WO2018019084A1 - 一种苯甲酰胺类化合物及其在制备抑制癌细胞增殖和/或治疗癌症的药物中的应用 - Google Patents

一种苯甲酰胺类化合物及其在制备抑制癌细胞增殖和/或治疗癌症的药物中的应用 Download PDF

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WO2018019084A1
WO2018019084A1 PCT/CN2017/091211 CN2017091211W WO2018019084A1 WO 2018019084 A1 WO2018019084 A1 WO 2018019084A1 CN 2017091211 W CN2017091211 W CN 2017091211W WO 2018019084 A1 WO2018019084 A1 WO 2018019084A1
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compound
cancer
cancer cell
benzamide
alkylcarbonyl
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French (fr)
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袁其朋
谢瑞
邓炳华
武新颖
陈光耀
刘霞
姚越
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北京化工大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C235/00—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms
    • C07C235/42—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups bound to carbon atoms of six-membered aromatic rings and singly-bound oxygen atoms bound to the same carbon skeleton
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

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  • This invention relates to a class of benzamide compounds and uses thereof.
  • HDAC zinc ion-dependent metalloproteinase family-histone deacetylase
  • Histone deacetylase HDAC
  • histone acetyltransferase HAT
  • Histone deacetylase HDAC
  • histone acetyltransferase HAT
  • the acetylation is in equilibrium.
  • the acetylation and deacetylation of histones is a way to regulate gene transcription.
  • the degree of acetylation of histones affects the expression of genes by affecting the structure of chromatin.
  • Overexpression of HDAC in tumor cells inhibits the expression of certain tumor suppressor genes.
  • a large body of literature indicates that inhibition of HDAC activity is effective in inhibiting tumor cell growth, metastasis, and invasion.
  • HDAC inhibitors have become important targets for anti-tumor effects, and one type of HDAC inhibitors with a benzamide structure has good oral bioactivity and anti-tumor activity and has attracted attention.
  • Tacedinaline (1, CI-994) shows certain inhibitory activity against HDAC, and has a broad spectrum of antitumor activity, becoming the first benzoic acid to enter clinical trials.
  • Amide HDAC inhibitor, CI-994 is currently used in combination with gemcitabine in phase II clinical trials for patients with solid tumors such as non-small cell lung cancer and colon cancer.
  • Sedabamide (2, Chidamide, CS055) is an approved oral anti-acetylase inhibitor developed by Shenzhen Microcore Biotechnology Co., Ltd., and the approved indication is recurrent and refractory peripheral T-cell lymphoma.
  • Sidabenamine is in the clinical stage for the treatment of lung cancer, stomach cancer, liver cancer and breast cancer.
  • benzamide compounds such as MS-275 (3), and MGCD-0103 (4) is in the clinical stage.
  • the inventors of the present invention synthesized a series of new skeleton structure benzamide compounds and evaluated their anti-tumor activity in vitro, and the compounds in this patent show It has stronger antitumor activity than the reference drugs CI994 and Chidamide (CS055).
  • the above structural formula is a benzamide compound that has been marketed or is in clinical research.
  • the present invention relates to the design and synthesis of a class of benzamides.
  • the present invention provides a benzamide compound which is more excellent in pharmacodynamic activity. The reason why the present invention can solve the above problems is achieved by the following technical solutions:
  • R 1 is hydrogen, fluorine, chlorine, bromine, iodine, alkyl, alkoxy, alkylcarbonyl, alkoxycarbonyl, alkanoamide, nitro, cyano, aryl, heteroaryl.
  • R 2 is hydrogen, fluorine, chlorine, bromine, iodine, alkyl, alkoxy, alkylcarbonyl, alkoxycarbonyl, alkanoyl, nitro, cyano, aryl, heteroaryl.
  • a benzamide compound according to a preferred embodiment characterized in that the benzamide compound From:
  • cancer cells or cancer include: human lung cancer cell A549, human gastric cancer cell MGC80-3, human liver cancer cell HepG2, human colon cancer cell HCT116, esophageal cancer, pancreatic cancer, rectal cancer, cervical cancer or ovarian cancer.
  • the invention provides a process for the preparation of a compound of formula (I), the process comprising the steps of:
  • R 1 is hydrogen, fluorine, chlorine, bromine, iodine, alkyl, alkoxy, alkylcarbonyl, alkoxycarbonyl, alkylamido, nitro, cyano, aryl or heteroaryl;
  • R 2 is hydrogen, fluorine, chlorine, bromine, iodine, alkyl, alkoxy, alkylcarbonyl, alkoxycarbonyl, alkanoyl, nitro, cyano, aryl or heteroaryl.
  • reaction step (c) uses a peptide condensing agent as a catalyst.
  • the peptide condensing agent is selected from the group consisting of benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate (BOP).
  • Example 1 Synthesis of a part of the compounds in the present invention
  • Step (1) Weigh 0.41 g (4.4 mmol) of phenol, 1 g (4.4 mmol) of methyl 4-bromomethylbenzoate, 1.21 g (8.8 mmol) of potassium carbonate, and 0.725 g (4.4 mmol) of potassium iodide in 100 ml of three mouths.
  • the round bottom flask was charged with 30 ml of methyl ethyl ketone, protected with nitrogen, magnetically stirred, heated in an oil bath to reflux with butanone, and the reaction time was 8 h.
  • the reaction was followed by a thin layer chromatography plate.
  • Step (2) 0.42 g (1.84 mmol) of the product obtained in the step (1) was dissolved in 15 ml of DMF, and added with BOP 0.97 g (2.2 mmol), triethylamine 0.74 g (7.32 mmol), and stirred at room temperature for 10 min. O-phenylenediamine 0.237 g (2.2 mmol), the reaction time was 3 h, and the reaction was followed by a thin layer chromatography plate.
  • Compound 6a was prepared in the same manner as Compound 5a except that o-phenylenediamine in step (2) was replaced with 3-fluoro-5-aminoaniline 0.28 g (2.2 mmol).
  • Compounds 12a, 15-18a are similarly prepared as compound 5a except that the starting phenol in step (1) is replaced by 4-fluorophenol (0.49 g, 4.4 mmol) (compound 12a); 4-methylphenol ( 0.48 g, 4.4 mmol) (Compound 15a); 4-methoxyphenol (0.55 g, 4.4 mmol) (Compound 16a); 4-ethylphenol (0.54 g, 4.4 mmol) (Compound 17a); 4-propyl Phenol (0.60 g, 4.4 mmol) (Compound 18a).
  • the cancer cells in the log phase are trypsinized into single cells, and then diluted to a cell density of (3 ⁇ 4) ⁇ 10 4 / ml with fresh medium in 96 wells.
  • the plate add 90 ⁇ L of cell liquid per well, incubate for 12 h at 37 ° C in a 5% CO 2 incubator, add the drug (diluted the synthesized compound to a series of different concentrations, and add different concentrations of the compound solution).
  • 3 replicate wells were repeated for each concentration.
  • A549 lung cancer cells in logarithmic growth phase were plated in 12-well plates, and 600,000 cells were plated per well. After 24 hours, the bottom of each well was streaked with a gun head, and then 8 ⁇ mol/L CS055, CI994, 12a, respectively. The cells were treated with 16a, and the DMSO-treated group was used as a blank control. After 48 hours, the cells were photographed with a microscope.
  • the A549 lung cancer cells in logarithmic growth phase were plated in 6-well plates, 3000 cells per well, and immediately treated with 2 ⁇ mol/L, 4 ⁇ mol/L, 8 ⁇ mol/L CS055, CI994, 12a, 16a, treated with DMSO.
  • As a blank control after 7 days, the medium of each well was removed, gently washed twice with PBS (phosphate buffer), fixed with 3.7% formaldehyde solution for 20 min, and gently washed twice with PBS, 0.1% crystal violet. The aqueous solution was stained for 30 min, washed three times with PBS, and the experimental results were observed and photographed.
  • PBS phosphate buffer
  • the aqueous solution was stained for 30 min, washed three times with PBS, and the experimental results were observed and photographed.
  • Example 5 Compound induced apoptosis of tumor cells
  • A549 lung cancer cells in logarithmic growth phase were plated in 6-well plates, 160,000 cells per well. After 24 hours, CS055, CI994 and 12a (concentration of 4 ⁇ mol/L and 8 ⁇ mol/L) were used to treat the cells.
  • the culture medium of each well was collected after 48 hours, and the cells of each well were collected by trypsinization, centrifuged at 1000 rpm for 5 min, the supernatant after centrifugation was removed, and then washed twice with PBS, and then 100 ⁇ L of 1 ⁇ binding buffer buffer was added, 5 ⁇ L of Alexa.
  • Fluor 488 annexin and 1 ⁇ L of propidium iodide were incubated for 15 min at room temperature in the dark.
  • the combination of Alexa Fluor 488 annexin and propidium iodide was used as a control group.
  • the samples were detected by MoFlo XDP flow cytometry. .
  • the cell flow pattern of tumor cell apoptosis induced by the compound shows that the compound 12a synthesized in the present invention can induce apoptosis of 64.4% of A549 lung cancer cells at a concentration of 8 ⁇ mol/L, while CS055 (sidabenamine) can only induce 22.42% of A549 lung cancer cells were apoptotic, and CI994 only induced apoptosis in 12.58% of A549 lung cancer cells.
  • the compounds synthesized by the present invention have a stronger ability to induce apoptosis of tumor cells.
  • A549 lung cancer cells in logarithmic growth phase were plated in 6-well plates, 160,000 cells per well. After 24 hours, CS055, CI994 and 12a (concentration of 2 ⁇ mol/L and 16 ⁇ mol/L) were used to treat the cells. Group, 48h After collecting the medium in each well, the cells were collected by trypsinization, centrifuged at 1000 rpm for 5 min, the supernatant after centrifugation was removed, and washed twice with PBS. The cells were fixed with 70% ethanol, washed twice with PBS, and RNase was added. Incubate at 37 ° C for 30 min in the dark, and the samples were detected by MoFlo XDP flow cytometry.

Abstract

一种苯甲酰胺类化合物及其在制备抑制癌细胞增殖和/或治疗癌症的药物中的应用。该化合物具有抑制癌细胞增殖的能力，达到治疗癌症的目的。尤其对抑制人肺癌细胞A549，人胃癌细胞MGC80-3，人肝癌细胞HepG2，人结肠癌细胞HCT116具有优异的抑制癌细胞增殖的活性。该化合物具有下列结构：其中R 1为氢，氟，氯，溴，碘，烷基，烷氧基，烷基羰基，烷氧基羰基，烷酰胺基，硝基，氰基，芳基或杂芳基；R 2为氢，氟，氯，溴，碘，烷基，烷氧基，烷基羰基，烷氧基羰基，烷酰胺基，硝基，氰基，芳基或杂芳基。

Description

一种苯甲酰胺类化合物及其在制备抑制癌细胞增殖和/或治疗癌症的药物中的应用 技术领域
本发明涉及一类苯甲酰胺类化合物及其用途。
背景技术
寻找和发现有效的非细胞毒抗肿瘤药物是目前抗肿瘤药物化学研究的一个重点，近年来，一类锌离子依赖性金属蛋白酶家族-组蛋白去乙酰化酶(histone deacetylase，HDAC)成为抗肿瘤药物设计的新靶点。
组蛋白去乙酰化酶(histone deacetylase，HDAC)和组蛋白乙酰转移酶(histone acetyltransferase，HAT)为真核细胞中广泛存在的的一类相互拮抗的蛋白酶，它们共同调控着组蛋白末端氨基酸残基的乙酰化，使之处于平衡状态。组蛋白的乙酰化和去乙酰化修饰为调控基因转录的一种方式，组蛋白的乙酰化程度通过影响染色质的结构进而影响基因的表达。在肿瘤细胞中HDAC过量表达，从而抑制了某些抑癌基因的表达。大量文献表明抑制HDAC的活性能有效地抑制肿瘤细胞的生长、转移和和侵袭。HDAC抑制剂已成为重要的抗肿瘤作用的靶标，其中有一类具有苯甲酰胺结构的HDAC抑制剂具有良好的口服生物活性和抗肿瘤活性而受到人们的关注。
许多苯甲酰胺类HDAC抑制剂正处于临床研究阶段，其中Tacedinaline(1，CI-994)对HDAC显示出一定的抑制活性，具有广谱的抗肿瘤活性，成为第一个进入临床试验的苯甲酰胺类HDAC抑制剂，CI-994目前在Ⅱ期临床试验中与吉西他滨联用治疗非小细胞肺癌、结肠癌等实体瘤患者。西达本胺(2，Chidamide，CS055)是深圳微芯生物科技有限公司开发的已获准上市的组蛋白去乙酰化酶口服抑制剂，批准的适应症为复发及难治性外周T细胞淋巴瘤，西达本胺对肺癌，胃癌，肝癌，乳腺癌等的治疗正处于临床阶段。还有许多苯甲酰胺类化合物如MS-275(3)，MGCD-0103(4)正处于临床阶段。为了获得更高活性的苯甲酰胺类HDAC抑制剂，本专利发明人合成了一系列新的骨架结构的苯甲酰胺类化合物，并对其体外抗肿瘤细胞活性进行评价，本专利中的化合物显示出比参比药物CI994和西达本胺(Chidamide,CS055)更强的抗肿瘤活性。
Figure PCTCN2017091211-appb-000001
上面结构式是已上市或正处于临床研究的苯甲酰胺类化合物
发明内容
本发明涉及一类苯甲酰胺类的设计与合成，为了克服现有技术的上述不足，本发明提供一种药效活性更优异的苯甲酰胺类化合物。本发明之所以能够解决上述问题乃是通过以下技术方案给予实现：
一种苯甲酰胺类化合物，其特征在于下列通式(I)所示的结构：
Figure PCTCN2017091211-appb-000002
其中R1为氢，氟，氯，溴，碘，烷基，烷氧基，烷基羰基，烷氧基羰基，烷酰胺基，硝基，氰基，芳基，杂芳基。
R2为氢，氟，氯，溴，碘，烷基，烷氧基，烷基羰基，烷氧基羰基，烷酰胺基，硝基，氰基，芳基，杂芳基。
作为一种优选方案所述的苯甲酰胺类化合物，其特征在于，所述的苯甲酰胺类化合物 选自:
表1本发明合成其中一部分的化合物
Figure PCTCN2017091211-appb-000003
Figure PCTCN2017091211-appb-000004
所述的化合物在制备抑制癌细胞增殖或治疗癌症药物中的应用。
其中癌细胞或癌症包括：人肺癌细胞A549，人胃癌细胞MGC80-3，人肝癌细胞HepG2，人结肠癌细胞HCT116，食管癌，胰腺癌，直肠癌，宫颈癌或卵巢癌。
本发明提供了通式(I)的化合物的制备方法，该方法包括以下步骤：
(a)将取代苯酚与化合物1缩合反应得到化合物2；
Figure PCTCN2017091211-appb-000005
(b)将化合物2水解为化合物3，并酸化为化合物4
Figure PCTCN2017091211-appb-000006
(c)将化合物4与化合物5进行缩合反应得到化合物6
Figure PCTCN2017091211-appb-000007
其中R1为氢，氟，氯，溴，碘，烷基，烷氧基，烷基羰基，烷氧基羰基，烷酰胺基，硝基，氰基，芳基或杂芳基；
R2为氢，氟，氯，溴，碘，烷基，烷氧基，烷基羰基，烷氧基羰基，烷酰胺基，硝基，氰基，芳基或杂芳基。
进一步，反应步骤(c)使用肽缩合剂作催化剂。
所述肽缩合剂选自苯并三氮唑-1-基氧基三(二甲基氨基)磷鎓六氟磷酸盐(BOP)。
附图说明
图1：化合物对肿瘤细胞迁移能力的影响
图2：化合物对肿瘤细胞单克隆形成能力的影响
具体实施方式
通过以下的具体实施方式，对本发明的上述内容再作进一步的详细解释及展述，使本领域的普通技术人员能够更加容易地理解本发明，但不应该将此理解为本发明所述主题的范围仅限于以下的实例及限制本发明的任何或所有权利，更不应该背离本发明的精神。
实施例1：合成本发明中的一部分化合物
N-(2-氨基苯基)-4-(苯氧基甲基)苯甲酰胺(化合物5a)的制备。
步骤(1)：称量苯酚0.41g(4.4mmol)，4-溴甲基苯甲酸甲酯1g(4.4mmol)，碳酸钾1.21g(8.8mmol)，碘化钾0.725g(4.4mmol)置于100ml三口圆底烧瓶，加入30ml丁酮，氮气保护，磁力搅拌，油浴加热至丁酮回流，反应时间为8h，期间用薄层层析板跟踪反应，展开剂为乙酸乙酯：石油醚＝1:6(体积比)。反应完成后向反应液加入3.5gNaOH(用30ml水溶解后加入)，再加入20ml甲醇溶液，油浴加热至回流，点板确定水解完全后，将反应也冷却至常温，冰浴下加入2mol/L的盐酸水溶液，析出大量固体，用砂芯漏斗抽真空 过滤得固体，并用水对固体进行清洗，抽干后再用石油醚清洗固体并将固体抽干。最终得白色固体0.83g，产率为：83％；
步骤(2)：称量步骤(1)得到的产物0.42g(1.84mmol)溶解于15ml DMF中，加入BOP 0.97g(2.2mmol)，三乙胺0.74g(7.32mmol)，常温搅拌10min后加入邻苯二胺0.237g(2.2mmol)，反应时间为3h，期间用薄层层析板跟踪反应，展开剂为乙酸乙酯：石油醚＝3:1(体积比)。反应完成后，向反应液中加入350ml乙酸乙酯萃取，倒入分液漏斗中，加入饱和碳酸钠水溶液洗3次，饱和氯化钠洗2次，有机相用无水硫镁干燥，过滤，旋蒸得固体，向固体中加入甲醇，过滤，用甲醇洗固体，用乙醚洗固体并抽干得灰白色固体0.35g，产率为：61％；mp 176.2-178.2℃；1H NMR(400MHz,DMSO-d6):δ＝9.70(s,1H),8.03(d,J＝7.8Hz,2H),7.60(d,J＝7.8Hz,2H),7.32(t,J＝7.8Hz,1H),7.21(d,J＝7.7Hz,1H),7.01(m,4H),6.82(m,J＝7.8Hz,1H),6.63(t,J＝7.2Hz,1H),5.22(s,2H),4.94(s,2H)；13C NMR(400MHz,DMSO-d6):δ＝68.50,114.84,116.11,116.23,120.83,123.28,126.47,126.66,127.15,127.93,129.50,134.03,140.58,143.12,158.12,165.05ppm；C20H18N2O2,MS(ES+)m/z:319.1442(M+H)+。
化合物6a与化合物5a相同的制备方法相似，只是将步骤(2)中的邻苯二胺替换为3-氟-5-氨基苯胺0.28g(2.2mmol)。
化合物12a,15～18a与化合物5a相同的制备方法相似，只是将步骤(1)中的原料苯酚分别替换为4-氟苯酚(0.49g，4.4mmol)(化合物12a)；4-甲基苯酚(0.48g，4.4mmol)(化合物15a)；4-甲氧基苯酚(0.55g,4.4mmol)(化合物16a)；4-乙基苯酚(0.54g,4.4mmol)(化合物17a)；4-丙基苯酚(0.60g,4.4mmol)(化合物18a)。
N-(2-氨基-4-氟基苯基)-4-(苯氧基甲基)苯甲酰胺(化合物6a)
mp 158.8-160.0℃；1H NMR(400MHz,DMSO-d6):δ＝9.61(s,1H),8.01(d,J＝6.7Hz,2H),7.59(d,J＝6.7Hz,2H),7.32(m,2H),7.05(m,4H),6.56(d,J＝10.7Hz,1H),6.38(m,1H),5.24(m,4H)；13C NMR(400MHz,DMSO-d6):δ＝68.48,101.28,102.09,114.83,119.19,120.83,127,12,127.94,128.48,128.58,129.50,133.89,140.60,145.41,145.53,158.10,159.83,162.20,165.30ppm；C20H17FN2O2,MS(ES+)m/z:337.1437 (M+H)+。
N-(2-氨基苯基)-4-((4-氟基苯氧基甲基)苯甲酰胺(化合物12a)
mp 196.0-197.8℃；1H NMR(400MHz,DMSO-d6):δ＝9.69(s,1H),8.02(d,J＝8.0Hz,2H),7.58d,J＝8.0Hz,2H),7.20(m,1H),7.15(m,2H),7.06(m,2H),6.99(d,J＝7.8Hz,1H),6.81(m,1H),6.62(t,J＝7.5Hz,1H),5.20(s,2H),4.93(s,2H)；13C NMR(400MHz,DMSO-d6):δ＝69.18,115.72,115.95,116.11,116.19,116.22,126.48,126.67,127.19,127.93,134.07,140.37,143.12,154.44,157.79,165.02；C20H17FN2O2,MS(ES+)m/z:337.1347(M+H)+。
N-(2-氨基苯基)-4-((4-甲基苯氧基甲基)苯甲酰胺(化合物15a)
mp 183.6-185.0℃；1H NMR(400MHz,DMSO-d6):δ＝9.68(s,1H),8.01(d,J＝8.1Hz,2H),7.57(d,J＝8.1Hz,2H),7.20(d,J＝7.7Hz,1H),7.11(d,J＝8.3Hz,2H),6.99(m,1H),6.93(d,J＝8.5Hz,2H),6.81(m,1H),6.62(t,J＝7.5Hz,1H),5.18(s,2H),4.92(s,2H),2.24(s,3H)；13C NMR(400MHz,DMSO-d6):δ＝20.05,68.57,114.70 116.10,116.22,123.27,126.46,126.66,127.09,127.89,129.49,129.82,133.95,140.75,143.12,155.99,165.04；C21H20N2O2,MS(ES+)m/z:333.1607(M+H)+。
N-(2-氨基苯基)-4-((4-甲氧基苯氧基甲基)苯甲酰胺(化合物16a)
mp 173.2-174.7℃；1H NMR(400MHz,DMSO-d6):δ＝9.68(s,1H),8.01(d,J＝8.0Hz,2H),7.57(d,J＝8.0Hz,2H),7.20(d,J＝7.6Hz,1H),6.98(m,3H),6.88(m,2H),6.81(m,1H),6.62(t,J＝7.5Hz,1H),5.15(s,2H),4.92(s,2H),3.71(s,3H)；13C NMR(400MHz,DMSO-d6):δ＝55.32,69.10,114.59,115.79,116.10,116.22,123.27,126.46,126.66,127.11,127.88,133.93,140.83,143.12,152.09,153.54,165.04ppm；C21H20N2O3,MS(ES+)m/z:349.1555(M+H)+。
N-(2-氨基苯基)-4-((4-乙基苯氧基甲基)苯甲酰胺(化合物17a)
mp 171.9-173.8℃；1H NMR(400MHz,DMSO-d6):δ＝9.68(s,1H),8.01(d,J＝8.1Hz,2H),7.57(d,J＝8.1Hz,2H),7.19(d,J＝7.7Hz,1H),7.14(d,J＝8.5Hz,2H),6.96(m,3H),6.81(m,1H),6.62(t,J＝7.5Hz,1H),5.19(s,2H),4.98(s,2H),2.54(m,2H)；1.56(m,2H),1.16(t,J＝7.5Hz,3H)；13C NMR(400MHz,DMSO-d6):δ＝15.87,27.26,68.57,114.71,116.10,116.23,123.28,126.46,126.66,127.08,127.90,128.64,133.95,136.04,140.77,143.08,156.17,165.03；C22H22N2O2,MS(ES+)m/z:347.1756(M+H)+。
N-(2-氨基苯基)-4-((4-丙基苯氧基甲基)苯甲酰胺(化合物18a)
mp 171.5-173.0℃；1H NMR(400MHz,DMSO-d6):δ＝9.68(s,1H),8.01(d,J＝8.0Hz,2H),7.58(d,J＝8.0Hz,2H),7.20(d,J＝7.7Hz,1H),7.12(d,J＝8.4Hz,2H),6.97(m,3H),6.81(d,J＝7.8Hz,1H),6.62(t,J＝7.5Hz,1H),5.18(s,2H),4.92(s,2H),2.48(d,J＝7.7Hz,2H)；1.56(m,2H),0.88(t,J＝7.4Hz,3H)；13C NMR(400MHz,DMSO-d6):δ＝13.56,24.27,36.35,68.57,114.60,116.09,116.21,123.27,126.45,126.65,127.09,127.90,129.22,133.96,134.39,140.76,143.11,156.21,165.03；C23H24N2O2,MS(ES+)m/z:361.1911(M+H)+。
实施例2：化合物抗肿瘤细胞增殖实验
(1)实验细胞系，选用四种癌细胞：人肺癌细胞A549，人胃癌细胞MGC80-3，人肝癌细胞HepG2，人结肠癌细胞HCT116。实验前，四种癌细胞被保藏在液氮中，首先取出癌细胞，在37摄氏度的水浴锅中使其快速升温到37摄氏度，将细胞液离心去除上层冻存液，细胞用培养基重新悬浮后，转移到24mL细胞培养瓶中，每瓶加入6ml培养基于37摄氏度，含5％CO2的培养箱培养，(其中人肝癌细胞HepG2，人胃癌细胞MGC80-3，在含10％胎牛血清的DMEM培养基中，用0.25％胰蛋白酶常规消化后传代培养。人肺癌细胞A549，人结肠癌细胞HCT116在含10％胎牛血清的RPMI-1640培养基中，用0.25％胰蛋白酶常规消化后传代培养。)等细胞生长达到培养瓶70％-80％左右时，先用PBS洗去培养基，用0.25％胰蛋白酶消化将细胞从培养瓶中脱落下来，加入新鲜培养基离心后，去除上层培养基，再加入新鲜培养基重悬后1：2～3传代。
(2)当细胞生长稳定后，将处于对数期的癌细胞用胰酶消化成单个细胞后，用新鲜培养基稀释到(3～4)×104个/ml的细胞密度，在96孔板中，每孔加入90μL的细胞液，在37摄氏度，含5％CO2的培养箱中培养12h，加药(把合成的化合物事先稀释到一系列不同的浓度，将不同浓度的化合物溶液加入到96孔板中，每个浓度重复3个复孔)，72小时后，96孔板中每孔加入10μL CCK-8溶液，再培养1小时后，用BIO-RAD酶标仪测定450nm波长处的吸光度值，通过Graphpad Prism 5软件拟合得到抑制曲线，最终得到IC50值。
表2：本发明所合成的化合物对不同癌细胞的抑制效果
化合物名称 A549 MGC80-3 HepG2 HCT116
CS055(西达本胺) 11.02±0.81 6.44±1.17 22.65±3.09 2.28±0.77
CI994 20.10±0.76 11.60±1.04 18.51±1.77 7.71±0.86
5a 6.76±0.20 5.67±0.82 6.70±0.73 4.89±0.29
6a 8.34±0.74 6.02±0.34 7.02±0.76 /
12a 5.37±0.21 4.97±0.65 3.84±0.54 3.86±0.39
15a 5.43±0.25 6.53±0.63 6.59±0.60 4.86±0.43
16a 5.53±0.21 3.48±0.33 4.20±0.51 2.02±0.23
17a 6.42±0.36 5.88±0.59 6.96±1.31 4.83±0.55
18a 5.21±1.27 4.42±0.67 4.75±0.89 3.23±0.73
表2的结果表明：本发明合成的苯甲酰胺类化合物对四种癌细胞具有比参比药物更强的抗肿瘤效果，尤其对于人肝癌细胞，本专利中合成的苯甲酰胺类化合物比参比化合物CI994的药效强2-5倍，比参比药物西达本胺的药效强3-6倍。
实施例3：化合物抗肿瘤细胞迁移实验
取对数生长期的A549肺癌细胞铺于12孔板，每孔铺60万个细胞，24h后用枪头在每孔底部划线得划痕，再分别用8μmol/L CS055，CI994，12a，16a处理细胞，以DMSO处理组为空白对照，48h后用显微镜拍照。
由图1可看出本发明合成的化合物12a,16a比参比药物CS055和CI994具有更强的抗癌细胞迁移的能力。
实施例4：化合物抗肿瘤细胞单克隆形成实验
取对数生长期的A549肺癌细胞铺于6孔板，每孔铺3000个细胞，立即用2μmol/L,4μmol/L，8μmol/L的CS055，CI994，12a，16a处理细胞，以DMSO处理组为空白对照，7天后，移去每孔的培养基，加入PBS(磷酸盐缓冲液)轻轻洗2遍，加入3.7％甲醛水溶液固定20min，再用PBS轻轻洗两遍，0.1％结晶紫水溶液染色30min，PBS洗3遍，观察实验结果并拍照。
由图2可看出本发明合成的化合物12a,16a比参比药物CI994具有更强的抗肿瘤细胞单克隆形成的能力。
实施例5：化合物诱导肿瘤细胞凋亡实验
取对数生长期的A549肺癌细胞铺于6孔板，每孔铺16万个细胞，24h后加入CS055，CI994和12a(浓度为4μmol/L和8μmol/L)处理细胞，以DMSO处理为对照组，48h后收集每孔的培养基，用胰酶消化收集各孔细胞，1000rpm离心5min，去除离心后的上清，再用PBS洗两遍，然后加入100μL1×binding buffer缓冲液，5μL of Alexa Fluor 488 annexin和1μL的碘化丙锭(100μg/ml)，避光室温孵育15min，以Alexa Fluor 488 annexin和碘化丙锭单染的组别作为对照组，样品用MoFlo XDP流式细胞仪检测。
由化合物诱导肿瘤细胞凋亡的细胞流式图可看出本发明合成的化合物12a在8μmol/L浓度下能够诱导64.4％的A549肺癌细胞发生凋亡，而CS055(西达本胺)仅能诱导22.42％的A549肺癌细胞凋亡，CI994仅能诱导12.58％的A549肺癌细胞凋亡。由此可见本发明所合成的化合物具有更强的诱导肿瘤细胞凋亡的能力。
实施例6：化合物阻断肿瘤细胞周期实验
取对数生长期的A549肺癌细胞铺于6孔板，每孔铺16万个细胞，24h后加入CS055，CI994和12a(浓度为2μmol/L和16μmol/L)处理细胞，以DMSO处理为对照组，48h 后收集每孔的培养基，用胰酶消化收集各孔细胞，1000rpm离心5min，去除离心后的上清，再用PBS洗两遍，细胞用70％乙醇固定，PBS洗两遍，加入RNA酶37℃避光孵育30min，样品用MoFlo XDP流式细胞仪检测。
结果显示无论在2μmol/L还是16μmol/L浓度下CS055和CI994对A549肺癌细胞的细胞周期基本无影响，而本发明合成的化合物12a在16μmol/L浓度下能将癌细胞周期阻断在G2/M期(加DMSO处理的对照组癌细胞处于G2/M期的比例为9.1％，经过16μmol/L化合物12a的处理后，处于G2/M期癌细胞的比例增加到37.74％)。

Claims (8)

  1. 一种苯甲酰胺类化合物，其特征在于下列通式(I)所示的结构：
    Figure PCTCN2017091211-appb-100001
    其中R1为氢，氟，氯，溴，碘，烷基，烷氧基，烷基羰基，烷氧基羰基，烷酰胺基，硝基，氰基，芳基或杂芳基；
    R2为氢，氟，氯，溴，碘，烷基，烷氧基，烷基羰基，烷氧基羰基，烷酰胺基，硝基，氰基，芳基或杂芳基。
  2. 苯甲酰胺类化合物，其特征在于，权利要求1所述的苯甲酰胺类化合物与无机酸、有机酸、无机碱或有机碱所形成的盐，与水或溶剂形成的水合物或溶剂合。
  3. 根据权利要求1所述的苯甲酰胺类化合物，其特征在于，所述的苯甲酰胺类化合物选自:
    Figure PCTCN2017091211-appb-100002
    Figure PCTCN2017091211-appb-100003
  4. 权利要求1至3中任意一项所述的化合物在制备抑制癌细胞增殖或治疗癌症药物中的应用。
  5. 根据权利要求4所述的应用，其中癌细胞或癌症包括：人肺癌细胞A549，人胃癌细胞MGC80-3，人肝癌细胞HepG2，人结肠癌细胞HCT116，食管癌，胰腺癌，直肠癌，宫颈癌或卵巢癌。
  6. 如权利要求1所述化合物的制备方法，其特征在于包括以下步骤：
    (a)将取代苯酚与化合物1缩合反应得到化合物2；
    Figure PCTCN2017091211-appb-100004
    (b)将化合物2水解为化合物3，并酸化为化合物4
    Figure PCTCN2017091211-appb-100005
    (c)将化合物4与化合物5进行缩合反应得到化合物6
    Figure PCTCN2017091211-appb-100006
    其中R1为氢，氟，氯，溴，碘，烷基，烷氧基，烷基羰基，烷氧基羰基，烷酰胺基，硝基，氰基，芳基或杂芳基；
    R2为氢，氟，氯，溴，碘，烷基，烷氧基，烷基羰基，烷氧基羰基，烷酰胺基，硝基，氰基，芳基或杂芳基。
  7. 如权利要求6所述的制备方法，其特征在于：反应步骤(c)使用肽缩合剂作催化剂。
  8. 如权利要求7所述的制备方法，其特征在于：所述肽缩合剂选自苯并三氮唑-1-基氧基三(二甲基氨基)磷鎓六氟磷酸盐。
PCT/CN2017/091211 2016-07-25 2017-06-30 一种苯甲酰胺类化合物及其在制备抑制癌细胞增殖和/或治疗癌症的药物中的应用 WO2018019084A1 (zh)

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