light yellow solid. Yield, 65%.1H NMR(400MHz,CDCl3)δ11.59(s,1H),10.58(s,1H),8.59(d,J＝7.7Hz,1H),8.17(dd,J＝9.0,4.5Hz,1H),7.94(s,1H),7.22–7.05(m,2H),6.82(d,J＝11.9Hz,1H),4.06(s,3H),3.13(d,J＝11.0Hz,2H),1.32(t,J＝7.3Hz,3H)。
(3) Synthesis of intermediate M-12
To a solution of intermediate M-11(1mmol,483mg) in dioxane was added N, N-diisopropyl-N-ethylamine (3mmol,388mg), and N, N.N-trimethylethylenediamine (1.5mmol,153 mg). The reaction was stirred at 100 ℃ for 2 hours and checked by TLC. After the reaction was complete, the reaction mixture was concentrated and the residue was purified by means of a silica gel Column (CH)2Cl2MeOH, 40:1) to give intermediate M-11. a red solid. Yield: 72 percent.1H NMR(400MHz,CDCl3)δ9.62(s,1H),8.63(s,1H),8.25(dd,J＝9.1,4.7Hz,1H),8.12(s,1H),7.30(s,1H),7.25–7.12(m,2H),6.64(s,1H),3.94(s,3H),3.31–3.18(m,2H),3.17–2.98(m,2H),2.85(s,3H),2.53(dd,J＝7.7,6.4Hz,2H),2.25(s,6H),1.25(t,J＝7.5Hz,3H).
(5) Synthesis of target product B-1
Tin dichloride dihydrate (10mmol, 2.11g) was added to a mixed solution of intermediate M-12(1mmol,566mg) in dichloromethane (8 mL)/methanol (8mL), and concentrated hydrochloric acid (2.1mL) was added dropwise with stirring. The reaction mixture was stirred at 50 ℃ for 5 hours, adjusted to pH 5 with ammonia and then adjusted to pH 7 with sodium carbonate. Filtering with diatomite, concentrating and drying the filtrate, and directly carrying out the next reaction.
The crude product from the previous step was dissolved in anhydrous dichloromethane (10mL) and N, N-diisopropyl-N-ethylamine was added. Acryloyl chloride (1.1mmol, 100mg) was added at 0 deg.C under nitrogen. After 30 minutes, add saturated sodium bicarbonate solution and quenchReaction, extraction with dichloromethane, combination of organic phases, washing with saturated brine and drying over anhydrous sodium sulfate. After concentration under reduced pressure, the residue was purified by silica gel Column (CH)2Cl2MeOH, 30:1) to obtain the target product B-1, a colorless solid. Yield: 60 percent.1H NMR(400MHz,CDCl3)δ10.05(s,1H),9.50(s,1H),9.13(s,1H),8.34(dd,J＝9.1,4.8Hz,1H),8.14(s,1H),7.29(s,1H),7.11–6.97(m,2H),6.75(s,1H),6.26(s,2H),5.72–5.54(m,1H),3.84(s,3H),3.23–3.02(m,2H),2.87(s,2H),2.69(s,3H),2.29(s,8H),1.28(t,J＝7.5Hz,3H).13C NMR(125MHz,CDCl3)δ163.19,159.38,157.71,155.76,155.29,145.42,136.65,135.80,132.22,130.70,126.93,126.09,118.74,118.51,113.72,113.47,105.91,104.58,57.34,56.09,47.66,45.38,43.56,7.52.HRMS(ESI)m/z):[M+H]+calculated for C27H33N7O3FSCl,590.2111；found,590.2124.Purity:99.9％(by HPLC).
The racemic compound B-1 was purified by chiral HPLC (chiral column:y352546, mobile phase: n-hexane (0.1% diethylamine): ethanol 75: 25, V/V) to give the two enantiomers B-1a and B-1B. The synthesis steps and methods of other targets in the series are similar to those of target compound B-1, and the structures and the spectra are shown in Table 2.
TABLE 2 Synthesis of ALK/EGFR dual-target inhibitors containing chiral sulfoxide structure
Antitumor activity test example: antitumor activity of pyrimidine derivative containing chiral sulfoxide group
For the activity test of the novel pyrimidine derivative containing the chiral sulfoxide group, the non-small cell lung cancer cell H1975 with high EGFR expression, the cell Pc9 and the H2228 with high ALK expression and the BaF3 cell are selected for the evaluation of the proliferation activity of the anti-tumor cell by the MTT method. The specific operation steps are as follows: inoculating tumor cells into 96-well culture plate at a cell density of 5 × 103～1*104Samples of test compound were added to each well after overnight incubation at 37 ℃ in an incubator with 5% carbon dioxide. After 72 hours of incubation, MTT was added for 4 hours, dissolved in DMSO, shaken and then detected in a microplate reader (570 nm). The half inhibitory concentration IC of the compound on non-small cell lung cancer cells H2228(ALK), H1975, Pc9 and the like50The values are shown in Table 3.
TABLE 3 in vitro anti-tumor testing of ALK inhibitors
Compound A-1a and compound A-1b were obtained from compound A1 by HPLC chiral separation (AD-H column, mobile phase: isopropanol/n-hexane, 40/60, flow rate: 0.5 mL/min); a-1a (retention time: 9.72 minutes); a-1b (retention time: 12.45 min). The isolation conditions for compounds A-2a, A-2b, etc. are similar to those for A-1a and A-1 b.
TABLE 4 in vitro anti-tumor test (IC) of EGFR/ALK target Compounds50μM)
Compound 7c is the best compound in the literature (European Journal of Medicinal chemistry2017,136, 497-510).
The chiral isomer of the compound B series is obtained by chiral separation of the racemate. For example, the compound B-3 was subjected to manual separation (AD-H chiral column; mobile phase: isopropanol/n-hexane, 40/60; flow rate: 0.5mL/min.) to give optically active two enantiomers (-) B-3a (retention time: 24.6 min.) and (+) B-3B (retention time: 27.0 min.). The chiral separation of the other compounds is similar.
Antitumor activity test example: liver microsome stability assay for compounds
The stability test of human liver microsomes is carried out on the compounds screened in the second round, and the results show that the molecular structure of the compounds greatly influences the stability. Among them, compounds in which ethyl or deuterated alkyl groups such as compounds B1 and B2 containing deuterium atoms or fluorine atoms are linked to sulfinyl groups exhibit good stability of liver microsomes.
Table 5 liver microsome stability assay of partial target compounds
Name of Compound
T1/2
CL(μL/min/mg)
Verapamil
28.0
123.7
Compound B1
223.3
15.5
Compound B2
161.8
21.4
Compound B4
125.2
27.7
Compound B5
50.5
68.7
Compound B6
26.3
131.9
Compound B7
32.3
107.2
Compound B8
49.1
70.5
Compound B9
31.9
108.7
Compound B10
37.7
92.0
Compound B11
52.0
66.7
Research and test example of in vivo antitumor experiment
Experimental animals BALB/c nude mice (5 weeks old, 18-20g), male SPF grade, purchased from Experimental animals technology, Inc., Viton, Beijing. Collecting EGFR double-mutation high-expression H1975 cells cultured to 3-5 generations and growing in logarithmic phase, digesting with pancreatin, and preparing into 1.0 × 10 with serum-free culture medium7Cell suspension at individual/ml concentration. The H1975 cell suspension was injected subcutaneously on the right side with a syringe and the cell seeding volume was 0.1 ml/cell. After the tumor formation, the diameter of the transplanted tumor is measured by a vernier caliper, and the tumor grows to 1000mm3Removing neck, killing mice, stripping tumor tissue block, and cutting into pieces with uniform size of about 4-6mm3The tissue blocks are inoculated to the right limb of a nude mouse in a partial armpit subcutaneously, animals are randomly divided into a negative control group and an administration group according to the weight and the tumor volume, the administration group is 4 groups, B3 is manually separated to obtain two enantiomers B3a and B3B (represented by (-) -P1 and (+) -P2 in the following figures) which are administered to each group according to 20mg/kg, the administration amount of B3a is 40mg/kg, and the other group is a positive control group which uses a third generation targeted drug oxcetin approved by the FDA in the United states for treating the non-small cell lung cancer. The tumor volume reaches 200mm3And (4) performing intragastric administration for one week. The negative control group was given an equal amount of physiological saline. The long and short diameters of the tumor tissue were measured with a vernier caliper and the body weight of the mice was recorded.
Observation ofIndexes are as follows: tumor volume TV 1/2 × a × b2(wherein a and b represent length and width, respectively)
TGI＝(TWNegative control-TWTreatment group)/TWNegative controlX 100%, TW value is mean tumor weight. The results of the in vivo antitumor experiments are shown in FIG. 2.
The results show that the two enantiomers B3a and B3B have good antitumor effect, and when the dosage is 20mg/kg, the tumor growth inhibition effect is obvious 1-2 days after the administration, and the tumor volume is not increased and is in a obviously descending trend. After the medicine is taken for 7 days, tumors are completely inhibited, and the tumor inhibition rate of the chiral compound B1a [ P1 ] is over 90 percent. The tumor inhibition rate of enantiomer B3B [ P2 ] reaches more than 95 percent, and the effect is equivalent to the administration dosage of 40mg/kg of B3 a. The body weight of each group of mice did not change significantly throughout the administration, and there was no abnormality in the behavior of the mice.
In the same way, we modeled H2228 tumor cells with high ALK expression and performed in vivo anti-tumor experiments in nude mice with compounds B3a and B3B. The results show that both compounds B3a and B3B were able to decrease tumor volume after administration, with tumor inhibition rates TGI of 75.3% and 85.9%, respectively. The body weight of each group of mice did not change significantly throughout the administration, and there was no abnormality in the behavior of the mice.
The embodiments of the present invention have been described in detail, but the present invention is not limited to the described embodiments. It will be apparent to those skilled in the art that various changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, and the scope of protection is still within the scope of the invention.